Abstract
K. Steece-Collier
Department of Neurology, Udall Center of Excellence, University of Cincinnati, Cincinnati, OH, USA
According to a recent hypothesis, neurodegenerative diseases are age-related diseases of specific brain regions that have developed relatively recently in evolutionary time in Homo sapiens (Ghika, 2008). Alzheimer's disease was the first to be thought of as a disease of “phylogenic regression” (Rapoport 1988, 1989), a concept comparing brain aging “involution” to the reversed phenomenon of darwinian evolution. There is recent scientific evidence that suggests that “premature aging” associated with neurodegeneration in humans involves phyllogenetically/evolutionarily recent cortical areas, that are more prone to cell death because of higher levels of plasticity due to more rapid evolution. It is hypothesized that premature aging of the “new” cortical areas may be due to ongoing evolution and as of yet “not fixed genome” in newly established cortical networks that have specific sensitivity to age and apoptosis, mutations, or loss of differentiation because of higher levels of plasticity and wider connections as secondary or integrative cortical areas (Ghika, 2008). These ideas are an intriguing way of thinking of neurodegenerative diseases; however, their validity remains to be determined. In the ASNTR Presidential Lecture for 2010, renowned human geneticist and molecular biologist Dr. John Hardy, UCL Institute of Neurology, London, will present a talk entitled “Whole Genome Analysis of Neurodegenerative Disease.” He will discuss genome-wide association studies and human disease genetic risk factors for neurologic diseases, highlighting in a portion of his talk evidence suggesting that Homo neanderthalensis contributed the H2 MAPT haplotype to Homo sapiens. The idea of paleoneurology may give a new perspective of seeing clinical signs in neurodegenerative diseases as “homo-specific syndromic presentations rather than patho-specific diseases” (Ghika, 2008).
J. F. Abisambra,* L. J. Blair,* C. Kraft,* S. Hill,* J. R. Jones,* J. Rogers,† J. Koren, III,* U. K. Jinwal,* A. G. Johnson,* K. Janesn-West,‡ J. Banko,† M. Muschol,* T. E. Golde,‡ E. J. Weeber,† and C. A. Dickey*
*Department of Molecular Medicine, USF Health Byrd Alzheimer's Institute, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology, USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
‡Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, FL, USA
In many diseases of aging, aberrant protein production is a common feature. The majority of these disease-associated proteins are also clients of the chaperone network. These abnormal clients are often prone to aggregation, forming pathologic inclusion bodies in neurodegenerative diseases. One of the chaperones that may be able to help offset the aggregation of these proteins is heat shock protein 27 kDa (Hsp27). Hsp27 exists in quiescent cells as large inactive multimers (200–800 kDa) that can be disassembled by phosphorylation at three serine residues (S15, S78, and S82) during stress, a process that has previously been shown to protect unfolded proteins from aggregating. The current information about a role for Hsp27 in regulating tau biology is restricted to in vitro data showing that Hsp27 interacts with tau and promotes its degradation. Therefore, we endeavored to define a mechanism of how Hsp27 could regulate tau aggregation and then determine for the first time whether acute overexpression of Hsp27 could reduce tau in transgenic mice and improve neuronal function. Using a novel adaptation of a technique that incorporates Ponceau S staining with near-infrared imaging, along with dynamic light scattering, we show in vitro that both wild-type and a mock phosphorylated form of Hsp27 can prevent tau from aggregating. Then, we investigated how these two Hsp27 variants might impact tau pathogenesis in vivo using the rTg4510 transgenic mouse model of human tauopathy. Our previous work showed that Hsp27 levels were not present in perinatal mice, suggesting that traditional transgenic overexpression might have deleterious developmental consequences. To overcome this, we generated adeno-associated viral (AAV) particles expressing each Hsp27 variant, which were injected into mice that were 1 month of age or older. We combined convection-enhanced delivery with AAV9 and intraperitoneal mannitol injections to facilitate maximal distribution of the intrahippocampal injection of each virus. We show that while distribution was widespread throughout the hippocampus, not all neurons expressed the vector contents; however, in the neurons that did express Hsp27, we found that wild-type Hsp27 reduced tau significantly, while the mock-phosphorylated mutant form of Hsp27 actually colocalized with tau to a greater extent, but caused tau levels to increase. Thus, we prove for the first time in vivo that Hsp27 is able to clear tau from the brain only when it is able to dynamically cycle its phosphorylation state. If this cycle is interrupted, as is the case with the mock-phosphorylated form, Hsp27 can still bind tau, but tau is actually stabilized. Finally, we show for the first time that rTg4510 mice have deficits in long-term potentiation (LTP) and that the reductions in tau caused by Hsp27 can restore these deficits, suggesting that therapeutic strategies aimed at increasing Hsp27 levels might be effective in tauopathies, and possibly other neurodegenerative diseases of proteotoxicity.
M. M. Acharya, L.-A. Christie, M. L. Lan, and C. L. Limoli
Department of Radiation Oncology, University of California, Irvine, CA, USA
Irradiation of the central nervous system (CNS) usually occurs under a clinical setting involving radiotherapy. Nearly all patients diagnosed with brain tumors (either primary or metastatic) undergo radiation as a primary treatment modality. While radiotherapy is beneficial, it is limited by the tolerance of normal tissue and resident stem cell pools to radiation injury, and can lead to the serious consequence of cognitive dysfunction, characterized by a range of learning and memory deficits. Thus, as a potential strategy to improve cognition after radiotherapy, we employed human embryonic stem cell (hESC) transplantation into the rat hippocampus shortly after cranial irradiation. hESCs were expanded and labeled (BrdU) in the pluripotent state prior to transplantation. Two-month-old male athymic nude rats were subjected to a clinically relevant dose (10 Gy) of head-only radiation and, at 2 days postirradiation, surgically grafted at four distinct sites along each hippocampi with cells (100,000 cells/site, 8 × 105cells/animal). Control (CON) and irradiated (IRR) rats receiving vehicle (sterile conditioned medium) served as sham surgery groups. Four months postgrafting, rats were habituated and trained on a novel place recognition task (NPR). As expected, irradiated rats (IRR) showed impaired novel place recognition compared to control animals. In contrast, irradiated rats receiving hESC grafts (IRR + hESC) did not differ from control animals (CON) and spent more time than expected by chance exploring the novel place (ANOVA, p = 0.031). To better understand the cognitive sequelae in our test groups, the total time spent exploring both objects during the familiarization phase (i.e., before the short-and long-retention intervals) was analyzed. Significant differences between the groups were found (ANOVA, p = 0.028); IRR animals tended to spend less time exploring during the familiarization phase compared to both the CON and IRR + hESC animals. These results suggested that hESC transplantation attenuated radiation-induced cognitive impairment by preserving short-term memory in a hippocampal-dependent spatial information task. Similar patterns of cognitive improvement were also observed in the 1-month postgrafting group. Histological examination of BrdU-immunostained sections from 1- and 4-month posttransplant animals revealed extensive migration of hESCs from the transplant site throughout the host hippocampus. hESCs were also found to exhibit significant homing to the neurogenic niche of the hippocampal subgranular zone. Unbiased stereology at 1 and 4 months postgrafting revealed 40% and 26% survival of transplanted cells, respectively. Ongoing analyses using dual immunofluorescence and confocal microscopy has thus far revealed that the grafted hESCs differentiated into neurons, astrocytes, and oligodendrocytes. These findings provide the first evidence that transplanted hESCs can survive, differentiate along neuronal lineages, and ameliorate cognitive impairments following cranial irradiation. These data also suggest that similar strategies can be tailored to minimize the adverse side effects associated with exposure to ionizing radiation.
Supported by a grant from the California Institute for Regenerative Medicine (CIRM) to C.L.L.
S. Acosta,* J. Jernberg,* C. D. Sanberg,† P. R. Sanberg,* B. Small,‡ C. Gemma,*§ and P. C. Bickford*§
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair and Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, USF Health, Tampa, FL, USA
†Natura Therapeutics, Inc., Tampa, FL, USA
‡School of Aging Studies, University of South Florida College of Medicine, Tampa, FL, USA
§James A. Haley Veterans Affairs Medical Center, Tampa, FL, USA
The process of aging is linked to oxidative stress, microglial activation, and proinflammatory factors, which are known to decrease cell proliferation and limit neuroplasticity. These factors may lead the transition from normal aging to more severe cognitive dysfunction associated with neurodegenerative diseases. We have shown that natural compounds such as polyphenols from blueberry, and green tea, and amino acids like carnosine are high in antioxidant and anti-inflammatory activity that decreases the damaging effects of reactive oxygen species (ROS) in the blood, brain, and other tissues of the body. Furthermore, we have shown that the combination of these nutrients (called NT020) creates a synergistic effect that promotes the proliferation of hematopoietic cells in vitro. We have also demonstrated that NT020 protects the brain from ischemic damage and increases the migration of neural stem cells to the site of injury. In the present in vivo study, we investigated the natural therapeutic potential of NT020 for improving learning and memory in aged rats. We also examined the effect of NT020 on neurogenesis in the major stem cell niches of the brain, the subgranular layer of the dentate gyrus and in the subventricular zone. To test this hypothesis, three groups were chosen to be treated with either NT020 or water gavage. Aged (20 months old) male Fisher 344 rats were treated with 135.0 mg/kg per day (n = 13) of NT020. Young (3 months old) (n = 10) and aged control (20 months old) (n = 13) male Fisher 344 rats were treated with water by oral gavage. All groups were treated for a period of 4 weeks. Rats were tested for learning on a Morris water maze and on the fifth day of training there were fewer aged impaired animals in the treatment group compared with controls. Using the cell cycle-regulating protein (Ki67), doublecortin (DCX), and OX6 antibody markers, neurogenesis, cell proliferation, and microglial activation were estimated in the dentate gyrus (DG) of young and aged animals. Cell proliferation was also examined in the subventricular zone (SVZ). Decreased number of OX6 MHC II-positive cells, increased neurogenesis, and increased number of proliferating cells were found in rats treated with NT020 in comparison with aged control rats. In sum, NT020 may promote health, proliferation, and maintenance of neurons in the aged animals and exert anti-inflammatory actions, which promote function in the aged stem cell niche.
This work was supported by USPHS grant AG04418 and the VAMRS. P.C.B. and P.R.S. are founders of NaturaTherapeutics, Inc.
M. Airavaara,* C. Richie,* P. J. Castillo,* M. J. Chiocco,* D. B. Howard,* J. Peränen,† C. Liu,‡ S. Fang,‡ Y. Wang,* B. J. Hoffer,* and B. K. Harvey*
**Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, USA
†Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
‡University of Maryland Biotechnology Institute, Baltimore, MD, USA
Mesencephalic astrocyte-derived neurotrophic factor (MANF) is a secreted protein that reduces endoplasmic reticulum (ER) stress and exhibits neuroprotective effects. We have previously reported that ex-ogenously administered recombinant MANF protein has neuroprotective effects against cerebral ischemia in rodents (Airavaara et al., JCN, 2009). In the current study, we used an adeno-associated virus expressing human MANF (AAV-MANF) to examine the effects of enhanced, endogenous MANF production in a rat model of stroke. AAV-MANF or an AAV vector expressing the green fluorescent protein (AAV-GFP) was administered into three cortical sites (AP −0.3, ML 5.5, DV −3.5—1.5, sites 1.5 mm apart) at a dose of approximately 1 × 1010 viral genomes per site in chloral hydrate-anesthetized rats. Administration of AAV-MANF significantly increased MANF protein expression, as determined by immunostaining and Western blot analysis, at 1 week after injection. In another set of animals, the right middle cerebral artery (MCA) was transiently ligated with a 10–0 suture for 60 min at 1 week after viral injections. Immunostaining shows a redistribution of MANF immunoreactivity 1 day after MCA occlusion. AAV-MANF pretreatment decreased infarction volume, body asymmetry, and neurological score compared to animals receiving AAV-GFP. Western blot analysis shows that AAV-MANF reduces ischemia-induced expression of binding immunoglobulin protein (BiP), a key regulator of the unfolded protein response (UPR). Given that MANF is a highly conserved protein present in nematodes to humans, we further examined the function of the orthologous protein (MANF-1) in Caenorhabditis elegans. Animals carrying a homozygous deletion in the manf-1 gene are viable and have a normal plate phenotype. However, these animals exhibited increased basal expression from a UPR-inducible GFP reporter, specifically, a BiP homolog promoter. Furthermore, animals with the manf-1 deletion failed to show the normal developmental arrest that occurs with chronic exposure to tunica-mycin, a chemical inducer of ER stress and UPR. Taken together, our data show that administration of AAV-MANF into rat cortex increases MANF production and reduces neurodegeneration induced by cerebral ischemia. This protection may involve the ability of MANF to alter BiP protein, a key regulator of the UPR.
J.-O. Andressoo,* J. Mijatovic,† A. Kuman,* C. Amberg,* S. Grealish,‡ M. Lindahl,* A. Björklund,‡ J. Lindholm,§ E. Castén,§ N. Kulesskaya,§ H. Rauvala,§ P. Piepponen,† and M. Saarma*
*Institute of Biotechnology, University of Helsinki, Helsinki, Finland
†Faculty of Pharmacy, University of Helsinki, Helsinki, Finland
‡Division of Neurobiology, Lund University, Lund, Sweden
§Neuroscience Center, University of Helsinki, Helsinki, Finland
Parkinson's disease (PD) affects over 1% of people over age of 60, has mostly unknown etiology, and is currently incurable. In PD midbrain dopamine (DA) neurons and their striatal projections specifically degenerate. The discovery of neurotrophic factors (NTFs) such as glial cell line-derived neurotrophic factor (GDNF), which can specifically support the survival of DA neurons, raised hope for the conceptually new treatment for PD. GDNF, together with conserved dopamine neurotrophic factor (CDNF) and mesencephalic astrocyte-derived neurotrophic factor (MANF), is the most potent NTF for DA neurons known to date. Some data also suggest that GDNF reduction upon aging may be causative of PD. Whether this is the case is still a matter of speculation. Phase I and II clinical trials with GDNF or its homologous protein neuturin (NRTN) for PD treatment have proved the principle but the results vary substantially in efficacy and in extent of adverse and side effects between different studies. A better understanding of GDNF biology in vivo may therefore help to improve its therapeutic use. Currently some fundamental questions have remained unanswered, including: (i) How does endogenous GDNF and its receptor GFRa1 affect the postnatal development of the brain dopamine (DA) system? (ii) Is embryonal or adult deletion of GDNF or GFRa1 sufficient to cause degeneration of DA neurons similar to Parkinson's disease (PD)? (iii) What is the role of GDNF and GFRa1 in maintaining the adult DA system? (iv) Are there alternate receptors for GDNF in the brain? (v) What is the role of GDNF/GFRa1 in memory, learning, and behavior? The above questions have remained difficult to address because knock-out (KO) mice lacking GDNF or its receptor GFRa1 die at birth due to the lack of kidneys and enteric nerves, whereas most of the essential brain connections mature postnatally. At birth, the DA system in GDNF KO mice is indistinguishable from the wt. To study the postnatal role of GDNF one has to generate GDNF and GFRa1 conditional KO (cKO) mice, where GDNF can be specifically deleted (e.g., only in the brain, enabling us to bypass the neonatal lethality). To avoid possible compensation of the absent factor in the KO model by alternative factors frequently observed in the KO animals and to assess the impact of GDNF levels in mice over- or underexpressing the gene products of interest would be equally valuable. Here we report generation of GDNF and GFRa1 cKO mice and knock-in mice overexpressing GDNF in naturally GDNF-producing cells (GDNF hypermorphs) as well as mice underexpressing GFRa1 in GFRa1 naturally expressing cells (GFRa1 hypomorphs). We report unpublished results from the first-round analysis on the roles of GDNF levels on postnatal brain function with the focus on the brain DA system.
E. Bible,* B. Solanky,* E. T. Ahrens,†‡ and M. Modo*
*Centre for Cellular Basis of Behaviour, Kings College London, London, UK
†Pittsburgh NMR Centre for Biomedical Research, Carnegie Mellon University, Pittsburgh, PA, USA
‡Department of Biological Sciences, Carnegie Mellon University, Pittsburgh, PA, USA
Tracking transplanted cells in vivo by MRI provides a means to study transplanted neural stem cells (NSCs) and potential mechanisms of repair longitudinally after brain injury. Reliably detecting transplanted cells on MR images remains difficult, however, due to the relatively low resolution of MR images for detecting small numbers of cells and the lack of contrast between transplanted cells and intact brain parenchyma. These problems can be overcome to some degree by the use of contrast agents to label cells prior to transplantation, but some of these have been found to adversely affect functional recovery. Ideally, therefore, a contrast agent for clinical applications would not contain metal particles to exert any deleterious effects on cellular functions. We therefore investigated a bimodal 19F contrast agent to label human neural stem cells (hNSCs) for detection by MRI and fluorescent microscopy. A comparison of agent incorporation in three different hNSC lines in vitro indicated that cell viability only dropped marginally with increasing agent concentration, but was generally within the same range as control values. We also established that the addition of 10% serum to media was essential during agent incorporation to minimize the presence of excess extracellular agent that would give false positives during imaging. The extent to which the agent was incorporated into cells, however, differed depending upon cell line. Cortical cell lines incorporated the agent, but a substantial amount remained extracellularly. In contrast, a striatal line incorporated the agent with minimal extracellular excess. These encouraging in vitro results now pave the way to evaluate the potential of this 19F contrast agent for the long-term in vivo monitoring of transplanted NSCs in stroke-lesioned animals.
E. Bible,* D. Y. Chau,† M. R. Alexander,† J. Price,* K. M. Shakesheff,† and M. Modo*
*Centre for Cellular Basis of Behaviour, Kings College London, London, UK
†Division of Advanced Drug Delivery, Centre for Biomolecular Sciences, School of Pharmacy, University of Nottingham, Nottingham, UK
After ischemic injury a cystic cavity is formed in the brain, surrounded by damaged/dying cells. Stem cell transplantation therapies have routinely involved injection of cell suspensions into the adjacent or contralateral surviving parenchyma. Although NSCs have been shown to migrate to the site of injury, the cystic nature of the cavity precludes reconstruction of lost tissue. Our previous work has demonstrated that NSCs can be transplanted directly into the lesion cavity on a microparticle scaffold system with the aim of providing structural support within the lesion to promote tissue regeneration from transplanted cells. Nevertheless, there was a lack of blood vessel ingrowth into the transplantation site and lesion area, which may affect the long-term survival of transplanted NSC. To overcome this problem, we have transplanted NSCs on microparticles that secrete vascular endothelial growth factor (VEGF) directly into the cyst to increase vessel ingrowth and ultimately cell survival in the grafted area. We have assessed in vitro the cell viability, proliferation, and differentiation on VEGF microparticles and found no negative effect from VEGF released from microparticles. In vivo transplanted cells were located at the lesion site 2 weeks after transplantation with an increase of collagen IV, a blood vessel membrane marker. We conclude that transplantation of cells on VEGF-releasing microparticles may provide a means to encourage revascularization of the lesioned area and aid the survival of transplanted cells for tissue regeneration in the cyst caused by stroke damage.
K. B. Bjugstad and A. Rachubinski
Department of Pediatrics, Neuroscience Program, University of Colorado Denver, Aurora, CO, USA
Neural progenitor cells (NPC) have been used with considerable success in adult models of neurodegeneration. Because of these encouraging results, it was hypothesized that NPC might be beneficial in pediatric models, such as Down syndrome (DS). If implanted early enough in the degenerative process, then NPC might alter the course of DS progression. Previously we reported on the emergence of developmental milestones in mouse pups that were implanted with either murine NPC (C17.2 line) or with saline on postnatal day 2 (PND2). We found that saline treatment delayed ear opening in disomic and trisomic pups, compared to unimplanted controls. In addition, disomic pups implanted with NPC were delayed in developing the negative geotaxis reflex. The current presentation follows up on some of these pups. Retired female breeders (11–12 months), implanted with saline or NPC on PND2 or nonimplanted, were tested in the conditioned taste avoidance (CTA) and in the novel object recognition (NOR) tasks. In CTA, mice learn to avoid a novel flavor during a second exposure after their first exposure elicited a feeling of nausea. All animals significantly preferred the novel flavor on the first exposure. With the second exposure, nonimplanted disomic mice learned the association between the novel flavor and the nausea. These mice avoided drinking the novel flavor whereas nonimplanted trisomic mice continued to show a preference, drinking more of the novel flavor than their average daily water consumption. PND2 implantation of saline or NPC appeared to impair the ability of the disomic mice to learn the association. Contrary to the results in disomic mice, NPC and, to a lesser extent, saline improved the ability of trisomic mice to learn the association. In NOR, the mice were allowed to explore two different novel objects. The following day, one of the objects was replaced by a new one and mice were again allowed to explore. The ratio of time spent exploring the objects was used as a measure of recognition. As before, the nonimplanted trisomic mice were impaired in this task whereas the nonimplanted disomic mice spent significantly more time exploring the new object compared to the old object. The only other group to spend a disproportionate amount of time exploring the new object was the saline-implanted trisomic mice. Disomic mice implanted with saline or NPC appeared impaired in their ability to recognize an old object, spending equal time with both objects. NPC implanted into the trisomic mice did not alter their behavior compared to the nonimplanted trisomic mice. Preliminary neuroanatomical analysis suggests that, at 12 months, there are very little or no surviving NPC in either the disomic or trisomic brains. The current data suggest that implanting NPC neonatally may not be as beneficial as the results obtained in adult models. Behavioral analyses suggest that the act of implanting, regardless of whether it is saline or NPC, could be altering behavior long term. In the case of trisomic mice, the act of implanting itself could be beneficial but probably should be carefully considered for normal disomic controls.
B. C. Blanchard, B. M. Pelka,* and J. R. Sladek, Jr.
Departments of Pediatrics and Neurology, University of Colorado Denver School of Medicine, Denver, CO, USA
Transplantation of dopamine (DA) neurons from fetal ventral mesencephalon (VM) has proven to be an effective ameliorative measure for the relief of symptoms caused by Parkinson's disease. In humans, fetal DA grafts placed into the host striatum sometimes have resulted in dyskinesias, suggesting an excess or unregulated release of DA from grafted neurons. We previously hypothesized that grafts that were placed into the substantia nigra (SN) may offer an opportunity for better afferent control than those placed into the striatum if neuritic outgrowth from the VM grafts to the striatum could be achieved. In prior studies in primates we showed that this is possible with the help of grafts of lateral ganglionic eminence (LGE) placed at progressively more rostral sites along the nigrostriatal pathway, with caudal LGE being placed closest to the VM graft and rostral LGE being placed more distal to the VM graft, resulting in the extension of neurites from the VM graft to the host striatum. Our current study attempts to demonstrate the existence of a developmental growth factor gradient of striatal trophic factors, from caudal striatum to rostral that is responsible for the extension of neurites during the establishment of the nigrostriatal pathway. Adult male rats were lesioned unilaterally with 6-OHDA, and then randomly assigned to one of the following four groups for transplant surgery: A) unilateral sham surgery, B) unilateral transplants of VM into SN, C) unilateral transplants of VM into SN in conjunction with caudal LGE placed closest to the VM graft and rostral LGE placed more distal to the VM graft, thus maintaining the proper anatomical orientation along the nigrostriatal tract, and D) unilateral transplants of VM into SN in conjunction with caudal LGE and rostral LGE placed in the reverse anatomical orientation along the nigrostriatal tract. Rotational behavior in response to amphetamine and apomorphine was assessed at 6, 12, and 18 weeks after transplant surgery. At the completion of behavioral testing animals were perfused with 4% paraformaldehyde and processed for immunohistochemistry. All animals were analyzed for potential DA fiber outgrowth from the VM grafts to the fetal LGE grafts in response to the orientation of the striatal grafts and will be described in detail at ASNTR 17.
Supported by NINDS Grant 1R21 NS065353-01.
F. Blandini,* L. Cova,† M.-T. Armentero,* P. Bossolasco,‡ E. Polli,‡ G. Nappi,*§ and V. Silani†
*Interdepartmental Research Center for Parkinson's Disease, IRCCS Neurological Institute “C. Mondino”, Pavia, Italy
†Department of Neurology and Laboratory of Neuroscience, University of Milan-IRCCS Istituto Auxologico Italiano, Milan, Italy
‡Fondazione Matarelli, University of Milan, Milan, Italy
§University of Rome “La Sapienza”, Rome, Italy
Progressive loss of dopamine-containing neurons of the nigrostriatal pathway is the pathological hallmark of Parkinson's disease (PD). Replacement of deficient neurotransmitter dopamine with its direct precursor, L-DOPA, is still the gold standard therapy for the disease. However, L-DOPA treatment just relieves PD motor symptoms, without modifying the course of the disease, and, in the long-term, is hampered by significant side effects. Neuroregeneration, based on the use of pluripotent stem cells, has been proposed as a new therapeutic strategy for PD. Controversial results, however, have been obtained so far, particularly with embryonic or neural stem cells, which has limited the translation of this approach into the clinical setting. As an alternative to heterologous embryonic or neural precursor cells, the use of autologous, bone marrow-derived mesenchymal stem cells (MSCs) has been recently proposed. These cells are virtually devoid of oncogenic potential and have differentiative multilineage capacity, including the ability to differentiate toward the dopaminergic phenotype, at least in vitro. The most interesting feature of MSCs, however, is represented by their immunoregulatory and neurotrophic properties. These cells may, therefore, have the capacity of modulating the neuroinflammatory response associated with PD while supporting surviving neurons, ultimately providing neuroprotection. We have transplanted undifferentiated, human MSCs into the striatum of rats previously lesioned with 6-hydroxydopamine (6-OHDA), a neurotoxin that induces profound neuronal loss in the nigrostriatal pathway. Following transplantation, a relative amount of MSCs acquired a neuroglial phenotype, as expressed by immunopositivity for glial fibrillary acid protein (GFAP), but only in animals bearing the nigrostriatal lesion. Transplanted animals showed increased survival of nigrostriatal dopaminergic neurons, which was associated with a reduction of the 6-OHDA-induced motor stereotypies (turning behavior). Increased survival of dopaminergic neurons in transplanted animals was associated with enhanced levels of neurotrophic factors, such as brain-derived neurotrophic factor (BDNF) and glial-derived neurotrophic factor (GDNF), in the nigrostriatal tract. These results support recent observations suggesting that acquisition of a neuronal phenotype, by grafted MSCs, is not required for the beneficial effect to manifest; indeed, this effect may be based on the anti-inflammatory/trophic effects MSC are likely to exert through the release of soluble factors. Furthermore, in line with the view that triggering endogeneous neuroregeneration (neurogenesis) may represent an alternative way to replace lost neurons, transplantation of MSCs promoted neurogenesis in the ependymal layer of the subventricular zone. In fact, rats that received the MSC graft showed increased cell proliferation in this area, as indicated by the increased number of cells positive for Ki-67 and proliferating cell nuclear antigen (PCNA), and enhanced migration of doublecortin-positive neuroblasts toward the lesioned striatum. In conclusion, the use of MSCs in the 6-OHDA model induced beneficial effects that may rely on multiple mechanisms, thereby supporting the therapeutic potential of this procedure for PD.
H. A. Boger, K. R. S. Reinert, and A.-C. Granholm
Department of Neurosciences and Center on Aging, Medical University of South Carolina, Charleston, SC, USA
Inflammation has been implicated in the pathology of several neurodegenerative diseases, including Parkinson's disease (PD). Studies using the endotoxin lipopolysaccharide (LPS), a potent inflammogen, show that systemic insults can trigger prolonged microglial activation and proinflammatory cytokine production, leading to delayed and progressive death of substantia nigra (SN) dopamine (DA) neurons, mimicking idiopathic PD. Therefore, the focus of this study is to assess time-dependent alterations in neuroinflammation, DAergic neurons, and neuronal signaling cascades in the SN following LPS administration. In this study, high-dose LPS (5 mg/kg, IP) or saline (0.9% NaCl) was administered to 8-month-old male c57Bl/6 mice. At 1, 3, 5, and 12 h postinjection, the SN was assessed for tyrosine hydroxylase (TH, DAergic marker), Iba-1 (pan-microglial marker), phospho-Erk (extracellular signal-regulated kinases signaling), and phospho-CREB (cAMP response element binding signaling). At 1, 3, and 5 h postinjection, high-dose LPS resulted in a significant reduction in SN TH-ir compared to saline controls, with a similar reduction in TH density observed at all three postinjection times. However, the high dose of LPS (5 mg/kg) resulted in the greatest reduction of SN TH-ir at 12 h postinjection, compared to all other groups. In regards to neuroinflammation, the SN of high-dose LPS-treated mice had an increase in microglia activation at all postinjection time points, with the greatest increase in activated microglia observed at 5 h post-LPS injection. By 12 h postinjection, high-dose LPS-treated mice exhibited activated as well as reactive microglia; the latter are known to produce inflammatory cytokines that can result in toxic damage to neuronal populations. These data demonstrate that the initial reduction in TH immunoreactivity observed after an LPS injection is not concomitant with morphological alterations in microglial cells. It is instead possible that the initial alteration in DA phenotype (TH reduction) may perpetuate an inflammatory response that persists and leads to further DAergic damage.
This work is supported by NIA PO1 AG023630.
C. V. Borlongan,* S. J. Yu,* Y. Kaneko,* H. Shojo,* E. C. Bae,* D. H. Park,* D. J. Eve,* P. R. Sanberg,* C. D. Sanberg,† P. C. Bickford,*‡ B. Roschek, Jr.,§ and R. S. Alberte§
*Department of Neurosurgery and Brain Repair, Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Natura Therapeutics, Inc., Tampa, FL, USA
‡James A. Haley Veterans Affairs Medical Center, Tampa, FL, USA
§HerbalScience Group LLC., Naples, FL, USA
Plant extracts have been indicated as potent therapeutic compounds in a number of neurological disorders. Here, we examined the prophylactic and restorative benefits of cacao and sage using both in vitro and in vivo models of stroke. For the in vitro study, we initially exposed primary rat cells to the established oxygen-glucose deprivation (OGD) stroke model followed by reperfusion under normoxic conditions, then added different cacao and sage concentrations to the cell culture media. Trypan blue cell viability results revealed specific cacao and sage dosages exerted significant therapeutic effects against OGD-induced cell death compared to cultured cells treated with extract vehicle. Based on these robust poststroke restorative benefits of the extracts in the cell culture study, we next embarked on testing the therapeutic effects of cacao and sage extracts when treatment commenced either 1 h prior to or 1 h after transient middle cerebral artery occlusion (MCAo). Significant reduction in ischemic cell death within the peri-infarct area coupled with better performance in routine motor and neurological tasks were demonstrated by stroke animals that received cacao or sage extracts prior to MCAo compared to those that received the extracts or vehicle after MCAo. Taken together, these results demonstrate that neuroprotective effects were afforded by plant extract treatment, and that the in vitro stroke paradigm partially approximates in vivo efficacy when considering prophylactic treatment for stroke. The novel finding here is that acute treatment (i.e., single oral administration) of the extracts produces robust neuroprotection against stroke.
P.R.S. and P.C.B. are cofounders of Natura Therapeutics.
S. Borysov,*† J. Wu,‡§ J. Padmanabhan,*† and H. Potter*†
*Department of Molecular Medicine, University of South Florida, Tampa, FL, USA
†USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
‡Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
§Electrophysiology Core at USF Health, University of South Florida, Tampa, FL, USA
Chromosome mis-segregation and defective neuronal plasticity implicated in Alzheimer's disease may result from amyloid-beta (Aβ)-induced microtubule dysfunctions. Microtubule-based motors are critical for the proper organization and functions of microtubule cytoskele-ton structures during a variety of cellular processes, including mitosis and the development of neuronal processes. Therefore, we hypothesize that Aβ abrogates functions of specific microtubule motors, thereby leading to aneuploidy and impaired neuronal plasticity. Aβ's interaction with motor proteins was studied by performing coimmunoprecipitation and affinity purification. Aβ's effects on the motors' activities were studied in an in vitro microtubule-dependent ATPase assay. Aβ's effects on the mitotic spindle were studied by using Xenopus egg extracts. Effects of the Eg5 inhibitor, Monastrol, on neuronal processes were studied by using a long-term potentiation (LTP) assay. Aβ induced defective spindle phenotypes, which are indicative of malfunctioning kinesin family motor proteins, Eg5, KIF4A, and MCAK. Consistently, Aβ directly associated with these mitotic motors, inhibited their activities in vitro, and abrogated their association with mitotic spindles. Interestingly, we showed that neurons express all of these motor kinesins, implying that their inhibition by Aβ may contribute to neuronal dysfunctions associated with Alzheimer's disease. In support of this, we showed that neuronal Eg5 is inhibited when neurons are exposed to elevated concentrations of Aβ. Further, treatment of hippocampus slices with the Eg5-specific inhibitor Monastrol significantly reduced LTP readings to levels comparable to the exposure with Aβ. The data herein support a unified explanation for aneuploidy and defective neuronal plasticity associated with Alzheimer's disease as resulting from Aβ direct inhibition of specific microtubule motors, including Eg5, KIF4A, and MCAK.
M. Brownlow, N. Carty, D. Williams, K. Nash, M. N. Gordon, and D. Morgan
Alzheimer's Research Laboratory, Department of Molecular Pharmacology and Physiology, School of Biomedical Sciences, University of South Florida College of Medicine, Tampa, FL, USA
Amyloid β (Aβ) deposition is one of the major neuropathological hallmarks of Alzheimer's disease (AD), so reducing amyloid burden may present a possible therapeutic strategy. One of the proteases involved in the degradation of Aβ peptides is neprilysin (Nep). In this study, we investigated the long-term effects of an intracranial administration of a recombinant adeno-associated viral vector (rAAV) expressing a Nep construct on amyloid deposition in APP/PS1 transgenic mice. Three constructs of Nep were employed. First, the native, membrane-bound form of Nep (Nep-N) was expressed under the control of the chicken β-actin promoter. In addition, a gene construct directing secretion of active Nep (Nep-S) and a gene construct containing a single point mutation at the enzymatic active site precluding proteolysis (Nep-M) were created. The rAAV vectors were injected bilaterally into the anterior cortex and hippocampus of 7-month-old mice, while noninjected transgenic mice were used as controls. Brain tissue was collected 9 months later when mice were 16 months of age. Immunohistochemical data suggest a reduction in Aβ staining in the hippocampus of mice injected with either the native or the secreted form of neprilysin compared with mice that received the proteolytically inactive Nep. Additional analyses of other markers of amyloid and microglial activation are ongoing. These findings suggest that increasing the expression of the Aβ-degrading enzyme neprilysin through gene therapy is a promising approach to the treatment of AD.
Supported by NIH grant AG 25509.
S. A. Busch,* J. A. Hamilton,† K. P. Horn,* F. X. Cuascut,* N. Lehman,† R. J. Deans,† A. E. Ting,† J. Silver,* and R. W. Mays†
*Department of Neurosciences, Case Western Reserve University, Cleveland, OH, USA
†Department of Regenerative Medicine, Athersys, Inc., Cleveland, OH, USA
There is substantial interest in stem cell transplantation after spinal cord injury. A phenomenon known as axonal dieback, the long-distance retraction of severed axons, occurs after many types of CNS injuries and infiltrating macrophages contribute directly to this process. We have developed an in vitro model in which adult rat dorsal root ganglion (DRG) axons are confronted with a gradient of increasing inhibitory proteoglycan and decreasing laminin. Growth cones in this environment develop a characteristic dystrophic morphology and, when contacted by activated macrophages, undergo dramatic axonal dieback. Endogenous NG2+ progenitor cells can stabilize axons after macrophage-mediated axonal dieback, but cannot prevent dieback. In this study, we sought to determine if culturing DRG neurons with rat multipotent adult progenitor cells (MAPC) or MAPC-conditioned media (MAPC-CM) could prevent macrophage-mediated axonal dieback. In the presence of MAPCs dystrophic axons became remarkably active. Macrophages contacted these axons extensively, but generally contacts were quickly broken and axonal retraction was prevented. Direct addition of MAPC-conditioned media to the time lapse dish enabled growth cones to extend further into the inhibitory rim, and occasionally robust branching was observed. Macrophages still contacted these axons, but did not induce axonal retraction. To determine the growth-promoting capability of MAPC-CM in vitro, we compared dissociated DRG neurons on a laminin substrate treated with MAPC-CM or control media and measured the longest neurite of every neuron. MAPC-CM-treated neurons exhibited a significant increase in outgrowth over control media and basal media conditions after 24 h. We next sought to determine if MAPC could prevent axonal dieback or promote regrowth of injured axons in vivo in a dorsal column crush model of spinal cord injury. We transplanted MAPC into the spinal cord immediately following injury and measured axonal position at 2, 4, and 7 days postinjury. The transplanted cells successfully integrated into the lesioned spinal cord tissue and associated with the endings of injured axons. Four days postlesion, MAPC-transplanted animals demonstrated a significant attenuation of axonal dieback normally observed at this time. Seven days postlesion, MAPC-transplanted animals showed a significant increase in the extent of axon extension into the lesion core compared to vehicle controls. Our results suggest that MAPC and MAPC-secreted factors prevent axonal dieback and promote neurite outgrowth both in vitro and in vivo after spinal cord injury.
M. M. Carballosa Gonzalez, L. Manoah, M. K. O'Connell, O. Furones-Alonso, H. M. Bramlett, and I. D. Hentall
Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA
Our laboratory has previously shown that the medulla's raphe magnus, which has a highly branched descending serotonergic projection, improves anatomical and behavioral recovery from an incomplete thoracic spinal contusion if stimulated for several days, starting within hours to a few days of the injury. We proposed that stimulation of divergent ascending serotonergic systems in the midbrain could have similar benefits in traumatic brain injury (TBI). Hence we tested the effect of 1 week of intermittent electrical stimulation in the dorsal raphe nucleus (DRN) or the median raphe nucleus (MRN) on sensorimotor and cognitive recovery after a moderate TBI. Adult male Sprague-Dawley rats (n = 50) received under isoflurane anesthesia a brief (18 ms) epidural pressure pulse (1.8–2.2 atm) applied through a fluid-coupling device over the lateral forebrain, or else received a sham operation. An epoxy-embedded battery-powered stimulator (about 2 g) with two-way remote readout and control was cranially implanted 4–6 h later. This delivered 5-min periods of alternating rest and stimulation (-30 μA, 1-ms pulses at 8 Hz) for 12 h of daylight to a Pt-Ir microelectrode in the midline DRN or MRN. Some rats had inactive stimulators as controls. At 6 weeks, hidden platform spatial learning and working memory were measured in a Morris water maze, and forelimb use asymmetry was quantified during rearing movement in a transparent cylinder, all assessed by ANOVA (p < 0.05) with Bonferroni post hoc comparison. Spatial learning in rats with TBI was superior on the second trial day when the DRN or MRN was stimulated. Forelimb use symmetry also recovered more in stimulated groups. At 14 weeks, brains were examined histologically. Anatomical analysis, presently confined to DRN, showed that stimulation bilaterally increased hippocampal volume relative to cortex (30%). Furthermore, preliminary data have demonstrated an increase in cAMP following stimulation of the MR. We propose that the enhanced sensorimotor and anatomical recovery is due to a widespread release of serotonin, leading to the increase in cAMP and the subsequent expression of neurotrophic and neuroprotective genes. In conclusion, sustained MRN or DRN activity can restore some kinds of motor and cognitive performance after TBI but may also have adverse effects. Surgery and hardware for deep brain stimulation (DBS) are readily adaptable to midbrain sites for early TBI, but benefits and drawbacks in relation to outcome predictors (injury status, stimulation modality) need further assessment.
Supported by USAMRMC W81XWH0810288.
P. M. Carvey, A. Patel, K. Collette, P. Roy, and B. Hendey
Department of Pharmacology, Rush University Medical Center, Chicago, IL, USA
We have previously demonstrated that treatment with several dopamine (DA) neurotoxins, including 1-methyl-4-phenyl 1,2,3,6 tetrahydropyridine (MPTP), 6-hydroxydopamine (6-OHDA), rotenone, paraquat, and prenatal lipopolysaccharide, leads to dysfunction of the BBB in rats and mice and that human postmortem PD tissue indicates BBB dysfunction. This dysfunction is associated with increased parenchymal entry of several BBB integrity markers including FITC-labeled albumin (FITC-LA; rats and mice), increased expression of alpha-v-beta 3 integrin (avβ3; a marker for angiogenesis; rats, mice, and humans), and is associated with microglial activation (rats, mice, and humans). We also have recently demonstrated that posttreatment of MPTP-treated mice with cyRGDfV peptide, which blocks avβ3, prevented the FITC-LA leakage, avβ3 upregulation, and microglia activation while preventing DA neuron loss (Patel et al. abstract this conference). This suggests that BBB dysfunction may be involved with the degenerative process and that the BBB dysfunction associated with toxin exposure can be repaired. We have also shown that MPTP-treated mice, but not control mice, exhibit increased entry of T cells and that the most profound entry was associated with T cells collected from MPTP-treated donors whereas less entry was observed from normal donors. This suggests the involvement of the peripheral immune system in disease progression that is apparently facilitated by the initial dysfunction in the BBB. We just recently conducted pilot studies suggesting that the peripheral administration of 1-methyl-4-phenylpyridinium (MPP+), a DA neurotoxin that normally does not enter the brain, led to further loss of DA neurons in MPTP-pretreated mice, suggesting that the BBB dysfunction allowed entry of water-soluble toxins from the periphery. This is consistent with our prior studies where we demonstrated that domperidone as well as benserazide exhibited increased entry into the rat brain following 6-OHDA lesions. Taken together, BBB dysfunction in PD is emerging as a contributor to disease pathogenesis. Thus, an initial insult to the DA neuron leads to inflammation that affects endothelial cells, leading to what appears to be compensatory angiogenesis and a breach of the BBB. This dysfunction can lead to increased entry of drugs as well as toxins that do not normally enter the brain as well as elements of the peripheral immune system, all of which can compromise effective therapy and/or mediate disease progression, respectively. Because the BBB dysfunction is punctate and encompasses only an estimated 1–2% of the nigra and striatum at any one time, this punctate leakage affords an opportunity for targeted delivery of water-soluble therapeutics that would only enter areas of active inflammation where the BBB is compromised.
Supported by The Kenneth Douglass Foundation and NS052414.
Y. H. Chiang, C.-F. Chang,† and K.-Y. Chen*†
*Department of Neurosurgery, Taipei Medical University Hospital, Taipei, Taiwan
†Department of Neurological Surgery, Tri-Service General Hospital, Taipei, Taiwan
Vascular endothelial growth factor (VEGF) has been shown to display neuroprotective effects in hypoxic-ischemic injury. Here, we investigated the neuroprotective capacity of VEGF in a rat model of stroke. To increase the in vivo transduction efficiency in the brain, we used a double-stranded adeno-associated virus 2 (dsAAV2) vector to deliver VEGF. A single administration of dsAAV2 vector carrying VEGF (dsAAV2-VEGF) 7 days before the onset of ischemia effectively decreased the infarct volume and the number of TUNEL-positive, cleaved caspase3-positive, and fluoro-Jade B-positive neurons. Next, we explored whether the dsAAV2-VEGF protected primary cultures of cortical neurons from hypoxia. After infection of dsAAV2-VEGF in primary cortical neurons 3 days before 24 h of hypoxia, the number of surviving cells was significantly increased and the activation of caspase 3 was reduced. We further examined the signaling pathway of VEGF's neuroprotective activity, the expressions of Akt, extracellular signal related kinases 1 & 2 (ERK-1/-2), and p38 were analyzed. Cortical neurons infected with dsAAV2-VEGF showed increased phosphorylated Akt in hypoxia while the expressions of phosphorylated ERK-1/-2 and p38 were without differences. These results revealed that exogenous expression of VEGF by dsAAV2 vector could attenuate ischemic brain injury through activation of Akt.
M. Coggiano, O. Dillon-Carter, J. Chen, S. Errico, and W. J. Freed
Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, DHHS, Baltimore, MD, USA
We have previously developed rodent CNS cell lines by transfecting primary cultures with truncated N-terminal variants of the SV40 large T antigen, from which the p53 binding C-terminal portion of the protein has been deleted (Truckenmiller et al., Cell Tissue Res., 1998). The purpose of the present study was to test the immortalizing properties of two versions of the N-terminal SV40 large T antigen fragment, T155g, coding for small t antigen, and T155c, which does not code for small t, driven by either the respiratory syncytial virus (RSV) or elongation factor (EF1) promoters. Four plasmids were created: EF1-T155g, EF1-T155c, RSV-T155g, and RSV-T155c. Rat primary mesencephalic cultures were transfected with one of the plasmids and maintained under neomycin selection for 1–3 weeks. Thirteen cell lines were produced, one from the EF1-T155g plasmid (designated ETG1), five from EF1-T155c (designated ETC1–5), three from the RSV-T155g plasmid (designated RTG1–3), and four from RSV-T155c (designated RTC1–4). All cell lines could be propagated indefinitely. While in log phase growth, many of the cell lines had a generally flattened, fibroblast-like appearance, while a few such as RTG1 and RTC3 were more compact with fine cytoplasmic extentions. All cultures contained cells that were positive for nestin, neurofilament (NF), and β-III-tubulin, although in no case were cells uniformly positive for any of these markers. The cell lines varied considerably in their properties; for example, RTC4 contained few nestin+ cells but many NF+ and β-III-tubulin+ cells, while RTG1 contained large numbers of nestin+ cells but few NF+ or β-III-tubulin+ cells. All cell lines tested were positive for glutamate decarboxylase GAD67 and chemokine receptor CXCR4, and negative for GAD65 by Western blotting. PCR was performed to examine incorporation of the various plasmid components into the cell's DNA. Six of the 13 lines were negative for both T155c and T155g, and moreover only one cell was positive for incorporation of either of the two promoters, RSV or EF1. Only one cell line, RTG1, was positive for both oncogene and promoter. All cell lines were positive for the selection marker. Therefore, only fragments of the immortalizing plasmid had been incorporated, suggesting that most of the cell lines had been spontaneously immortalized. Because mutations in p53 have been proposed as the mechanism responsible for spontaneous immortalization of rodent cells, we examined the cell lines for p53 mutations, and none were detected. We also examined the 13 cell lines for functional p53 activity, using a transfection assay with a plasmid containing the p21 promoter. Significant p53 activation by adriamycin was detected in 7 of 12 cell lines tested, including RTG1. Therefore, transfection does not consistently lead to incorporation of complete plasmids under the present conditions. The reason for partial plasmid integration is unknown; however, in the present study, the plasmids were linearized prior to transfection, while in previous experiments, linearization was not employed.
Research supported by the IRP of NIDA, NIH, DHHS.
T. J. Collier,* C. E. Sortwell,* L. Madhavan,* B. F. Daley,* K. Johe,† and K. L. Paumier*
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Neuralstem, Inc., Rockville, MD, USA
The present study sought to determine whether undifferentiated, nonimmortalized, expanded neural progenitor (NP) cells promote the survival of mature and developing midbrain dopamine (DA) neurons in vitro and in vivo. First, we conducted several cell culture experiments utilizing NP cells derived from the developing rat midbrain (mNP) to determine their effects on primary rat DA neurons. Results from our in vitro studies indicate that undifferentiated mNP cells promote the survival of developing DA neurons by secreting soluble factors that are supportive to DA neurons and sufficient to protect against toxin exposure. ELISA and Western blots detected significant levels of brain-derived neurotrophic factor (BDNF), neurotrophin 3 (NT-3), and pleiotrophin (PTN) in mNP cell lysates, suggesting these factors may be involved in the survival effects produced by undifferentiated mNP cells. Next, we explored the potential of mNP cells to prevent the loss of mature DA neurons in an in vivo neuroprotection paradigm using a rat intrastriatal 6-hydroxydopamine (6-OHDA) partial lesion model. We conducted two separate transplantation experiments utilizing undifferentiated mNP cells derived from rat or human. At 8 weeks postgrafting, the rat mNP cells elicited a significant sparing of tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SN compared to vehicle controls. To determine whether mNP-mediated neuroprotection was the result of signals/cues specific to the midbrain region, or could be induced by NP cells derived from other regions of the central nervous system (CNS), we evaluated undifferentiated human NP cells (Neuralstem, Inc.) derived from either midbrain (hMB) or spinal cord (hSC). At 4 weeks postgrafting, there was a significant decrease in the number of SN TH-ir neurons in the hSC-implanted subjects (57% of intact side) and VEH (61% of intact side) conditions compared to the hMB condition (90% of intact side), which suggests that transplanted hMB NP cells selectively protected host DA neurons. Our findings support the view that undifferentiated mNP cells express properties that support neuroprotection and repair. Furthermore, our limited survey suggests that NP cells may differ in their properties based on the region from which they are derived, and that those derived from midbrain may be optimal for repair of the nigrostriatal DA system.
Supported by NS58830, the Udall Center of Excellence in Parkinson's Disease Research at the University of Cincinnati.
M. M. Daadi,* J. Klausner,* R. Bhatnagar,* G. Sun,* B. Rutt,† and G. K. Steinberg*
*Department of Neurosurgery and Stanford Stroke Center, Stanford University School of Medicine, Stanford, CA, USA
†Department of Radiology, Stanford University School of Medicine, Stanford, CA, USA
Cell transplantation is a promising therapeutic intervention for human neurological disorders. Of clinical interest is the development of noninvasive neuroimaging approaches to track transplanted cells and to monitor their survival and actions on stroke-damaged brain regions. These imaging technologies will be invaluable to research and development of therapeutic strategies, as well as to stroke patient management. In the present study, we used magnetic resonance imaging (MRI) to follow the stroke evolution and tissue repair and to track the fate of grafted human embryonic stem cell (hESC)-derived neural stem cells (NSCs) in the stroke-damaged brain. Self-renewable and multipotent NSCs were isolated from hESC based on their responsiveness to mitogenic growth factors, including epidermal growth factor, fibroblastic growth factor, and leukemia inhibitory factor. The NSCs uniformly expressed nestin, vimentin, and 3CB2 (radial glial marker). For the MRI, cultured NSCs were incubated with MR-compatible contrast agent superparamagnetic iron oxides (SPIO) in the presence of poly-l-lysine prior to cell transplantation for 3 days in vitro. To induce stroke, rats were subjected to middle cerebral artery occlusion for 65 min. Labeled cells were suspended at a concentration of 50,000 cells/μl and stereotaxically grafted into the stroke boundary zone, 2 weeks poststroke. Graft survival was monitored using MRI on a weekly basis during the first 2 weeks and biweekly thereafter for 3 months. Volumetric analysis of the stroke and the NSC grafts demonstrated a marked diminution of the lesion over time. The precise locations of the stroke and grafts were confirmed histologically using Prussian blue staining. The fate of the grafted cells was analyzed in confocal microscopy using the human nuclear marker in conjunction with various neural lineage markers, including nestin for neural precursors, glial fibrillary acidic protein for astrocytes, β-tubulin for neurons, and galactocerebrocyde for oligodendroglial cells. Our study demonstrates that NSC grafts prevent tissue loss and that MRI provides a reliable means to monitor and analyze in real time the stroke evolution in relation to NSC engraftment.
H. B. Dodiya,* Y. Chu,* T. G. Beach,† C. H. Adler,‡ C. W. Olanow,§ R. T. Bartus,¶ and J. H. Kordower*
*Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
†Banner Health Care, Sun City, AZ, USA
‡Neurology, Mayo Clinic, Scottsdale, AZ, USA
§Neurology, Mt. Sinai Medical Center, New York, NY, USA
¶Ceregene Inc., San Diego, CA, USA
Intraputaminal trophic factor therapy, especially the delivery of the genes or proteins for glial-derived neurotrophic factor (GDNF) or neurturin, has been proposed as a therapeutic strategy for Parkinson's disease (PD). The common approach employed delivers the trophic factor gene or protein to the terminal fields in the putamen where the therapeutic protein binds to receptors located upon remaining nigrostriatal fibers. The trophic factor/receptor complex is then retrogradely transported to nigral perikarya where gene changes occur that invoke pro-dopaminergic repair and protective molecular cascades. To date, all clinical trials have been performed in relatively advanced PD patients where the status of the residual nigrostriatal system has been poorly characterized. The present study examined the putamen of 24 PD cases with disease durations between 1 and 27 years and compared the tyrosine hydroxylase (TH) and dopamine transporter (DAT) staining with that seen in 10 age matched controls. In cases with 1–2 years' duration, the breadth and intensity of TH and DAT were similar and approached that seen in age-matched controls. In most cases in which disease duration was 3–4 years, there was a clear diminution in TH and DAT staining, but numerous fibers could be observed throughout the entire extent of the putamen. In cases of 5 years' duration or longer, there was a precipitous decrease in both TH and DAT staining. In all cases of this duration, staining for TH and DAT was limited to the occasional individual fiber throughout, at minimum, the dorsal half of the putamen and in most cases this staining pattern was seen throughout the dorsal three quarters of the putamen. In many cases appreciable dopaminergic fiber staining was limited to the most ventral aspect of the putamen and to fibers traversing in the most medial aspect of the putamen en route to the caudate nucleus. Optical density measurements suggest profound DA innervation loss within 5 years of disease duration. These data, coupled with stereologically estimated melanin-containing and TH-ir neurons within the substantia nigra, support the concept that PD is an axonopathy and trophic factor therapy that relies exclusively on retrograde transport mechanisms and faces significant challenges due to the absence of viable dopaminergic fibers in all but the earliest PD cases.
A. D. Ducray,* R. Liechti,* P. Jensen,*† S. Di Santo,* C. Jensen,‡ A. Raabe,* M. Meyer,† and H. R. Widmer*
*Department of Neurosurgery, University of Berne, Berne, Switzerland
†Department of Anatomy & Neurobiology, Institute of Medical Biology, SDU-Odense University, Odense, Denmark
‡Department of Immunology & Microbiology, Institute of Medical Biology, SDU-Odense University, Odense, Denmark
The epidermal growth factor superfamily member fetal antigen 1/delta-like (FA1/dlk) has been reported to be a growth and/or differentiation factor expressed in cells during development. Furthermore, FA1/dlk has been demonstrated to be expressed in the ventral mesencephalon of rats and humans. The present study aimed at investigating the detailed expression pattern of FA1/dlk-immunoreactive (-ir) cells in the midbrain of adult rats and in the 6-hydroxydopamine (6-OHDA) rat model of Parkinson's disease. For that purpose adult rats received a unilateral injection of 6-OHDA either into the ascending mesotelencephalic pathway or into the striatum. One month later the brains were processed for histological analyzes. In line with our previous observations we found that FA1/dlk-ir cells were predominantly distributed in the substantia nigra pars compacta (SNc) and ventral tegmental area. Colocalization experiments revealed that most of the tyrosine hydroxylase (TH)-ir neurons also expressed FA1/dlk in the SNc, assuming that these cells are projection neurons. This latter assumption was further substantiated by colocalization of FA1/dlk with retrogradely transported fluorogold injected into the striatum. The unilateral 6-OHDA lesions resulted in a marked loss of both FA1/dlk-ir and TH-ir neurons in the SNc. Notably, this loss of FA1/dlk-ir cells was less pronounced in the intrastriatally lesioned rats (by 50%) compared to animals with lesions of the ascending mesotelencephalic pathway (by 85%). Accordingly, innervation of TH-ir and FA1/dlk-ir fibers was severely reduced in the lesioned striata. Importantly, we detected that the number of striatal FA1/dlk-ir cells in the denervated striatum was significantly increased (by 70%) compared to the unlesioned side. The observation that no TH-ir cells were detected in the denervated striata in response to the lesions reflects that these cells are not dopaminergic neurons. Moreover, our preliminary observations revealed that these FA1/dlk-ir cells were not newly generated, indicating that FA1/dlk may be upregulated in already existing cells in response to the lesions. Taken together, our findings show that FA1/dlk expression is differentially modulated in the nigrostriatal sytem in response to 6-OHDA lesions and indicates that FA1/dlk may play an important role in Parkinson disease.
S. B. Dunnett, C. M. Kelly, and A. E. Rosser
Cardiff University, Cardiff, South Wales, UK
Validation of human cells for clinical transplantation, whether primary fetal tissues or stem cells, first requires demonstration of functional efficacy in animal models of the human disease. For the grafts to survive, it is necessary to prevent rejection by the host immune system, which is particularly challenging in “xenotransplantation” across the species barrier. Effective immune suppression has traditionally been achieved either (a) by chronic treatment with immunosuppressant drugs (such as cyclosporine A), although such drugs typically cause significant side effects on long-term administration, or (b) implanting into immunocompromised hosts (such as nude rats or mice), although such animals are highly susceptible to infection and require full barrier maintainence. These constraints make both strategies unsuitable for long-term studies of cell survival, differentiation, and behaviour over more than 3 or 4 months. In order to evaluate alternative sources of human primary fetal (hPF), fetal neural progenitors (hFNP), and embryonic stem (hES) cells for transplantation in neurodegenerative diseases of the brain, we have recently published a new method—“neonatal tolerization”—that allows long-term survival of human neuronal tissues in the rodent brain without the need for chronic immunosuppression [Kelly et al., Nature Methods, 6(4):271–273; 2009]. In this presentation we report on updated studies demonstrating equal efficacy of tolerization in mouse as well as rat hosts. “Neonatal tolerization” involves inoculating the pups during the first postnatal week, when their immune system is still developing, with tissues similar to that subsequently used for transplantation in adulthood. Typically, 100,000 cells are injected IP on postnatal day P0 or P1, followed by implanting neural cells as a dissociated cell suspension into the brain when the pup reaches adulthood, 6–10 weeks following tolerization. In the absence of neonatal tolerization, all grafts are fully rejected within 3–5 weeks. Chronic treatment with 10 mg/kg/day cyclosporine A yields ~75% surviving grafts over 12 weeks, but the drugs cause increasing side effects, requiring that the animals be euthanized on welfare grounds. Following a single neonatal tolerization treatment, we achieve effective 87% transplant survival in the adult brain at 40 weeks (the longest duration so far investigated). Tolerization is effective with human hPF, hFNP, and hES cells in both rats and mice. Any human brain tissue is effective—neonatal inoculation with hPF, hFNP, or hES cells permits equivalent levels of survival of human hPF, hFNP, and hES in any combination. Peripheral tissues (such as liver) are less effective. Although most studies have used human cortical tissue implanted into the intact striatum, we see appropriate differentiation of striatal dopamine- and cyclic AMP-regulated phosphoprotein (DARPP+) and tyrosine hydroxylase (TH+) neurons within striatal and nigral hPF grafts in the relevant excitotoxic and 6-hydroxydopamine (6-OHDA) lesion models. Neonatal tolerization now allows long-term survival, differentiation, and function of various cell types transplanted to the brain over extended periods of time required for systematic developmental and behavioral analysis.
G. El-Akabawy, A. Jeffries, J. Price, and M. Modo
Institute of Psychiatry, Kings College London, London, UK
Transplantation of neural stem cells is a promising therapeutic approach for neurodegenerative diseases including Huntington's disease (HD). HD is an autosomal dominant neuropsychiatric disorder that leads to a progressive loss of medium-sized spiny neurons (MSNs) in the striatum. DARPP-32 (dopamine and cyclic AMP-regulated phosphoprotein, 32 kDa) is transcribed in 98% of the MSNs. To establish an effective cell therapy for HD, the differentiation of human neural stem cells (hNSCs) into more specific neuronal phenotypes is required. To this end, it is possible to use small molecules acting on particular pathways involved in neuronal differentiation to enhance this process. By adding purmorphamine, a sonic hedgehog signaling pathway agonist at a concentration of 1 μM for 7 days, we were able to significantly increase the neural differentiation of a striatal hNSC line (STROC05) by nearly threefold. Conversely, this increase in neural differentiation was concomitant with a decrease in astrocytes (GFAP+ cells). Upon long-term differentiation (21 days), purmorphamine further increased the number of neurons and also specifically increased the number of DARPP-32-positive cells. These results indicate that small synthetic molecules, such as purmorphamine, can increase the differentiation of NSCs. It is anticipated that enhancing DARPP-32 differentiation will eventually provide a more efficacious cell therapy in animal models of HD.
G. El-Akabawy, I. Rattray, B. Solanky, R. Gale, G. Bates, and M. Modo
Institute of Psychiatry, King's College London, London, UK
Cell replacement therapies are currently a very promising new approach for the treatment of a wide variety of neurodegenerative disorders, including Huntington's disease (HD). To establish stem cell transplantation as an effective therapeutic approach for HD, animal models that mimic the behavioral and histopathological disorder of this disease are crucial. Transgenic R6/2 mice, expressing exon 1 of the human mutant HD gene, develop many aspects of human HD and thus provide a suitable model to establish whether transplanted cells can ameliorate this type of neurodegeneration. R6/2 mice of both sexes were randomly allocated into two groups (n = 10 each) either receiving unilateral stereotaxic injection of 120,000 human striatal neural stem cells (hNSCs) into the striatum or a vehicle injection at 7 weeks of age. A behavioral testing battery consisting of the rotarod, swimming T-maze, and passive avoidance was used to evaluate functional impairments over 6 weeks postgrafting. Magnetic resonance structural imaging was performed prior to transplantation and at 13 weeks of age prior to perfusion fixation, in addition to spectroscopy prior to the last time point. Transplanted cells did not improve motor impairments, but a significant improvement was observed on the swimming T-maze. Magnetic resonance spectroscopy revealed a trend of increased concentrations of various brain metabolites (phosphocreatine, creatine, taurine, and glutamine). Collectively, these results suggest that hNSCs have a potential to impact HD-related neurodegeneration, but that future refinement of the current study may be required to ensure a more efficacious therapeutic outcome.
J. D. Elsworth,* N. Wallingford,† S. Diano,† B. A. Morrow,* R. H. Roth,* and D. E. Redmond, Jr.*
*Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA
†Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT, USA
Excessive loss of nigrostriatal dopamine neurons is the proximate cause of Parkinson's disease (PD). There is overwhelming evidence that oxidative stress occurs in PD and little doubt that it leads to damage of the dopamine neurons that originate in the substantia nigra. Both methamphetamine and MPTP (1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine) are specifically toxic to nigrostriatal dopamine neurons in the adult human and adult nonhuman primate through mechanisms involving oxidative stress. We have found, however, that in young monkeys, there is a marked resistance of dopamine neurons to both methamphetamine and MPTP. Specifically, we have found that methamphetamine and MPTP significantly reduced striatal dopamine concentration (by 80% and 95%, respectively) in adults while causing no significant loss in young monkeys. Altered pharmacokinetics do not appear to provide an explanation for these age-dependent differences, as the same dose of methamphetamine actually results in higher CNS drug levels in the young monkey. The transient protection of dopamine neurons to neurotoxicity has raised interesting questions concerning differences in the induction and/or handling of oxidative stress in developing dopamine neurons. Thus, we are examining several specific mechanisms that would provide an explanation for the resistance of young monkeys to methamphetamine and MPTP, as this may shed light on the plasticity and regenerative capacity of the dopamine system at different stages of primate development, and provide drug targets to enable the early-in-life protection from damage to dopamine neurons to be reinstated later in life. Uncoupling proteins (UCPs) are inner mitochondrial membrane proteins that regulate mitochondrial homeostasis. Recent evidence suggests that UCP2 plays a positive physiological role by regulating reactive oxygen species (ROS) production, and that its activity is neuroprotective and antiaging. These data have led to the “uncoupling to survive” hypothesis of aging (Brand, Exp. Gerontol. 35:811–820; 2000). We have examined the expression of mRNA for UCP-2 in the substantia nigra of adult and young St Kitts green monkeys by real-time PCR. A significant twofold higher expression of UCP2 occurred in young compared with adult monkeys, suggesting that this might be a key protective mechanism in the young dopamine system that is suppressed later in life. Recent work (Andrews et al., J. Neurosci. 29:14057–14065; 2009) has shown that administration of the hormone, ghrelin, protects against MPTP toxicity by a UCP2-dependent mechanism. Thus, ghrelin may be a novel therapeutic UCP-dependent strategy to reduce dopamine neurodegeneration that is associated with PD and aging.
Supported by NS056181 (J.D.E.).
T. Federici,* J. Riley,* K. Johe,† T. Freeman,‡ E. Feldman,§ J. Glass,* and N. Boulis*
*Emory University, Atlanta, GA, USA
†Neuralstem, Inc., Rockville, MD, USA
‡University of South Florida, Tampa, FL, USA
§University of Michigan, Ann Arbor, MI, USA
Amyotrophic lateral sclerosis (ALS) is a progressive and fatal neurodegenerative disease characterized by rapid loss of muscle control and eventual paralysis due to the death of spinal and cortical motor neurons. Preserving the survival of motor neurons in the spinal cord might prolong the survival of ALS patients. Neural stem cells derived from human fetal spinal cord have previously been shown to prolong survival in animal models of ALS. When transplanted into the spinal cord of rodents, the grafted cells formed synaptic contacts on host motor neurons and expressed multiple growth factors, including glia-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF). This trial will test the safety of the injection process and the implanted stem cells into the spinal cords of ALS subjects. The strategy for cellular transplantation is derived from our collective experience with rodent and porcine spinal cord cell grafting and human spine and spinal cord surgery, as well as the experience of others with human spinal cord transplant protocols. The use of a spine-mounted platform will ensure that no shearing occurs on the spinal cord. The initial cohorts of subjects will undergo lumbar injections in order to minimize the potential risks of transplant. Treatments will consist of a dose escalation of 5 unilateral to 10 bilateral injections of 10 μl each into the lumbar enlargement. By injecting the cells at 3–4-mm intervals, the intention is to deliver the therapy to multiple segments that are important to functional outcome. Once the subjects receiving the lumbar transplantation are shown to be safe, the same surgical approach will be extended to cervical enlargement of new subjects. Prior to and after the transplant, subjects will be required to remain on immunosuppressive therapy. Subjects will be followed until death. This is the first human stem cell trial to treat ALS in the US.
A. M. Fortress,* M. Buhusi,* K. L. Helke,† and A.-C. Granholm*‡
*Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA
†Department of Comparative Medicine, Medical University of South Carolina, Charleston, SC, USA
‡Center on Aging, Medical University of South Carolina, Charleston, SC, USA
Down syndrome patients develop neuropathological hallmarks of Alzheimer's disease (AD) by their fourth decade of life; one such pathology is the degeneration of basal forebrain cholinergic neurons (BFCNs). BFCNs extend their axons to the hippocampus where they bind nerve growth factor (NGF) at its high-affinity receptor, TrkA. The NGF/TrkA complex is then endocytosed and retrogradely transported to the cell soma in the basal forebrain in order to promote cholinergic neuron survival. Loss of NGF transport to the BF is correlated with both cognitive deficits and BFCN degeneration, posing an important role for this system in memory processing in AD and Down syndrome. Recent evidence has provided that the precursor to NGF, pro-NGF, is the predominant form of NGF in AD and is capable of binding to the low-affinity NGF receptor, p75. This interaction preferentially activates cell death signaling by recruiting the coreceptor sortilin. Currently, the working hypothesis is that disrupted retrograde signaling of NGF/TrkA and failure of pro-NGF metabolism in certain disease states results in reduced trkA activation and elevated p75 response, resulting in a propelling degeneration of the cholinergic neurons. To better understand this mechanism, we conducted a life span study in a mouse model of Down syndrome, the Ts65Dn, to determine how the TrkA to p75 ratio and upstream pro-NGF metabolism may correlate with behavior during aging and in a disease state. We examined p75, TrkA, plasmin, and sortilin in 4-, 8-, 12-, and 18-month-old male normosomic and trisomic mice that were tested for memory deficits in the water radial arm maze. Based on previous work, we propose that dysmetabolism of pro-NGF leads to preferential activation and upregulation of p75 that acts in concert with downstream NGF mechanisms to decrease the NGF/TrkA interaction and prevent the survival promoting signal in the BFCNs. To our knowledge, this is the first insight to modeling BFCN degeneration in a mouse model of Down syndrome using the novel mechanism of pro-NGF-induced alterations.
This work is supported by NIA AG012122.
L. Freeman* and A.-C. Granholm*†
*Department of Neuroscience, Medical University of South Carolina, Charleston, SC, USA
†Center on Aging, Medical University of South Carolina, Charleston, SC, USA
The “Western Diet” has been found to be a main culprit for obesity, type II diabetes, cancer, and cardiovascular issues. However, its effects on the brain and cognition are not currently well understood. Previous work from our laboratory has shown that a 10% hydrogenated coconut oil and 2% cholesterol diet resulted in impaired performance on the 12-day water radial arm maze, increased cholesterol and triglyceride levels, and decreased dendritic microtubule associated protein 2 (MAP2) staining in the hippocampus. We have also recently shown that each component of the “Western Diet” alone (saturated fat, trans fat, or cholesterol) resulted in alterations in hippocampal morphology and serum triglyceride/cholesterol levels. A reduction in dendritic integrity and fatty acid metabolism combined with an increase in microgliosis as measured by antibodies against MAP2, 5-lipoxygenase (5-LOX), and OX-6 (MHC class II 1α), respectively, revealed damaging effects after just 8 weeks on the various diets. Our current study has further evaluated the damaging effects of the “Western Diet” to cognition and hippocampal morphology using the 10% hydrogenated coconut oil and 2% cholesterol diet for 6 months starting at the age of 7 months in Fischer 344 rats. We are further probing the mechanisms behind diet-induced damage to the brain and have found that inflammation plays a role in altering the blood–brain barrier, allowing cytokines to flood into the brain and cause damage. In this long-term study, serum was collected in order to analyze triglyceride and cholesterol levels, inflammatory markers were measured, and immunofluorescence studies on the hippocampus have been performed. These experiments have revealed a compromised blood–brain barrier, release of interleukin-1 into the brain parenchyma, and dendritic damage for those animals receiving the hydrogenated coconut oil and cholesterol diet compared to control. Together, this provides novel evidence for a role of inflammation in diet-induced neurodegeneration.
S. Garbuzova-Davis,*†‡§ M. Louis,* R. Woods, III,* L. Wells,* S. K. Klasko,# and P. R. Sanberg*†‡§¶
*Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
‡Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
§Department of Pathology and Cell Biology, University of South Florida College of Medicine, Tampa, FL, USA
¶Department of Psychiatry, University of South Florida, Tampa, FL, USA
#University of South Florida College of Medicine, Tampa, FL, USA
In Sanfilippo syndrome type B (mucopolysaccharidosis MPS III B), a deficiency of alpha-N-acetylglucosaminidase (Naglu) enzyme leads to accumulation of heparan sulfate (HS), a glycosaminoglycan (GAG), within cells and to eventual progressive cerebral and systemic organ abnormalities. We have previously shown that human umbilical cord blood cells ameliorated pathological changes after intravenous (IV) injection into Naglu-deficient mice, probably due to continuous delivery of the missing enzyme. Although administered cells were found widely distributed among different brain structures, the mechanism of cell migration to the brain is still unclear. One possibility is that cell migration to lesioned areas of the brain occurs due to “signaling” substances in damaged tissues, substances that attract the transplanted cells. Another possible mechanism of transplant cell migration may be crossing of a damaged blood–brain barrier (BBB). However, no data exist about BBB condition in Sanfilippo. The aim of this study was to determine functional and structural BBB condition in a mouse model of Sanfilippo type B at different stages of disease. BBB functionality was determined by testing for vascular leakage with Evans Blue (EB) and albumin. BBB structural characteristics were identified by electron microscopy (EM). EB dye was IV injected into Naglu mice to assess BBB integrity at early or late stage disease. Wild-type mice (controls) were also injected at the same ages. After 30 min, mice were euthanatized and the brains examined for EB leakage. Immunohistochemical staining for albumin was also performed in serial brain sections. Results showed EB and albumin vascular leakage in various brain structures of early and late symptomatic Naglu mice, males and females. The majority of blood vessel leakage was detected in areas of the cerebellum, cerebral cortex, hippocampus, and midbrain. More leakage was found in late than in early symptomatic mice. Ultrastructural analysis of brain microvessels in Naglu mice showed severe damage to endothelial cells and pericytes. Additional immunohistochemical analysis for GM3 ganglioside in brain sections of Naglu mice revealed GAG accumulation within the vascular endothelium. These results indicate functional and structural BBB damage in a mouse model of MPS III B. Diminished BBB integrity likely occurred due to GAG accumulation in endothelium of the brain microvasculature. Determining structural and functional BBB damage in MPS III B is important not only for examining cell transplant migration, but is also crucial to understanding additional mechanisms of disease pathogenesis and to developing pharmacological and cellular treatments.
Supported by The Children's Medical Research Foundation.
C. Gemma,*†‡ J. Morganti,*† J. T. Rogers,§ L. Wells,* A. D. Bachstetter,¶ J. K. Harrison,# E. J. Weeber,§ and P. C. Bickford*†‡
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
‡James A. Haley Veterans Administration Medical Center, Tampa, FL, USA
§Murine Neurobehavior and Cellular Electrophysiology Laboratories Alzheimers Research Institute, University of South Florida, Tampa, FL, USA
¶Department of Cell & Molecular Biology, Northwestern University Feinberg School of Medicine, Chicago, IL, USA
#Department of Pharmacology and Therapeutics, University of Florida, College of Medicine, Gainesville, FL, USA
In recent years, extensive evidence has shown that inflammation develops in the brain during aging and Alzheimer's disease (AD). However, the causes and consequences of specific brain inflammatory responses are still not well understood. Fractalkine (CX3CL1), the CX3C chemokine, is the exclusive ligand for the CX3C receptor (CX3CR1) and has recently been identified as a key neuroimmune regulatory molecule. This chemokine is expressed on neurons while CX3CR1 is found on microglia, and the interaction between fractalkine and CX3CR1 is thought to maintain microglia in a resting state, thereby suppressing the neurotoxic activity of these cells. Decreased protein levels of fractalkine have been found in the aged brain, as well as in the plasma of patients with AD. It is our hypothesis that a disruption in fractalkine/CX3CR1 signaling is a key trigger of microglial activation during aging, which leads to cognitive impairment and suppression of neurogenesis. Recently we have shown that neurons and microglia are actively involved in a cross-talk to regulate the production of new neurons through Fractalkine/CX3CR1 signaling. Our study showed that loss of function of CX3CR1 in young adult rodents, mice and rats, resulted in a significant decrease in hippocampal neurogenesis, while the administration of exogenous fractalkine reversed the decline in neurogenesis associated with aging. IL-1 receptor antagonist protected against the decrease in hippocampal neurogenesis induced by blocking CX3CR1 function. The functional significance of adult neurogenesis is not fully understood. It has been suggested that the age-related decrease in hippocampal neurogenesis together with the decline in other types of synaptic plasticity contributes to a disruption in memory function. A great deal of evidence suggests that brain inflammation plays an important role in neuronal plasticity, as revealed by the fact that microglia toxicity and inflammatory cytokines are implicated in the impairment in long-term potentiation (LTP), a model system for the neural mechanism underlying hippocampus-dependent memory, in aged rats. It has been shown that age-related increases in endogenous IL-1β (IL-1β concentration is accompanied by an impaired expression of LTP). Consistent with this finding it has been demonstrated that IL-1β inhibits LTP in dentate gyrus, CA1, and CA3. To investigate whether CX3CR1 deficiency leads to impairment in other forms of synaptic plasticity we analyzed the induction and maintenance of LTP in the CA1 region of CX3CR1 null mice (3 months old). Acute hippocampal slices were made from CX3CR1-deficient mice and C57BL/6 mice. LTP was induced with high frequency stimulation (HFS). The HFS consisted of two 1-s pulses at 100 Hz separated by 20 s at half max with an interburst interval of 10 s. Following HFS, we recorded fEPSPs at half max for an additional 60 min. Our preliminary data showed that deficiency in CX3CR1 causes a decrease in LTP. These findings further confirm our hypothesis that CX3CR1 deficiency negatively regulates neuronal plasticity.
This work was supported by the USPHS grants (AG004418 P.C.B.; AI058256 J.K.H.) and the VA Medical Research Service.
C. Gill,* T. David,† P. Konrad,‡ M. Bliton,§ M. Tramontana,¶ and D. Charles†
*Stritch School of Medicine, Loyola University Chicago, Chicago, IL, USA
†Department of Neurology, Vanderbilt University Medical Center, Nashville, TN, USA
‡Department of Neurosurgery, Vanderbilt University Medical Center, Nashville, TN, USA
§Center for Biomedical Ethics and Society, Vanderbilt University Medical Center, Nashville, TN, USA
¶Department of Psychiatry, Vanderbilt University Medical Center, Nashville, TN, USA
Parkinson's disease (PD) affects 1 million Americans, and no available intervention slows disease progression. Bilateral deep brain stimulation (DBS) of the subthalamic nucleus has gained acceptance as a safe, effective, and cost-effective treatment for the symptoms of advanced PD. Recent studies mandate testing DBS in early PD because it (1) is more effective than medication alone, (2) may impart neuroprotection, and (3) may prevent development or worsening of motor fluctuations. We are therefore conducting a single-blind, randomized pilot trial in 30 patients with Hoehn & Yahr Stage II idiopathic PD, aged 50–75, who have been on medication for 6 months to 4 years. They must be without motor fluctuations or wearing-off, psychiatric disease, previous brain operation, or dementia. The informed consent process includes four meetings with study physicians and completion of a questionnaire documenting study understanding and reasons for participation. On day 1 of an 8-day inpatient medication washout evaluation, patients complete an 8-h ON/OFF diary, UPDRS testing, autonomic testing, and measures of gait in the ON state. Following completion of ON measures, subjects are washed out and measured daily for 1 week. Subjects also undergo neuropsychological testing for adverse events and quality of life. Randomization [15 best medical therapy (MED), 15 DBS + MED] occurs after baseline evaluation. Identical follow-up inpatient evaluations occur at 6-month intervals for 2 years. Subject enrollment was conducted from 2006 to 2009, during which 329 individuals contacted study personnel. Sixty-five attended information sessions, and 37 provided informed consent. Of these, one withdrew consent before baseline and six failed screening due to dementia (1), greater than Hoehn & Yahr II (3), abnormal MRI (1), and psychiatric disease (1). Participants who underwent baseline are 28 males and 2 females, aged 60.0 years (range 50–74), who have been on medication 2.0 years (range 0.5–4). The average full UPDRS score ON medication is 37.7. Participants' average UPDRS III scores are 14.4 ON medication and 26.9 OFF medication. To date, there are no withdrawals and 13 have completed participation. Most (80%) patients stated one of their top motivations to participate was to benefit future patients, while for 40% this was the primary reason. Whether DBS is disease modifying remains highly controversial among neuroscientists and neurologists, and will only be resolved in a carefully designed clinical trial. In our trial we achieved rapid recruitment, and participants appear representative of early PD patients in general. The results of this trial, regardless of outcome, will improve the design of the definitive phase III trial. Any therapy that reduces motor complications or positively modifies disease progression will have a tremendous impact on the millions dealing with the prospect of a relentlessly progressive disease.
S. E. Gombash,*† T. J. Collier,* B. F. Daley,* S. L. Wohlgenant,* N. D. Levine,* B. T. Terpstra,*† R. J. Mandel,‡ F. P. Manfredsson,‡ and C. E. Sortwell*§
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Graduate Program in Neuroscience, University of Cincinnati, Cincinnati, OH, USA
‡Department of Neuroscience, University of Florida, Gainesville, FL, USA
§Division of Translation Science and Molecular Medicine, Michigan State University, East Lansing, MI, USA
Neurotrophic factors are integrally involved in the development of the nigrostriatal system and, in combination with viral vector therapies, possess therapeutic potential for Parkinson's disease (PD). Our laboratory and others have previously shown that the trophic factor pleiotrophin (PTN), a 15.3-kDa cell surface and extracellular matrix protein, is vital for the development, maintenance, and repair of the nigrostriatal dopamine system. Our goal is to optimize PTN gene transfer in order to provide meaningful protection of the nigrostriatal system in PD. We have previously demonstrated that striatal overexpression of PTN that recapitulates peak developmental levels provides significant neuroprotection for tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the substantia nigra (SN), significantly increases TH-ir neurite density in the striatum, and can reverse functional deficits in forepaw use following a toxic insult. Previous work utilizing glial cell line-derived neurotrophic factor (GDNF) gene transfer has shown that optimal functional restoration can be achieved via simultaneous trophic factor overexpression in both the SN and striatum (Kirik et al., 2000). The present study was conducted to determine the optimal PTN transduction site(s) within the nigrostriatal system for maximal neuroprotective and neurorestorative effects. Adult, male, Sprague-Dawley rats were unilaterally injected in the striatum, SN, or both structures with recombinant adeno-associated virus serotype 2/1 (rAAV2/1) expressing either PTN and green fluorescent protein (GFP) or GFP alone. Four weeks following vector injection all rats received unilateral intrastriatal injections of 6-hydroxydopamine (6-OHDA). All rats were evaluated for extent of forelimb asymmetry prior to vector injection and immediately before and 4 weeks following the 6-OHDA injection. Stereological analyses of transduction volume, SN TH-ir neuron survival, and striatal TH-ir neurite density are ongoing. Our results to date suggest that striatal PTN overexpression produced by PTN gene transfer can provide morphological neuroprotection and promote functional recovery in the nigrostriatal system. Pending results will provide valuable information regarding optimal surgical PTN transduction parameters in order to inform our ultimate goal of testing the efficacy of PTN overexpression in PD patients.
Supported by NS058682 (C.E.S.), the James J. and Joan A. Gardner Family Center for Parkinson's Disease and Movement Disorders, and the Morris K. Udall Center of Excellence for Parkinson's Disease Research at the University of Cincinnati NS058830 (T.J.C.).
J. T. Greenamyre, J. Cannon, R. Drolet, E. Hoffman, M. Horowitz, and P.-G. Mastroberardino
*Pittsburgh Institute for Neurodegenerative Diseases and Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Our laboratory began to develop the rotenone model in the late 1990s because there was a growing suspicion that Parkinson's disease (PD) might be associated with systemic mitochondrial defects. Rotenone, unlike MPTP, produces uniform, systemic complex I inhibition. To our surprise, chronic systemic inhibition of complex I—to a degree similar to that reported in platelets from PD patients—produced selective nigrostriatal degeneration. Even more remarkably, rotenone-treated rats developed α-synuclein-positive cytoplasmic inclusions, similar to Lewy bodies, in nigral dopaminergic neurons, overcoming for the first time a key limitation of other available in vivo models. Moreover, rotenone provided the first proof-of-concept that a systemic defect in mitochondrial function could lead to selective nigrostriatal neurodegeneration. Of course, genetic studies subsequently confirmed this view. And although the rotenone model was developed initially to test the “mitochondrial hypothesis” of PD, given the epidemiological links to pesticide exposure, it was also of interest that rotenone is a pesticide. Since the first description of the model, our laboratory has found that the rotenone model accurately recapitulates many other features of PD, including: a moderate systemic level of complex I impairment; oxidative damage; accumulation and aggregation of endogenous, wild-type α-synuclein; α-synuclein- and polyubiquitin-positive Lewy bodies and Lewy neurites; apomorphine-responsive behavioral deficits; early and sustained activation of microglia; oxidative modification and translocation of DJ-1 into mitochondria in vivo; impairment of the nigral ubiquitin-proteasome system (UPS) function with accumulation of poly-ubiquitinated proteins; accumulation of iron in the substantia nigra through a mechanism involving transferrin and transferrin receptor 2; α-synuclein pathology in enteric neurons and functional deficits in gastrointestinal (GI) function, including gastroparesis. It is also worth noting that the transferrin-dependent mechanism of iron accumulation we discovered in the rotenone model was subsequently found to be operative in human PD. In other words, the rotenone model predicted what we would find in PD. It is also worth noting that, based on our initial report, epidemiological studies began to look at the potential role of exposure to rotenone per se as a risk factor for development of human PD. While the number of individuals exposed to rotenone and other “botanicals” is small compared to other classes of synthetic pesticides, a recent study found an odds ratio of 5.9 and another found an odds ratio for rotenone of 10.9. Thus, the rotenone model informed subsequent epidemiological studies, which have suggested a potential role for rotenone in some cases of PD. Given the apparent accuracy and predictive value of the rotenone model, we are now beginning to test pharmacological and viral-mediated genetic therapeutic strategies for PD.
J. G. Green,*† L. J. Cloud,* and A. R. Noorian*†
*Department of Neurology, Emory University School of Medicine, Atlanta, GA, USA
†Center for Neurodegenerative Disease, Emory University School of Medicine, Atlanta, GA, USA
Since its original description, Parkinson's disease (PD) has been known to be associated with dysfunction referable to multiple levels of the neuraxis, not merely dopamine neurons in the substantia nigra pars compacta. Recent clinical and neuropathological data have renewed interest in nonmotor and other “nonnigral” aspects of the disease (psychiatric and cognitive dysfunction, sleep abnormalities, autonomic dysfunction, and digestive problems). Associated with this revival is the recognition that extranigral symptoms and pathology must be taken into account when proposing experimental model systems and may provide unique opportunities for therapeutic intervention in PD. We have recently begun investigating the effects of PD on the gastrointestinal (GI) tract in patients and various animal models of parkinsonism. Our evidence confirms that GI symptoms are very common in PD patients and manifest early in the course of this chronic neurodegenerative movement disorder. PD is associated with α-synuclein aggregation in the enteric nervous system (ENS) that lines the GI tract, and enteric Lewy bodies appear to be more common in proximal GI segments. In parallel, we have examined several animal models of PD, including rotenone-treated rats, MPTP-treated mice, VMAT2 low-expressing mice, and α-synuclein transgenic mice. Each model system has abnormalities in GI function, some that are concordant with those seen in PD and others that are not. Intriguingly, α-synuclein transgenic mice display an age-related decrease in gastric and colonic motility in the absence of overt or severe motor symptoms. The GI deficit does not appear to be related to either nonspecific GI pathology or neurodegeneration in the enteric nervous system. However, in mice of all ages, there is accumulation of α-synuclein in and around the ENS similar in distribution and quality to that seen in human PD. Enteric synuclein accumulation in transgenic mice mirrors Lewy body pathology in PD and GI innervation from the dorsal motor nucleus of the vagus nerve. Immunostaining approaches indicate that synuclein is colocalized with choline acetyltransferase, but not nitric oxide synthase or vasoactive intestinal peptide, further supporting a possible vagal localization. The results implicate the vagus nerve as a potential modifier of GI dysfunction in parkinsonism. Given the extensive anatomical ramifications and multifaceted role of the vagus nerve, it may provide an intriguing target for anti-parkinsonian therapies, not merely for GI symptoms, but for PD as a whole. Timely intervention may be facilitated by early detection of GI dysfunction in animal model systems and vulnerable patient populations.
R. Grier,*† B. Hattiangady,*† V. K. Parihar,*† B. Shuai,*† and A. K. Shetty*†
*Medical Research and Surgery Services, VA Medical Center, Durham, NC, USA
†Department of Surgery (Neurosurgery), Duke University Medical Center, Durham, NC, USA
Chronic temporal lobe epilepsy (TLE), typified by spontaneous recurrent motor seizures (SRMS) and cognitive deficits, is one of the common neurological disorders. The morphological changes associated with chronic TLE include abnormal sprouting of dentate mossy fibers, reductions in the numbers of GABAergic inhibitory interneurons, astrocytic hypertrophy, and greatly waned dentate neurogenesis. Reelin, an extracellular matrix glycoprotein expressed by most GABAergic interneurons in the cortex and the hippocampus, has been shown to be reduced during the early phase of epilepsy (i.e., shortly after an episode of acute seizures or status epilepticus). While reelin is well known as a regulator of the neuronal migration and positioning in the developing brain, it is believed to have multiple functions in the adult brain. These include modulation of the synaptic plasticity, stimulation of the dendritic and dendritic spine growth, and the apt migration of newly born neurons in neurogenic sites such as the dentate gyrus and the subventricular zone. Because TLE is associated with substantial reductions in the numbers of different subclasses of GABAergic hippocampal interneurons, we investigated whether any link exists between the loss of a subclass of GABAergic interneurons that express reelin, the frequency of spontaneous seizures, and cognitive function in a rat model of chronic TLE. We induced status epilepticus (SE) in young adult male F344 rats via graded intraperitoneal injections of kainic acid (KA). To reduce the mortality related to SE, diazepam (5 mg/kg) was injected at 1 h after the first stage V seizure and rats were allowed to survive for 7 months. Another group of age-matched rats served as controls. The extent of chronic epilepsy (characterized by behavioral spontaneous seizures) in these rats was measured via direct observation (8 h/week; 32 h/month) by two independent observers. At 6 months post-SE, rats in both groups were examined for cognitive function using a water maze test (WMT). All rats were perfused at the end of the WMT and the brain sections (every 20th section) through the hippocampus were processed for reelin immunostaining. Following this, numbers of reelin+ interneurons were quantified in each of the three regions in the hippocampus (dentate gyrus and CA1 & CA3 subfields) using the optical fractionator method in StereoInvestigator (Microbrightfield Inc.). While all KA-treated rats developed chronic TLE characterized by SRMS, the frequency of SRMS varied between animals, which facilitated their classification into rats with greater frequency of SRMS and rats with a lower frequency of SRMS. Stereological quantification revealed that the numbers of reelin+ neurons decline significantly in all three regions of the hippocampus in chronic TLE with maximal loss in the CA1 subfield. The overall reduction was 34% in the dentate gyrus, 68% in the CA3 subfield, and 95% in the CA1 subfield. Comparison of the frequency of SRMS with the loss of reelin+ interneurons did not suggest any link between these two phenomena in TLE, as rats with both greater and lower frequencies of SRMS exhibited similar loss of reelin+ interneurons. However, reelin+ interneuron loss was greater in rats that exhibited significant impairments in spatial learning and memory function in the WMT. Taken together, these results suggest that while greater loss of reelin+ subpopulation of interneurons does not enhance the frequency of SRMS, it does have an adverse effect on memory function in the WMT. These results also imply that reduced reelin expression is one of the contributory factors to impaired learning and memory function in chronic TLE. It is possible that reduced reelin mediates these effects via inappropriate migration of newly born neurons in the dentate gyrus, resulting in loss of access to the learning and memory network for these newly born neurons. In this context, exogenous reelin application might be helpful for alleviating learning and memory dysfunction in chronic TLE.
Supported by National Institute of Neurological Disorders and Stroke (NS 054780 to A.K.S.) and the Department of Veterans Affairs (Merit Award to A.K.S.).
J. Hardy
Reta Lilla Weston Laboratories and Department of Molecular Neuroscience, UCL Institute of Neurology, London, UK
Whole genome analysis of major neurologic disorders is now consistently finding genetic risk factors for disease. In my talk, I will detail whole genome analyses of Alzheimer's disease (AD), Parkinson's disease (PD), motor neuron disease, and progressive supranuclear palsy. I will make several points: (1) we are now able to find, in a systematic fashion, any genetic risk factor for any neurologic disease; (2) these analyses are beginning to show that in each disease, the risk factor genes map onto specific biochemical pathways; (3) some genes, and most clearly the tau (microtubule associated protein tau, MAPT) locus on chromosome 17, are risk loci for many diseases, including, for MAPT, PD and progressive supranuclear palsy certainly, and AD (probably). I will discuss the unusual structure of the MAPT locus in European populations and speculate on its evolutionary history and also describe that likely mechanism for its disease association is through differences in gene expression between different MAPT haplotypes.
B. K. Harvey,* M. T. Airavaara,* J. Hinzman,† M. J. Chiocco,* H. Shen,* D. B. Howard,* G. A. Gerhardt,† B. J. Hoffer,* and Y. Wang*
*National Institute on Drug Abuse, Baltimore, MD, USA
†University of Kentucky, Lexington, KY, USA
Neurodegeneration involves multiple cellular responses to injury or disease. Following an ischemic brain injury, one of the cellular responses that occurs is the excessive release of glutamate into the extracellular space, which leads to excitoxicity and cell death. In the current study, we examined the ability of an adeno-associated viral vector expressing the rat glutamate transporter-1 (AAV-GLT1) to decrease ischemia-induced elevation in extracellular glutamate and the associated ischemic degeneration in the brain. We first characterized the AAV-GLT1 vector and confirmed protein expression by Western blotting and immunostaining in primary cortical cultures. We next demonstrated functionality of AAV-GLT1 by measuring glutamate clearance in the rat striatum. Sprague-Dawley (SD) rats received intrastriatal injections of AAV-GLT1 or AAV-RFP (red fluorescent protein) followed 3 weeks later by in vivo electrochemical measurements of glutamate clearance using a novel microelectrode array configured for l-glutamate. Animals that received AAV-GLT1 showed a significantly higher glutamate clearance rate compared to AAV-RFP injected animals. After demonstrating the ability of AAV-GLT1 to alter extracellular glutamate levels, we tested the vector for neuroprotective effects in a rat model of stroke. AAV-GLT1 or AAV-GFP (green fluorescent protein) was administered to the right fronto-temporal cortex at three sites adjacent to the first bifurcation of the right middle cerebral artery (MCA). Three weeks after viral injection, the right MCA was transiently occluded (MCAo) by ligation with a 10–0 suture below the bifurcation for 60 min. Two days after MCAo, infarction volume was measured by triphenyltetrazolium chloride staining in 2-mm brain sections. Animals receiving AAV-GLT1 had significantly less infarction than those treated with AAV-GFP. In a separate set of animals at 3 weeks after viral injection, the tonic levels of extracellular glutamate in the lesioned cortex were measured using microdialysis fractions collected at 20-min intervals starting from 1 h prior to MCAo. MCAo increased extracellular glutamate levels, which were significantly attenuated by AAV-GLT1, compared to AAV-GFP. Overall, our data support that AAV-GLT1 transduction can attenuate ischemic injury, possibly through a reduction of extracellular glutamate in the lesioned rat cortex.
T. G. Hastings, V. Van Laar, A. Dukes, A. Mishizen, A. Mortimer, and D. Hauser
Pittsburgh Institute for Neurodegenerative Diseases and Department of Neurology, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
Oxidative stress and mitochondrial dysfunction have been implicated in the pathogenesis associated with Parkinson's disease (PD). Even though pathology in PD is not restricted to dopamine neurons, it is the dopaminergic neurons of the nigrostriatal pathway that are most susceptible to degeneration in PD. The enhanced vulnerability of this population may be linked to the presence of dopamine itself, where under oxidative conditions the catechol ring of dopamine will oxidize to form reactive oxygen species and dopamine quinones. The electrophilic quinones react readily with the thiol group of protein cysteinyl residues, resulting in covalent modification of proteins that may be detrimental to the cell. Dopamine exposure has been shown to be toxic both in cell culture and in vivo, causing selective damage to dopamine neurons that correlated with the extent of intracellular DA oxidation and protein modification. Exposure to dopamine quinone has resulted in mitochondrial dysfunction, suggesting alterations in critical proteins. Recently, the Hastings' laboratory has utilized proteomics techniques such as two-dimensional gel electrophoresis and mass spectrometry to identify proteins modified by reactive metabolites of dopamine. Results from our laboratory and others have identified critical mitochondrial proteins as well as familial PD-linked proteins as targets for the dopamine quinone. Oxidative modification of proteins are likely to lead to altered protein function, disrupted protein-protein interactions, accelerated protein degradation, and/or protein aggregation. The susceptibility of certain proteins to modification suggests they may play a role in the vulnerability of dopaminergic neurons, as well as the pathophysiology of PD, in general.
I. D. Hentall, S. S. Burns, M. L. Rodriguez, A. Irvine, L. Manoah,* and M. M. Carballosa-Gonzalez
*The Miami Project to Cure Paralysis, University of Miami School of Medicine, Miami, FL, USA
Many neurotrophic or neuroprotective cellular and molecular mechanisms are known in mammals. Some, we propose, are under coordinated central neural control and activated by signs of neuro-trauma, such as pain or injury-released molecules. The serotonin-releasing brain stem nuclei may constitute such centers. Most spinal cord serotonin (5-hydroxytryptamine, 5-HT) is released by descending fibers from the nucleus raphe magnus (NRM). We stimulated the NRM of unrestrained rats with a small (<2 g), cranially implanted wireless stimulator through a tungsten microelectrode (impedance 0.5 megohm at 1 kHz). Stimulation began 1–2 h after a moderate weight-drop contusion injury at segment T8 under isoflurane anesthesia. Monophasic pulse trains (8 Hz, 1 ms, −30 μA; charge-density 1.5 nC/cm2), using skull screws for anodal current return, were given in 5-min alternating on-off periods for 12 daylight hours for 1–8 days (mean 3.1). Motor recovery emerged significantly better than in controls. Thus, at 8 weeks, Basso, Beattie & Bresnahan (BBB) score was 14.2 (n = 12), compared with 12.8 (n = 11) in rats with inactive implants and 12.5 (n = 6) in rats without implants. BBB subscore, gridwalk speed, footprint (toe spread only), and fore paw mechanical allodynia (not hind paw) similarly improved. More days of NRM stimulation gave a greater increase in BBB score and subscore (rank order test, p < 0.05). Postmortem, at 14 weeks, myelin staining with Luxol fast blue 5 mm above the lesion (but not below) and 5-HT immunostaining 5 mm below the lesion (but not above) showed improved staining after NRM stimulation; cavity and contused tissue sizes were unchanged. Identical stimulation (mean 5.9 days) in the periaqueductal gray (PAG), a major source of NRM afferents, raised 8-week BBB scores to 12.6 (n = 7) versus 11.2 in a no-implant cohort (n = 7). Footprint toe-spread was significantly improved, but not gridwalk performance and not forelimb mechanical allodynia. Histologically, 14 weeks after spinal cord injury (SCI), total tissue around the injury segment was increased, and my-elination was greater both rostral and (unlike with the NRM) caudal by 5 mm from the epicenter; injury cavity and contused tissue volume was again unchanged. Analysis of 5-HT terminal density remains to be completed. We can account for these beneficial effects by a combination of the following: (i) sustained enhancement of analgesia and motor pattern generation by raphe spinal activity, yielding rehabilitational benefits; (ii) neuroprotective and neurotrophic effects of 5-HT, possibly mediated by 5-HT7 receptors and their elevation of cAMP (preliminary findings show increased spinal cord cAMP with NRM stimulation); (iii) peptides coreleased with 5-HT, such as thyrotropin-releasing hormone (TRH), which has documented benefits after SCI; (iv) NRM axons' greater intrinsic propensity to survive and regrow after SCI. The PAG effect is likely mediated by the NRM. The PAG is a safe, proven target for neurosurgical pain treatment by deep brain stimulation (DBS), which suggests a potential treatment for early, incomplete SCI.
J. Jäderstad,* L. M. Jäderstad,* J. Li,‡ S. Chintawar,§ C. Salto,† M. Pandolfo,§ V. Ourednik,‡ Y. D. Teng,¶ R. L. Sidman,‡ E. Arenas,† E. Y. Snyder,# and E. Herlenius*
*Department of Women & Child Health, Karolinska Institutet, Stockholm, Sweden
†Department of Medical Biochemistry & Biophysics, Karolinska Institutet, Stockholm, Sweden
‡Department of Neurology, Beth Israel Deaconess Medical Center, Boston, MA, USA
§Service de Neurologie, Hôpital Erasme, University Libre de Bruxelles, Brussels, Belgium
¶Department of Neurosurgery, Brigham & Women's Hospital, Boston, MA, USA
#Burnham Institute for Medical Research, La Jolla, CA, USA
How grafted neural stem cells (NSCs) and their progeny integrate into recipient brain tissue and functionally interact with host cells is as yet unanswered. We report that, in organotypic slice cultures analyzed by ratiometric time-lapse calcium imaging, current-clamp recordings, and dye-coupling methods, an early and essential way in which grafted murine or human NSCs integrate functionally into host neural circuitry and affect host cells is via gap-junctional coupling, even before electrophysiologically mature neuronal differentiation. The gap junctions, which are established rapidly, permit exogenous NSCs to influence directly host network activity, including synchronized calcium transients with host cells in fluctuating networks. The exogenous NSCs also protect host neurons from death and reduce such signs of secondary injury as reactive astrogliosis. To determine whether gap junctions between NSCs and host cells may also mediate neuroprotection in vivo, we examined NSC transplantation in two murine models characterized by degeneration of the same cell type (Purkinje neurons) from different etiologies, namely, the nervous and SCA1 mutants. In both, gap junctions (containing connexin 43) formed between NSCs and host cells at risk, and were associated with rescue of neurons and behavior (when implantation was performed before overt neuron loss). Both in vitro and in vivo beneficial NSC effects were abrogated when gap junction formation or function was suppressed by pharmacologic and/or RNA inhibition strategies, supporting the pivotal mediation by gap-junctional coupling of some modulatory, homeostatic, and protective actions on host systems as well as establishing a template for the subsequent development of electrochemical synaptic intercellular communication. During the meeting we will present new data regarding the importance of gap junctions in the host response and stem cell rapid and long-term interaction.
S. Jergova, M. Varghese, S. Gajavelli, I. Hentall, and J. Sagen
The Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA
A serious outcome of peripheral nerve injury is the development of chronic pain, which does not respond well to conventional treatment. Therefore, there is a need for novel treatment approaches. One of the hypothesized mechanisms underlying chronic neuropathic pain is increased hyperexcitability of spinal dorsal horn neurons due to loss of inhibitory γ-aminobutyric acid (GABA)ergic interneurons. Previous findings in our laboratory have shown that neural progenitor cells (NPCs) predifferentiated to a GABAergic phenotype can partially reverse neuropathic pain symptoms and reduce spinal hyperexcitability when transplanted into the dorsal horn of nerve-injured rats. The effect of transplanted cells may be mediated through GABA receptors. To test this hypothesis, pharmacological manipulation of GABA signaling was used. The effects of GABAA receptor antagonist bicuculline (BIC) and GABAB receptor antagonist CGP35348?(CGP) and a GABA reuptake inhibitor SKF 89976A were evaluated in rats with a peripheral nerve injury and spinal GABAergic NPCs transplants. One week following unilateral chronic constriction injury (CCI) of the sciatic nerve, rats exhibited increased sensitivity to noxious (hyperalgesia) and innocuous (allodynia) stimulation of the ipsilateral hind paw. Spinal application of either BIC or CGP increased firing of wide dynamic range (WDR) neurons to thermal stimuli in CCI rats. Antagonist treatments also caused enhanced responses of Aβ and C fibers to transcutaneous stimulation. CCI rats received spinal transplants of predifferentiated GABAergic NPCs from primary cultures by microinjection into the lumbar dorsal horn, ipsilateral to the injury, or control microinjections of phosphate-buffered saline (PBS). NPC-treated animals showed decreased hyperalgesia and allodynia, 1 week posttransplantation. By contrast, PBS-injected CCI rats continued to display robust hyperalgesia and allodynia. Also, in PBS-injected CCI rats, WDR neurons exhibited exaggerated responses to peripheral cutaneous stimulation. In animals with NPC transplants, WDR neuron responses to cutaneous stimulation were markedly reduced. Systemic (IP) or topical spinal application of BIC and CGP attenuated the antinociceptive effect of the NPC transplants, indicating a GABAergic-mediated antinociception of the spinal transplants. Increased antinociception was observed after injection of SKF 89976A in NPC-transplanted rats, suggesting a GABAergic-mediated mechanism of the transplants. By contrast, SKF 89976A alone had only a moderate effect in PBS-injected CCI rats. These results suggest that a loss of spinal GABAergic inhibition mediates nerve injury-induced chronic pain. A primary mechanism of the effect of the transplants in the current study is release of GABA. Direct delivery of GABA to the spinal dorsal horn using cell-based or possibly gene therapy may be ideal for the long-term management of chronic pain.
Supported by NS51667.
V. Joers,*† K. Seneczko,* N. Goecks,* T. J. Kamp,§ K. Brunner,* H. Simmons,* J. Holden,*‡ and M. E. Emborg*†‡
*Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
†Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI, USA
‡Department of Medical Physics, University of Wisconsin-Madison, Madison WI, USA
§Department of Medicine, University of Wisconsin-Madison, Madison, WI, USA
Parkinson's disease (PD) is characterized by the degeneration of dopaminergic nigrostriatal neurons and is classically defined as a movement disorder. Yet PD patients experience other symptoms, such as pain, sleep disturbances, autonomic dysfunction, and cognitive deficits. These nonmotor symptoms frequently present in PD patients; however, they can vary in severity and time of onset across individuals. Furthermore, traditional dopamine (DA) replacement therapies for PD patients do not provide relief of these symptoms. At this time preclinical models of PD characterizing the nonmotor components, such as neurodegeneration of the autonomic nervous system resulting in dysautonomias, are missing and, in consequence, development of new treatments is difficult. In this study we aimed to develop a cardiac dysautonomia model in rhesus monkeys and generate a toolbox of methods that can be utilized to assess neurodegeneration in the heart and possible therapies. We hypothesized that systemic administration of 6-hydroxydopamine (6-OHDA) would induce cardiac dysfunction in the monkeys through neurodegeneration of the ganglionic catecholaminergic neurons innervating the heart. Five adult male rhesus monkeys (ages 5–7 years old, 7–9 kg) were evaluated before and after intravenous administration of 6-OHDA through clinical evaluations, EKG, ultrasound, blood pressure monitoring, troponin, and catecholamine blood chemical assays. In addition, in vivo monitoring of neurodegeneration of the catecholaminergic terminals in the heart was achieved with positron emission tomography (PET) using the nor-epinephrine analog 11C-meta-hydroxyephedrine (MHED). Baseline PET scans of the normal monkeys displayed normal MHED uptake in the heart wall, specifically a larger uptake in the left ventricle walls. PET images obtained 7 days post-6-OHDA revealed an extensive decrease in MHED uptake that persisted up to 3 months, confirming loss of noradrenergic innervation. These results suggest that intravenous administration of 6-OHDA induces neurodegeneration of catecholaminergic neurons innervating the heart and can be used to model heart autonomic dysfunction in PD. In future studies we plan to use the cardiac dysautonomia model to evaluate therapies.
D. Joseph,* J. Trecki,* E. Yehling,* H. Hathaway,† T. Mhyre,* H. J. Federoff,* and K. Maguire-Zeiss*
*Department of Neuroscience, Georgetown University Medical Center, Washington, DC, USA
†Department of Pharmacology, Georgetown University Medical Center, Washington, DC, USA
Parkinson's disease (PD) is an age-related neurodegenerative disorder typified by the loss of substantia nigra pars compacta dopamine neurons (DAN), the presence of large proteinaceous synuclein-positive inclusions, and activated microglia. Evidence also exists for oxidative damage including lipid peroxidation, protein nitration, and changes in antioxidant defense mechanisms. The etiology of sporadic PD is still unknown, but several lines of evidence suggest that misfolded α-synu-clein promotes inflammation and oxidative stress that engenders neurodegeneration. Recent work from our laboratory has demonstrated in synuclein transgenic mice increased microglial activation in the substantia nigra that markedly antedates DAN cell loss. This activation is thought to be the result of a direct interaction with α-synuclein because cultured microglia are activated following treatment with α-synuclein in a manner that is partially mediated through pattern recognition receptor (PRR) engagement. Here we show that various conformers of synuclein have disparate effects on microglia morphology and PRR ligation. We also report an increase in antioxidant response genes in the substantia nigra of synuclein-overexpressing mice compared with nontransgenic animals. This increase occurs as early as 1 month of age prior to any neuropathological changes, suggesting that early oxidative events may incite later pathology.
L. Kelly,*† M. Newman,*† J. H. Kordower,*‡ and R. Bakay*†
*Department of Neurosurgery, The Graduate College, Rush University Medical Center, Chicago, IL, USA
†Department of Pharmacology, The Graduate College, Rush University Medical Center, Chicago, IL, USA
‡Department of Neuroscience, The Graduate College, Rush University Medical Center, Chicago, IL, USA
Previous findings from our laboratory indicate that human umbilical cord blood (hUCB), particularly the fraction of CD133 stem cells, yields significant behavioral and histological improvements over human embryonic stem cells (hESC) in a rodent model of Parkinson's disease (PD). In comparison to hESCs, hUCBs are a heterogeneous population rich in hematopoietic stem and progenitor cells, lack ethical implications, can be collected easily and noninvasively, are more immune immature than other adult stem cells, and have over two decades of history treating various clinical diseases. One concern about hUCB cells is their propensity to migrate out of the transplant site. The hypothesis is that combining transfection with a neurotrophic factor prior to transplantation with hUCB cells will enhance the retention, dopamine (DA) phenotype, and restorative properties of the cells at the graft site. The present study examined the effects of striatal CD133 transplantation with and without pretreatment with striatal glial cell line-derived neurotrophic factor (GDNF) transfection. The specific aim of our study was to determine the viability and potential of CD133s in a GDNF-enriched environment to repair, replace, or regenerate the loss of DA in the striatum of bilaterally 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-treated parkinsonian-like primates. To test the hypothesis, cynomolgus monkeys (n = 10) were lesioned with both a right intracarotid and systemic injection of MPTP. The monkeys were symptomatically stable for over a year before being transfected with GDNF (n = 5) or needle sham lesioned (n = 5) 1 month prior to transplantion with CD133 stem cells (n = 10) into the right putamen. Timed arm-reach tasks and clinical rating scores on/off l-dopa were performed weekly and before sacrifice. In this model of PD, at least 60% of DA neurons in the SNpc are lost, with greater than 80% depletion of DA levels in the striatum, and full PD-like motor deficits are observed by 3 weeks postlesion. One monkey from each group was sacrificed at 1 month after transplantation and the remainder at 4 months. The brains, livers, spleens, bone marrow, lymph nodes, and peripheral blood were taken from all primates for immunohistochemistry. Tissue sections were stained for tyrosine hydroxylase (TH), dopamine transporter (DAT), neuronal nuclei (NeuN), CD133 human nuclei (CD133), or double-labeled for DAT and CD133 for cell counts. All subjects demonstrated improvement in both behavioral assessments. The greatest statistically significant improvement was ipsilateral to the graft. The most fundamental finding thus far is that, when transplanted into a GDNF-enriched striatum, more CD133 stem cells remain at the injection site.
Z. Z. Khaing,*1 S. A. Geissler,*1 T. Schallert,† R. J. Grill,‡ and C. E. Schmidt*
*Department of Biomedical Engineering, The University of Texas at Austin, Austin, TX, USA
†Department of Psychology, The University of Texas at Austin, Austin, TX, USA
‡Department of Integrative Biology and Pharmacology, The University of Texas Health Science Center, Houston Medical School, Houston, TX, USA
Cervical spinal cord injuries (SCIs) can result in loss of hand functions, which are among the most devastating deficits in patients. Impairment is typically chronic because adult mammalian axons in the CNS do not normally regenerate after injury. This regeneration failure is attributed in substantial part to components of the scar tissue and myelin-associated inhibitors within the injury site. Previously, we have shown that degradation-resistant hyaluronic acid (HA) hydrogels can inhibit astrocyte activation and proteoglycan deposition after SCI in adult rats. It is not known whether the modified scar tissue afforded by the presence of HA hydrogels can support axonal growth and functional improvement. HA alone is generally non-cell adhesive. Therefore, here we examined the effects of unmodified HA hydrogels compared to HA hydrogels modified with laminin (HA/LN) after acute lateral hemisection of the cervical spinal cord in adult rats. A gelfoam-only group was included as control. A number of behavioral tests (limb use during vertical-lateral rearing and exploration of the wall of a cylindrical enclosure, vibrissae-elicited forelimb placing, and grid walk tests) were used to assess forelimb function for 6 weeks after SCI. All tests readily detected SCI-induced deficits relative to noninjury over the 6-week period. Using the cylinder test, we found that animals treated with HA/LN hydrogel reached higher and explored more using their affected forepaw than HA or gelfoam-only animals. This improvement in the HA/LN implant group was statistically significant starting at week 2 and escalated through week 4. Relative to gelfoam-only SCI rats, no significant beneficial effects were detected in the HA or HA/LN groups in the grid walk or vibrissae-elicited forelimb placing tests. Histological and axon tract tracing analyses are currently under way to determine the type and extent of axonal regeneration. Results presented here suggest that HA-based scaffolds, modified with cell adhesive proteins, may hold great potential for repair strategies after spinal cord injury.1These authors contributed equally.
This work was supported by The Gillson Longenbaugh Foundation and The David Van Wagener Spinal Cord Fund of the Joy to The World Foundation to C.E.S.
C. E. Khodr, Y. Han, and M. C. Bohn
Neurobiology Program, Department of Pediatrics, Children's Memorial Research Institute, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
α-Synuclein (α-syn) gene mutations, multiplications, and promoter variants have been linked to familial forms of Parkinson's disease (PD). α-syn is an abundant protein in brain and a major component of Lewy bodies, intracellular inclusions characteristic of PD. We have previously reported effective in vivo silencing of ectopically expressed human (hu) α-syn in rat striatum and substantia nigra (SN) using lentiviral and adeno-associated viral (AAV) vectors, respectively, harboring a hu α-syn-specific short hairpin (sh) RNA under control of the H1 promoter. In the SN, an unexpected reduction in tyrosine hydroxylase (TH) immunoreactivity (ir) was observed in the region of the SN where hu α-syn was silenced. Nevertheless, hu α-syn gene silencing reversed the hu α-syn-induced forelimb use deficit. These findings suggest a partial rescue effect of hu α-syn gene silencing on forelimb use despite an apparent toxic effect on dopamine (DA) cells. To further investigate this conundrum, the effects of hu α-syn and silencing vector expression on distribution of DA projections to striatum were studied. Adult, male Sprague-Dawley rats received a unilateral SN injection of AAV-hu α-syn and AAV-α-syn shRNA and striatal-projecting DA neurons were labeled using retrograde transport of fluorogold (FG). One group of rats (prelabeled group) received bilateral striatal FG injections 1 week before injection of AAV into SN to determine whether treatment reduces the number of SN DA neurons that send axonal projections to a defined site in the striatum. Another group of rats (postlabeled group) received bilateral striatal FG injections 5 days before euthanasia to label DA neurons in the SN that send axonal projections to the striatal site at the end of the experiment. Rats were euthanized at 9 weeks postinjection and brains were stained for TH-ir and hu α-syn-ir, and the numbers of FG-positive cells in SN were counted bilaterally. In the prelabeled group, FG-positive cells in the injected SN (269 ± 39, n = 4) were significantly reduced compared to control SN (456 ± 60, n = 4; p ≤ 0.05). In contrast, no decrease in FG labeled DA neurons was observed in the postlabeled group. However, when FG counts were divided into subregions of the SN (i.e., transduced or nontransduced DA neurons), FG labeled DA neurons were reduced in the virus-transduced region in both groups (p ≤ 0.05). However, the percent of FG-positive cells in nontransduced SN compared to uninjected SN was higher in the postlabeled group (106.7 ± 12.3%, p ≤ 0.05, n = 5) than in the prelabeled group (69.5 ± 8.8%, n = 4). These data suggest there is an initial toxic effect of hu α-syn and α-syn shRNA on nigrostriatal projections and that neighboring DA neurons respond by enhancing their projections to striatum. Ongoing experiments are examining the effects of treatments on DA levels and the effects of silencing hu α-syn overexpression in SN using a second generation silencing vector design.
Supported by grants from the Department of Defense NO06079001 and NIH grants NS054989 and T32 NS041234.
F. Klempin, L. D. Peterson, and D. A. Peterson
Center for Stem Cell and Regenerative Medicine and Department of Neuroscience, The Chicago Medical School, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
Neurogenic niches of the adult brain contain various cell components, including neural stem cells, uncommitted and lineage committed neural progenitor cells, mature neurons, astrocytes, microglial, and endothelial cells. Cells of the neurogenic niche express a variety of signaling molecules, many of which have been shown to modulate proliferation and differentiation. However, this neurogenic activity is spatially restricted to a very small portion of the brain. Despite much study, it appears that other brain regions, such as the functionally important cerebral cortex, do not produce neurons under normal conditions. This may be due to the absence of neural progenitor cells or the lack of supporting neurogenic signals in the nonneurogenic regions. A lack of neurogenic signals is supported by the observation that neither migrating neuroblasts arising from neurogenic areas nor grafted neural progenitor cells differentiate into cortical neurons. We have identified the existence of endogenous immature proliferating cells within the entorhinal cortex that remain phenotypically uncommitted. We hypothesized that these cells remain uncommitted due to the lack of instructive differentiation signals in the mature cortical environment. To test this hypothesis, we used a retroviral gene delivery approach to either suppress glial fate or instruct neuronal fate. To suppress glial fate, a retroviral vector containing a dominant-negative form of Olig2 (Olig2 VP16-GFP; construct a gift from M. Goetz) was delivered to the entorhinal cortex. Expression of the dominant-negative construct shifted the fate of infected cells to production of astrocytic and neuronal phenotypes relative to GFP expression alone. By 5 days of expression, some 10% of infected cortical neural progenitor cells expressed the mature neuronal marker NeuN and appeared morphologically indistinguishable from adjacent cortical neurons. Similarly, retroviral delivery of Pax6 shifted the fate of infected cells to a neuronal phenotype relative to GFP alone. In both cases, most of the resulting new neuronal cells also expressed the neurotransmitter glutamate. Thus, there exists a population of cortical neural progenitor cells that are susceptible to lineage instruction to produce neurons. As there is a series of instructive signals guiding cell fate during development, we expect that efficiency of neuronal fate commitment will be improved by introducing a combination of signals in an appropriate temporal sequence. Ongoing work is investigating additional signal expression and assessing the longevity and functional integration of these newly produced neurons.
Supported by NIH AG020047 and DOE DE2009174 to D.A.P.
M. A. Lane,* K.-Z. Lee,† D. D. Fuller,† and P. J. Reier*
*Department of Neuroscience, University of Florida College of Medicine and McKnight Brain Institute, Gainesville, FL, USA
†Department of Physical Therapy, University of Florida College of Public Health and Health Professions, Gainesville, FL, USA
A significant potential for natural recovery exists within the injured spinal cord (SCI), and a challenge is to optimally interface therapeutic interventions with post-SCI neuroplasticity. This requires an understanding of the neural circuitry involved and the range of spontaneous recoveries under different injury conditions. To address these issues, we have been investigating the spinal neural circuitry related to diaphragm function and respiratory changes following cervical SCI above or within the phrenic motoneuron (PhMN) pool. Neuroanatomical findings have demonstrated the presence of a population of spinal prephrenic interneurons (PINs), some of which receive descending projections from brain stem regions (VRC) subserving inspiratory drive to PhMNs. Correlative extracellular electrophysiological data suggest some PINs exhibit activity in phase with PhMNs and are responsive to hypoxic challenge in synchrony with elevated PhMN activity. These data support the possibility that PINs may play a role in some aspects of spontaneous recovery of ipsilateral PhMN function following a C2 hemisection lesion (C2Hx). To date, such neuroplasticity has been thought to be mediated entirely by uninjured, contralateral VRC fibers. Although there is a recovery of ipsilateral hemidiaphragm activity following C2Hx, prior findings suggest that ventilatory improvement was modest. Diaphragm electromyography (EMG) recordings 12 weeks post-C2Hx also revealed little progressive change beyond initial recovery, and anterograde tracing of VRC projections showed reduced innervation of ipsilateral PhMNs. To obtain a further measure of respiratory drive to these neurons, experiments were performed in which ipsilateral (left) and contralateral (right) diaphragm EMG activity was recorded following left or right phrenicotomies. Transection of the ipsilateral phrenic nerve resulted in a silencing of left diaphragm activity, whereas a compensatory increase in EMG response was seen contralaterally. Contralateral phrenicotomy silenced the right hemidiaphragm; however, only a minimal change in left diaphragm EMG recordings was observed. Absence of a significant ipsilateral response suggests that ventilation in the C2Hx rat is more reflective of compensatory rather than restorative neuroplasticity. We next explored to what extent respiratory function is affected after a clinically more representative contusion injury at the level (C3/4) of the PhMN pool. Plethysmographic measurements of breathing frequency and tidal volume were obtained under baseline room air conditions and during CO2 (hypercapnic) challenge. Terminal recordings of diaphragm activity also were obtained. Both physiological measures revealed a progressive loss in the capacity to increase respiratory drive in response to hypercapnia. These studies reveal differential degrees of neuroplasticity relative to predominantly white matter (C2Hx) and combined white and gray matter (contusion) damage.
Supported by 1 R01 NS054025 and The Anne and Oscar Lackner Chair (P.J.R.), and the Paralyzed Veterans of America (M.A.L.).
B. W. M. Lau,*†§1 S.-Y. Yau,*†§1 T. M. C. Lee,†‡ Y.-P. Ching,*†§ S.-W. Tang,* and K.-F. So*†§¶
*Department of Anatomy, Li Ka Shing Faculty of Medicine, The University of Hong Kong, Pokfulam, PR China
†The State Key Laboratory of Brain and Cognitive Sciences, The University of Hong Kong, Pokfulam, PR China
‡Laboratory of Neuropsychology, The University of Hong Kong, Pokfulam, PR China
§Research Centre of Heart, Brain, Hormone and Healthy Aging, Faculty of Medicine, The University of Hong Kong, Pokfulam, PR China
¶Joint laboratory for Brain function and Health (BFAH), Jinan University and The University of Hong Kong, GuangZhou, PR China
Adult neurogenesis occurs in the dentate gyrus of hippocampus and subventricular zone (SVZ). Deficit of hippocampal neurogenesis brings impairment of spatial learning and memory, which suggests the physiological role of new born neurons in memory formation. In contrast, functional significance of new neurons born in SVZ remains elusive. Recent studies showed that neurogenesis in the SVZ is increased by pheromone exposure from the opposite sex and neurogenesis is essential for normal mating behavior of female mice. These results suggest that SVZ-derived new neurons may take part in reproductive function. The current study aimed at exploring the relationship between adult neurogenesis and male sexual behavior. Adult male Sprague-Dawley rats were treated with corticosterone and/or paroxetine (an antidepressant) for 2 weeks. These two drugs were shown to suppress and promote neurogenesis, respectively. Mating behavior was assessed after the treatment, and bromodeoxyuridine immunohistochemistry was used to identify new cells. Neural circuit activation of the mating-related pathway was assessed by c-fos immunostaining. To further confirm necessity of neurogenesis in male mating behavior, inhibition of neurogenesis was performed by intracerebroventricular infusion of cytostatic cytosine arabinose (Ara-c) and sexual behavior test was carried out. From cell quantification and mating behavior test, corticosterone treatment inhibited neurogenesis in the olfactory epithelium and SVZ, and simultaneously an inhibited sexual performance was found. Conversely, paroxetine treatment increased newborn neurons and enhanced the sexual performance in corticosterone-treated animals. When Ara-c was used to suppress neurogenesis, the sexual performance was suppressed. In addition, a decrease in cell proliferation was associated with a decreased level of c-fos expression in regions related to mating, including medial amygdala, bed nucleus of stria terminalis, and medial preoptic areas. Taken together, the results suggest that neurogenesis plays a role in male reproductive behavior in rodents. Alteration of neurogenesis rate may be a potential treatment option for sexual dysfunction.
1Both authors contributed equally to this work.
D. C. Lee, J. Rizer, M.-L. Selenica, P. Reid, C. Kraft, A. Johnson, L. Anderson, M. N. Gordon, C. A. Dickey, and D. Morgan
Byrd Alzheimer's Institute, Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
Inflammation and microglial arguably contribute to Alzheimer's disease (AD) pathology. Evidence shows that pathological hallmarks, notably amyloid-beta (Aβ) and hyperphosphorylation of tau activate microglia. However, it is less well understood how certain inflammatory events modulate Aβ and tau pathology. These studies were designed to elucidate the role of acute inflammation and its effects on tau pathology. We used transgenic rTg4510 mice, which express the P301L mutation (4R0N TauP301L) and initiate tau pathology between 3 and 5 months of age. We injected either saline or lipopolysaccharide (LPS; 5 μg per site; two sites) into the right frontal cortex and hippocampus of 5-month-old mice rTg4510 or nontransgenic littermate mice. One week postinjection, brains were harvested and analyzed by immunohistochemistry for markers of microglial activation and phospho-tau species using unbiased image analysis. Microglial activation was measured by induction of CD45 (general activation marker) and alternative activation markers YM1 and arginase. LPS induced significant activation of CD45 and arginase activation. YM1 was further exaggerated in transgenic mice treated with LPS. Expression of phospho-tau Ser199/202 and Ser396 was observed in tau-laden area of transgenic mice only and increased in mice that received LPS compared to saline injections. However, mature tangles (silver positive) remained unaffected by LPS administration. These data suggest that inflammatory stimuli can facilitate tau pathology and that tau impacts the certain alternative activation markers. Coupled with prior results demonstrating clearance of Aβ by similar LPS injections, these results suggest that inflammation may have opposite effects on amyloid and tau pathology, possibly explaining the failures (to date) of anti-inflammatory therapies in AD patients.
This work was supported by P01 AG 04418, R01AG 15490, R01 AG18478, and P50 AG 225711, Alzheimer's Association (NIRG-06-27099), the NIA (AG031291), The Abe and Irene Pollin Fund for CBD from CurePSP.
J. W. Lee,* S. Gajavelli,† D. Asmar,* and J. Sagen†
*Department of Biology, William Paterson University, Wayne, NJ, USA
†Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA
Pharmacological compounds provide minimal alleviation from neuropathic pain, with a short-lasting effect. Recent human genetic data show ion channels play a major role in pain perception. Absence of gene encoding for voltage-gated Nav 1.7 renders patients incapable of sensing pain; abnormal expression causes development of erythro-melalgia. Neurotoxins that block opening of voltage-dependent calcium channels, ω-conotoxin, also provide short-lasting alleviation from mechanical and thermal hyperalgesia. Synthetic conotoxin, Ziconotide, is currently being used for pain management in terminal cancer patients. Advancements in molecular techniques permit insertion/attachment of genes of analgesic neuropeptides to viral vectors. Sub-dermal or intraneural injections will retrogradely transport the viral-peptide complex to dorsal root ganglia (DRGs) for continual replication/synthesis of neuropetides using host nuclear machinery. The goal of the present study is to examine long-term usefulness of transfecting the DRGs with adenovirus-ω-conotoxin complex to inhibit peripheral nociceptive signal transduction and possibly activation of second-order nociceptive neurons in the spinal cord. Adult male Sprague-Dawley rats and C57/6J mice were injected with 2–5 μl of adenovirus-ω-conotoxin complex into the left sciatic nerve. Seven days later, animals were tested for formalin behavior. Intraplantar injection of formalin (5%, 30–50 μl) was made in the ipsilateral hindpaw, and the numbers of paw flinches were recorded at 5-min intervals for 60 min. Controls received injections of adenovirus only. Animals were perfused trans-cardially with cold saline and paraformaldehyde solutions, and spinal cords and DRGs were removed for ω-conotoxin peptide immunohistochemistry. The number of infected DRG neurons was correlated with the analgesic behaviors. Animals injected with ω-conotoxin expressed significantly fewer numbers of paw flinches during formalin phase II compared to control (conotox: 207 ± 53, control: 67 ± 22). The significance was more pronounced during the phase IIa compared to phase IIb. There was no significant difference in paw flinch behaviors during formalin phase I between control and the conotoxin group. Neuropathic pain is a complex condition that is difficult to treat with current pharmacological agents. Recent advancements in molecular techniques make it possible to create genetically engineered viruses to deliver genes of interest to select population of neurons. Such targeted delivery may provide a better therapeutic window with minimal side effects compared to current drugs. As such, the present study examined the effect of transfecting DRGs with ω-conotoxin in reversing peripheral neuropathic pain.
B. Li,* A. Fagan,† X. Liu,* M. E. Gonzalez-Toldo,* C.-S. Piao,* S. Zhang,* and L.-R. Zhao*†
*Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
†Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Alzheimer's disease (AD) is the major cause of dementia and the fifth leading cause of death in the elderly in the US. Although the cause of AD remains uncertain, substantial evidence shows that toxic amyloid-beta (Aβ) peptide plays a critical role in the progress of this devastating disease. Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) are the hematopoeitic growth factors regulating blood cell production and bone marrow stem cell mobilization. Recently, we have demonstrated the therapeutic effects of SCF in combination with G-CSF (SCF + G-CSF) on chronic stroke. The purpose of this study is to determine whether SCF + G-CSF can reduce the burden of Aβ deposits and to improve cognitive function in a mouse model of AD. APP/PS1 mice were used as the mouse model of AD. To track bone marrow-derived cells (BMDCs) in the brain, the bone marrow of the APP/PS1 mice was destroyed by 900 rad X-ray and was replaced with the bone marrow harvested from mice expressing green fluorescent protein (GFP) at the age of 7 months. Six weeks after bone marrow transplantation, mice were randomly divided into a saline control group and an SCF + G-CSF-treated group based on a water maze test. SCF (100 μg/kg) + G-CSF (50 μg/kg) or equal volume of saline was subcutaneously administered for 12 days. Circulating bone marrow stem cells (CD117+ cells in blood) were determined using flow cytometry 1 day after the final injection. Nine months after treatment, spatial learning and memory were evaluated with the water maze test. At the age of 18 months, mice were sacrificed. Brain sections were processed for immunohistochemistry to determine Aβ deposits (4G8 antibody), microglia (Iba1 antibody), and GFP-expressing BMDCs in the brain. The level of CD117+ cells in peripheral blood was significantly elevated by SCF + G-CSF, indicating that the treatment paradigm is effective in mobilizing bone marrow stem cells. In the water maze test, a significant short time in the escape latency was observed in the mice treated with SCF + G-CSF, suggesting that the treatment induces a long-term improvement in spatial learning and memory. In histological data, we found that the number of senile plaques was significantly reduced in SCF + G-CSF-treated mice and that SCF + G-CSF-treated mice showed a significant high number of GFP-Iba1-expressing cells surrounding Aβ plagues, suggesting that SCF + G-CSF treatment reduces Aβ deposits and that this reduction may be through enhancing the clearance of senile plaques by bone marrow-derived microglia. SCF + G-CSF treatment can delay AD progress by reducing the Aβ load and improving cognitive function. These data would help in developing a new treatment to improve health of AD.
This study was supported by The Franks' Fundation and Louisiana Gene Therapy Research Consortium.
C. A. Lieu,* V. Murthy,† R. B. Mailman,† and T. Subramanian*
*Departments of Neurology and Neural & Behavioral Sciences, The Pennsylvania State University College of Medicine and Hershey Medical Center, Hershey, PA, USA
†Department of Pharmacology, The Pennsylvania State University College of Medicine and Hershey Medical Center, Hershey, PA, USA
The high-affinity D1 dopamine receptor full agonist dihydrexidine (DHX) had been shown to cause profound attenuation of parkinsonism in MPTP-treated primates. Unfortunately, further studies of DHX in Parkinson's disease (PD) patients demonstrated that it had limited oral bioavailability, short duration of action, and caused profound hypotension when given intravenously. Here we describe the behavioral effects of a novel D1 full agonist EFF0311 (an analog of DHX) in rats rendered hemiparkinsonian (HP) by stereotactic injection of 6-hydroxydopamine (6-OHDA) into the medial forebrain bundle. All HP rats demonstrated >7 rotations/min on apomorphine testing on two separate occasions 2 weeks apart to demonstrate stable complete lesioning. In experiment 1, we hypothesized that EFF0311 will induce a longer duration of action and increased efficacy when compared to the equivalent DHX dose. Animals were treated with various doses of EFF0311 (0.1, 0.3, and 1 mg/kg) or DHX (1 mg/kg) (n = 4 per group, SC). After injection, animals were tested for contralateral rotational behavior over 360 min. In experiment 2, we hypothesized that the rotational effects of EFF0311 are mediated by activation of D1 receptors, and that blockade of D1 receptors will prevent these actions. Animals were initially treated with vehicle, D1 antagonist SCH39166 (1 mg/kg) or D2 antagonist remoxipride (3 mg/kg) (n = 2–3 per group). After 15 min, animals were injected with EFF0311 at 1 mg/kg. Contralateral rotational behavior was monitored for 180 min. In experiment 1, we found that EFF0311 induced a dose-dependent effect. Furthermore, 1 mg/kg of EFF0311 induced a longer duration of action and increased potency when compared to the equivalent DHX dose. Average total number of rotations over 360 min was 4, 370, and 3,659 with 0.1, 0.3, and 1 mg/kg EFF0311, respectively. DHX (1 mg/kg) induced 1,026 total rotations (3× less than 1 mg/kg EFF0311). In experiment 2, the D1 antagonist SCH39166 completely blocked EFF0311-induced rotation, whereas the D2 antagonist remoxipride slightly increased turning (1,972 vs. 1,370 for vehicle). These results suggest that EFF0311 has selectivity for D1 receptors and, compared to almost all earlier D1 agonists, a longer duration of action. This combination of desirable pharmacological properties and longer duration of action may make this compound very useful for studies of pharmacotherapeutic interventions in PD. For example, the relative role of different dopamine receptor subtypes in the induction and/or reversal of drug-induced and graft-induced dyskinesias are controversial, and sometimes limited by the drugs available to study this critical aspect of current PD treatment. Future studies will investigate if EFF0311 can significantly ameliorate parkinsonism and reduce or prevent dyskinesias in preclinical models of PD, and how these behaviors are affected by D1 receptor activation. In addition, it has been proposed that use of drugs with the properties of EFF0311 might also have antidegenerative effects that could slow progression of PD. EFF0311 thus represents a tool to advance our knowledge in these areas, and possibly lead to novel therapeutics for PD.
Grant support: MH082441, MH040537, PA Keystone Innovation Grant (R.B.M.), NS042402 and PA Tobacco settlement fund (T.S.).
S.-Z. Lin,* W.-C. Shyu,* S.-P. Liu,* D.-C. Chen,† H.-J. Wang,* C.-C. Lin,§ Y.-J. Liu,¶ and C.-H. Tsai‡
*Center for Neuropsychiatry and Graduate Institute of Immunology, China Medical University & Hospital, Taichung, Taiwan
†Department of Neurosurgery, China Medical University & Hospital, Taichung, Taiwan
‡Department of Neurology, China Medical University & Hospital, Taichung, Taiwan
§Department of Radiology, China Medical University & Hospital, Taichung, Taiwan
¶Department of Engineering, Fong-Gia University, Taichung, Taiwan
We have demonstrated that peripheral blood hematopoietic stem cell (PBSC, CD34+) transplantation improved the neurological deficit after old cerebral infarction in a previous animal study. We next examined the feasibility and efficacy of using intracerebral implantation of PBSCs mobilized by granulocyte-colony stimulating factor (G-CSF) to treat patients suffering from an old stroke. We prospectively randomized 36 patients suffering from an old cerebral infarction [middle cerebral artery territory as documented on the T2 weight image (T2WI) of MRI] between 6 months and 5 years of onset who had initial scores on the National Institute of Health stroke scale (NIHSS) of between 9 and 20. After determining eligibility, we randomly assigned them into three groups: G-CSF + PBSC implantation group (n = 15), G-CSF-alone group (n = 3), and control group (with usual care) (n = 15). The G-CSF + PBSC-treated and G-CSF-alone group received subcutaneous G-CSF injections (15 μg/kg per day) for 5 consecutive days. The G-CSF + PBSC-treated group also received stereotactic implantation of CD34+ immunosorted PBSCs mobilized by G-CSF. We used the following clinical scales to assess neurological recovery: National Institute of Health Stroke Scale (NIHSS), European Stroke Scale (ESS), ESS Motor Subscale (EMS), and Barthel Index (BI). In this trial, all clinical information and evaluation data from each patient were assessed blindly by the data recorders and clinicians. Patients have been followed for 12 months. The primary outcomes were assessed by group percentage changes between baseline and 12-month follow-up in mean group scores on four clinical scales. As the secondary end point, we also assessed neurological functional activity with 18fluorodeoxyglucose positron emission tomography (18FDG-PET) to measure the metabolic uptake of FDG, monitored the white matter tract integrity using diffusion tensor tractography (DTI) in MRI, and examined the electrophysiological conductance of corticospinal system (CSS) of motor evoked potential (MEP) by transcranial magnetic stimulation (TMS) in the cortical areas surrounding the ischemic core. No severe adverse effects were seen in G-CSF + PBSC-treated or G-CSF-alone patients, who had received subcutaneous G-CSF injections (15 μg/kg) for 5 consecutive days immediately after an episode of acute cerebral infarction. All patients completed the 6-month follow-up. In the G-CSF + PBSC-treated group, we stereotactically transplanted about 4–8 × 106 CD34+-immunosorted PBSCs through three trajectories to the peri-infarcted area of the brain. The whole operative procedure of intracerebral implantation did not aggravate the inflammation and ischemia. According to the purity of CD34+ cells after immunosor-ting, we subdivided the G-CSF + PBSC-treated patients into two subgroups, including subgroup A: percentage of CD34+ cells >90% and subgroup B: percentage of CD34+ cells <90%. Compared to subgroup B, G-CSF-alone, and control group, subgroup A of G-CSF + PBSC-treated patients achieved greater neurological improvement at the 12-month follow-up in NIHSS (G-CSF + PBSC group G-CSF group 59%, control group 36%), ESS (G-CSF + PBSC group G-CSF group 33%, control group 20%), EMG (G-CSF + PBSC group GSF-group 106%, control group 58%), and BI (G-CSF + PBSC group G-CSF group 120%, control group 60%). In a neurological functional activity assessment conducted with 18FDG-PET, significant improvement in cerebral uptake of FDG over the peri-infarcted area was apparent in subgroup A of G-CSF + PBSC-treated subjects compared with subgroup B, G-CSF alone and control group. The relationship between metabolic activity and the clinical motor stroke scale (EMS) was found to correlate closely upon examination by simple linear correlation analysis. Significant increment of corticospinal tract fiber density ratio (CTFDR) assessed by DTI was found in subgroup A of PBSC-treated subjects compared to subgroup B and control. Furthermore, in the CSS integrity evaluation by TMS, reappearance of electrophysiological response of motor evoked potential (MEP) was noted only in subgroup A of G-CSF + PBSC-treated subjects. Finally, we also concluded that rehabilitation should be a major adjunct for the enhancing neurological improvement of patients after PBSC transplantation. This randomized control study demonstrates that a therapeutic strategy using G-CSF combined with autologous implantation of PBSCs mobilized by G-CSF transplantation for old stroke patients is safe, feasible, and shows preliminary evidence of improved neurological outcomes.
W. D. Lindsay,*§ M. McCormick,* S. Clarke,† and C. G. van Horne*‡
*Division of Neurosurgery, Caritas St. Elizabeth's Medical Center, Brighton, MA, USA
†Ithaca College, Ithaca, NY, USA
‡Tufts Medical School, Boston, MA, USA
§University of Connecticut, Storrs, CT, USA
While many studies have reported the clinical benefits of deep brain stimulation (DBS) on the motor symptoms of Parkinson's disease (PD), few have addressed the effects on speech. Some investigators have demonstrated improvements in speech, while others reported either no effect or a worsening of the dysarthria. Furthermore, there is a lack of consistency in the methodological evaluation of speech as it pertains to PD and DBS in particular. The goal of our project is to create a computer-assisted algorithm to record and measure changes in speech with objective and quantifiable metrics. Our subjects include PD patients who have been implanted with bilateral DBS electrodes targeting the subthalamic nuclei, as well as age-matched controls. A total of 63 recordings have been obtained from 24 subjects: 17 PD patients with DBS and 7 controls. Speech was digitally recorded while patients performed standardized speech tasks, including the recitation of individual polysyllabic words, semantically unpredictable sentences, and the rainbow passage. Identical tasks were performed before and after routine DBS adjustments. Speech samples were processed with a psychoacoustic bark filter bank, allowing direct comparison of intensity changes over a range of frequencies. The intensity data were collected in 10-ms bins and signal averaged over a moving 50-ms window. Rates of change in intensity were calculated as the difference between signal averaged intensities at 50-ms intervals, to avoid signal averaging interdependence. Three-dimensional graphical displays were reconstructed using the rate of change data for the multiple frequency bands. Graphical analysis demonstrated differences in rates of change of intensity for some of the DBS adjustments. These differences correspond to subjective evaluations of speech by observers. A more quantitative analysis is under way to identify the areas of speech output that are most vulnerable to deterioration by PD, which may also be susceptible to changes in DBS programming. In regards to patient care, this tool for speech assessment could provide clinicians with an objective means of optimizing DBS parameters.
X.-Y. Liu,* A. Fagan,† M. E. Gonzalez-Toledo,* S. Zhang,* and L.-R. Zhao*†
*Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
†Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Cerebral autosomal dominant arteriopathy with subcortical infarcts and leukoencephalopathy (CADASIL) is the most common form of inherited stroke and vascular dementia. This disease is caused by mutations of Notch 3 gene. To date, no drug can inhibit the progression of this disease. Recently, we have demonstrated that stem cell factor (SCF) in combination with granulocyte-colony stimulating factor (G-CSF) (SCF + G-CSF) has therapeutic effects on chronic stroke. The aim of this study was to determine the effects of SCF + G-CSF on remodeling cerebral vasculature and improving cognitive function in a mouse model of CADASIL disease. Transgenic mice carrying the human mutant Notch 3 gene were used as the mouse model of CADASIL disease. To track bone marrow-derived cells, the bone marrow expressing green fluorescent protein (GFP) was transplanted into CADASIL mice 1 month before SCF + G-CSF treatment. SCF + G-CSF or an equal volume of saline was subcutaneously administered for 5 days and the same treatment was repeated an additional four times with 1–4-month intervals. Spatial learning and memory were evaluated twice by a water maze test after the initial treatment. Mice were sacrificed at the age of 21–23 months. Brain sections were processed for immunohistochemistry and immunohistochemical double labeling was determined with confocal imaging. We observed that the cerebral blood vascular density in both cortex and striatum was significantly increased by SCF + G-CSF treatment. In addition, the number of GFP-expressing cells in the corpus callosum and striatum of SCF + G-CSF-treated mice was greater than those of controls, and many of these GFP+ cells also coexpressed endothelial marker, CD31. Interestingly, cerebral amyloid angiopathy (CAA) (CD31+/4G8+) was also observed in the aged CADASIL mice, and the CAA was dramatically prevented by SCF + G-CSF treatment. Further, the degeneration of smooth muscle cells on the small arterial wall was significantly inhibited in SCF + G-CSF-treated mice. Finally, SCF + G-CSF-treated mice spent a significantly shorter time to find the platform than saline controls during water maze tests. In conclusion, these data suggest that SCF + G-CSF treatment can inhibit the pathological progress in CADASIL mice by enhancing the cerebral blood supply, preventing CAA formation, and improving cognitive function. This study provides a new route for developing therapeutic strategies for improving health of CADASIL disease.
This study was supported by The CADASIL Foundation of America and Louisiana Gene Therapy Research Consortium.
L. Madhavan,* B. F. Daley,* B. L. Davidson,‡ R. L. Boudreau,‡ J. W. Lipton,§ A. Cole-Strauss,† and T. J. Collier‡
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA
‡Department of Internal Medicine, University of Iowa, Iowa City, IA, USA
§Division of Translational Science & Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
Neural stem/precursor cell (NPC) based therapeutic strategies for Parkinson's disease (PD) are generally divided into two approaches: cell transplantation and endogenous cell stimulation. Realistically, future PD cell therapies will most likely involve combining these two approaches, a theme of our current research. Our previous studies in a 6-hydroxydopamine (6-OHDA) rat model of PD suggest a “synergy” between transplanted and endogenous NPC actions (Madhavan et al., J. Comp. Neurol., 2009; Madhavan et al., Neuropharmacology, in press). In particular, our work indicates that NPC implantation before the 6-OHDA insult can stimulate an endogenous NPC response, and that this response is associated with nigrostriatal neuroprotection and amelioration of behavioral deficits (spontaneous paw placement in cylinder). Further, the transplanted NPCs expressed certain molecules [glial-derived neurotrophic factor (GDNF), sonic hedgehog (SHH), and stromal-derived factor 1a (SDF1a)] providing a potential molecular basis for the observed phenomenon. Currently, we are investigating mechanisms underlying the phenomenon by examining the roles of (a) endogenous NPCs and (b) above-mentioned graft-expressed factors. Specifically, we are studying the role of endogenous NPCs, by inhibiting host NPC proliferation and neurogenesis using cytosine-d-arabino-furanoside (AraC). Behaviorally, AraC-infused animals that were subsequently grafted with NPCs performed significantly better on the cylinder task than sham controls, but were also significantly worse than NPC-grafted animals that had received no prior AraC infusion. Tyrosine hydroxylase (TH) cell counts through the substantia nigra support the behavioral data. These results indicate that endogenous NPCs are contributing, in part, to the observed NPC-mediated neuroprotection. In parallel, the roles of graft-expressed GDNF and SHH are also being determined by using RNA interference (RNAi) techniques. As a beginning step we have successfully designed and tested multiple short hairpin RNA (shRNA) constructs against rat GDNF, moved the most efficient constructs into feline immunodeficiency virus (FIV) vector, and optimized viral infection and GDNF knock-down rate in NPCs. Also, shRNAs targeting SHH have been designed and tested for transfer into FIV. As a next step, NPCs in which either GDNF or SHH, or both, have been silenced will be transplanted into host rats to determine whether or not they contribute to the observed NPC-mediated neuroprotection and endogenous response to transplantation.
Supported by NIH grant NS055295, NS58830 the Udall Center of Excellence in Parkinson's Disease Research at University of Cincinnati, and The Gardner Family Center at the University of Cincinnati.
S. Z. Malik,* M. Lewis,† M. Haskins,† T. Van Winkle,† C. H. Vite,† and D. J. Watson†
*Department of Neurosurgery, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
†Department of Clinical Studies, School of Veterinary Medicine, University of Pennsylvania, Philadelphia, PA, USA
In the adult rodent brain, neural progenitor cells migrate from the subventricular zone (SVZ) of the lateral ventricle towards the olfactory bulb (OB) in a track known as the rostral migratory stream (RMS). A similar pathway has been described in other species but not in cats and dogs. Here, we describe the RMS in canine and feline brains. The RMS was found in Nissl-stained sagittal sections of adult canine and feline brains, as a prominent, dense, continuous cellular track beginning at the base of the anterior horn of the lateral ventricle, curving around the head of the caudate nucleus and continuing ventrally to the olfactory peduncle before entering the olfactory tract and bulb. To determine if cells in the RMS were proliferating, the thymidine analog 5-bromo-2-deoxyuridine (BrdU) was administered and detected by immunostaining. BrdU-immunoreactive cells were present throughout this track. The RMS was also immunoreactive for markers of proliferating cells, progenitor cells, and immature neurons (Ki-67, nestin, and doublecortin), but not for NeuN, a marker of mature neurons. Identification and characterization of the RMS in canine and feline brain will facilitate studies of neural progenitor cell biology and migration in large-animal models of neurologic disease.
This work was supported in part by University of Pennsylvania Department of Neurosurgery funds (D.J.W.), NIH DK54481, DK25759, and RR02512 (M.H.), and the Ara Parseghian Medical Research Foundation (C.H.V.).
F. P. Manfredsson,*† W. W. Hauswirth,‡ V. Chiodo,‡ and R. J. Mandel*†
*Department of Neuroscience, University of Florida, Gainesville, FL, USA
†Powell Gene Therapy Center, University of Florida, Gainesville, FL, USA
‡Department of Ophthalmology, University of Florida, Gainesville, FL, USA
Recombinant adeno-associated virus (rAAV) has proven to be a useful gene-transfer utility in the CNS. However, successful transgene expression is dependent on relatively high titer viral preparations. One significant limitation in the level of transgene expression is the ubiquitination of viral capsid proteins. This phenomenon, occurring on tyrosine residues of the capsid protein, promotes degradation of the vector before it reaches the nucleus, and interferes with transduction. Previous studies have shown improved transduction efficiencies utilizing vectors containing mutations at these tyrosine sites, both in vitro and in various organ systems in vivo. Here we present the results from striatal injections of rAAV type 2 expressing the green fluorescent protein (GFP) under a strong constitutive chicken β-actin/cytomegalovirus enhancer promoter hybrid. Adult female Sprague-Dawley rats were stereotaxically injected in the striatum with 2 μl of vectors carrying capsid mutations ranging from a single tyrosine residue up to seven mutated residues. The results indicate that all mutated variants resulted in increased neuronal transduction spread, and increased GFP expression in transduced cells when compared to a wild-type rAAV2 control. In addition, several of the mutant combinations tested resulted in both anterograde and retrograde detection of GFP in various midbrain nuclei. It is unlikely that the observed increase in transduction efficiency is the result of improved infection efficiency. Rather, the expression efficacy of infectious vector genomes is improved, thus requiring less viral particles per cell to enable transgene detection. These results are promising in that the use of tyrosine mutant vectors provides for the use of lower titer viral preparations in the CNS. Consequently, the use of less viral particles may be beneficial if one considers potential immune responses that are known to occur by readministration of the viral capsids. Furthermore, if one considers the targeting of large volume areas in clinical applications, such as the caudate/putamen in Parkinson's disease, improving transgene transduction properties certainly will be invaluable when a majority of this area needs to express the transgene.
L. M. Mercier,* J. C. Vavrousek,* K. Salazar,* B. E. O'Steen,* J. W. Meyer,* D. D. Fuller,† P. J. Reier,* and M. A. Lane*
*Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
†Department of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
It is well established that spontaneous recovery of ipsilateral diaphragm function occurs in adult rats following complete lateral C2 hemisection (C2Hx). This “restorative neuroplasticity” coincides with a contralateral “compensatory neuroplasticity.” The extent of ipsilateral recovery is limited, however, and its contribution to overall breathing behavior has not been extensively investigated. In addition, little is known about possible associated changes in phrenic neural circuitry. Diaphragm activity ipsi- and contralateral to injury was assessed via electromyography (EMG) recordings at 2–12 weeks post-C2Hx and overall ventilatory function was evaluated at weekly intervals using plethysmography. Preliminary results show that restored ipsilateral function contributes modestly to ventilation following injury. Instead, contralateral activity plays a more significant role. This was confirmed by transection of the phrenic nerve (phrenicotomy) either ipsi- or contralateral to C2Hx. While ipsilateral phrenicotomy has little effect on respiration, contralateral phrenicotomy results in pronounced ventilatory deficits. Combined anterograde and transneuronal retrograde tracing [with biotin dextran amine (BDA) and pseudorabies virus (PRV), respectively] was used to examine changes in phrenic circuitry following C2Hx lesions. Inspiratory drive to phrenic moto-neurons (PhMNs) derives from mono- and polysynaptic intraspinal pathways from ventral respiratory column (VRC) medullary neurons. Injection of BDA into the VRC showed reduced bulbospinal input to PhMNs ipsilateral to injury, and associated prephrenic interneurons. Retrogradely tracing phrenic pathways ipsilateral to injury with PRV also revealed decreased connectivity between interneurons and PhMNs. In contrast, PRV tracing of the phrenic pathways contralateral to injury shows some recruitment of interneurons above the injury with projections to contralateral PhMNs. These collective results reveal changes in the phrenic motor system contralateral to C2Hx that likely reflect compensatory plasticity and underscore its importance in maintenance of breathing postinjury. Studies aimed at enhancing PhMN recovery ipsilateral to C2Hx and optimizing restorative plasticity need to consider how such treatments also may affect compensatory plasticity. We have recently begun studies using delivery of rolipram alone, or in combination with transplantation of neural progenitors, to promote repair and optimize functional recovery. Preliminary evidence suggests that rolipram delivery alone significantly improves respiratory outcome within 1 week post-C2Hx, but these effects do not persist. Combination treatment with cell transplantation suggests that respiratory outcome can be enhanced 4–5 weeks postinjury, which may represent a more lasting functional improvement.
Supported by Paralyzed Veterans of America (M.A.L.), Anne and Oscar Lackner Chair (P.J.R.), and the NIH (R01 NS054025; P.J.R.).
R. F. Mervis,*† S. Aradi,‡ V. Lozano,‡ R. A. Long,‡ J. Kotick,†§ A. Winkler,¶ M. Shah,†# A. Lozano,‡ S. Scheff,** and E. Mufson††
*Center of Excellence for Aging & Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†NeuroStructural Research Labs, Inc., Tampa, FL, USA
‡The Honors College, University of South Florida, Tampa, FL, USA
§University of Miami School of Medicine, Miami, FL, USA
¶University of South Florida College of Medicine, Tampa, FL, USA
#University of Florida College of Medicine, Gainesville, FL, USA
**Sanders-Brown Center on Aging, University of Kentucky, Lexington, KY, USA
††Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
Cognitive dysfunction in Alzheimer's disease (AD) correlates more strongly with synaptic loss and disruption of brain circuitry rather than senile plaque and neurofibrillary tangle neuropathology. Mild cognitive impairment (MCI) is a prodromal stage of AD. Quantification of dendritic branching and spines (the loci of synaptic communication) in MCI and AD defines the integrity of brain circuitry. We evaluated dendritic parameters in layer II–III pyramids from formalin-fixed cortical tissue harvested from individuals diagnosed with no cognitive impairment (NCI), MCI, or AD. We assessed dendritic parameters of the pyramidal neurons from three different neocortical regions: the superior frontal cortex (SFx, Brodmann area 9), the parietal cortex (PCx, areas 39–40), and the inferior temporal cortex (ITx, area 21). Tissue blocks were Golgi stained, all slides coded, and layer II–III pyramids randomly selected for dendritic branching and spine analysis of the basilar dendritic arbor. Results showed that MCI and AD elicited variable effects on dendritic branching and spines from the different cortical brain regions. With respect to branching, Tx neurons appeared to be the most vulnerable, losing 21% of their arbor in MCI and another 5% in AD (down to 74% of the NCI group). By contrast, in PCx neurons branching lost only 4% in MCI, but an additional 10% in AD (down to 86% of the NCI group). Neurons of the frontal cortex actually showed a neuroplastic increase in branching in MCI (+16%), but then in AD lost a dramatic 36% of their arbor (to 80% compared to the NCI group). Evaluation of dendritic spines showed that Fx neurons were resilient and showed little spine loss in MCI, but then had significant subsequent spine loss in AD. PCx neurons initially lost significant spines in MCI but not significantly additional more in AD. Tx neurons lost significant spines both in MCI and then additionally in AD. These results point to the heterogeneity of the cortex with respect to the initial effects of MCI vis-à-vis the subsequent impact of AD. The results suggest that the Tx and PCx show primarily only atrophic changes whereas in the frontal cortex the neurons show an initial compensatory neuroplastic response (in an attempt to maintain circuitry?) prior to subsequent AD-related atrophy.
Supported by AG14449.
T. R. Mhyre,* K. A. Maguire-Zeiss,* and H. J. Federoff†
*Department of Neuroscience, Georgetown University Medical Center, Washington, DC, USA
†Office of the Executive Vice President and Executive Dean, Georgetown University Medical Center, Washington, DC, USA
Alzheimer's disease (AD) and Parkinson's disease (PD) are the leading neurodegenerative disorders of aging that affect over 6 million Americans and cost the US economy over $100 billion annually. However, the etiologies of the sporadic forms of these diseases are currently unknown and clinical diagnosis occurs only after significant neuron loss and pathology have taken place. Mounting evidence suggests that these diseases begin early in life and may affect multiple cell populations and tissues. We propose that the pathogenesis and progression of these diseases involve systemic processes that are reflected in multiple biological compartments and cell types, including neurons and leukocytes. Furthermore, we hypothesize that the leukocyte population will provide important information regarding diagnosis, pathogenesis, and potential therapies. These initial pilot studies were designed to examine the utility of peripheral leukocytes in biomarker discovery in neurodegenerative diseases. Using a number of biomolecular profiling approaches, we set out to discover differentially expressed molecules within peripheral blood using the following clinical populations: probable PD without dementia, mild cognitive impairment (MCI), probable AD, and nondemented age- and gender-matched control subjects. Peripheral blood samples were collected from our cohort of subjects and isolated whole blood RNA and leukocyte proteins were subjected to further analysis. We identified a number of differentially expressed transcripts and proteins among our cohorts. A more detailed analysis of our transcriptomic data identified a number of targets in multiple pathways affected in each disease. Principal components analyses of these targets were capable of differentiating each separate disease group from the control group. Our results address whether disease-specific alterations in gene transcripts and proteins in peripheral populations are reflective of generalized molecular and cellular alterations that may provide important information regarding disease action within the CNS. These pilot studies demonstrate the clinical and scientific utility of biomolecular profiling of peripheral leukocytes in neurodegenerative diseases such as PD and AD. Furthermore, the data represent potential biological signatures of these diseases and, importantly, demonstrate the feasibility of examining peripheral leukocyte biomarkers as predictors or reporters of CNS diseases. We are now in the process of undertaking larger scale studies to aid in the detection of early MCI/AD and PD.
This work was supported in part by the NIH (AG025354, NS052151, AG030753) and TATRC/DOD (W81XWH-09-1-0103).
C. Miller,* P. R. Sanberg,*†‡§¶ N. Kuzmin-Nichols,# and S. Garbuzova-Davis*†‡§;
*Center of Excellence for Aging & Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
‡Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
§Department of Pathology and Cell Biology, University of South Florida College of Medicine, Tampa, FL, USA
¶Department of Psychiatry, University of South Florida, Tampa, FL, USA
#Saneron CCEL Therapeutics, Inc., Tampa, FL, USA
Amyotrophic lateral sclerosis (ALS) is a fatal disease characterized by motor neuron degeneration in the spinal cord and brain. Activated microglia and reactive astrocytes might contribute to motor neuron damage by releasing proinflammatory cytokines. Current treatments for ALS are only palliative. Cell therapy shows promise as a treatment for ALS. A recent study by Garbuzova-Davis et al. (2008) compared three different doses (10 × 106, 25 × 106, and 50 × 106) of intravenously injected mononuclear human umbilical cord blood (MNC hUCB) cells into G93A (superoxide dismutase-1) SOD1 mice. Results showed that the dose of 25 × 106 cells was most effective in increasing mouse life span and delaying disease progression, likely through modulation of the immune inflammatory response. However, MNC hUCB anti-inflammatory properties are unknown. The aim of this study was to determine dose effects of MNC hUCB cells upon activated spinal cord microglia in ALS mice. After behavioral tests, mice were sacrificed and cervical/lumbar segments of the spinal cord were removed, fixed, and cut into coronal sections. Immunohistochemical staining of ramified microglia was performed using antibody IBA1 and staining of activated microglia was performed using MHC class II antibody OX6. Microglia density (MD) was measured in the gray matter of the ventral horn region of spinal cord for both cell types. The spinal cord MD was compared between cell-treated, nontreated (media injected), nontreated [cyclosporine (CsA) injected], and C57BL/6J control mice. Consistently reduced ramified microglia density was noted in mice receiving 25 × 106 cells in both the cervical (p < 0.01) and lumbar (p < 0.05) spinal cord. ALS mice injected with the 10 × 106 or 50 × 106 cell dose showed no significant differences from nontreated G93A control mice. Surprisingly, exceptionally higher microglia densities were observed in mice receiving 50 × 106 cells, similar to densities in the nontreated G93A mice, possibly due to an immune conflict between microglia and the MNC hUCB cells introduced. Anticipated results should show reduced activated microglia density among mice receiving 25 × 106 cells. Our results confirm that the 25 × 106 MNC hUCB cell dose, previously shown most beneficial in regards to mouse lifespan and disease progression, also provides the most anti-inflammatory benefit through inhibition of activated microglia.
Supported in part by NIH STTR (Phase I) grant IR41NS46870-01A1. S.G.D. is a consultant and P.R.S. is a cofounder of Saneron CCEL Therapeutics, Inc. S.G.D. and P.R.S. are inventors on a cord blood-related patent application.
A. J. Monahan,*† Z. Ling,‡ A. Miley,† and P. M. Carvey*
*Rush University, Chicago, IL, USA
†Union University School of Pharmacy, Jackson, TN, USA
‡National Health Research Institute, Taiwan
Parkinson's disease (PD) is estimated to affect 4 million people worldwide. While treatments are available to address the symptoms of PD, the etiology of the disease is still up for debate. Nonfamilial PD studies have been approached from the standpoint of adult exposures, suggesting that the etiology of PD begins in adulthood. Data from our laboratory, however, support the idea that prenatal exposures may be risk factors for PD. When gravid female rats were exposed to the bacterotoxin lipopolysaccaharide (LPS) at embryonic day 10.5 (E10.5), offspring were born with reduced numbers of dopamine (DA) neurons, less striatal DA, increased DA activity, and life-long increases in tumor necrosis factor-α (TNF-α) and increased numbers of microglia. In vitro studies from our lab suggests that prenatal LPS exposure at E10.5 alters the ability of the DA neurons to develop process extensions necessary to reach neurotrophic-rich target regions and that an alteration(s) in a soluble factor(s) expressed within both the developing mesencephalon and striatum may be responsible for the effect. The mechanism through which these effects occur, however, is unknown. We hypothesize that prenatal LPS exposure within the gravid female rat leads to elevations of proinflammatory cytokines such as interleukin-6 (IL-6), IL-1β, and tumor necrosis factor-α (TNF-α) and in turn effects development of the nigrostriatal pathway by altering the expression of neurotrophic factors. We exposed gravid female rats to Hank's balanced salt solution (HBSS) or LPS at E10.5 and collected the maternal serum (MS), amniotic fluid (AF), and whole brains from embryos. The results indicate that IL-6 was elevated within MS [F(3, 11) = 870.27, p < 0.001] and amniotic fluid [F(3, 11) = 9.32, p< 0.05], whereas IL-1β, on the other hand, was elevated in the MS [F(3, 11) = 8.18, p < 0.001], AF [F(3, 11) = 39.07, p < 0.001], and brain tissue [F(3, 11) = 3.7, p < 0.001]. TNF-α, however, was only found to be elevated in MS [F(3, 11) = 47.41, p < 0.001]. In addition, nigrostratial cocultures were established from tissue harvested at E14.5 and 72 h later were exposed to various doses of IL-6, IL-1β, and TNF-α to assess the effects of proinflammatory cytokines on expression of neurotrophic factors, glial-derived neurotrophic factor (GDNF), and brain-derived neurotrophic factor (BDNF). The results indicate that after 24 h of treatment, IL-1 β produced a statistically significant dose-dependent increase in GDNF protein [F(5, 23) = 7.33, p < 0.001, using Tu-key's post hoc] while TNF-α produced a dose-dependent decrease (p < 0.05) in GDNF. There were no observed dose-dependent changes in expression of BDNF. In conclusion, these results suggest that exposure to prenatal LPS results in increases in proinflammatory cytokines within MS, AF, and brain that can impact the expression of GDNF. Alteration of neurotrophic factors, such as GDNF, during this critical period of nigrostriatal pathway development could result in altered DA cell development, leading to permanent cell loss. Future studies will further explore the mechanism whereby proinflammatory cytokines alters neurotrophic factor expression in the developing nigrostriatal pathway.
Supported by DOD and the Kenneth Douglass Foundation.
J. Morganti,*† K. Nash,§ A. Bachstetter,¶ P. Bickford,†‡ and C. Gemma†‡
*Center for Excellence in Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
‡James A. Haley Veterans Affairs Hospital, Tampa, FL, USA
§Alzheimer's Research Laboratory and Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
¶Northwestern University Feinberg School of Medicine, Chicago, IL, USA
In the context of aging and neurodegeneration, prolonged microglial activation has been shown to contribute to and further exacerbate pathological conditions. It is therefore relevant to examine molecular signaling that may alter or reduce the levels of activated microglia. Chemokines generally function as chemotactic mediators of cells with corresponding receptors. Fractalkine (FKN) is unique among the chemokine family due to its ability to exist not only as a soluble factor but also in a membrane-bound form. FKN signaling is achieved via its sole receptor CX3CR1, which in the brain is expressed primarily in microglia. Through its dual characteristics as a chemokine (soluble and membrane bound) FKN-CX3CR1 signaling may factor into preserving a resting phase of microglia. Recently, we have shown that an inhibition FKN-CX3CR1 signaling increases activated microglia leading to a disruption in neurogenesis. Furthermore, in vitro models have shown FKN ability to regulate known proinflammatory molecules such as interleukin (IL)-1β, IL-6, and tumor necrosis factor-α (TNF-α), thus acting as a potential anti-inflammatory signal. Currently, little is known regarding the signaling functions of these two forms of FKN. To elucidate differences in action among these forms, three AAV9 vectors, each encoding one of three types of mouse FKN, native (nFKN), membrane-bound (mFKN), and soluble (sFKN), were created. These vectors were examined for their differential role in modulating inflammation and neurogenesis.
This study was funded by USPHS grant #AG0448 and the VA Medical Research Fund.
Z. Nan, L. Belur, D. Wolf, R. Gunther, C. Whitley, R. S. McIvor, and W. C. Low
University of Minnesota, Minneapolis, MN, USA
Current treatments for lysosomal storage disorders involve enzyme replacement therapy (ERT) such as iduronidase (IDUA) for treating Hurler's syndrome. This approach can ameliorate systemic deficits, but current ERT delivered intravenously does not allow therapeutic enzymes to cross the blood-brain barrier, and thus patients such as those with Hurler's syndrome continue to exhibit progressive deterioration in cognitive performance. An alternate approach for correcting enzyme deficiencies in the brain associated with lysosomal storage disorders is the delivery of genes encoding the enzyme of interest into the brain using nonreplicating viral vectors. In the present study we examined the biodistribution of the adeno-associated viral vector AAV5 to deliver the gene encoding fluorescent protein (GFP) for visualization within the central nervous system. AAV5-GFP was injected stereotaxically into the right lateral ventricle of 5-day-old mice or adult mice. The brains of these animals were examined at 4 months after injection to determine the biodistribution of GFP-expressing cells in different regions of the central nervous system. We observed that AAV5-GFP expression was more extensive when injected into the ventricles of neonates compared to adults. Areas of brain with high expression of AAV5-GFP for both groups include the hippocampal formation, striatum, olfactory bulb, frontal cortex, and in ependymal cells surrounding the ventricles. Our results demonstrate that AAV5 can achieve a widespread distribution within the central nervous system when injected into the ventricles of the neonatal and adult brain, and may therefore be suitable as a vector for gene delivery to correct neurological disorders where there is a deficiency of specific enzymes.
K. Nash,* M. J. Berg,† P. Reid,* M. C. Cardenas-Aguayo,† N. Marks,† M. Gordon,* and D. Morgan*
*Byrd Alzheimer Institute, Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA †Center for Dementia, Nathan Kline Institute, New York University, Orangeburg, NY, USA
Recombinant adeno-associated viruses (rAAV) provide tools to manipulate gene expression of proteins relevant to neurodegeneration. Stem cells provide a potential method for cell replacement therapy. Here we examine rAAV's ability to transduce neural precursor cells (NPC) from murine E13 embryonic brain areas destined to differentiate into areas vulnerable to neurodegeneration. Transduction with rAAV enables modification of gene expression in NPC cells for potential therapeutic applications. We evaluated transduction efficiency for rAAV serotypes 1, 2, 5, 8, and 9 encoding green fluorescent protein (GFP) with a cytomegalovirus (CMV)/chicken β-actin promoter, in cultured NPCs derived from anterior brain destined to become primary neurons or glia. Transduction efficiency was assessed by fluorescence/phase-contrast, cell differentiation by immunocytochemistry, and toxicity by colony counts on long-term culture. NPCs were cultured 0–30 days in serum-free media plus epidermal growth factor (EGF) to limit terminal differentiation in the presence of 103–105 viral genomes (VG) per cell. Transgene expression was observed 72 h postinfection with rank order of efficiency of1> 2> 9> 8> 5. This compares with minimal detection after 10 days with intracerebral injections. Stable expression of the transgene was detected for up to 30 days and significant toxicity was only seen at higher viral titers. Data provide an in vitro model to rapidly determine infectivity of viral preparations for which none currently exist for most serotypes. We demonstrate that rAAV are excellent vehicles for modification of gene expression in NPC for potential therapeutic applications.
F. Nasiri Nezhad, M. E. Collado, L. M. Collado, S. Liu, D. Collante, L. Rusakova, M. Varghese, S. Jergova, S. Gajavelli, and J. Sagen
Pope Life Center, Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA
Spinal cord injury (SCI) pain is clinically difficult to treat and few effective treatments exist. Marine cone snails produce numerous selective and potent peptides that have CNS activity, and several have shown promise for treatment of severe pain. FDA-approved Prialt® is a synthetic version of naturally occurring peptide ω-conotoxin (MVIIA) found in the piscivorous marine snail Conus magus. Another peptide conantokin G (Con G) from Conus geographus has been a subject of Phase I clinical trials. Both these peptides are significantly antinociceptive when injected intrathecally in rat pain models. ω-Cono-toxin specifically blocks the presynaptic N-type Ca2+ channels (Cav2.2) while Con G blocks postsynaptic N-methyl-D-aspartic acid (NMDA) receptors. However, continuous administration of pharmacological agents is often associated with undesirable side effects. Recent findings in our laboratory indicated that an intrathecal combination of Con G and MVIIA resulted in robust synergistic antinociception without attendant motor side effects in a SCI pain model, in contrast to additivity in other pain models. These findings suggest that SCI pain is uniquely vulnerable to this peptide combination, and may be good candidates for gene therapy in SCI pain management. To explore the recombinant potential of these peptides, viral constructs that encode ω-conotoxin MVIIA and Con G were generated. MVIIA is amidated; hence, the cDNA was ligated to the signal sequence of mammalian amidating enzyme, peptidyl a monooxygenase (ssPAM), in order to facilitate correct processing and packaging of the active peptide. Glutamate residues in Con G are carboxylated and, in mammals, γ-gluta-myl carboxylase encoded by the gene Ggcx is responsible for the modification and expressed in all tissues. Hence, recombinant Con G in mammalian cells is expected to undergo posttranslational modification in all cells. Because antibodies to these unique peptides are not commercially available, it was necessary to generate custom antibodies. In Fluorophore-Linked Immunosorbent Assays (FLISA) with synthetic peptides, the resultant antibodies showed specificity and a concentration-dependent reaction. The anti-MVIIA antibody recognized cells expressing recombinant MVIIA using immunocytochemistry and the anti-Con G antibody recognized venom granules in a section of the Conus geographus venom duct. These data suggest that the antibodies are authentic and can be used to identify both natural and synthetic MVIIA and Con G, respectively. Transduction of rat spinal cord with MVIIA resulted in robust expression of MVIIA for at least 4 weeks evaluated thus far after injection, without apparent adverse effects. These findings provide an avenue to explore conopeptide-based gene therapy as a potential treatment for chronic SCI pain.
Supported by the Ralph Wilson Medical Research Foundation.
M. B. Newman,* J. L. Zinnanti,† W. J. Zinnanti,‡ and R. A. E. Bakay*
*Department of Neurosurgery, Rush University Medical Center, Chicago, IL, USA
†Department of Biology, Beckman Institute, California Institute of Technology, Pasadena, CA, USA
‡Department of Neurosurgery, Penn State College of Medicine, Hershey, PA, USA
Glutaric aciduria type I (GA-I) is a rare organic acid autosomal recessive human disorder, in which there is an inborn error in the mitochondrial catabolism of l-lysine, l-hydroxylysine, and l-tryptophan that is caused by a deficiency of the enzyme glutaryl coenzyme-A dehydrogenase (Gcdh). In humans and in a mouse model of GA-I, the accumulation of glutaric acid (GA), 3-hydroxyglutaric acid (3-OH-GA), glutarylcarnitine, and glutaconic acid is present in blood, urine, CSF, or brain tissue. Both GA and 3-OH-GA are thought to induce imbalances in glutamatergic and GABAergic neurotransmission pathways, and 3-OH-GA may act through the excitotoxic NMDA receptors to produce a neurotoxic effect in the brain. Clinically, the disease course is not noticed until an acute encephalopathic crisis occurs, which is usually precipitated by an infectious disease, immunization, or surgery during infancy or childhood. The current treatment is administration of riboflavin that assists in the breaking down and processing of proteins, and a strict low-protein diet; however, approximately one third of children with GA-I do not respond to this diet regime. Even those individuals under dietary management may still have metabolic crises and other detrimental effects. Currently, there are no approved therapies for patients with GA-I disorder beside the strict diet. However, allogeneic and autologous stem cell transplants have been included as part of the overall therapy for some patients with other metabolic disorders (mucopolysaccharidoses and leukodystrophies). Therefore, the objective of this study was to investigate and observe the transplantation of neural-induced CD133 (N-CD133) stem cells, from human umbilical cord blood, into Gcdh knockout (-/-) mice, an animal model for GA-I. N-CD133 cells were transplanted in Gcdh-/- mice (1) before placement on high lysine diet, (2) after placement on this diet and with obvious motor deficits, and in age-matched (3) wild-type (WT) mice for controls. In addition, some (4) WT and (5) Gcdh-/- mice received no cell transplants. Life span (survival analysis) was compared across all groups to determine the N-CD133 cells' overall effect on mice. Behavioral motor function data were measured using a quantitative neurological scale, which consisted of a 1 to 6-point scale that corresponded to motor movement of hindlimb, intermittent dystonia to complete paralysis of the animal. Immunohistochemistry and stereological assessment of brain tissue for N-CD133 cells, properties, viability, and location, as well as number of medium spiny neurons in the striatum and the activity of β-galactosidase, was performed. To date, the N-CD133-transplanted, high lysine diet, Gcdh-/- mice of both conditions had extended life span compared to high lysine diet Gcdh-/- mice with no cell transplants, and there was no difference compared to either condition of the WT mice. Behavioral motor assessment showed improved function for both conditions of the N-CD133-transplanted Gcdh-/- mice compared to the nontransplanted Gcdh-/- mice. We are currently assessing the immunohistochemistry results and will present these at the meeting.
Supported in part by International Organization of Glutaric Aciduria (IOGA) created by Steven Goodman's laboratory.
J. B. Nikas*† and W. C. Low*‡§
*Department of Neurosurgery, Medical School, University of Minnesota, Minneapolis, MN, USA
†Pharmaco-Neuro-Immunology Program, Medical School, University of Minnesota, Minneapolis, MN, USA
‡Graduate Program in Neuroscience, Medical School, University of Minnesota, Minneapolis, MN, USA
§Department of Integrative Biology and Physiology, Medical School, University of Minnesota, Minneapolis, MN, USA
Nuclear magnetic resonance (NMR) spectroscopy has emerged as a technology that can provide information regarding quantitative levels of metabolites within organ systems in vivo. This technology permits the monitoring and analysis of metabolites associated with normal and pathophysiological states. Because of the resolution of current high field NMR spectrometers there is an abundance of metabolic compounds that can be used to assess progression of disease states and response to therapy. Conventional statistical tools are not designed to handle the complexity and volume of data that can be generated by NMR spectroscopy, and new approaches that can accomplish that task with speed and accuracy are required. In this study, we introduced the novel concept of employing a clustering method to render a differential diagnosis in a given disease state. We developed mathematical approaches that rendered the aforementioned novel concept a reality, and we devised three tests to assess the suitability and the accuracy required for diagnostic purposes of the four clustering methods we investigated (K-means, Fuzzy, Hierarchical, and Medoid Partitioning). To accomplish this goal, we studied the R6/2 transgenic mouse model of Huntington disease (HD) to determine the concentrations of metabolites within the striatal area of the brain. Thirteen R6/2 HD transgenic mice as well as 17 wild-type (WT) mice were studied using high field in vivo proton NMR spectroscopy at 9.4 Tesla. We tested all four clustering methods: 1) with the original 30 mice, 2) with 31 unknown mice, whose status had been determined via genotyping, and 3) with the ability to separate the original 13 R6/2 mice into two age subgroups (8 and 12 weeks old). Supervised by ROC curve analysis, Fuzzy and K-means clustering passed all three stringent tests with a total accuracy of 100% [positive likelihood ratio approximating infinity (1/0 = ∞) and negative likelihood ratio equal to zero (0/1 =0)], proving that they may be used for diagnostic purposes. Our aforementioned mathematical approaches are independent of HD and NMR spectroscopy (i.e., they may be applied to any other disease state using any other technology).
M. M. Pabon,*‡ J. Jernberg,* C. E. Hudson,*† A. Bachstetter,§ C. Gemma,*†‡ and P. C. Bickford*†‡
*Center of Excellence for Aging and Brain Repair, Department Of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†James A. Haley Veterans Affairs Hospital, Tampa, FL, USA
‡Department of Cell and Molecular Biology, Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
§Northwestern University Feinberg School of Medicine, Chicago, IL, USA
A primary pathological component of Parkinson's disease (PD) is brain inflammation. Activation of microglia, the cellular component of the neuroinflammatory response, is capable of producing neuronal damage through the production of bioactive molecules such as cytokines, as well as reactive nitrogen and oxygen species. The inflammatory response in the brain is normally tightly regulated in part by neurons, which produce molecules that can suppress the activation of microglia. Fractalkine (FKN) is a neuronally produced chemokine that has a critical role in the regulation of excessive microglia activation. With age there is a loss of FKN and this may make microglia primed for excessive activation, which is known to occur in age-related neurodegenerative diseases including PD. While loss of FKN has been shown to result in excessive microglia activation, it is not known if addition of exogenous FKN beyond otherwise physiologically normal levels of FKN will be therapeutically beneficial following a neurotoxic insult. To test the hypothesis that the FKN signaling pathway could be a therapeutic target for neurodegeneration produced by microglia activation, we used the intrastriatal 6-hydroxydopamine (6-OHDA) model of PD. Briefly, this model uses 6-OHDA (20 μg/4 μl) infused into the left striatum of 3-month-old F344 male rats (coordinates: AP +1, ML +3, and V −4.5). Immediately after the 6-OHDA a stereotaxically implanted cannula, connected to an osmotic minipump, was affixed to the skull to allow for a sustained delivery of FKN for 28 days into the site of the lesion [Alzet model 2004: pumping rate 0.25 μl/h (±0.05 μl/h); total volume 1.0 ml]. To establish a dose response for FKN, 3 ng of FKN, 30 ng of FKN, 90 ng of FKN, or 90 ng of heat-inactivated FKN (as a control) was infused via osmotic minipump. After 28 days the rats were anesthetized followed by transcardial perfusion with phosphate-buffered saline (PBS), then with 4% paraformaldehyde in PBS. Immunohistochemistry was performed for every third 40-μm-thick tissue sections for tyrosine hydroxylase (TH) and MHC class II (OX-6) throughout the entire dorsal striatum. Using the cavalieri method of unbiased stereology to calculate the TH-negative lesion volume revealed that exogenous soluble FKN was neuroprotective, resulting in a significantly smaller lesion. Importantly, FKN produced a dose-dependent decrease in activated microglia, supporting the proposed mechanism of action. These findings demonstrated that FKN plays a neuroprotective role in 6-OHDA-induced dopaminergic lesions and it might be an effective therapeutic target for many neurodegenerative diseases, including Alzheimer' disease and PD, where inflammation plays an important role.
This work was supported by the VA Medical Research Service (P.C.B.).
V. K. Parihar,*† B. Hattiangady,*† B. Shuai,*† and A. K. Shetty*†
*Medical Research and Surgery Services, VA Medical Center, Durham, NC, USA
†Department of Surgery (Neurosurgery), Duke University Medical Center, Durham, NC, USA
The Gulf war illness (GWI) in Persian Gulf War-1 (PGW-1) veterans manifests as a set of nonspecific symptoms with emphasis on central nervous system (CNS) impairments. The CNS impairments include cognitive dysfunction, memory loss, and depression and anxiety. During the PGW-1, the veterans were exposed to a mixture of biological and chemical environments. First, to protect against the nerve agents, the troops were given pyridostigmine bromide (PB) as a prophylactic treatment. Second, pesticides such as N, N-diethyl-m-tolu-amide (DEET) and permethrin were widely used by troops to combat insects/rodents in the region. From these viewpoints, it is supposed that the neurological symptoms exhibited by Gulf war veterans are owed to a synergistic interaction of chemicals PB, DEET, and permethrin. Considering the suggested functions of hippocampal neurogenesis in learning, memory, and mood, we investigated whether the cognitive dysfunction seen in GWI is linked to a greatly declined hippocampal neurogenesis. A larger cohort of male Sprague-Dawley (3-month-old) rats was exposed daily to chemicals DEET (40 mg/kg/day, dermal), permethrin (0.13 mg/kg/day, dermal), and PB (1.3 mg/kg/day, oral) for 28 days. A second cohort of age-matched rats received vehicle during the same period and a third cohort of age-matched rats served as naive controls. One day after the above exposure regimen, subgroups of rats from all groups received four IP injections of 5′-bromodeoxyuridine (BrdU; one injection every 6 h over an 18-h period) and were perfused at 6 h after the last injection for analyses of the production of new cells per day. Three months after the exposure, additional subgroups of rats were given 12 injections of BrdU (once daily over 12 days; 100 mg/kg) and perfused at 1 month after the last BrdU injection to ascertain the long-term effects of the chemical exposure. Separate subgroups of rats from all groups were tested for learning and memory function using a water maze test (WMT) at 2 months after the exposure regimen. These rats were also examined for the extent of depression using the forced swim test (FST). Quantification of BrdU+ cells in subgranular zone-granule cell layer (SGZ-GCL) revealed that the overall production of new cells/day diminishes by 40% at 1 day after the exposure regimen, in comparison to control groups (p < 0.001, n = 5/group). Measurement of the numbers of doublecortin (DCX)-positive newly born neurons in the SGZ-GCL revealed a similar trend. Furthermore, analyses of newly born cells in the SGZ-GCL at 3 months after the exposure regimen revealed that the reductions seen in the production of new cells at an early time point persists for prolonged periods, as rats exposed to chemicals exhibited 45% reduction in the production of new cells (p < 0.01; n = 5/group). Counting of DCX+ newly born neurons also showed a similar trend (p < 0.001). Analyses using a WMT demonstrated learning and memory impairments in rats exposed to chemicals (n = 6/group). Delayed learning was evidenced by increased latency (in seconds) to find the hidden platform in all seven training sessions, in comparison to the vehicle group. Impaired memory was revealed by reduced dwell time in the target area (p < 0.05), fewer entries into the target area (p < 0.05) and increased latency to reach the target area (p < 0.001) in the memory retrieval (probe) test conducted at 24 h after the last training session. The FST revealed that, in rats exposed to chemicals, the overall depressive-like behavior was 51% greater than the vehicle group (p < 0.01). Thus, 28-day exposure to a combination of GWI-related chemicals (PB, DEET, and permethrin) greatly diminishes hippocampal neurogenesis for prolonged periods, and impairs learning, memory, and mood functions. Considering the purported role of hippocampal neurogenesis, it appears that a greatly declined hippocampal neurogenesis underlies learning and memory impairments and depression in GWI.
Supported by a Gulf War Research Grant from the Department of Veterans Affairs (A.K.S.).
A. Patel,* B. Desai,* P. M. Carvey,*† and B. Hendey*
*Department of Pharmacology, Rush University Medical Center, Chicago, IL USA
†Department of Neurological Science, Rush University Medical Center, Chicago, IL, USA
Parkinson's disease (PD) is a progressive neurodegenerative disorder, characterized by dopamine (DA) neuron loss in the substantia nigra pars compacta (SNpc). Inflammatory mediators such as TNF-α and VEGF are upregulated in PD and are known to promote angiogenesis; the sprouting of new vessels from preexisting vessels. In previous animal studies using toxin-induced models of PD, we showed that areas exhibiting blood-brain barrier (BBB) disruption [assessed using FITC-labeled albumin (FITC-LA) leakage into brain] also exhibited increased expression of integrin β3, a marker for angiogenesis. We also showed upregulation of β3 integrin in autopsy samples from PD patients, suggesting angiogenesis may play a role in toxin induced as well as idiopathic PD. Using the MPTP mouse model of PD, we tested whether the angiogenic inhibitor, cyclo Arg-Gly-Asp-D-Phe-Val (cyRGDfV), was effective in reducing BBB dysfunction, preventing DA neuron loss and neuroinflammation. Mice were injected with MPTP (10 mg/kg q 1 h × 4) or saline (SAL). Twenty-four hours later, mice in each group were treated with SAL, cyRGDfV (100 μg/50 μl) or cyRADfV (a negative control) two times/day for 3 consecutive days. Twenty-four hours following their last treatments, the animals were assessed for FITC-LA leakage, DA neuron loss [tyrosine hydroxylase immunoreactive (TH-ir) cell counts], and microglia activation (Iba-1 immunoreactive cell counts) in SNpc. MPTP treatment reduced TH-ir cell counts by 32% compared with SAL-treated mice [F(4, 20) = 17.032, p < 0.001]. The TH-ir cell counts in MPTP+ cyRGDfV-treated mice were unchanged relative to SAL-treated mice, but were markedly increased relative to MPTP only and MPTP+ cyRADfV-treated mice (p < 0.001), indicating that the angiogenic inhibitor completely blocked DA neuron loss. MPTP and MPTP+ cyRADfV-treated mice exhibited areas of punctate leakage of FITC-LA into the SNpc and striatum, suggesting barrier compromise, whereas cyRGDfV posttreatment prevented barrier compromise. MPTP and MPTP+ cyRADfV-treated mice exhibited marked increases in microglia cell counts that were completely prevented in MPTP- cyRGDfV-treated mice. cyRGDfV blocked TH-ir cell loss, FITC-LA leakage, and microglial activation, indicating that angiogenesis may underlie the neuroinflammation-mediated disruption of the BBB and DA neuron loss. This suggests that antiangiogenic factors currently available or in cancer clinical trials may slow disease progression.
Supported by NS052414 and Kenneth Douglas Foundation.
K. L. Paumier,* A. D. Siderowf,† P. Auinger,‡ D. Oakes,‡ A. J. Espay,§ F. J. Revilla,§ A. Sahay,§ and T. J. Collier*
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Department of Psychiatry, University of Pennsylvania, Philadelphia, PA, USA
‡University of Rochester School of Medicine and Dentistry, Rochester, NY, USA
§The Neuroscience Institute, Department of Neurology, Movement Disorders Center, University of Cincinnati, Cincinnati, OH, USA
It recently has been demonstrated that administration of antidepressants such as tricyclics (TCAs) and selective serotonin reuptake inhibitors (SSRIs) modulates the signaling pathways involved in cell survival and plasticity. Additionally, preliminary data from our laboratory indicate that the TCA, amitriptyline (AMI), provides significant protection for DA neurons in a rat model of PD; and accordingly, may hold promise as both a treatment for PD-associated depression and the progression of the DA degeneration that occurs in PD. The primary objective of this study was to assess whether early antidepressant treatment has disease-modifying effects in PD patients. We performed a patient-level meta-analysis utilizing existing prospectively collected data from six completed clinical trials [from the Parkinson's Study Group (PSG) and the NIH Exploratory Trials in Parkinson's Disease Project (NET-PD)] to examine the extent to which antidepressant treatment in PD patients delays the “need for dopaminergic therapy” or changes the degree of motor impairment and disability. A total of 2,064 patients compiled from both treatment and placebo groups were included in our study. Of these, 451 were taking some form of antidepressant. The primary outcome “time to dopaminergic therapy” was reported as days to initial use of dopaminergic therapy (levodopa, dopamine agonists, etc.). Preliminary results indicate that subjects taking any form of antidepressant showed a lower probability of requiring dopaminergic therapy than those not taking antidepressants (HR = 0.6, p = 0.0002). Furthermore, this effect was not specific to a particular class of antidepressant as subjects taking TCAs (HR = 0.5, p = 0.0009), SSRIs (HR = 0.6, p = 0.001), or atypical antidepressants (HR = 0.5, p = 0.02) showed a slower rate of time to dopaminergic therapy compared to those not taking antidepressants. The secondary outcome, degree of motor impairment and disability, was reported as annualized change in total UPDRS score from baseline to final visit. There was no significant difference in UPDRS scores for subjects taking any form of antidepressant (mean 11 point worsening) compared to those not taking antidepressants (mean 14 point worsening; p = 0.15). However, there was a significant difference in UPDRS scores for patients taking atypical antidepressants (mean 7 point worsening) compared to those not taking antidepressants (mean 14 point worsening; p < 0.05). These results show that antidepressants, regardless of class, significantly slow the time to dopaminergic therapy in a population of early PD patients. Also, subjects treated with atypical antidepressants exhibit a lesser degree of motor impairment and disability than those not taking antidepressants. Combined, these results suggest antidepressant therapy can provide additional therapeutic benefit for early PD patients beyond treatment of depressive symptoms, thus providing a rationale for early intervention.
Supported by Parkinson's Study Group (PSG) and the Udall Center of Excellence in Parkinson's Disease Research at the University of Cincinnati (NS58830).
A. I. Persson,*¶ R. Jandial,# K. Shchors,§¶ S. Masic,*†¶ A. Ho,# S. Vandenberg,§ T. P. Nicolaides,†¶ K. Nguyen,*¶ S. Yakovenko,*¶ M. Prados,‡ A. Alvarez-Buylla,‡ G. I. Evan,§ W. A. Weiss,*†‡¶ and E. Y. Snyder#
*Department of Neurology, University of California, San Francisco, CA, USA
†Department of Pediatrics, University of California, San Francisco, CA, USA
‡Department of Neurosurgery & Brain Tumor Research Center, University of California, San Francisco, CA, USA
§Department of Pathology, University of California, San Francisco, CA, USA
¶Helen Diller Family Comprehensive Cancer Center, University of California, San Francisco, CA, USA
#Burnham Institute for Medical Research, La Jolla, CA, USA
Cancer stem cell biology has typically focused on cells that already exist in glioma; it has less extensively explored the cell-of-origin in a manner that allows for the rigorous in vivo clonal analysis necessary to begin distinguishing between fundamental models and mechanisms of cerebral oncogenesis. Determining whether a tumor is polyclonal versus monoclonal would suggest a nonhierarchical versus hierarchical process, respectively. To trace gliomagenesis, starting in the premalignant brains of newborn mice fated to develop tumors in adulthood, we applied unbiased retroviral-mediated lineage and fate mapping techniques in a well-characterized model for glioma. Identical, equipotent neural stem cells (NSCs) in the subventricular zone (SVZ) of newborn GFAP-Ras mice were transduced in situ using a replication-incompetent retrovirus carrying a complex oligonucleotide library comprised of 108 unique genetic inserts, each of which could be subsequently identified by PCR at the single cell level, allowing unequivocal assignment of clonal relationships between cellular progeny that shared these “tags.” Glioma proved to be of polyclonal, nonhierarchical NSC origin with cell nonautonomous influences. Tumor-founding NSCs were initially indistinguishable from those yielding normal structures. Any cells with a proliferative advantage emerged late in the process while already in the tumor. These data speak against the classic view of cancer stem cells and the models for oncogenesis that they predict. Furthermore, prior to developing tumors, NSCs evinced an altered differentiation trajectory from principally a neuronal fate to one of inappropriate gliogenesis, resulting in a paucity of neurons in the olfactory bulbs, the normal principal output of SVZ NSCs. These findings suggest that oncogenesis may impact fundamental normal developmental programs. Indeed, to the list of processes that can impair adult neurogenesis, cerebral oncogenesis may now need to be added. Small-molecule MEK inhibitors restored neurogenesis in vivo, with both murine and human glioma cells subsequently showing reduced self-renewal and a switch to neuronal differentiation, suggesting this NSC-based gliomagenic process to be reversible.
A. Rachubinski, S. K. Cornelius, and K. Bjugstad
University of Colorado, Denver, CO, USA
Down syndrome (DS), the most common genetic cause of intellectual disability, is due to the triplication of human chromosome 21. One of the most incapacitating results of this overexpression of genes is the impairment in cognition, especially in learning acquisition, spatial tasks, and association tasks. Much of the cognitive decline associated with DS occurs in postnatal development. While current DS treatments are directed at restoring already impaired cognitive functions, a treatment used earlier in development may postpone or prevent the emerging impairments. Neural progenitor cells (NPC) have been used in a wide range of disorders with promising results. The present study sought to determine if NPC could be used as a long-term treatment when implanted neonatally, before the DS brain undergoes abnormal postnatal changes. The Ts65Dn mouse model of DS, which has delayed development and cognitive impairments, was used. On postnatal day 2 (PND2), trisomic and disomic littermate control male mice received a bilateral implantation into the hippocampus of 100,000 NPC (C17.2 cell line) or saline. An additional control group was left unimplanted. At 14 weeks, the now adult mice began a series of behavior tests designed to assess functioning in learning and memory regions of the brain most affected in DS. First, mice were tested in the single trial Plus maze, a spontaneous alternation task that uses novel alternations to gauge spatiotemporal memory. To further evaluate spatial memory, the Morris water maze (MWM) was used, which required mice to remember the location of a hidden platform in a pool. Finally, conditioned taste avoidance (CTA) was used to evaluate association learning, a classical conditioning task that relies on making a correlation between a novel flavor (conditioned stimulus, CS) and a feeling of nausea. In the Plus maze, trisomic mice were significantly impaired (p < 0.05). While not significant, trisomic mice treated with either NPC or saline had an increase in the number of novel alternations compared to untreated trisomic mice, suggesting a potential for treatment to improve cognition in this task (p > 0.05). Trisomic mice remained impaired in the acquisition of the MWM and no significant effect of NPC or saline was found, regardless of karyotype (p > 0.05). In CTA, all groups of mice learned the association and consumed significantly less CS on the second exposure compared to the first exposure, although the reduction for trisomic mice was less than the disomic mice (p < 0.05). Implantation of either NPC or saline did not significantly affect whether mice reduced their consumption of the CS on second exposure. Based on this general assessment of behavior, treatment with NPC did not significantly improve cognitive function in trisomic mice. Interestingly, the implantation of either saline or NPC may have had a positive effect, albeit insignificant, on the simple spatial task (plus maze). However, mice failed to show improvement in complex spatial tasks (MWM) and association learning (CTA). It is possible that penetration of the brain at PND2 elicits a response that is still measurable 14 weeks later, potentially mediated by the injection-induced growth factor release at a critical time in development. The implications of such a response may offer a new approach to treating conditions early in development.
A. Rao, R. Ramachandra, K. Venkiteswaran, C. A. Lieu, and T. Subramanian*
Departments of Neurology and Neural and Behavioral Sciences, Pennsylvania State University College of Medicine, Hershey, PA, USA
Transplantation of fetal ventral mesencephalic (FVM) tissue has been extensively tested and shown to ameliorate the symptoms associated with Parkinson's disease (PD). However, the efficacy of FVM transplantation requires life-long systemic long-term immunosuppression and may be susceptible to neurodegeneration. hRPEC have been shown to secrete several neurotrophic factors, immunomodulators, and l-dopa. We have previously shown that hRPEC cografts are able to protect striatal FVM grafts from immunological rejection for a period of 18 days. In the present study we investigated the hypothesis that the immunosuppressive and neurotrophic properties of hRPEC will help to protect the mFVM striatal graft and provide long-term recovery of function. Rats were rendered hemiparkinsonian by stereotactic injection of 6-hydroxydopamine into the medial forebrain bundle/substantia nigra. At 3 and 5 weeks, animals were exposed to apomorphine (0.2 mg/kg, SC) to verify extent of nigrostriatal degeneration. Animals with >7 rotations/min were utilized in this study. hRPEC were grown in culture and attached to collagen microcarriers and mFVM tissue was dissected from E13.5 mouse embryos, then made into a single cell suspension for transplantation. Rats were separated into three groups: group 1—FVM-hRPEC cotransplant, group 2—hRPEC attached to collagen microcarriers, and group 3—mFVM tissue with daily cyclosporine immunosuppression. The total cell count of each graft was kept constant, as was the approximate number of microcarriers transplanted. Apomorphine-induced rotations, Vibrissae evoked forelimb placement test, and stepping test were utilized to track behavioral improvement and the tests were administered every 2 weeks for a period of 3 months. Apomorphine-induced rotation results show that group 1 animals had significant behavioral improvement (p = 0.0344) compared to their pretransplant rotational scores. Groups 2 and 3 animals also showed improvements in rotational scores to a lesser extent but not to the extent (p = 0.0707 and p = 0.0611, respectively). Group 1 animals showed significant improvement in the forelimb placement test with a t-test value of p = 0.0065. Group 2 and group 3 also showed improvement, although the p-values were not significant (p = 0.221 and p = 0.260, respectively). Taken together, these results suggest that the hRPEC cografts can successfully protect striatal mFVM grafts from host immune rejection up to 3 months and provide functional improvement against parkinsonism.
This study was funded in part by NIHRO1NS42402, HRSADI BTH06321, and a grant with the Pennsylvania Department of Health using Tobacco Settlement Funds. The Pennsylvania Department of Health specifically disclaims responsibility for any analyses, interpretations, or conclusions. The authors also acknowledge partial funding of this project by the Penn State University Brain Repair Fund.
I. Rattray,* R. Gale,† B. Solanky,* G. Bates,† and M. Modo*
*Institute of Psychiatry, King's College London, London, UK
†Medical and Molecular Genetics, King's College London, London, UK
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder characterized by the occurrence of severe brain atrophy coupled with the development of deficits in motor ability and cognitive abnormalities. The transgenic R6/2 mouse line has been developed to model this disorder preclinically. Although a well-described mouse line, to date there has not been a comprehensive description of age-related changes in multiple behavioral measures, which are then correlated with neuropathological abnormalities assessed through serial, in vivo high-resolution magnetic resonance imaging (MRI). For this purpose, 20 male and 20 female R6/2 mice and wild-type littermates (WT, CBAxC57Bl/6) were used. Mice were exposed to serial in vivo MRI to quantify changes in regional brain volumetry and T2 relaxivity, along with MR spectroscopy to determine the striatal metabolite profile. For MR measures, mice are scanned at 4, 8, 12, and 14 weeks of age. Between scanning sessions mice were tested for motor performance on the rotarod, for anxiety-like behavior in an open field, and cognitive performance in the passive avoidance test and cued/spontaneous alternation behavior in a swimming T-maze. At the final MRI session, mice were culled and brains taken for postmortem quantification of pathological status. R6/2 mice developed motor deficits between 4 and 8 weeks of age compared to WT mice, as determined by the rotarod test. Correlating such behavioral changes with the various in vivo MR measures will provide a more detailed description of the functional and neuropathological changes that occur in this preclinical HD mouse model.
A. C. Rising,* E. Denovan-Wright,† and R. Mandel*
*Department of Neuroscience, Powell Gene Therapy Center, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL, USA
†Department of Pharmacology, Dalhousie University, Halifax, Nova Scotia, Canada
Huntington's disease (HD) is a neurodegenerative disease whose behavioral, motor, and cognitive symptoms progress as the individual ages. HD is caused by a poly-glutamine expansion in the huntingtin protein. The exact function of WT huntingtin is not known. Similarly, the mechanism by which the poly-glutamine expansion causes the lethal disease is not fully understood. The cloning of the HD gene has allowed the creation of various mouse models of this disease and greatly increased our knowledge of the disease itself. Here we examine the behavioral, histopathological, and anatomical characteristics of the CAG140 knock-in HD mouse model. This model has the repeat region of the first exon that has been replaced with the human version that contains 140 glutamine repeats. We have longitudinally examined the rearing behavior, accelerating rotarod, constant speed rotarod, and open field behavior for the heterozygote, homozygote, and their wild-type littermates and have found a significant deficit in the behavior of the afflicted mice. However, unlike the human disease, these behaviors do not appear to progressively get worse over time. Histopathologically, the CAG140 mice may model the HD disease better than the behavior observed in the mice. Neuronal intranuclear inclusions (NIIs) is a hallmark of the disease in humans as well as other HD models. We show here that the NIIs in the CAG140 mice significantly increase as the mice age. As in the human disease, we show that the CAG140 mice also have a significant decrease in specific mRNA transcripts localized to the striatum. Lastly, these HD mice show a progressive decrease in their cortical thickness, similar to what has recently been shown in humans with HD. The mice did not show a significant change in weight in any of the groups.
J. Rossignol,*† K. D. Fink,* K. K. Davis,* S. Cheng,* S. A. Lowrance,* J. J. Matchynski,* L. Lescaudron,‡§ and G. L. Dunbar*†
*Field Neurosciences Institute, Saginaw, MI, USA
†Program in Neuroscience, Central Michigan University, Mt. Pleasant, MI, USA
‡INSERM U643, ITERT, CHU, Nantes, France
§Faculté des Sciences et des Techniques, Université de Nantes, France
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder. Although only palliative treatments (e.g., tetrabenzadine) are currently available, promising new approaches (e.g., siRNA and stem cell therapy) offer significant hope. Presently, only experimental treatments using transplanted embryonic/fetal cells have proven effective for slowing the progression of HD. However, ethical concerns, problems of availability of embryos, tumor formation, and rejection may limit the long-term viability of embryonic stem cell treatments. Mesenchymal stem cells (MSCs) exhibit the ability to differentiate into neural lineages, representing an attractive source for cell replacement therapy in neurological disorders, such as HD. Neural differentiation of MSCs is more difficult, but their ability to release neurotrophic factors (BDNF, NGF, CNTF) and their anti-inflammatory properties may be of benefit in neurological diseases by protecting host neurons from dysfunction and death. However, survival of transplanted MSCs has been a concern in some studies. Our previous work (Rossignol et al., JCMM; 2009) suggested that reducing the number of cell passages may increase survivability. The goal of this present study was to compare the efficiency of low versus high number of passages of cultured cells from two sources (bone marrow and umbilical cord blood cells) when transplanted into the striatum of R6/2 mouse model of HD. Bilateral intrastriatal transplants (Tp; 400,000 cells/striatum) of MSCs in R6/2 mice were performed when the mice were 5 weeks old. A high passage (> 40) and a low passage (<7) populations of MSCs from both bone marrow and umbilical cord blood were used for the Tps. Following surgery, all mice were tested on a battery of tasks, which included assessments of clasping, rotarod, and the Morris water maze (MWM) performance. The animals were sacrificed by transcardial perfusions at 12 weeks of age (7 weeks after the transplantation). All R6/2 mice showed deficits on the rotarod tasks, with those receiving the low-passaged MSCs having slightly longer latencies to fall, relative to those receiving the high-passaged MSCs. Similarly, the MWM results revealed a decrease in distance swum to find the hidden platform for all HD mice transplanted with MSC Tps. Immunohistochemistry revealed that MSCs were viable within the striatum at 7 weeks post-Tp, with GFAP astrocytes concentrated in the vicinity of the Tp site. MSCs tended to remain within the area of transplantation, and only a few MSCs expressed the neuronal marker NeuN. These results indicate transplantation of MSCs is able to reduce behavioral deficits in the R6/2 model of HD and support our previous work that fewer passages tend to increase survivability and efficacy. These results suggest that MSCs may provide a promising therapeutic approach for treating HD.
M. Saarma, P. Lindholm, J.-O. Andressoo, J. Peränen, and M. Lindahl
Institute of Biotechnology, University of Helsinki, Helsinki, Finland
In Parkinson's disease (PD) dopaminergic (DA) neurons located in the substantia nigra progressively degenerate and die. Available PD treatments are symptomatic and unable to slow down or stop neurodegeneration. Neurotrophic factors (NTFs) are critically important for the maintenance of neurons, and during trauma they are able to protect and induce the repair of neurons. Glial cell line-derived neurotrophic factor (GDNF) and its homologous protein neurturin (NRTN) are able not only to protect, but also to induce the repair of DA neurons in animal models of PD. Although both proteins work well in animal models of PD they also have limitations—they are unable to pass the blood-brain barrier and therefore for the treatment they should be delivered directly into the brain. In addition, they diffuse very poorly in the brain tissue and GDNF did not prevent the α-synuclein-induced dopaminergic neurodegeneration in the genetic rat model of PD. Part of these limitations may be overcome by using the new neurotrophic factor recently discovered in our laboratory. Cerebral dopamine neurotrophic factor (CDNF) and its homologous protein mesencephalic astrocyte-derived neurotrophic factor (MANF) together with the invertebrate homologous protein form a novel family of evolutionarily conserved NTFs. Analysis of the crystal structure revealed that MANF and CDNF proteins consist of two domains: an amino-terminal saposin-like domain and a natively unfolded carboxy-terminal domain with a single disulphide bond, revealing a structurally novel group of proteins. The N-terminal saposin-like domain is able to bind lipids. We have demonstrated that CDNF and MANF can protect and repair midbrain dopamine neurons in rodent models of PD more efficiently and specifically than any other known neurotrophic factor. CDNF and MANF diffuse much better in the rodent brain tissue than GDNF and in addition have a unique property to bind oxidized phospholipids. The latter is the hallmark of neurodegeneration that is also known to contribute to the pathogenesis of PD. Thus, there are at least four naturally occurring NTFs that can not only slow down neurodegeneration, but can also repair and regenerate diseased DA neurons. Without doubt, GDNF and NRTN, but in particular CDNF and MANF, have a potential in the treatment of PD. We are currently making efforts to take the new and most promising neurotrophic factor CDNF into Phase I clinical trials for PD. To understand the physiological roles for the new NTFs in vivo, we have created conventional CDNF-deficient mice, as well as a CDNF conditional knockout (cKO) mouse model where CDNF can be removed from specific tissues or brain areas. We will discuss unpublished results on the roles of CDNF with the focus on the brain DA system.
K. Salazar,* L. M. Mercier,* J. C. Vavrousek,* B. E. O'Steen,* J. W. Meyer,* D. D. Fuller,† M. A. Lane,* and P. J. Reier*
*Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
†Department of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
High cervical spinal cord injury often results in some compromise of the phrenic motor system and subsequent diaphragm dysfunction. However, experimental studies have revealed some spontaneous recovery after C2 hemisection lesions. While previous work has focused on this hemilesion model, less attention has been given to more clinically frequent cervical contusion injuries occurring at the level of the phrenic motoneuron pool (C3–C5), which innervates the diaphragm—the primary respiratory muscle. Given the experimental and translational advantages such a model could present related to spinal cord repair and plasticity, the present study was carried out to determine the feasibility of producing such lesions and the degree of enduring respiratory compromise that can be achieved. Adult female Sprague-Dawley rats received either midline or lateral cervical contusion injuries at C3/4 (infinite horizon; 150–250 kilodynes). Plethysmography was used to assess ventilation (e.g., breathing frequency and tidal volume) both prior to injury and on a weekly basis thereafter. Ventilatory measurements were made under baseline (breathing normoxic, normo-capnic air) and hypercapnic (normoxic gas containing 7% CO2) conditions. All animals were then left to recover for 1–12 weeks postinjury. At the end of the study, animals were anesthetized for terminal measurements of diaphragm activity. The diaphragm was exposed and bilateral EMG recordings made in spontaneously breathing animals under baseline and hypercapnic conditions. Anterograde (biotin dextran amine) and transynaptic retrograde tracing (pseudorabies virus) was used to examine the changes in phrenic circuitry following injury. All contusion injuries resulted in substantial gray matter compromise, extending into the ventral horns. Ventilatory function was not significantly altered following injury. However, there were impairments in the response to challenge (hypercapnia) 10 weeks postinjury. Terminal diaphragm EMGs revealed significantly reduced responses to hypercapnia 12 weeks post-SCI. Transneuronal tracing results showed increased interneuron labeling in the ventral gray matter, rostral to the lesion epicenter, 1 week postinjury. However, preliminary evidence suggests this altered pattern of labeling may not be persistent. In addition, anterograde tracing of respiratory neurons within the medulla revealed that projections in both the ventrolateral and ventromedial white matter are compromised by injury. Experiments are under way to test the therapeutic efficacy of rolipram delivery in this injury model, to enhance anatomical repair and improve ventilatory function.
Supported by Paralyzed Veterans of America (M.A.L.), Anne and Oscar Lackner Chair (P.J.R.), and the NIH (R01 NS054025; P.J.R.).
P. R. Sanberg,* D. J. Eve,* D.-H. Park,*† C. B. Borlongan,* C. D. Sanberg,‡ and J. D. Sinden§
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery, Korea University Medical Center, Korea University College of Medicine, Seoul, Korea
‡Saneron CCEL Therapeutics, Inc., Tampa, FL, USA
§ReNeuron Ltd., Guildford, UK
Stem cell implantation has previously shown some promise for the treatment of neurological disorders, such as stroke. The timing of administration is believed to play an important role, with optimal results being obtained at the 2-day point with some cell lines after stroke. We therefore implanted an immortalized fetal cortical stem cell (CTX0E03) or vehicle into a small number of animals (n = 4–8), 2 days after transient middle cerebral artery occlusion, and performed behavioral testing, such as the elevated body swing test prior to implant. Three days later, behavioral testing was repeated and the animals were perfused with saline and the infarct size was determined by 2,3,5-triphenyltetrazolium chloride (TTC) staining. We observed a significant improvement in the elevated body swing test (p < 0.05; t-test) in the cell-treated animals, as well as increased survival (0 dead in cell-implanted vs. 50% in vehicle; p < 0.05; chi-square test). The improvement in elevated body swing test was also found to correlate with a reduced infarct size (p < 0.05; regression analysis), although due to the small sample size, infarct size was not significantly different. These results provide evidence that the CTX0E03 stem cell shows promising results with respect to behavioral improvements and reduced damage in the treatment of early stroke rats, suggesting that further research should be performed to characterize this response, as a possible treatment for stroke.
This study was supported by ReNeuron Ltd, USF, and Saneron CCEL Therapeutics, Inc. P.R.S. is on the Scientific Advisory Board of ReNeuron, and is a Founder of Saneron.
M.-L. B. Selenica, D. C. Lee, M. Gordon, and D. Morgan
USF Health Byrd Alzheimer Institute, Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
Evidence indicates that cells of monocytic origin, microglia and infiltrating monocytes/macrophages, are activated in the brains of AD patients and transgenic mouse models. A number of recent studies have found that some of the activated cells near amyloid deposits in APP transgenic mice are derived from circulating monocytes. Our recent data confirm the identification of periplaque macrophages as being at least partially derived from circulating cells. The aim of this study is to identify the factors that mediate infiltration of circulating monocytes into the brains of nontransgenic mice. We used the classical M1 response in order to monitor the infiltration of peripheral monocyte/macrophage population into the brain compare to saline-injected cohort. M1 response results in tissue damage, inflammation, and pathogen destruction in the tissue. M1 cocktail consisted of TNF-α, IL-12, and IL-1 and was injected bilaterally into dentate gyrus of hippocampus and frontal cortex by CED injections. Two days following the injections of M1 cocktail, we purify CD11b+ bone marrow monocytes from donor mice ubiquitously expressing GFP using magnetic sorting. GFP-CD11b+ monocytes were injected into nontransgenic mice via intracardiac puncture. We injected 5 × 106 cells in each mouse and a volume of 100 μl. We have previously shown that age is also an important factor in determining the infiltration of monocytes due to stimuli, and we used 12-month-old mice for this purpose. Twenty-four hours later the brains were harvested and evaluated for whether the mixture of cytokine cocktail induced the infiltration of GFP-CD11b+ monocytes into the brain. Half of the brain was used for flow cytometry and the other half was immersed in 4% PFA, sectioned, and used for fluorescence double labeling. By using both analyses we also stain for various expression markers such as GFP, CD45, CD11b, YM1, and Marco in order identify the phenotypes of the infiltrating cells. Our results will be useful in deciding the factors that play a role in enhancing or impairing monocyte infiltration, as a therapeutic approach in the treatment of AD patients.
This work was supported by Ag15490, Ag 25509.
M. Shahaduzzaman,* J. E. Golden,*† S. M. Green,* D. Rowe,† K. R. Pennypacker,*† and A. E. Willing*†
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
Administration of human umbilical cord blood (HUCB) cells subsequent to oxygen glucose deprivation (OGD) significantly increases neuronal survival and improves behavioral outcomes in an animal model of stroke. The cellular mechanisms causing the observed benefits are not fully understood. In this study we applied proteomics-based analysis techniques to both in vitro and in vivo stroke models to examine HUCB pharmacodynamics. Microarray studies were performed on RNA sampled from neurons cocultured with HUCB and subjected to OGD. Quantitative PCR (RT-PCR) followed by immunohistochemistry was performed to verify these results. To identify shared transcription factor binding sites (TFBS), the promoter region of genes altered in neuron coculture with HUCB cells subsequent to OGD were analyzed by Genomatix software (Gene2Promoter v4.7.0). The expression of significant genes was then examined through immunoblotting and quantitative analysis of incorporated radioactivity in the Typhoon 9400 Image Quant analyzer. Under OGD conditions, HUCB cells cocultured with neuronal cells significantly increased neuronal cell viability compared to the neuronal cells cultured alone (p < 0.001). Twenty-four genes from the microarray results were selected and the expression profiles were confirmed by PCR. There were significant elevations in mRNA expression of peroxiredoxin 5 (Prdx5, p < 0.05), vascular cell adhesion molecule 1 (Vcam1, p < 0.01), chemokine ligand 1 (Cxcl1, p < 0.01), paired box 6 (Pax6, p < 0.05), doublecortin-like kinase 1 (Dclk1, p < 0.05), protein phosphatase 3 regulatory subunit B (Ppp3r1, p < 0.01), and stathmin-like 4 (Stmn4, p < 0.05) in neurons with either coculture or OGD conditions. When Prdx5 protein expression was verified with immunohistochemistry in vivo, expression was upregulated in the ipsilateral hemisphere in comparison to the contralateral hemisphere (p < 0.05) in rats subjected to middle cerebral artery occlusion (MCAO) and infused with HUCB cells 48 h later; Prdx5 was not expressed in animals that underwent MCAO and treatment with vehicle (PBS). Protein expression of the other identified genes is currently under investigation, as is identification and analysis of common TFBS. The results of these experiments suggest that HUCB cells alter the gene expression profile of neurons to enhance neuronal survival following OGD exposure in both in vitro and in vivo models. There may be a common signaling pathway that modifies the acute gene expression profile induced by ischemia. If such a pathway exists, HUCB therapy would be a promising treatment for stroke and other forms of brain injury.
This study was supported in part by NIH Grant #R01NS52839 (A.E.W.). A.E.W. is a consultant to Saneron CCEL Therapeutics, Inc. and is an inventor on cord blood patents licensed to Saneron.
P. K. Shetty,*† F. Galeffi,*† M. Sadgrove,*† and D. Turner*†
*Medical Research and Surgery Services, VA Medical Center, Durham, NC, USA
†Division of Neurosurgery, Duke University Medical Center, Durham, NC, USA
Severe hypoglycemic damage to neurons can occur during ischemia or during insulin therapy for type I diabetes. Depending on the severity, hypoglycemia may cause irritability, impaired concentration and reaction times, unconsciousness, focal neurological deficit, and a seizure, confirming the constant supply of blood glucose is very important for maintaining normal brain function. Glucose is partially metabolized in astrocytes to pyruvate and then lactate, which is then primarily extruded to the extracellular space, supplementing direct glucose utilization in neurons for energy production. Either exogenous lactate or pyruvate can sustain hippocampal synaptic function during hypoglycemia. Here we hypothesize that pyruvate pretreatment, prior to glucose deprivation, can preserve neuronal and synaptic function for an extended period of hypoglycemia by increasing the stored glycogen content in hippocampal slices. We prepared transverse hippocampal slices (400 μm) from male rats and they were kept in oxygenated (95% O2/5% CO2) artificial cerebrospinal fluid (ACSF) at room temperature (22° C) for 1 h for recovery and then incubated with 10 mM pyruvate at 36°C for 2–6 h. Field excitatory postsynaptic potentials (fEPSPs) were recorded from stratum radiatum of the CA1 region after stimulating the Schaffer collateral/commissural fibers. Simultaneously we also monitored the changes in NAD(P)H fluorescence as a function of metabolic activity. Low glucose condition was induced by modified ACSF containing a decreased glucose level (2.5, 1.25, or 0 mmol/L). The effects of glycogenolysis inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (10 μmol/L DAB) and adenosine AD1 receptor antagonist 8-cyclopen-tyl-1,3-dipropylxanthine (100 nmol/L DPCPX) were tested after acquiring the stable baseline recording for least 10 min. Pyruvate pretreatment (i.e., only during slice incubation) significantly prolonged the tolerance of slices to various levels of hypoglycemia. The decrease in NAD(P)H fluorescence during glucose deprivation (2.5 mmol/L) was significantly less in pyruvate-pretreated slices (-16.9 ± 2.2%) compared to control slices (-26.7 ± 1.5%) after 60 min. In the presence of adenosine antagonists the fEPSP amplitudes were significantly maintained for a longer period of time in pyruvate pretreated slices (~30 min vs. control 10 min) in glucose-free medium. Pyruvate pretreatment led to a direct enhancement of glycogen stores (~threefold), which then declined during glucose deprivation as glycogen was metabolized for energy. The tolerance to glucose deprivation after pyruvate pretreatment was prevented by glycogenolysis inhibitors. These results indicate that the pyruvate pretreatment significantly increased the energy buffering capacity of hippocampal slices during glucose deprivation by enhancing internal glycogen stores. These observations indicate that pyruvate treatment can be an effective strategy to prevent neuronal dysfunction during glucose deprivation.
H. Shojo,*† Y. Kaneko,† N. Adachi,* and C. V. Borlongan†
*Department of Legal Medicine, Interdisciplinary Graduate School of Medicine and Engineering, University of Yamanashi, Chuo, Yamanashi, Japan
†Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
Traumatic brain injury (TBI) causes massive brain damage and severely impairs neurological functions immediately after the insult, thereby limiting the therapeutic window for neuroprotective strategies. However, a progressive secondary damage has been recently recognized in TBI, suggesting a potential therapeutic target. Here, we examined the occurrence of apoptosis in the TBI brain. Following a lateral moderate fluid percussion TBI model in adult rats, microarray analyses detected apparent changes in the expression levels of apoptosis-related genes that revealed time-dependent expression patterns for 75 genes in the lateral cortex. The altered genes involved in TBI-induced apoptosis included interleukin-1α (IL-1α), IL-1β, and tumor necrotic factor (TNF), which immediately increased and reached the maximum levels at 3 h following the injury. At 48 h, most of the upregulated apoptosis-related genes were significantly attenuated. Accompanying this surge of cell death genes after TBI was a neurostructural pathologic hallmark of apoptosis characterized by DNA fragmentation and apoptotic cells in the lateral cortex of the impacted hemisphere. Caspase-3-positive cells in the TBI brain were initially sporadic after 3 h, but these apoptotic cells subsequently increased and populated the cerebral cortex at 6 and 12 h, and gradually reached a plateau after 48 h. Interestingly, the expression profile of CD68 macrophage-labeled cells closely resembled that of apoptotic cells after TBI, implicating the role of inflammatory signaling pathway in the progression of apoptotic cell death. These results taken together indicate that TBI induced upregulation of apoptosis-related genes concomitant with the detection of apoptotic brain pathology during the 3-48-h postinjury period, which was likely mediated by inflammation. Therapies designed at abrogating apoptosis and/or inflammation may prove effective when initiated at the subacute TBI phase.
A. Smith,†‡§ B. Giunta,‡§ P. C. Bickford,†,†† M. Fountain,¶#** J. Tan,*†‡§ and R. D. Shytle†§**
*Silver Child Development Center, Department of Psychiatry and Behavioral Medicine, University of South Florida, Tampa, FL, USA
†Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
‡Neuroimmunology Laboratory, Department of Psychiatry and Behavioral Medicine, University of South Florida, Tampa, FL, USA
§Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
¶Center for Entrepreneurship, College of Business, University of South Florida, Tampa, FL, USA
#Department of Industrial and Managing Systems Engineering, College of Engineering, University of South Florida, Tampa, FL, USA
**Department of Psychiatry and Behavioral Medicine, University of South Florida, Tampa, FL, USA
††Research Service, Veterans Administration Hospital, Tampa, FL, USA
Prevention of amyloidogenic processing of precursor protein with the use of natural phytochemicals capable of enhancing α-secretase activity may be a therapeutic approach for treatment of neurodegenerative diseases, including Alzheimer's disease (AD) and HIV-associated dementia (HAD). We have recently shown promising preclinical results with the use of green tea polyphenol (—)-epigallocatechin-3-gallate (EGCG) in mouse models of both diseases. However, the translation into clinical use has been problematic, primarily as a result of poor bioavailability and inefficient delivery to the central nervous system (CNS). EGCG has been shown to possess potent antioxidative capacity. Furthermore, we have shown that it is able to promote non-amyloidogenic processing of amyloid precursor protein (APP) by upregulating α-secretase, thus preventing brain beta amyloid plaque formation, a hallmark of AD pathology and common finding in HIV infection. In this preliminary study, we investigated the ability of one preformulation method to improve the oral bioavailability of EGCG. We found that forming EGCG nanolipidic particles improves the neuronal (SweAPP N2a cells) α-secretase-enhancing ability in vitro by up to 91% (p < 0.001) and its oral bioavailability in vivo by more than twofold over free EGCG.
K. E. Soderstrom,* J. A. O'Malley,† N. D. Levine,† C. E. Sortwell,† T. J. Collier,† and K. Steece-Collier*
*Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
†Department of Neurology, Udall Center of Excellence at University of Cincinnati, Cincinnati, OH, USA
The delivery of embryonic dopamine grafts to the parkinsonian striatum has yielded variable efficacy and less symptomatic benefit than would be predicted from the degree of dopamine replacement provided. This lack of success may be in part due to pathological changes within the parkinsonian striatum. Specifically, severe dopamine deficiency associated with advanced Parkinson's disease (PD) results in marked loss of dendritic spines on striatal medium spiny neurons (MSN), the primary target sites for nigral dopamine and cortical glutamate neurons. The loss of these critical input sites could interfere with the ability of grafted neurons to reestablish the appropriate connections needed for therapeutic benefit. Dendritic spine loss occurs via dysregulation of intraspine Cav1.3 L-type Ca2+ channels and can be prevented, in animal models, by administration of the calcium channel antagonist, nimodipine. To determine if preventing dendritic spine loss in a grafted parkinsonian rat would result in improved therapeutic benefit and diminished side effects, we analyzed unilaterally parkinsonian, dopamine-grafted Sprague-Dawley rats receiving either nimodipine (normal spine density) or vehicle (severe spine atrophy) treatment. Preservation of MSN dendritic spines did not provide additional benefit in grafted animals for levodopa-induced rotational asymmetry. However, there was a significant improvement in grafted rats with preserved spine density compared with those with severe spine atrophy in vibrissae-induced forelimb use, levodopa-induced dyskinesias, and graft-induced dyskinesias. Chronic nimodipine did not affect graft survival, as no significant difference was seen in the number of surviving grafted cells between grafted groups. However, grafted rats with preserved spine density did show significantly increased fiber density in the reinnervated striatum, suggesting that an increase in normal structural contact sites within the striatum of these rats may have promoted the outgrowth and/or stability of dopamine terminals from grafted neurons. Taken together, these results suggest that preserving MSN dendritic spines in the parkinsonian striatum results in improved behavioral efficacy and the reduced occurrence of side effects in dopamine-grafted rats.
B. Solanky,* Y.-L. Chung,† M. O. Leach,† P.-W. So,* and M. Modo*
*Kings College London, London, UK
†Institute of Cancer Research UK, Cancer Research UK, EPSRC Cancer Imaging Centre, London, UK
Neural stem cells (NSCs) have been identified as a potential therapeutic solution for many neurodegenerative diseases. However, the ability to monitor their presence and differentiation in vivo remains a major challenge. In particular, their transition from NSCs to mature functioning cells is poorly documented, but could be essential to ensure that therapy is effective. We here explored high-resolution magnetic resonance spectroscopy (MRS) to identify and quantify specific metabolites that are present in undifferentiated and differentiated NSCs to elucidate potential biomarkers to identify NSCs and their differentiation. On these high-resolution MRS scans, undifferentiated neural stem cells were metabolically more active than differentiated cells, with metabolite intensities being higher across the spectrum. As expected, this was most striking in the choline region. The highly proliferative nature of stem cells resulted in a markedly higher phospho-choline level compared to differentiated cells. In addition to this difference, important changes were recognized in the myo-inositol, glucose, and NAA regions. MRS of in vitro cell extracts therefore provides a potential way to characterize a specific neural stem cell marker or “signature.” If translated into in vivo studies this could prove to be a robust, noninvasive technique to assess the differentiation of transplanted cells in vivo.
C. H. Song,* J. J. Kim,* J. Kim,†‡ S. H. Kim,†‡ and Y. G. Kim§
*Department of Obstetrics and Gynecology, Chosun University, Gwangju, Korea
†Department of Anatomy, School of Medicine, Chosun University, Gwangju, Korea
‡JB Stem Cell Institute Inc., College of Medicine, Chosun University, Gwangju, Korea
§Biology, Natural Sciences, Chosun University, Gwangju, Korea
To develop the potential enhancement of cellular adhesion, proliferation, and differentiation of human umbilical cord blood mesenchymal stem cells (hUCB-MSCs), trophogel, a three-dimensional matrix derived from placenta, was used as a substrate and their interactions were studied by comparing with two-dimensional culture systems. Furthermore, to identify what strategy allowed for maximum enhancement of the growth capabilities from MSCs, we determined the potential effects of placental extracts (PLx) and antioxidants on proliferation of hUCB-MSCs. UCB-MSC cells were seeded at 5,000 cells/cm2 (5 × 104cells/6 plate) in culture medium using trophogel. Then cell adhesion, spreading, and proliferation were assayed, and total cellular RNA was extracted to allow for determination of the cell surface marker expression in hUCB-MSCs. von Kossa stain and oil red O were used to assess the differentiation of hUCB-MSC on trophogel into osteo-blasts and adipocytes. The proliferative capacity of MSCs was determined by utilizing four commercial placental hormones [chorionic gonadotrophin (hCg), dexamethazone (Dex), estrogen (Es), and progesterone (Pg)] and four antioxidants [ascorbic acid (Aa), curcumin (Cur), resveratrol (Rr), and pycnogenol (Pyc)] including placental extracts (Plx) with nonradioactive spectrophotometeric quantification assays using Wst-1 proliferaton assay kit (Roche, Germany). The issues associated with hUCB-MSC adhesion and spreading were significantly greater on trophogel compared to control on surfaces cultured in vitro for 2 h. The proliferation growth was also dramatically increased compared to both negative (plastic tissue culture polystyrene: 2.5×) and positive controls (collagen 1: 1.1× and laminin: 1.6×) on day 5 cultures. UCB'MSC expressed oct4 when grown on trophogel, a tran-scriptional binding factor present in undifferentiated cells with high proliferative capacity with increased mRNA levels and these patterns were clearly dependent upon the culture timing. This expression correlated with an increase in differentiation efficiency towards osteogenic and adipogenic phenotypes based on oil red O and Von Kossa staining. In addition, the results strongly suggest that Es and Pg with or without Plx hormones may be useful stimulants for ex vivo expansion of hUCB-MSCs. In addition, ascorbic acid was also shown to be a good candidate for this hypothesis. Furthermore, the Plx supplemented with basic fibroblast growth factor (bFGF) increased proliferation of MSCs significantly. We conclude that bFGF and Plx may play a critical part in the recruitment of MSC function and promotion. Finally, we assume that trophogel may contribute to solving the problems associated with the proliferative capacity and differentiation of hUCB-MSCs.
C. H. Song,*† J. J. Kim,* and J. J. Kim§
*Department of Obstetrics and Gynecology, School of Medicine, Chosun University, Gwangju, Korea
†JB Stem Cell Institute Inc., Chosun University, Gwangju, Korea
‡Biology, Natural Sciences, Chosun University, Gwangju, Korea
§Department of Anatomy, Chosun University, Gwangju, Korea
These days hUCB-MSCs are generating huge interest because of their therapeutic implications in the physiological regeneration processes and transplantation therapies. The dominant hUCB-MSCs may serve as potential therapeutic agents in regenerative medicine, apart from their immunosuppressive effects and their potential to generate multiple cell lineages. The selection of dominant hUCB-MSCs is a prerequisite for cell survival and also a key factor for the differentiation of MSCs against senescence. To facilitate such a selection, we devised a novel Stem Cell Scoring System (SCSS) based on multilineage differentiations, growth rates, morphologies, passages of cell cycle, immunophenotypic analysis, differentiation, senescence-associated gene expression, ROS formation, and senescence-associated siRNA gene including SA-β-Gal. The high-scoring dominant cells (group A; score 7–10) survived 16 passages when compared to 12 passages in the low-scoring group (group C; score 1–4). The multilineage differentiations in vitro of the neurogenic, adipogenic, and osteogenic lineages were 100%, 57.1%, and 37%, respectively, and the differentiations of all the lineages decreased with increasing passages with differentiation of the adipogenic and osteogenic lineages decreasing more than that of neurogenic lineage. Quantification of MSCs at the levels of the cell cycle inhibitors showed that their senescence is accompanied by increased expression of p21 and MMP1 as indicated by lower SCSS, but TRF1 is not associated with the senescence in this system. Low scoring cells of SCSS showed increased SA-β-gal staining and produced more ROS when compared with high-scoring cells of SCSS. The expression of siRNA also correlated well with the scores of the new system. The expressions were more inhibited in high-scoring cells than in low-scoring cells. These findings suggest that high-scoring hUCB-MSCs on SCSS may be useful agents in regeneration processes or in transplantation therapies at the levels of various tested agreements.
S. Song,*† S. Song,‡ C. Chuanhai,§ K. Li,*† V. Sava,*† G. Arendash,¶ and J. Sanchez-Ramos*†
*Department of Neurology, University of South Florida College of Medicine, Tampa, FL, USA
†James A Haley Veterans Affairs Hospital, Tampa, FL, USA
‡Feinberg School of Medicine, Northwesten University, Chicago, IL, USA
§Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
¶Department of Biology, University of South Florida College of Medicine, Tampa, FL, USA
Greater understanding of mechanisms that regulate neurogenesis may ultimately lead to enhanced brain self-repair following trauma, stroke, and neurodegenerative diseases. In this report, we studied the effects of stereotaxic microlesions on neurogenesis in multiple regions of adult mice brain. Over the last few years it has become clear that neurons are generated continuously from adult stem cells in “germinal zones” of the adult brain in rodents. The adult brain retains a reservoir of neural progenitors in the dentate gyrus (DG) of the hippocampus and the rostral subventricular zone (SVZ) that can proliferate in response to ischemic injury, trauma, hypoxia, epilepsy, focal injury, and to other stimuli. In addition, bone marrow-derived cells may contribute to brain repair in several ways, including transdifferentiation into, or fusion with, neurons. The acupuncture needle (microneedle) was inserted briefly with stereotaxic guidance into specific brain regions (cortex, hippocampus, striatum, substantia nigra, and cerebellum) of C57BL/6J mice and in brains of chimeric mice with green fluorescent protein (GFP)-expressing bone marrow. The primary objective was to determine the extent to which insertion of a microneedle would increase generation of new neurons and glia in nonneurogenic zones. BrdU was used simultaneously to birth-date newly born stem/progenitor cells. To determine the extent to which newly born neural cells in various brain regions were derived from bone marrow, we used GFP+ chimeric mice. After transient stereotaxic insertion of the sterile microneedle, mice were euthanized at 1, 2, and 3 weeks and processed for immunohistochemistry. Antibodies directed against neuronal proteins (TuJ1, NeuN), astrocytic proteins (GFAP), and BrdU or GFP revealed many double-labeled neural cells in cortex, hippocampus, striatum, midbrain, and cerebellum, suggesting that neural stem/progenitor cells or bone marrow-derived stem cells could be induced to differentiate into neurons or neuron-like cells. This method generates a microlesion that results in increased migration of bone marrow-derived cells to the site; many of these cells appear to differentiate into neurons in regions where new neurons generally do not form. Understanding the signaling induced by transient microinjury may shed light on the regulation of neurogenesis but may also elucidate possible neuroprotective mechanisms attributed by some researchers to deep brain stimulation (DBS).
R. P. Stroemer,* N. Gorenkova,† E. Smith,*† E. Tang,* J. Sinden,* and M. Modo†
*ReNeuron Ltd., Guildford, UK
†King's College London, London, UK
Stroke remains one of the most promising targets for stem cell therapy as patients currently have very few therapeutic options. A thorough preclinical testing of neural stem cell lines, however, is required prior to translation into a clinical setting. To this end, we transplanted 450,000 human neural stem cells (hNSCs) prepared from the CTX0E03 clonal cell line into a rat model of stroke (transient middle cerebral artery occlusion). We used magnetic resonance imaging (MRI) to ensure all animals had an ischemic lesion and to help target implants into surviving tissue, rather than the lesion cyst, 3 weeks following the stroke. In addition to intraparenchymal injections, one group of animals received an equal amount of cells implanted intra-ventricularly. Stroked animals with intraparenchymal vehicle injections, as well as normal controls, provided comparisons to evaluate the effects of hNSC implantation. Intraparenchymal implants recovered dysfunctions on the bilateral asymmetry test, the footfault test and asymmetric circling on the rotameter. Intraventricular grafts did not recover any of these deficits. All animals with stroke damage, irrespective of their treatment, did not learn the water maze 12 weeks postgrafting. Serial MRI measures prior to transplantation, as well as 1, 4, and 12 weeks postgrafting, charted the progression of anatomical changes due to the stroke and transplanted cells. These results indicate that hNSC implants recover behavioral dysfunction over a 3-month time frame and that this effect is specific to their site of implantation.
I. Strömberg, N. Nevalainen, M. Chermenina, A. Rehnmark, P. Schouten, E. Berglöf,* and F. Marschinke
Department of Integrative Medical Biology, University of Umeå, Umeå, Sweden
Glial cell line-derived neurotrophic factor (GDNF) is a potent compound for ventral mesencephalic dopamine neurons. GDNF promotes survival of dopamine neurons and induces sprouting. However, little is known about the development of the nigrostriatal dopamine system in the absence of GDNF. Therefore, this study was conducted to investigate the development and long-term survival of the nigrostriatal dopamine system in Gdnf knockout, heterozygous, and wild-type ventral mesencephalic (VM)-lateral ganglionic eminence (LGE) cografts implanted to the lateral ventricle of wild-type hosts. The results revealed that similar number of tyrosine hydroxylase (TH)-positive neurons was found at 3 months postgrafting in all genotypes. At 6 months postgrafting, both the VM and the striatal cograft had degenerated when derived from Gdnf knockout fetuses, while surviving transplants derived from heterozygous and wild-type tissues could be demonstrated. The TH-positive innervation of the striatal cograft was sparse and widespread in the Gdnf knockout grafts at 3 months, while it was dense and patchy in wild-type-derived cografts. The dense TH-positive zones overlapped with areas demonstrating dense expression of DARPP-32 (dopamine cAMP-regulated phosphoprotein of 32,000 kDa). In transplants derived from Gdnf heterozygous tissue, a mismatch between TH- and DARPP-32-dense patches in the LGE cografts was often found, while in the Gdnf knockout-derived transplants, no or very sparse DARPP-32-positive cells were found. Thus, the nigrostriatal dopamine system develops without the presence of GDNF, although the nerve fiber innnervation of the striatum appears to be malformed, and GDNF is needed during adulthood to maintain the nigrostriatal system.
Y. Su,* C.-S. Paio,* M. E. Gonzalez-Toledo,* and L.-R. Zhao*†
*Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
†Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) belong to the family of hematopoietic growth factors. At present, it remains poorly understood whether SCF and G-CSF play a role in the central nervous system. Recently, we have demonstrated that the receptors for both SCF and G-CSF are constitutively expressed in neurons of the adult brain, that SCF and G-CSF can pass through the blood–brain barrier, and that SCF in combination with G-CSF (SCF + G-CSF) improves sensorimotor functional recovery when systemically administered in the phase of chronic stroke. However, how SCF + G-CSF improves functional outcome for chronic stroke remains entirely unknown. Here we tested our hypothesis that SCF and G-CSF have a direct effect in the neurons to promote neurite outgrowth and to enhance neuronal network formation. In an in vitro study, SCF and G-CSF were added to primary cortical neuron cultures and neurons were fixed and processed either for immunohistochemistry to visualize neurites or for neurite outgrowth assay after 2–3-day culturing. Neurite outgrowth was determined by three independent methods: traditional measurement of the neurite length, identification of neurite density with ImageJ software, and using a Neurite Outgrowth Quantification Assay Kit. We found that SCF + G-CSF has a synergistic effect on neurite outgrowth and that neuronal network formation was significantly enhanced by SCF + G-CSF. To determine whether PI3/AKT signaling was involved in SCF + G-CSF-induced neurite outgrowth, PI3/AKT phosphorylation was determined with Western blotting, and the inhibitor for PI3/AKT signaling, LY294002, was added to the neurons. We found that PI3/AKT signaling was activated by SCF + G-CSF and that PI3/AKT kinase inhibitor diminished the effect of SCF + G-CSF on neurite outgrowth. In an in vivo study, SCF + G-CSF was administered subcutaneously for 7 days beginning at 3 months after cortical ischemia. Dendritic spines in the cortex peri-infarct cavities were determined with Golgi staining. We observed that spine density adjacent to the infarct cavities was increased by SCF + G-CSF treatment and that a 63% increase in mushroom-type spines in the apical dendrites of pyramidal neurons peri-infarct cavities was seen in SCF + G-CSF-treated mice. These data suggest that SCF and G-CSF have direct effects on neurite extension, neural network formation, and neuronal circuit remodeling and that PI3/AKT signaling is required for SCF and G-CSF-induced neuronal network formation. Our findings provide insights into the contribution of hematopoietic growth factors, SCF and G-CSF, on neuronal network development and remodeling, which may be critical for their effects on brain repair during chronic stroke.
This study is supported by American Heart Association, CADASIL foundation of America, Malcolm Feist Endowment for Cardiovascular Research, and Louisiana Gene Therapy Research Consortium. We thank Neurostructural Research Lab for dendritic spine staining and analysis.
D. J. Surmeier
*Department of Physiology, Feinberg School of Medicine, Northwestern University, Chicago, IL, USA
Parkinson's disease (PD) is a pervasive, aging-related neurodegenerative disease whose cardinal motor symptoms reflect the loss of a small group of neurons—dopaminergic neurons in the substantia nigra pars compacta (SNc). Mitochondrial oxidative stress is widely viewed as responsible for this loss, but why these particular neurons should be stressed is a mystery. Using transgenic mice that expressed a redox-sensitive variant of green fluorescent protein targeted to the mitochondrial matrix, it was discovered that the unusual engagement of L-type calcium channels during normal autonomous pacemaking created an oxidative stress in SNc dopaminergic neurons, but not in adjacent dopaminergic neurons in the ventral tegmental area. Knocking out DJ-1, a gene associated with an early onset form of PD, exacerbated this stress. The oxidative stress induced by calcium induced flickering in the mitochondrial membrane potential that was attributable to reversible opening of a protein with properties similar to those of the mitochondrial membrane transition pore. Because calcium entry through L-type channels is not essential for the normal function of SNc dopaminergic neurons and can be effectively antagonized with drugs approved for human use, these results point to a novel neuroprotective strategy for PD.
C. Swanson,*† E. Du,* J. Raschke,* V. Bondarenko,* D. Johnson,‡ J. Johnson,*†‡ and M. E. Emborg*†§
*Preclinical Parkinson's Research Program, Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
†Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI, USA
‡School of Pharmacy, University of Wisconsin-Madison, Madison, WI, USA
§Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
Current evidence suggests that activation of the peroxisome proliferator activated receptor-γ (PPAR-γ) can modulate neuroprotection against Parkinson's disease (PD). PPAR-γ agonists such as rosiglita-zone and pioglitazone have been shown to be neuroprotective in MPTP mouse models of PD. Our laboratory has recently shown that chronic oral administration of pioglitazone induces neuroprotection against MPTP intoxication in a primate model. Based on this information we continued to investigate PPAR-γ activation as a therapeutic target for PD. Eli Lilly Ltd. has synthesized a novel PPAR-γ agonist, LY554862, which has the potential to be more effective than pioglitazone as it has higher brain penetration. We therefore examined the effects of oral administration of LY554862 to MPTP-treated mice. LY554862 (30 mg/kg) or vehicle was orally administered daily to C57BL/6 and antioxidant response element (ARE)-hPAP reporter mice 3 days prior to MPTP or PBS administration (30 mg/kg, IP × 5 days). ARE-hPAP reporter mice were sacrificed 7 days after the last MPTP injection and following the last dose of LY554862. C57BL/6 mice were sacrificed 21 days after the last MPTP injection and following the last dose of LY554862. Results revealed: a) significantly higher expression of tyrosine hydroxylase (TH) in the striatum determined by Western blot in animals treated with LY554862 + MPTP compared to animals treated with MPTP alone, b) significantly higher stereological cell counts of TH-ir nigral neurons in LY554862 + MPTP-treated mice compared to vehicle + MPTP-treated mice, c) an increase in striatal levels of dopamine, DOPAC, and HVA, in LY554862 + MPTP-treated mice compared to vehicle + MPTP-treated mice using HPLC-ECD, yet this increase did not reach statistical significance, d) no difference between treatment groups in hPAP activity in the striatum and substantia nigra, e) no difference in MAO-B levels between vehicle + MPTP-treated mice compared to LY554862 + MPTP-treated mice. We are continuing target validation studies using RT-qPCR to assess LY554862′s mechanisms of action in conferring neuroprotection against MPTP intoxication. Our results strongly suggest that oral administration of LY554862 has protective effects against the MPTP administration and could be a potential therapy in neurodegenerative disorders such as PD.
Support for this study was provided by the Michael J. Fox Foundation for Parkinson's disease (M.E.E.), T32 AG000213 (CS.), and Grant P51 RR000167 from NIH-NCRR to the Wisconsin National Primate Research Center, University of Wisconsin-Madison, and Eli Lilly Ltd for graciously providing LY554862.
J. Syka, D. Buckiova, T. Chumak, and J. Popelar
Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
The inner ear is an isolated, multicompartmental organ. To apply drugs directly to the cochlea carries the risk of causing irreversible damage. A less invasive approach is to apply a drug as a part of a nanoparticle (NP) to the middle ear and transport it through the round window membrane (RWM) to the cochlea. Six types of unloaded NPs were tested for such a purpose: liposomes (University of Helsinki, Finland), polymersomes (University of Southampton, UK), silica NPs (Abo Akademi University, Turku, Finland), hyperbranched polyly-sines (EPFL, Lausanne, Switzerland), poly(lactide-co-glycolide) PLGA NPs (KTH, Kista, Sweden), and lipid core nanocapsules (University of Anger, Anger, France). NP size ranged between 30–100 nm. NPs were tagged with a fluorescent dye (rhodamine, DiO, fluorescein, CdSe/CdS quantum dots). NPs were applied to the RWM on a small piece of gelfoam in ketamine (35 mg/kg) and xylazine (6 mg/kg) anesthetized rats or mice, either as a single injection of a 5–10-μl suspension or using continuous delivery with an Alzet microosmotic pump (100–200 μl of NP suspension for 7–14 days). The animals' hearing thresholds were periodically assessed using auditory brain stem responses (ABR), while the physiological state of the outer hair cells was assessed by recording distortion-product otoacoustic emissions (DPOAEs). Histological evaluation was performed at the end of the experiment when the animals were sacrificed and their auditory bullas fixed in paraformaldehyde (4%) and decalcified. Paraffin-embedded sections of the cochlea (10 μm) were stained with Alexa Fluor 488-labeled phalloidin and DAPI. A confocal microscope (Zeiss 510 DUO) was used for analysis of the samples. All types of NPs were identified within 24 h in the cochlea. They entered the cytoplasm of cells in the organ of Corti (hair cells and supporting cells), neurons of the spiral ganglion, and cells of the lateral wall. Their distribution in the inner ear structures was, however, very different with most NPs entering the spiral ganglion. NPs did not cause any distinct morphological damage in the inner ear nor any pronounced changes in hearing thresholds or DPOAE amplitudes. Our results suggest that nanoparticles can function as a suitable drug transport tool to the inner ear.
Supported by grants AV0Z50390512, GACR 309/07/1336, LC 554, and Nanoear NMP4 -CT-2006-02556.
E. Sykova,*† P. Jendelova,*† A. Hejcl,*† N. Kozubenko,*† and T. Amemori*†
*Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
†Department of Neuroscience and Centre for Cell Therapy and Tissue Repair, Charles University, Prague, Czech Republic
Mesenchymal stem cells (MSCs), olfactory ensheathing glia (OEGs), and neural progenitor cells (PNCs) have the capacity to migrate towards lesions and induce better regeneration. MSCs, OEGs, or PNCs labeled in culture with iron oxide nanoparticles were transplanted into rats with a balloon-induced spinal cord compression lesion (SCI). In vivo MRI, used to track their migration and fate, proved that MSCs, OEGs, as well as PNCs migrated into the lesion. We compared the effects of implanted PNCs, OEGs, or MSCs with the implantation of a mononuclear fraction of bone marrow cells (BMCs) or the injection of granulocyte colony-stimulating factor (G-CSF). Seven days after SCI, the rats received either PNCs, MSCs, OEGs, BMCs, or G-CSF intravenously or intraspinally. All implanted animals had significantly smaller lesions and better scores on BBB (motor) and plantar (sensory) tests. In further studies, hydrogels or nanofiber scaffolds seeded with stem cells were implanted into rats with acute SCI. The implants reduced scar formation bridged the lesion and increased functional recovery. Autologous BMC implantation was also used in a Phase I/II clinical trial in patients with acute and chronic SCI (n = 34). The results show that implantation is safe and has a beneficial effect if administered in the first 1–4 weeks after cervical injury by catheterization of a. vertebralis (i.e., close to the injury site). In a model of chronic SCI, at 5 weeks after injury, HPMA-RGD hydrogels [N-(2-hydroxypropyl)-methacrylamide with attached amino acid sequences—Arg-Gly-Asp] were implanted into the lesion, either with or without seeded MSCs. Animals with chronic SCI served as controls. The animals were behaviorally tested using the BBB (motor) and plantar (sensory) tests once a week for 6 months. Subsequently, the animals were sacrificed 6 months after SCI, and the spinal cord lesions evaluated histologically. Behavioral analysis showed a statistically significant improvement in rats with combined treatment, hydrogel, and MSCs, compared with the control group (p < 0.05). No significant improvement was found in rats treated with hydrogel implantation only. Further, combined therapy prevented tissue atrophy (p < 0.05). The hydrogels were infiltrated with axons myelinated with Schwann cells. Blood vessels and astrocytes also grew inside the implants. MSCs were present in the hydrogels even 5 months after implantation. We conclude that 5 weeks after injury, HPMA-RGD hydrogels seeded with MSCs can successfully bridge a spinal cord cavity and provide a scaffold for tissue regeneration. This treatment leads to functional improvement even in chronic SCI.
Supported by AVOZ50390703, 1M0538, GAAV-IAA500390902.
B. T. Terpstra,*‡ J. W. Lipton,†¶ T. J. Collier,* H. Muratsubaki,§ N. D. Levine,* S. L. Wohlgenant,* A. D. Cole-Strauss,* K. L. Paumier,*‡ S. E. Gombash,*‡ and C. E. Sortwell *¶
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA
‡Graduate Program in Neuroscience, University of Cincinnati, Cincinnati, OH, USA
§Department of Clinical Chemistry, Kyorin University, Tokyo, Japan
¶Division of Translation Science and Molecular Medicine, Michigan State University, East Lansing, MI, USA
Elevated serum urate (UA) has been associated with a slower rate of PD progression as well as a decreased risk of PD. However, it has yet to be determined whether serum UA is a biomarker or a disease-modifying compound. Evidence from our laboratory indicates that systemic treatment with inosine, a UA precursor, is protective in the 6-hydroxydopamine (6-OHDA) rodent model of Parkinson's disease (PD). However, when administered peripherally to a rodent, inosine has the potential to act via several metabolites including UA and allantoin. The purpose of the current study was to determine the active metabolite involved in the neuroprotective effect of systemically administered inosine in the 6-OHDA rodent model of PD. Male Sprague-Dawley rats received subcutaneous matrix-driven pellets for compound delivery. Animals received 200 mg total dose of inosine, inosine plus the urate oxidase inhibitor potassium oxonate (KOx), allantoin, or no pellets (sham) prior to intrastriatal 6-OHDA. Plasma levels of inosine, hypoxanthine, xanthine, UA, and allantoin were measured 4 days following the pellet implantation. Animals were assessed prior to and following 6-OHDA for forelimb asymmetry. After 6 weeks the striatum was analyzed for levels of DA and metabolites and the substantia nigra (SN) was analyzed for tyrosine hydroxylase-immunoreactive (TH-ir) neuron number utilizing unbiased stereology. Inosine or allantoin administration resulted in significant accumulation of plasma allantoin (p < 0.001) whereas administration of inosine with KOx prevented this accumulation. Further, administration of inosine plus KOx resulted in significant increases in plasma UA and xanthine (p < 0.05). Both inosine and allantoin administration significantly ameliorated forepaw asymmetry (p < 0.001); however, preventing the conversion of UA to allantoin completely eliminated this effect. Additionally, both inosine and allantoin treatment resulted in significant sparing of TH-ir neurons in the SN (p = 0.02) whereas inhibition of the metabolism of UA to allantoin abolished this nigral cell sparing. No significant differences in the levels of DA and its metabolites in the striatum were observed between groups. These results are the first to indicate that the end product of inosine metabolism in rodents, allantoin, is a potent neuroprotective compound in a parkinsonian model and suggests that allantoin, not UA, is involved in the neuroprotective capacity of inosine.
Supported by the James J. and Joan A. Gardner Family Center for Parkinson's Disease and Movement Disorders and the Morris K. Udall Center of Excellence for Parkinson's Disease Research at the University of Cincinnati NS058830 (T.J.C.).
D. R. Wakeman,*† J. Bober,‡ J. R. Sladek, Jr.,§ C. Leranth,‡ R. J. Samulski,¶ E. Y. Snyder,*† and D. E. Redmond, Jr.#
*Graduate Program in Biomedical Sciences, University of California at San Diego, La Jolla, CA, USA
†Stem Cells and Regeneration, The Burnham Institute for Medical Research, La Jolla, CA, USA
‡Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT, USA
§Department of Pediatrics, University of Colorado School of Medicine, Denver, CO, USA
¶Gene Therapy Center & Department of Pharmacology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA
#Departments of Psychiatry and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
Human fetal and embryonic stem cell derived neural precursor cells (hNPC) have been suggested as a renewable alternative substrate to fetal dopaminergic neurons/progenitors for transplantation in Parkinson's disease (PD). However, little is known about the long-term potential of these grafts to differentiate and/or integrate into anatomically correct loci in the adult, nonhuman primate brain. We transplanted undifferentiated hfNPC that were derived from human fetal forebrain subventricular zone homotopically into the ventral midbrain of MPTP-lesioned monkeys. In the same animals, we concomitantly delivered striatal, glial-derived neurotrophic factor (GDNF) by an AAV serotype5 vector. The fate of grafted cells was assessed after 11 months in vivo. Donor soma remained predominantly within the ventral mesencephalon in the area of injection and extended numerous, morphologically relevant, monoamine fiber types including both smooth and beaded varicose profiles. While these neurofilament-positive neurites appeared to project in multiple directions, they also coursed in trajectories, often circuitous, to adjacent disease-relevant targets, such as the substantia nigra. Donor processes paralleled tyrosine hydroxylase-positive fibers of the host nigrostriatal tract, but did not differentiate substantially into fully mature, striatally integrated, A9 dopaminergic neurons. This work demonstrates that hNPC are capable of generating neuronal phenotypes long term, retain the capacity to direct axonal projections with trajectories comparable to the intrinsic nigrostriatal pathway, and respond to specific local endogenous signaling cues in the adult dopamine-depleted primate brain, suggesting that the adult primate brain retains specific axonal guidance cues and maintains a permissive environment for xenotransplantation of hNPC and possible pathway construction.
D. Williams, D. Lee, M. N. Gordon,* and D. Morgan
Byrd Alzheimer Research Center, Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
The inducible form of nitric oxide synthase (NOS2) is activated during times of inflammation; however, its role during neurodegenerative diseases is less well understood. When APPSwDI mice are crossed with NOS null mice, they accumulate Aβ amyloid pathology, develop tau pathology, and exhibit neuron loss in hippocampus (Colton et al., J. Neurosci. 28:1537; 2008). Thus, APPSwDI/NOS-/- mice may represent a unique murine model of Alzheimer's disease (AD). Here, we evaluate the role of NOS2 during classical microglial activation. Mice with NOS2 null background and their wild-type littermates were injected with either saline or a proinflammatory cocktail consisting of tumor necrosis factor-α (TNF-α), interlukin-1 β (IL-1β), and interlukin-12 (IL-12), which has been shown to illicit proinflammatory type response in microglia. The intracranial injections were made bilaterally in hippocampus (HPC) and frontal cortex (CX). Three days posttreatment the brains were harvested and dissected with half of the brain destined for biochemical analysis and half being used for immunochisto-chemistry. We evaluated general markers of microglial activation, in addition to alternative activation markers. We observed an upregulation of CD45+ and YM1+ cells in the CX and HPC of mice treated with the proinflammatory cocktail compared to the saline-treated groups. Additionally, the activation response by the proinflammatory cocktail was significantly greater in the NOS2 null mice than in their wild-type littermates. The presence of NOS2 appears to limit certain inflammatory responses and thus may be protective during times of neuroinflammation. Understanding the exact role of NOS2 may help elucidate underlying mechanisms of neurodegeneration in diseases like AD.
Supported by AG04418, AG15940, AG18478.
D. Yu,*†‡ G. Song,§ C.-S. Poon,§ and Y. D. Teng*†‡
*Department of Neurosurgery, Harvard medical School/BWH/CHB, Boston, MA, USA
†Department of PM&R, Harvard medical School/SRH, Boston, MA, USA
‡Division of SCI Research, VA Boston Healthcare System, Boston, MA, USA
§Harvard-MIT Health Science & Technology Program, MIT, Cambridge, MA, USA
Respiratory disorders, major complications of cervical spinal cord injury (CSCI), are the leading cause of morbidity and mortality of clinical spinal cord trauma. We previously reported that human neural stem cells (hNSCs) improved general sensorimotor functions in SCI rats mainly through staging neuroprotection and neuroplasticity. Because the respiratory system is known for possessing high capacity for functional and anatomical compensation, we hypothesized that post-CSCI respiratory abnormalities may be an “appropriate” therapeutic target for the treatment of prototype hNSCs. We tested our hypothesis by examining the effects of hNSCs implanted into the hemicontused C5 spinal cord and explored the roles of hNSCs in functional repair, tissue reconstitution, respiratory neuroprotection, and neural network reorganization. Female Sprague-Dawley rats were anesthetized before giving C5 unilateral laminectomy and severe hemicontusive injury (10 g × 50 mm). hNSCs were injected into the injured cervical cord via a glass pipette 3 days after CSCI (50K cells/μl; 2 μl/per injection; three injections/rat at injury epicenter and spinal cord regions immediately rostral and caudal to the epicenter; n = 11). The respiratory parameters were monitored by plethysmography 1 day post-CSCI and weekly afterwards for 4 weeks. Neurobehavioral studies were conducted in parallel to evaluate general motosensory recovery. Neural tracers of biotin dextran amine and fluorogold were injected into the spinal cord and diaphragm to evaluate the structural reorganization after treatment. EMG of diaphragm and the phrenic neurogram were recorded at 10 weeks post-CSCI. In the end, spinal cord tissue was collected for histopathology and immunocytochemistry analyses. Treatment with hNSCs restored respiratory function starting 7 days post-SCI, which was demonstrated by sustained recovery in tidal volume (Vt), respiratory frequency (f), minute ventilation (Ve), and EMG and phrenic neurogram restoration. Conversely, vehicle-treated control CSCI rats (n = 10) bore long-term respiratory deficits that our C5 SCI model typifies (Choi et al., J. Neurosci.; 2005). Spinal cords receiving hNSC treatment revealed large-scale tissue reconstitution as well as neurite reorganization, though long distance regeneration of descending axons was not confirmed. These data indicate that hNSCs provide potent functional repair for the mammalian respiratory system. Our findings suggest that for the potential clinical application of prototype hNSCs, it is pivotal to identify neurological disorders with underlying pathophysiologies that can effectively respond to the multifaceted impacts of stem cells.
Supported by VA, NIH, CIMIT, and Massachusetts SCI Cure Fund.
S. J. Yu,* Y. Kaneko,* E. C. Bae,* S. Platt,† and C. V. Borlongan*
*Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Small Animal Medicine & Surgery, College of Veterinary Medicine, University of Georgia, Athens, GA, USA
Stroke is a major cause of death and disability in adults. Ischemic injury in the brain results in neuronal cell death, involving both necrosis and apoptosis. Stem cell transplantation has been proposed for treating brain diseases, including stroke. The placenta has been recognized as a source of nonembryonic stem cells, which could be harvested from humans and rats. Here, we examined dog placenta cells (DPCs) as an equally good source of stem cells. We also explored the potential of DPCs to exert benefits in an in vitro model of stroke. Dog placenta cells were grown in well-defined cell culture media. Established phenotype markers of embryonic stem cells (e.g., Oct4, Nanog, SSEA4, CXCR4) and neural lineage (e.g., nestin, MAP2, NeuN, GFAP and O4) were employed to reveal stemness and cell fate over time. In parallel studies, primary rat neurons/astrocytes were exposed to oxygen-glucose deprivation (OGD), an established ischemic stroke model, and subsequently cocultured with DPCs. Cell viability was measured by immunocytochemistry and trypan blue to determine neuroprotective effects of DPCs. Moreover, in order to reveal the mechanism of action underlying therapeutic benefits of DPCs, we examined the expression of heat shock protein (hsp) 27, a recently discovered member of the heat shock protein family and implicated as neuroprotective against stroke. At early and prolonged (>8) passages, DPCs expressed stable embryonic stem cell markers, and when grown in neutrally inducing defined media, started to exhibit neuronal, astrocytic, and oligodendrocytic phenotypes. Under the OGD condition, primary DPCs dose-dependently reduced cell death in primary rat cells. Moreover, hsp 27 was strongly expressed under the OGD condition, suggesting the role of hsp in this observed neuroprotection. The present results indicate that DPCs represent as a source of stem cells that can be differentiated at least towards the neural lineage. In addition, DPCs promote neuroprotection against stroke, possibly via hsp 27 upregulation. In concert with advancing the use of cell therapy in humans, we are also cognizant of testing this novel treatment in humans' best friend—the dog, which exhibits diseases similar to humans, such as stroke.
Y. Zhu,*1 H. Hou,*1 A. Ruscin,*† T. Mori,*‡ C. Gemma,§ D. Obregon,* J. J. Jin,* N. Dragicevic,¶ P. Bradshaw,¶ P. Bickford,§ B. Giunta,*† T. Town,#** and J. Tan*†#
*Rashid Laboratory for Developmental Neurobiology, Silver Child Development Center, University of South Florida College of Medicine, Tampa, FL, USA
†Neuroimmunology Laboratory, Department of Psychiatry & Behavioral Medicine, University of South Florida College of Medicine, Tampa, FL, USA
‡Departments of Medical Science and Pathology, Saitama Medical Center/Saitama Medical University, Kawagoe, Saitama, Japan
§Department of Neurosurgery and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
¶Department of Biology, College of Arts and Science, University of South Florida, Tampa, FL, USA
#Department of Neurosurgery and Biomedical Sciences, Cedars-Sinai Medical Center, Los Angeles, CA, USA
**Department of Medicine, David Geffen School of Medicine, University of California, Los Angeles, Los Angeles, CA, USA
Past Alzheimer's disease (AD) epidemiologic and pathologic studies indicate CD45 dysregulation within the brain and periphery. We report transgenic mice overproducing Aβ, but CD45 deficient (PSAPP/CD45-/-), closely mimic the neuropathologies of clinical AD. Specifically, we found: 1) increased intra- and extracellular soluble and insoluble Aβ (p < 0.05), 2) decreased plasma soluble Aβ (p < 0.001), 3) alternated microglial phenotype with increased neurotoxic cytokine expression (TNF-α and IL-1β), and, most importantly, 4) increased neuronal loss in PSAPP/CD45-/- mice compared to PSAPP/CD45+/+ littermates (p < 0.001). This may result from microglial modulation from an Aβ phagocytic to a proinflammatory phenotype upon CD45-/- ablation as observed in vitro and in vivo. These events positively correlated with neuronal loss as revealed by immunohistochemistry, decreased ratio of Bcl-xL to Bax, increased activated caspase-3, and mitochondrial dysfunction. These data suggest PSAPP/CD45-/-mice represent a relevant AD model, and specific promotion of CD45-mediated microglial clearance as a novel target for AD treatment.
1These authors contributed equally to this work.