Abstract
Evan Y. Snyder
Burnham Institute, La Jolla, CA, USA
A number of emerging tools & technologies appear poised to transform regenerative medicine. Given that ASNTR has always been at the forefront of neural repair, starting with its early focus on transplantation and now broadening its scope to embrace all means of restoring function to the dysfunctional or maldeveloped CNS, we have convened a symposium to expose attendees to some of the most prominent of these cuttingedge approaches. Speakers will address:
S. A. Acosta*†, J. Jernberg*, M. Cole*, A. Schlunk*†, R. D. Shytle*†, J. Tan*†‡, C. Sanberg§, P. Sanberg*†, G. Gemma*†‡, and P. C. Bickford*†‡
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University South Florida, Tampa, FL, USA
†Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
‡James A. Haley VA Hospital, Tampa, FL, USA
§Natura Therapeutics, Inc., Tampa, FL, USA
It has been shown that cognitive decline during aging may be linked to oxidative stress, microglia activation, and proinflammatory factors, which limit neuroplasticity. These factors may lead the transition between mild cognitive declines of normal aging to learning and memory dysfunction, or severe neurodegenerative diseases. Previously, we have shown that nutrients found in natural compounds such as blueberry, green tea, and carnosine contain certain vitamins high in antioxidants and flavonoids that may be able to decrease the damaging effects of oxidation, inflammation and cell death in the blood, brain, and other tissues of the body. (R. D. Shytle, 2007). Furthermore, we have shown that the combination of these nutrients (called NT 020) creates an additive and synergistic effect that promotes the proliferation of hematopoietic cells (P. Bickford, 2006). In the present in vivo study, we aimed to investigate the natural therapeutic potential of NT 020 for improving learning and memory in aged rats. To test this hypothesis, three groups were chosen to be treated with NT 020 gavage, or water gavage. Aged (20 months old) male Fisher 344 rats were treated with 135.0 mg/kg per day (n = 13) of NT 020. Aged control (20 months old) male Fisher 344 rats were treated with water (n = 13). Young F344 rats (3 months old) (n = 10) were used as a young control group and were treated with water through oral gavages. All groups were treated for a period of 4 weeks. Morris water maze (MWM) was used to evaluate the effects of natural compounds of NT 020 on spatial learning and memory by young and aged rats. The performance on the MWM was measured over 5 days of training, four trials per day, using a computer tracking software (Noldus) and assessed for the cumulative search error. This type of measurement was chosen for our data because it mainly reveals the age-related cognitive impairments while restricting bias due to swimming capability. The results confirmed that aged rats fed with the water gavages showed impaired performance on the water maze compared to the young rats. Furthermore, the NT 020 oral gavages treatment ameliorates the performance deficit found in aged rats on the control gavages. Thus, the aged rats fed with NT 020 performed significantly different from the aged control rats. The results indicate that dietary supplementation of NT 020 can be used to treat and improve the age-related deficit in learning and memory as evaluated by the spatial navigation task on the MWM. Although further work is necessary, it appears that NT 020 may promote the health, proliferation, and maintenance of neurons and has potential as a therapeutic agent for an aging population to improve learning and memory.
This work was supported by the National institute of Health (AG04418, AG024165A, MH070430,) and the VA Medical Research Service.
M. Airavaara*, B. Harvey*, M. Chiocco*, D. Howard*, J. Peränen†, M. Saarma†, Y. Wang*, and B. Hoffer*
*National Institute on Drug Abuse, I.R.P., Baltimore, MD, USA
†Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Finland
Mesencephalic astrocyte-derived neurotrophic factor (MANF), also known as arginine-rich, mutated in early stage of tumors (ARMET), is a secreted protein that reduces endoplasmic reticulum (ER) stress. Our previous results suggest that exogenous MANF has neuroprotective effects against cerebral ischemia, possibly through the inhibition of cell necrosis/apoptosis in cerebral cortex. We previously found that cortically administered recombinant MANF protein reduced infarction volume, decreased body asymmetry and neurological score, and increased locomotor activity after transient middle cerebral artery occlusion (MCAo) in rats. The purpose of this study is to examine the ability of a double-stranded adeno-associated viral vector expressing human MANF (AAV-MANF) to confer neuroprotective effects in a rodent model of stroke. The efficacy of double-stranded AAV-MANF was first examined in vitro. We found that AAV-MANF increased MANF immunoreactivity in primary cortical neurons. AAV (serotype 7)-MANF or an AAV(7) vector expressing the green fluorescent protein (AAV-GFP) was next administered into three cortical sites (middle 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. One week after injection, MANF immunoreactivity was increased in the cortex of animals that received AAV-MANF but not AAV-GFP. The right middle cerebral artery (MCA) was ligated with a 10-O suture for 60 min 1 week after the injections. We found that AAV-MANF pretreatment decreased body asymmetry in 20 trials when analyzed 2 days after MCAo (AAV-MANF = 15.8 ± 1.3; AAV-GFP = 8.8 ± 0.5; p = 0.023). Brains subsequently were removed and sliced into 2-mm-thick coronal sections for triphenyltetrazolium chloride (TTC) staining. Administration of AAV-MANF significantly reduced the cerebral infarction volume (AAV-MANF = 107 ± 19 mm3; AAV-GFP = 168?20 mm3, p = 0.0447). In addition, the area of the largest infarction in a slice was found to be significantly reduced in AAV-MANF-treated rats (13 ± 2 mm2, p = 0.0276) compared with AAV-GFP-treated rats (18 ± 1 mm2). In another set of animals, we found that the neurological score was decreased in AAV-MANF-treated rats on days 2, 7, and 14 after MCAo (p < 0.05, two-way ANOVA). Taken together, our data show that administration of AAV-MANF into rat cortex increases MANF immunoreactivity and reduces neurodegeneration induced by cerebral ischemia.
R. H. Andres*, A. V. Pendharkar*, R. Guzman*, A. De†, T. M. Bliss*, E. McMillan‡, C. N. Svendsen*‡, S. S. Gambhir†, H. R. Widmer§, T. Wallimann¶, and G. K. Steinberg*
*Department of Neurosurgery, Stanford University, Stanford, CA, USA
†Molecular Imaging Program, Stanford University, Stanford, CA, USA
‡The Waisman Center, University of Wisconsin-Madison, Madison, WI, USA
§Department of Neurosurgery, University of Berne, Berne, Switzerland
¶Department of Cell Biology, instSwiss Federal Institute of Technology, Zurich, Switzerland
Creatine kinase (CK) catalyzes the reversible transphosphorylation of creatine (Cr) by ATP. The CK/phosphocreatine (PCr) system therefore plays a central role in cellular energy buffering and energy transport, particularly in cells with high and fluctuating energy demands. Given the high expression of CKs in the developing central nervous system, we hypothesize that Cr supplementation might improve the metabolic state of neural stem cells (NSCs). In the present study, we investigated the effects of Cr on survival, migration, and differentiation of mouse NSCs isolated from the subventricular zone (mNSCs) and human NSCs derived from the fetal cortex (hNSCs). We found both the brain-specific cytosolic (BB-CK) and the ubiquitous mitochondrial (uMt-CK) isoform of CK expressed in mNSC and hNSC. Accordingly, Cr supplementation at 5 mM for 7 days resulted in higher ATP levels in mNSCs and hNSCs, compared to untreated controls (p < 0.01). Chronic Cr exposure of mNSCs and hNSCs resulted in a dose-dependent increase in neurosphere size and total cell numbers with a maximal effect at 5 mM after 7 days in vitro (p < 0.05). Analysis of bromodeoxyuridine (BrdU) incorporation did not reveal a significant increase in proliferation activity, but Cr-treated cultures contained less cells with immunoreactivity for active caspase-3 (p < 0.05), pointing towards an antiapoptotic effect of Cr. In a modified Boyden chamber assay, both Cr-pretreated mNSCs and hNSCs demonstrated an improved migratory potential (p < 0.01) after 60 min, compared to untreated controls. In line with this finding, we found that Cr-pretreated, GFP-labeled mNSCs showed improved migration to the ischemic brain area after intrastriatal transplantation in a distal MCA occlusion stroke model in C57/Bl6 mice (p < 0.05). In a next step, we examined the effects of Cr on differentiation of NSCs. Chronic Cr exposure at 5 mM resulted in higher neuronal cell numbers (TuJ1+, p < 0.05) at the expense of the glial fate (GFAP+, p < 0.05) after differentiation conditions in vitro for 5 days. In addition, we found disproportionally higher numbers of GABA-immunoreactive cells in the Cr-treated groups (p < 0.05), suggesting that Cr acts as a differentiation factor for specific neuronal subpopulations. In conclusion, our findings suggest that the CK/PCr system is critically involved in maintaining the energy metabolism of NSCs. Chronic Cr supplementation resulted in increased cellular ATP levels, inhibited apoptosis during in vitro expansion, and improved NSC migration in vitro and in vivo. Cr exposure also promoted the differentiation of NSCs towards the neuronal lineage, particularly supporting the GABAergic phenotype. Cr pretreatment of NSCs might therefore offer new ways for improving cell replacement approaches for stroke and other diseases of the nervous system.
A. D. Bachstetter*†, J. Jernberg*, A. Schlunk‡, C. E. Hudson§, P. C. Bickford*†§, and C. Gemma*§
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, Tampa, FL, USA
†Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
‡Honors College USF, Tampa, FL, USA
§James A. Haley VA Hospital, Tampa, FL, USA
Adult neurogenesis is a lifelong process by which relatively few cells are added into two restricted regions of the brain. Integration of the cells into the existing neuronal circuitry, with the unique properties involved in the maturation of these cells, is possibly critical to the acquisition and retrieval of new memories. With the chronological aging of the organism a process of cellular senescence occurs throughout the body, a portion of which is independent of primary alterations to the stem cells; instead, it appears to be dependent on the environment where the cells reside, and is in part regulated by inflammation. Microglia, the resident immune cells in the brain, are neuroprotective but chronic activation of the microglia, such as the chronic activation that occurs with advanced age, can promote neurotoxic inflammation. Recently, it has been shown that CX3CR1, a chemokine receptor for the neuronal expressed ligand fractalkine, is critical for regulating microglia and blocking excessive microglia activation. The hypothesis of this study was that with age a disruption in signaling through CX3CR1/fractalkine occurs that results in chronic inflammation leading to the decrease in neurogenesis that occurs with age. We used two model systems to test this hypothesis: 1) a mouse model with a loss of the fractalkine receptor; and 2) a pharmacological rat model in three age groups, using an intracerebroventricular infusion via an osmotic minipump of either a blocking antibody to the fractalkine receptor, or recombinate fractalkine and the appropriate controls. We then used multiple thymidine analogs to label proliferating cells at different time points during the treatment. Through unbiased stereological quantification of the thymidine analogs and endogenous markers of neurogenesis, we addressed two main questions: 1) Is CX3CR1/fractalkine signaling important for maintaining adult hippocampal neurogenesis? 2) Could a disruption in CX3CR1/fractalkine signaling contribute to the decrease in hippocampal neurogenesis associated with aging? The results of this study demonstrated three main findings. First, loss of function of CX3CR1 in young adult rodents, mice and rats, resulted in a significant decrease in hippocampal neurogenesis. Second, administration of exogenous FKN reversed the age-related decrease in hippocampal neurogenesis. Finally, IL-1β receptor antagonist protected against the decrease in hippocampal neurogenesis induced by blocking CX3CR1 function. Our findings demonstrate for the first time that changes in fractalkine signaling in the hippocampus occur, as a result of normal aging, which can be restored by the addition of recombinant ligand. Futhermore, this study indicates that fractalkine/CX3CR1 signaling has a critical role in maintaining hippocampal neurogenesis. We found that we could model the age-related affects on neurogenesis in young rats by a blocking antibody directed at the fractalkine receptor. Using this model we demonstrated that the mechanism by which fractalkine affects neurogenesis is most likely indirect through the regulation of IL-1β. As it appears that the fractalkine receptor on microglia is functional with age, CX3CR1 might be an effective therapeutic target for aging and age-related neurodegenerative diseases.
This work was supported by the National Institutes of Health (AG024165A, AG004418) and the VA Medical Research Service.
S.-H. Baek*, E.-K. Kim*§, H. Lee‡, S. K. Park¶, K. J. Lookingland†‡, J. L. Goudreau*†‡, S. W. Kim¶, and S.-W. Yu*†
*Department of Neurology and Ophthalmology, Michigan State University, East Lansing, MI, USA
†Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA
‡Neuroscience Program, Michigan State University, East Lansing, MI, USA
§Department of Food Science and Human Nutrition, Michigan State University, East Lansing, MI, USA
¶Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, Seoul, Korea
The brain as one of the least regenerative organs represents an ideal target for stem cell replacement therapy; however, poor survival of engrafted stem cells remains one of the biggest obstacles. Understanding the mechanisms that regulate survival and death of neural stem cells is critical to the formulation of efficient stem cell-based therapies. We have recently reported that hippocampal neural (HCN) stem cells derived from the adult rat brain are insulin dependent for proliferation and undergo a nonapoptotic, autophagic cell death following insulin withdrawal (Stem Cells, 26:2602, 2008; Autophagy, in press). Insulin-deprived HCN cells exhibit morphological and biochemical markers of autophagy, including massive formation of autophagic vacuoles, accumulation of Beclin 1 and the type II form of microtubule-associated protein 1 light chain 3 (LC3) without evidence of apoptosis. Suppression of autophagy by knockdown of Atg7 reduces cell death, whereas promotion of autophagy with rapamycin augments cell death in insulin-deficient HCN cells. These data reveal a causative role of autophagy in insulin withdrawal-induced HCN cell death. On the other hand, staurosporine induces robust activation of caspase-3 and nucleosomal DNA fragmentation, suggesting that HCN cells have intact apoptotic capability despite the apparent absence of apoptosis following insulin withdrawal. Taken together, our study demonstrated that autophagy is the primary cell death mechanism in insulin-deficient HCN cells, and provide a bona fide model of autophagic cell death in apoptosis-intact mammalian cells. In order to identify the molecular mechanisms that link autophagy to cell survival and death in HCN cells, we performed whole genome microarray analysis following insulin withdrawal and found that 412 genes showed significant changes in expression level. Wnt inhibitory factor (Wif) was one of the upregulated genes, suggesting that negative regulation of Wnt pathway may underlie HCN cell death. Consistent with this hypothesis, recombinant Wnt proteins and pharmacologic inhibition of GSK-3β (a negative regulator of Wnt pathway) both increased cell survival in insulin-deprived HCN cells. These results suggest that understanding the mechanisms governing autophagy of adult neural stem cells may provide novel strategies to improve the survival rate of transplanted stem cells for treatment of neurodegenerative diseases. Furthermore, these studies will bring to light the role of autophagy in loss of endogenous stem cells in neurodegenerative diseases.
B. Behrouz*, K. Lookingland*†, and J. Goudreau*†‡
*Neuroscience Program, Michigan State University, East Lansing, MI, USA
†Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA
‡Department of Neurology, Michigan State University, East Lansing, MI, USA
Parkinson's disease (PD) is a neurodegenerative disorder that causes severe motor impairments due to progressive loss of nigrostriatal (NS) dopamine (DA) neurons. Abnormal DA metabolism has been proposed to underlie the degeneration of these neurons, but not all DA neurons are affected to the same extent in PD. While there is severe loss of NSDA neurons, hypothalamic tuberoinfundibular (TI) DA neurons remain intact. The reason for this differential susceptibility amongst DA neurons is unknown. This differential susceptibility is mimicked in the 1-methyl,4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model, with NSDA neurons highly susceptible and TIDA neurons fully protected. The immediate response of these neuronal populations suggests that TIDA neurons initially respond to MPTP treatment in a similar fashion to NSDA neurons, but rapidly recover. These experiments demonstrate that the recovery of TIDA neurons from MPTP-induced DA loss is dependent on synthesis of new proteins following MPTP treatment. One of the genes upregulated in TIDA neurons following MPTP treatment is the protective protein parkin. Mutations in this gene cause autosomal recessive PD and increased expression of this protein infers protection to cells in a variety of models, leading to the hypothesis that parkin upregulation may contribute to the recovery of TIDA neurons following MPTP treatment. This hypothesis is tested by viral delivery of parkin shRNA in the arcuate nucleus of mice in order to transiently knock down (KD) the expression of parkin in TIDA neurons. TIDA neurons of mice receiving parkin shRNA lentivirus show stunted recovery of DA in response to MPTP treatment, suggesting parkin may be essential for the recovery of these neurons. Elucidating factors that are responsible for differential susceptibility to mitochondrial complex I inhibition may be further translated into neuroprotective strategies that can prevent the ongoing degeneration of DA neurons in PD.
M. A. Berk, U. Salli, R. Ramachandra, K. Venkiteswaran, and T. Subramanian
Departments of Neurology and Neural & Behavioral Sciences, The Pennsylvania State University Hershey Medical Center and College of Medicine, Hershey, PA, USA
Human embryonic stem cells (hES) hold promise for significant therapeutic advantage as a viable cell-based therapy of Parkinson's disease. hES have been shown to differentiate into dopaminergic neurons and integrate into the host striatum after transplantation to provide continuous dopamine replacement. hES may serve as a better option than other surgical interventions, such as deep brain stimulation (DBS), that require battery replacements, suffer mechanical failures, and frequent adjustments according to disease progression and patient aging. Striatal xenotransplants of hES have demonstrated the ability to survive, function, and restore behavioral asymmetry in 6-OHDA-lesioned hemiparkinsonian rats but required systemic immunosuppression to prevent immune rejection and were associated with the occurrence of teratomas. We have previously shown that striatal xenotransplants of human retinal pigment epithelial (hRPE) cells do not undergo immune rejection up to 18 months, provide local immunoprotection, and prevent fetal ventral mesencephalic striatal xenotransplants from host rejection. Using a similar paradigm, we tested whether hRPE cotransplants will protect hES striatal xenotransplants in nonimmunosuppressed rats. Bilateral striatal transplants were performed in two separate groups of rats, each receiving six grafted sites, three in each hemisphere. Group 1 received hES (24,000 cells/4 μl/site) cografted with hRPE (10,000 cells/4 μl/site). Group 2 received hES alone (24,000 cells/4 μl/site). Animals in both groups were euthanized 18 days posttransplantation. Their brains were sectioned and histologically examined. Specific human nuclear protein immunostaining confirmed successful targeting of the hES and hRPE grafts within the striatum and design based unbiased stereological counts indicated nearly 100% cell survival of hES cells among group 1 animals receiving the hES + hRPE cell cotransplants. In comparison, only 19.8% graft survival was noted in group 2 animals receiving only hES xenografts. To enable easy identification of the hRPE cells we prelabeled them with green fluorescent protein and attached hRPE cells to collagen microcarriers before transplantation. Cresyl violet staining of the cotransplanted sites indicated healthy grafts with minimal increase of surrounding cellularity, suggesting a lack of host immune response. There was no evidence of teratomas at any of the graft sites or anywhere else in the brain. Our results show that hES grafts are dramatically protected from host immune rejection by cografted hRPE cells and such cografts may provide a survival advantage to transplanted hES cells on their own. This study provides additional proof of the principle of the therapeutic strategy of using hRPE cell cografts to mitigate the need for systemic immunosuppression and as a tool to provide trophic support for clinical CNS transplantation.
This study was funded in part by the NIH NINDS RO1NS42402, HRSA DIBTH0632, PA Tobacco Settlement Funds Biomedical Research Grant, PSUHMC Movement Disorders Brain Repair Fund, and NCCAM R21 AT001607.
G. M. Bernal, 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
The adult brain contains specialized and spatially restricted germinal centers where ongoing neurogenesis is supported. These neurogenic niches contain various cell components, including neural stem cells, uncommitted and lineage committed neuroblasts, 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. Vascular endothelial growth factor (VEGF) has been shown to be a potent mitogen following delivery to the hippocampal dentate gyrus and is upregulated in response to injury. However, it is not required to maintain baseline neurogenic proliferation. VEGF has also been shown to mediate the increase in hippocampal neurogenesis following antidepressant medication as well as modulating excitability in mature neurons. Understanding potential interactions of neurogenic factors will not only elucidate regulation of neurogenesis in the germinal centers, but may also provide insights necessary to recruit stem cells to contribute to repair. In the present study, we analyzed VEGF distribution and found complete colocalization with GFAP-positive cells. As reported previously, the VEGF receptor Flk1 was found almost exclusively on both double cortin-positive neuronal progenitor cells and mature dentate granule neurons. This relationship suggests that astrocytes may influence both developing and mature neurons through VEGF expression. However, we demonstrate that Flk1 is also expressed in a small population of GFAP-positive cells that also are known to express VEGF. This population of GFAP-positive cells is also Sox2 positive, indicating by this combination of expression that these cells are type 1 neural stem cells. Thus, neural stem cells are VEGF/Flk1 positive, but lose expression of VEGF with neuronal lineage progression. The coexpression of VEGF and its receptor in this neural stem cell population suggests that stem cell maintenance, as distinct from proliferation of intermediate progenitor cells, could be accomplished through autocrine/paracrine mechanisms of VEGF signaling. VEGF expression is reduced with age, suggesting that limited availability of VEGF may contribute to the age-related decline in neurogenesis.
Supported by NIH AG022555 to D.A.P.
K. B. Bjugstad*, K. Lampe†, D. S. Kern*, and M. Mahoney†
*Department of Pediatrics, University of Colorado Denver, Aurora, CO, USA
†Department of Chemical and Biological Engineering, University of Colorado, Boulder, CO, USA
Recent studies suggest that repairing the brain is dependent on specificity and timing. Neurodegenerative tissue, but not the healthy surrounding tissue, can benefit from external sources of additional neurotrophic factors for a limited time. Biodegradable polymers can be a medium for treating specific areas with neurotrophic factors for a determined period depending on site of implant, rate of factor release, and rate of degradation. The objectives of the study were to assess the biocompatibility of poly(ethylene) glycol (PEG)-based hydrogels implanted into the brains of healthy rats and to evaluate the release of growth factor from the hydrogels over a 2-month period. Thirty-eight male rats were implanted bilaterally with a 10-mm 20% w/v PEG strand engineered to degrade relatively fast (EVA), slow (KL1), or not at all (CE5N; nondegradable). Strands were laid down at a 23° angle such that they bridged the midbrain to the striatum. Rats were analyzed at 24 h, 1 week, 1 month, or 2 months postsurgery. Histological analyses were performed for evidence of astrogliosis (GFAP) and microglial response (CD68) compared to sham-implanted rats. Sham- and EVA-implanted sites had more astrocytes than the KL1 or the CE5N implant sites. Furthermore, the astrocytes in the sham and EVA groups tended to have a reactive morphology. CD68+ cells were found in greater frequency at the KL1 and CE5N sites; however, the morphology of the microglia at the sham and EVA implant sites were, again, more reactive in appearance. Because of the apparent lower immunoreactivity of the slower degrading KL1, this hydrogel was used to evaluate glial cell-derived neurotrophic factor (GDNF) and brain-derived neurotrophic growth factor (BDNF) release rates. Strands of KL1 were incorporated with GNDF or BDNF micro-particles and implanted into the brains of female rats. The implant sites were evaluated histologically for the presence of GDNF and BDNF and measured by intensity profiles of the region surrounding the implant site. Initial measures suggest that GDNF was being released from the KL1 hydrogel for 2 months after implant, with increased levels of GDNF up to 50 μm from the implant site. The current data suggest that the biocompatibility of PEG-based hydrogels is partially dependent on the rate of degradation in the brain, with faster degradation rates associated with greater immunoreactivity. It also indicates that PEG-hydrogel strands could be a viable medium for releasing growth factors into a specifically defined area.
Support: Michael J. Fox Foundation, NIH/NINDS RO1-NS052597-01 and NS40822-01A1.
V. Bobek*, K. Kolostova*, D. Pinterova*, M. Boubelik*‡, O. Raska§, R. Rokyta§, and M. Jirkovska†
*Department of Tumor Biology, Third Faculty of Medicine, Charles University Prague, Czech Republic
†Institute of Histology and Embryology, First Faculty of Medicine, Charles University Prague, Czech Republic
‡Institute of Molecular Genetics, Academy of Sciences of Czech Republic, Prague, Czech Republic
§Department of Normal, Pathological and Clinical Physiology, Charles University Prague, Czech Republic
Little is known about the differences in gene expression within the spinal cord injury lesion. Our goal was to describe the dynamics of the regeneration process following spinal cord injury (SCI) using plasmid transfer of cDNA vascular endothelial grown factor (pVEGF) and nerve growth factor (pNGF) to enhance angiogenesis within the lesion. The balloon compression technique was used to produce spinal cord injury in adult Wistar rats. Plasmid DNA (pVEGF, pNGF) was administered directly at the site of lesion immediately after the balloon compression. Rat spinal cord paraffin-embedded tissue samples with and without plasmid DNA injections were analyzed morphologically and immunohistochemically, and from the stabilized spinal cord tissue RNA was isolated. qRT-PCR was used to confirm the plasmid expression pattern along the whole thoracic part of the spinal cord on days 1, 2, 3, 7, and 21. The gene expression value of Ki-67, PECAM, FGF2, NRG1, ADAM8, SCn11A (IB4), VEGF, and NGF was evaluated within the regeneration process of the traumatized tissue. qRT-PCR data were analyzed by multimarker analysis system Genex. CatWalk system was used for behavioral testing. There were no behavioral differences shown between tested animals after VEGF and NGF plasmid administration (single dosage and combination dosage) even in the gene expression or immunohistochemical judgment. There was no significant difference between pNGF and pVEGF groups in the gene expression data tested over the proposed timeline. VEGF expression was elevated in the caudal part of the spinal cord. The opposite was found for NGF, which was expressed in the upper part of the spinal cord lesion more frequently than in a control nontraumatized tissue. A wider gene collection describing changes in neural repair process after plasmid administration will be tested soon.
H. A. Boger, L. D. Middaugh, and A.-Ch. Granholm
Department of Neurosciences and the Center on Aging, Medical University of South Carolina, Charleston, SC, USA
Glial cell line-derived neurotrophic factor (GDNF) is a potent neurotrophic factor promoting the survival and health of dopaminergic (DA) neurons in the nigrostriatal pathway during development and adulthood. Previous studies from our laboratory have shown that male GDNF+/- mice demonstrate accelerated age-related decline in motor function and the nigrostriatal DA system. However, to date no studies have been conducted to assess sex differences in terms of GDNF dependence. Therefore, the purpose of this study was to compare male and female GDNF+/- mice at 3 and 12 months of age in terms of motor activity and DAergic function. Initial studies suggest that as early as 3 months of age, female GDNF+/- mice are hyperactive compared to their age-matched wild-type (WT) controls, whereas male GDNF+/- mice do not differ from male WT mice at this age. On the contrary, 12-month-old GDNF+/- mice exhibit reduced motor activity compared to WT mice regardless of sex. However, 12-month-old female GDNF+/- mice are hyperactive compared to male GDNF+/-mice. Microdialysis studies have been conducted to assess extracellular DA in the striatum. GDNF+/- mice demonstrated higher baseline concentrations of extracellular DA at 3 months of age, regardless of sex, compared to WT mice. Female mice displayed the highest level of striatal DA release in the first 15 min following high potassium (60 mM K+) stimulation, while the male mice did not exhibit a peak DA release response until 30 min after stimulation, with male GDNF+/-mice displaying a greater time-dependent increase in DA release (30 min) compared to WT mice (15 min). Extracellular striatal DA measurements in 12-month-old male and female GDNF+/- mice are currently being studied. These data demonstrate both genotypic and sex differences in response to K+ evoked DA release, and suggest that at least at the younger age studied (3 months), male mice are more affected by the gene deletion than female mice, in terms of DA neurotransmission. The male mice exhibited a delayed DA response to K+ compared to female mice, which displayed increased response in the microdialysis paradigm used.
This work is supported by P50 DA016511 (H.A.B.) and P01 AG023630 (A.-Ch.G.).
M. Chermenina, N. Nevalainen, F. Marschinke, A. Rehnmark, E. Berglöf, and I. Strömberg
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
Glial cell line-derived neurotrophic factor (GDNF) is a potent factor for the ventral mesencephalic dopamine neurons. To study the importance of GDNF on the dopamine system during development, fetal tissue from gdnf gene-deleted mice was used. Fetal tissue from ventral mesencephalon and the striatum were dissected from wild-type, heterozygous, and knockout mice for the gdnf gene, and were thereafter grafted into the lateral ventricle of adult wild-type mice. Hence, the aims with this study were to investigate the effects of GDNF on survival of dopamine neurons and dopaminergic innervation of the cografted striatum. The transplanted tissues were evaluated by imunohistochemistry at the time points of 3, 6, and 12 months. At the longer time points of 6 and 12 months, neither the ventral mesencephalon nor the striatal grafts had survived when derived from the gdnf gene-deleted tissue. In transplants evaluated at 3 months, the number of tyrosine hydroxylase (TH)-immunoreactive neurons did not differ between the genotypes. Interestingly, the TH-positive nerve fiber innervation of the striatal cografts was found in the transplants derived from wild-type as well as in some gdnf heterozygous tissues as dense nerve fiber patches, leaving some areas TH negative. In the striatal cografts derived from gdnf gene-deleted tissue, a sparse pattern of innervation covering a larger volume was obtained. In conclusion, GDNF is important for long-term survival of grafted nigrostriatal cografts, and for formation of a proper dopaminergic innervation of the striatum in a patch/matrix pattern.
F. Cicchetti*†, S. Saporta‡#, R. A. Hauser§, M. Parent**††, M. Saint-Pierre*, P. R. Sanberg¶#, X. J. Li‡‡, J. R. Parker§§, Y. Chu¶¶, E. J. Mufson¶¶, J. H. Kordower¶¶, and T. B. Freeman¶#
*Centre de Recherche du CHUL (CHUQ), Université Laval, Québec, Canada
†Department of Medicine, Université Laval, Québec, Canada
‡Department of Pathology and Cell Biology, University of South Florida, Tampa, FL, USA
§Department of Neurology, University of South Florida, Tampa, FL, USA
¶Department of Neurosurgery, University of South Florida, Tampa, FL, USA
#Center of Excellence for Aging and Brain Repair, University of South Florida, Tampa, FL, USA
**Department of Pathology and Cell Biology, Groupe de Recherche sur le Système Nerveux Central (GRSNC), Université de Montréal, Montreal, Canada
††Faculty of Medicine, Université de Montréal, Montreal, Canada
‡‡Departments of Genetics and Neurology, Emory University, Atlanta, GA, USA
§§Department of Pathology, Louisville Health Sciences Center, Louisville, KY, USA
¶¶Department of Neurological Sciences and Center for Brain Repair, Rush University Medical Center, Chicago, IL, USA
The brains of three female patients with Huntington's disease (HD) who underwent transplantation of embryonic striatal anlagen into the striatum a decade earlier were examined histologically. Surviving grafts were identified bilaterally in two of the subjects, and displayed classic striatal projection neurons and interneurons. Grafts demonstrated more robust pathological changes than observed in either the host brains or in transplants from a similar case, previously analyzed 18 months posttransplantation. Genetic markers of HD were not expressed within the graft. Medium spiny neurons within the genetically unrelated grafts exhibited pronounced morphological changes in comparison with interneurons. Degenerating medium spiny neurons received glutamatergic cortical input. Microglia specifically targeted patches of striatal tissue within grafts. These results combined suggest that disease-specific pathology attenuates long-term graft survival, and raises uncertainty about this potential therapeutic approach for the treatment of HD. Furthermore, these findings have important implications regarding the pathogenesis of HD.
L. M. Collado, M. E. Collado, A. Hama, S. Gajavelli, and J. Sagen
Miami Project to Cure Paralysis, University of Miami, Miami, FL, USA
Some pharmacological interventions for chronic pain require continuous administration, which may be associated with undesirable side effects. The synthetic equivalent of the naturally occurring ω-conotoxin MVIIA found in the piscivorous marine snail, Conus magus, and ser-histogranin (SHG), a synthetic equivalent of naturally occurring peptide NMDA receptor antagonist found in adrenal chromaffin cells, are significantly antinociceptive when injected intrathecally into rat pain models. However, the use of ω-conotoxin is limited by severe motor side effects at higher doses, while analgesic effects of SHG diminish with increasing dose. Potentially these side effects could be reduced by delivering therapeutic molecules to a specific region in a continuous and sustained fashion. Approaches such as injection of naked genes into the CNS leading to host expression of the peptide gene product, or transplanting cells engineered to produce the peptide, could be employed. The current study evaluated the possibility of re-combinantly delivering genes encoding naturally derived antinociceptive peptides in rat pain models. Rats were intrathecally injected with naked DNA and tested in the formalin test, a screening model for tonic pain. In rats treated with DNA encoding SHG, formalin-evoked pain-related flinching behavior was attenuated. However, rats treated with DNA encoding ω-conotoxin MVIIA continued to display pain-related behavior. Thus, a cell- or vector-based delivery may be necessary to optimize delivery of the peptides. In order to accomplish this, AAV-2 viral constructs encoding peptides and monomeric red fluorescent protein (mRFP), to prospectively identify the cells were generated. The constructs were transfected into human bone marrow cells, bovine chromaffin cells, and neural precursor cells. RT-PCR revealed that the “ω-conotoxin-mRFP” and “SHG-mRFP” mRNA were synthesized as expected. Live cell imaging demonstrated red fluorescence was vesicular, suggesting that encoded proteins were in the secretory pathway. Cells modified by this method may be used as “biological minipumps” of analgesic peptides for long-term treatment of chronic pain. Furthermore, recombinant AAV vectors may also be used for direct injection into CNS pain modulatory areas.
Supported by The Ralph Wilson Medical Research Foundation Grant.
M. M. Daadi, A. Arac, Z. Li, G. Sun, J. C. Wu, and G. K. Steinberg
Department of Neurosurgery, Stanford University, Stanford, CA, USA
Ischemic brain injury in newborn infants represents a major cause of cerebral palsy, mental retardation, and epilepsy. Currently, there are no effective interventions to improve the chronic sequelae of perinatal asphyxia. Stem cell-based therapy has the potential to replace the necrotic tissue caused by hypoxia-ischemia (HI) and to restore function. Promising preclinical studies in experimental stroke models have demonstrated the efficacy of stem cells derived from various sources, including bone marrow, cord blood, and central nervous system. In most of these studies, grafted animals showed some degree of functional recovery. Although these findings are promising, little is known about how stem cell transplantation therapy achieves these outcomes. In the present study, we sought to investigate the efficacy of human neural stem cell progeny (hNSCs) derived from human embryonic stem cells (hESCs) in the rat model of neonatal HI and the effect of these cells on axonal sprouting and on the inflammatory and cellular immune responses. The hNSCs were isolated from the hESCs and perpetuated using serum-free media supplemented with epidermal growth factor, basic fibroblast growth factor, and leukemia inhibitory growth factor. Twenty-four hours after the induction of HI, animals were grafted with a single cell suspension of hNSCs (4.5 × 105) into three sites in the forebrain. The animals were evaluated 5 weeks after transplantation for their sensorimotor skills in the cylinder and in the rotarod tests. Our results showed that during the forth week after transplantation, HI transplanted animals significantly improved in their use of the contralateral impeded forelimb (p < 0.05). The hNSC grafts significantly ameliorated the locomotor deficits in the rotarod test (p < 0.05). Anterograde neuronanatomical tracing revealed significant contralesional sprouting in the stroke-damaged cortex, striatum, and thalamus. Flow cytometric analysis demonstrated that stroke induced a threefold increase in the CD3+, CD4+ T cells and no significant changes in the CD8+ and CD11b+ cells in the peripheral immune system of the vehicle group. Graft-treated animals manifested a significant decrease (p < 0.05) in the CD3+, CD4+ T lymphocytes, suggesting an anti-inflammatory effect. Post mortem histopathological analysis of grafted hNSCs identified with a human-specific nuclear marker, demonstrated good survival, dispersion, and differentiation in the stroke-damaged tissue. Interestingly, the transplanted animals showed a twofold increase in Iba1+ microglia with no apparent infiltration or reaction against the grafts. These preliminary results suggest that hNSCs transplants enhance axonal sprouting and could modulate the peripheral immune system.
H. B. Dodiya*, D. Kirik†, R. Mandel‡, J. Stansell, III*, A. Bjorklund†, and J. H. Kordower*
*Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
†Brain Repair and Imaging in Neural Systems (B.R.A.I.N.S) Unit, Lund University, Lund, Sweden
‡Department of Neuroscience, University of Florida, Gainesville, FL, USA
As a prelude to translational and clinical trials in animal models and patients with neurodegenerative diseases, especially Parkinson's disease, the present study examined the transduction of cells within the basal ganglia following injections of different adeno-associated virus (AAV) serotypes encoding for green fluorescent protein (GFP). In this experiment, six normal young adult cynamologus monkeys were employed. These monkeys received a single 10-μl injection of AAV1-GFP, AAV5-GFP, or AAV8-GFP into the caudate nucleus and putamen bilaterally in a pattern that resulted in each serotype being injected into at least four sites in the striatum. In addition, AAV1-GFP and AAV8-GFP were injected into the substantia nigra. GFP-immunohistochemistry revealed excellent transduction rates for each AAV serotype. Statistical analysis on the stereological estimates of GFP-ir cells within the striatum revealed AAV5-GFP transfect the significantly higher number of cells than AAV8-GFP (p < 0.039) and there was no significant difference between AAV5-GFP and AAV1-GFP (p = 0.348). Consistent with this result, according to Cavalieri estimates AAV5-GFP resulted in a much larger GFP-expressing area than AAV8-GFP (p < 0.05) and again there was no significant difference between AAV1-GFP and AAV5-GFP distribution area in the striatum. Each serotype transduced neurons effectively in the striatum (above 95% GFP+ cells were also positive for NeuN) while very poor gliotropism activity was observed for each serotype (below 4% GFP+ cells were also positive for GFAP). Within the substantia nigra, AAV1-GFP injections resulted in 83,402 ± 17,809 GFP-immunoreactive cells and AAV8-GFP injections resulted in 68,826 ± 17,501 GFP-immunoreactive cells, numbers that did not differ from each other (p > 0.05). GFP-positive fibers were observed in GPe and GPi, which confirms the ability of anterograde transportation for these serotypes. The current data suggest that AAV5 and AAV1 are superior to AAV8 for gene delivery to the nonhuman primate striatum.
E. M. Emborg*†, J. Johnson‡, C. Swanson*, M. Dobbert*, V. Joers*, K. Brunner*, J. Kemnitz*, and T. Ziegler*
*Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison WI, USA
†Department of Medical Physics, University of Wisconsin-Madison, Madison WI, USA
‡School of Pharmacy, University of Wisconsin-Madison, Madison WI, USA
Pioglitazone, a thiazoledionedione currently FDA approved as an antidiabetic treatment, has been proposed as a neuroprotective therapy for neurological disorders. We have previously demonstrated in nonhuman primates that daily oral pioglitazone dosing protects against the functional and anatomical effects of the parkinsogenic toxin MPTP. In this study we continue our clinical translation analysis of this compound by analyzing the ability of different doses of pioglitazone to penetrate the CSF. Five adult female rhesus monkeys (5–6 kg) received once daily oral administration of placebo, 2.5, 5.0, or 7.5 mg/kg of pioglitazone for 7 days. There was a lapse of 1 week between a change in dose and plasma and CSF sampling to allow for steady state. Plasma and CSF samples were obtained at each baseline and 5 h after the last dose of pioglitazone. Levels of pioglitazone were evaluated by HPLC methods. In plasma, comparison between pioglitazone levels showed, as expected, that administration of 5 mg/kg induced higher levels than 2.5 mg/kg. Yet, in four of the five monkeys 7.5 mg/kg dosing did not result in consistently higher increases of pioglitazone plasma levels compared to 5 mg/kg. These data suggest increased drug elimination with 7.5 mg/kg. CSF levels of pioglitazone compared to plasma showed more individual variability. A dose of 2.5 mg/kg induced nondetectable levels in two of the five animals. Both 5 and 7.5 mg/kg had consistent measurable levels in CSF. Four of the five monkeys had higher CSF levels of pioglitazone after receiving 5 mg/kg compared to 7.5 mg/kg. The results confirmed the ability of pioglitazone to penetrate CSF and suggest that in rhesus monkeys 5 mg/kg was an efficient dose to consistently induce detectable higher levels of pioglitazone in plasma and CSF.
This study was supported by a grant from The Michael J. Fox Foundation, and performed at the Wisconsin National Primate Research Center (NIH-NCRR base grant RR000167).
D. J. Eve*, J. Musso, III*, D.-H. Park*†, V. Bui*, A. Smith*, D. Cameron*, C. Oliveira‡, K. Pollock‡, A. Hope‡, M.-O. Baradez‡, J. D. Sinden‡, and P. R. Sanberg*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery, Korea University Medical Center, Korea University College of Medicine, Seoul, Korea
‡ReNeuron Ltd., Guildford, UK
There is increasing evidence that stem cells may exert their benefit via release of trophic factors that can modulate the surroundings to make them more “friendly” to survival, rather than via cell replacement. We have previously shown that the conditionally immortalized neural stem cell line CTX0E03 secretes vascular endothelial growth factor (VEGF) into its surrounding media. Further measurements of specific factors by Enyzme-Linked Immunosorbent Assay (ELISA) demonstrates related changes in factor release with a positive correlation between VEGF, endostatin, and tissue inhibitor of metalloproteinases (TIMP-1) concentrations. VEGF, endostatin, and TIMP-1 have been linked to cell proliferation and angiogenesis. We have used CTX0E03 conditioned media in an in vitro angiogenesis model, which demonstrates growth of vessel-like projections from an aortic ring dependent on the media conditions. Our preliminary studies suggest that there is an optimal concentration of VEGF with respect to the average distance of a projection from the aortic ring. The optimal concentration for distance would appear to be different from the optimal concentration that determines the thickness of the projections, because only the combined diluted and antibody media significantly reduced vessel thickness. Further characterization of this response with the use of antibodies for VEGF and endostatin are ongoing. These studies provide in vitro evidence that transplantation of the CTX0E03 cells is likely to influence both angiogenesis and neurogenesis as suggested by other studies.
This study was funded by ReNeuron Ltd, Guildford, UK, and P.R.S. is on the Scientific Advisory Board of ReNeuron.
T. Federici*, B. Raore*, J. Taub*, K. Johe†, and N. Boulis*
*Emory University, Atlanta, GA, USA
†Neuralstem, Inc., Rockville, MD, USA
Amyotrophic lateral sclerosis (ALS) leads invariably to death in 2–5 years from the progressive weakness, which results from the loss of motor neurons. In particular, the loss of upper cervical spinal cord and brain stem lower motor neurons lead to the loss of diaphragm function and control of the upper airway, triggering aspiration and respiratory compromise. Preserving the survival of motor neurons in this area of the spinal cord might prolong the survival of ALS patients. A stem cell line, NSI-566RSC (Neuralstem, Inc.), derived from human fetal spinal cord, has previously been shown to prolong survival in rodent models of ALS and ischemic spastic paraplegia. The objective of this study was to assess potential toxicity, adverse effects, or morbidity arising from, or associated with, the transplantation of NSI-566RSC cells in a large animal model. We have developed a device and technique for safe and accurate injection of stem cell grafts into the human spinal cord. The size and morphological similarity of the swine and human spines renders the pig optimal for safety and feasibility studies of grafting approaches in the spinal cord. A total of 15 female Gottingen minipigs, divided into three experimental groups, underwent multiple unilateral stereotaxic injections of NSI-566RSC or vehicle into the C3–C6 segments of the spinal cord. All animals received intravenous Tacrolimus (0.025 mg/kg) administration twice daily during the course of the study. Animals were observed weekly for 28 days for signs of motor and sensory dysfunction as well as general morbidity. Sensory function was assessed by the presence of the withdrawal response to a mechanical stimulus. Motor function was evaluated following the Tarlov's score. Blood was collected on days 0 and 28 for hematology and serum chemistry. Finally, full necropsy was performed and spinal cords analyzed for graft survival. This study was performed under Good Laboratory Practice (GLP) conditions. There was no mortality or surgical complications associated with the study. There was no obvious effect of the procedure on blood chemistry parameters. All animals experienced transient motor dysfunction limited to the hind limbs, but returned to normal function by day 14. Finally, graft survival was demonstrated by the presence of human nuclear antigen (HuNu)-positive cells (stem cell grafts) primarily around or near needle tracks. Additional histological analyses are undergoing. We were able to establish toxic thresholds for cell density and number of injections using NSI-566RSC cells and surgical techniques and tools specifically developed for this purpose. Overall, multiple unilateral injections of NSI-566RSC into the spinal cord proved to be feasible and safe. The present data enabled an Investigational New Drug (IND) application to the Food and Drug Administration (FDA) for a trial of NSI-566RSC spinal cord transplant therapy.
A. M. Fortress*, K. L. Helke‡, and L. Granholm*†
*Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA
†Center on Aging, Medical University of South Carolina, Charleston, SC, USA
‡Department of Comparative Medicine, Medical University of South Carolina, Charleston, SC, USA
Learning and memory impairments occurring with normal aging and Alzheimer's disease are associated with degeneration of the basal forebrain cholinergic neurons (BFCNs). BFCNs extend their axons to the hippocampus (HPC) and are dependent on nerve growth factor (NGF) for survival and maintenance. Cholinergic terminals in the hippocampus bind NGF at its high-affinity receptor, trkA, and the trkA–NGF complex is transported to the BFCN cell bodies via classical retrograde transport mechanisms. Absence 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. The precursor to NGF, pro-NGF, is capable of binding to the low-affinity NGF receptor, p75; and this binding has been demonstrated to induce cell death in vitro. Previous work in our laboratory has shown that systemic administration of NGF increases the expression of the high-affinity NGF receptor, trkA, in the BF and enhances memory performance. Currently, the working hypothesis is that disrupted retrograde signaling of NGF with aging [i.e., diminished phospharylated extracellular signal-regulated kinase (pERK)] results in reduced trkA activation and elevated p75 response, resulting in a propelling degeneration of the cholinergic neurons. To test this hypothesis, 24-month-old rats were given bilateral stereotaxic intrahippocampal injections of pro-NGF (10 μg combined over two sites) and were then sacrificed 24 h or 1 week postinjection. Twenty-four hours after pro-NGF injection, immunohistochemical assessment of basal forebrain sections revealed: 1) pERK-ir was reduced, 2) p75 was elevated, and 3) p75 and sortilin were colocalized. One week following the pro-NGF injection, Western blots revealed a twofold increase in p75, sortilin, and pro-NGF. Combined, these data represent the first evidence of the effects of pro-NGF in vivo and provide new insight in the field of aging and neurodegeneration. We propose that pro-NGF-induced upregulation of p75 and sortilin are correlated with memory deficits following the pro-NGF administration, and that this alternative receptor activation may contribute to the BFCN degeneration that has been demonstrated in the aged rodent brain.
Supported by AG10755.
L. Freemansp*, and A.-Ch. 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,” containing high levels of saturated fat, hydrogenated fat (“trans fats”), and cholesterol is detrimental to many aspects of health. However, the effects of a Western diet on the brain are currently not understood. We have previously shown this type of diet to be detrimental to performance on a spatial memory task. To further these studies, we have investigated the effects of the diet on the hippocampus in isolation. Therefore, we grafted hippocampal tissue from embryonic day 18 rats to the anterior eye chamber of 16-month-old host animals that were fed either a control, normal rat chow diet or a 10% hydrogenated coconut oil + 2% cholesterol diet (HF/HC) for 6 weeks. Additionally, we tested the role of IL-1 in the diet-induced inflammatory effects. One eye per rat received an IL-1 inhibitor (IL-1Ra, Kineret) and the other served as a saline control. The HF/HC diet lead to a marked reduction in hippocampal transplant growth, and this effect was blocked by the IL-1 inhibitor. Graft morphology was evaluated with hematoxylineosin staining and preliminary results showed differences in cellular organization between treatments. The HF/HC diet appeared to be detrimental for organotypic development of the hippocampal grafts compared to the control diet, but IL-1Ra attenuated this organizational effect. Grafts were evaluated with immunofluorescence to measure activated microglia, neurogenesis, and vascularization. Preliminary results suggested that the grafts in hosts receiving the HF/HC diet exhibited an altered morphology compared to controls. This will impact treatment strategies for degenerative disorders of aging, because dietary intake has been found to play a role in their onset and progression. The use of IL-1Ra also provides a potential therapeutic for aging disorders and graft survival in elderly hosts.
Supported by AG04418.
S. Garbuzova-Davis*†‡§, Y. Xie*, O. Zayko*, 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, 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, College of Medicine, Tampa, FL, USA
Sanfilippo syndrome type B is an inherited disorder caused by a deficiency of the alpha-N-acetylglucosaminidase (Naglu) enzyme in the degradative pathway of heparan sulfate (HS). Our previous studies showed that a single administration of mononuclear human umbilical cord blood (MNC hUCB) cells into the cerebral ventricle of presymptomatic Naglu mice or intravenous cell delivery at different stages (early symptomatic or late stage) of the disease had a beneficial effect, probably due to enzyme delivery into the enzyme-deficient mutant mice. Transplanted mutant mice showed cognitive improvement and decreased disease-related hyperactivity even when cells were administered in the late stages of the disease. After intracerebroventricular or intravenous administrations of MNC hUCB cells, the cells were found widely distributed within and outside the CNS. HS accumulation was significantly reduced in the liver and spleen of Naglu treated-mice. However, most of the observed behavioral benefits in Naglu mice were limited to the first few months after cell transplantation, possibly due to declining production of the missing enzyme over time. In consideration of this point, our preclinical translational study was designed to determine the effect of multiple intravenous transplantations of MNC hUCB cells into early symptomatic Naglu mice during a period of 6 months. Results showed significant behavioral improvements in mutant mice of both sexes with repeated cell transplants. Additionally, neuronal architecture in the hippocampus was improved in Naglu mice. Immunohistochemical analysis showed more migrating cells in the brains of multiple-cell-treated mutant mice than in animals receiving a single cell injection. The majority of cells were found in the cerebellum, medulla, cerebral cortex, thalamus, and olfactory bulb of multiple-cell-treated mutant mice and some cells expressed nestin. Administered cells were also widely distributed in the abdominal organs and peripheral blood. Another advantage of repeated cell injections was significantly reduced HS accumulation in the livers of Naglu mice versus single-cell-treated or nontreated mutants. Thus, multiple intravenous administrations of MNC hUCB cells into Naglu mice have a prolonged beneficial effect compared to a single administration, most likely due to continuous enzyme delivery into the enzyme-deficient mutant mice. However, despite promising results, determining the ideal hUCB cell subpopulation for transplantation remains a critical point in developing a cell transplant strategy for Sanfilippo type B. Our recent studies using administration of volume-reduced nucleated hUCB cells and monocytes/macrophage-derived hUCB cells showed potential benefit. Advantages and disadvantages of these cell administration treatments will be discussed. Overall, results of our preclinical translational studies demonstrate the potential of hUCB cells in the treatment of Sanfilippo Syndrome type B. This treatment might be also suitable for genetic disorders such as glutaric acidemia, Friedreich's ataxia, and others.
Supported by the Children's Medical Research Foundation, Lauren's Hope Foundation, and the International Organization Glutaric Acidemia. S.G.D. is a consultant and P.R.S. is a cofounder of Saneron CCEL Therapeutics, Inc.
M. Ghosh*, L. M. Tuesta*, R. Puentes*, B. Monaco*, A. El Maarouf‡, U. Rutishauser‡, and D. D. Pearse*†
*The Miami Project to Cure Paralysis, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
†Department of Neurological Surgery, University of Miami Leonard M. Miller School of Medicine, Miami, FL, USA
‡Department of Cell Biology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Schwann cell (SC) implantation following spinal cord injury (SCI) has been demonstrated to support axonal growth and remyelination as well as improve locomotor recovery and therefore has significant clinical potential as a cell-based repair strategy. The efficacy of SC implants, however, but may be limited due to their inability to migrate outwards from the lesion site and thus associate with and guide axons into and from the injury site as well as reach distant regions of demyelination. Here, we investigated whether the genetic expression of polysialyl transferase (PST) to produce cell surface polysialic acid (PSA) on neural cell adhesion molecule (NCAM) to reduce cell–cell interactions could promote their migration across the inhibitory glial scar after SCI and increase their ability to support corticospinal axon growth, remyelination repair, and functional recovery. Adult rats with a moderate T8 spinal cord contusion received lentiviral vector-transduced green fluorescent protein (GFP) or PST-GFP SCs at 1 week postinjury by injection directly into the injury epicenter suspended in either medium or Matrigel, which supports improved cell survival. Immunofluorescence staining was performed on spinal cord tissue at 10 weeks postimplantation to examine cell survival, migration, axonal growth, and remyelination. The results revealed significant rostrocaudal migration of the PST-SCs (in media) between 2 and 5 mm from the implantation/lesion site compared to the control GFP SCs, which remained confined to the injury site. BDA-anterograde tracing from the motor cortex showed extensive corticospinal axon growth within and surrounding the implantation site only with PSA-modified SCs. Behavioral analysis of PST-GFP-SC implanted animals revealed significant improvement in open-field locomotor performance and hind paw placement on the grid walk test compared to GFP-SC controls. In contrast, when PSA-expressing SCs were implanted in Matrigel, they exhibited migration that was comparative to GFP controls and were unable to improve functional outcome. These results suggest that chemoattractant cues, such as growth factors within the Matrigel, may override any positive migratory cues for these PSA-modified SCs within the host spinal cord. In addition, migration of PSA-SCs appears to be critical for the ability of the cells to improve behavioral recovery.
Funding: NYS Department of Health, the Craig H. Neilsen and Fa Bene Foundations.
R. Golshani*, S. Savant-Bhonsale‡, L. M. Tuesta*, J. Louro*, T. Wilson*, S. Patel*, and D. D. Pearse†
*Miami Project to Cure Paralysis, University of Miami Miller School of Medicine, Miami, FL, USA
†Department of Neurological Surgery, University of Miami Miller School of Medicine, Miami, FL, USA
‡Theradigm, Inc., Baltimore, MD, USA
Within the United States over 200,000 individuals live with traumatic spinal cord injury (SCI); 11,000 new injuries occur each year. The implantation of cells to either replace or bridge lost tissue at the injury site to provide anatomical and functional restoration has been to date a promising reparative strategy. The neural progenitor cell (NPC) is among the most recent of cell therapies to be used for SCI. This multipotent cell type is capable of differentiating into both neurons and glia, potentially providing both neuroreplacement and neurorepair. In the current investigation we examined the survival, migration, differentiation, and axon growth support of human fetal spinal cord-derived NPC survival when implanted 1 week following a clinically relevant cervical (C5) contusion in adult athymic rats. In these studies, we examined whether approaches aimed at improving NPC survival—(1) administration of the immunosuppressant FK506, (2) implantation of NPCs within healthy tissue adjacent to rather than within the injury epicenter, or (3) conditioning the cells to a low O2 environment in culture—could affect these anatomical outcomes compared to epicenter injections of NPCs alone. Animals were either treated with or without the immunosuppressant FK506 and received either (i) direct injection of 400,000 NPCs into the injury epicenter or (ii) injections of 100,000 cells in each of four locations 4 mm rostral and caudal to the injury epicenter, bilaterally. NPCs were either cultured in low or normal O2 conditions prior to implantation. Four weeks postgrafting, injured spinal cord tissue was obtained and stained for human-specific nuclear antigen-Numa to characterize NPC survival and their migration from the original site of deposition. Implantation of NPCs either into or adjacent to the injury site led to good survival/proliferation and rostral–caudal migration from the implant site (up to 4 mm respectively). The concurrent administration of FK506 with NPC implants provided a significant approximately twofold enhancement in NPC survival/proliferation (p < 0.01) as well as extended their distance of migration (p < 0.05). To examine in vivo differentiation of NPCs into specific neural lineages, we employed confocal microscopic analysis of costained NPCs for Numa with either CC1 (oligodendrocytes) or GFAP (astrocytes). We identified colocalization of numerous Numalabeled NPCs with both CC1 and GFAP, indicating that the implanted NPCs were able to differentiate into both oligodendrocytes and astrocytes. Ongoing work will investigate the neuronal and glial differentiation potential of low oxygen NPCs. In conclusion, the therapeutic efficacy of NPC implants depends upon their ability to survive, migrate, differentiate, and integrate into new or existing neural networks to provide anatomical and functional restoration. In these studies we have shown NPCs with the administration of FK506 survive well within the hostile milieu of the injured spinal cord, exhibit extensive rostral–caudal migration (properties that are enhanced by FK506), and are able to differentiate into different glial cell lineages. Future studies will determine the potential of implanted NPCs to give rise to neurons and their ability to integrate and restore function to the injured host system.
Work supported by: Theradigm Inc., The Miami Project to Cure Paralysis, and The Buoniconti Fund.
S. E. Gombash*‡, A. Cole-Strauss†, J. W. Lipton†, T. J. Collier*, K. Steece-Collier*, B. T. Terpstra*‡, A. L. Spieles-Engemann*‡, B. F. Daley*, S. L. Wohlgenant*, V. B. Thompson†‡, R. J. Mandel§, F. Manfredsson§, 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 Neuroscience, University of Florida, Gainesville, FL, USA
Neurotrophic factors are integrally involved in the development of the nigrostriatal system and therefore possess therapeutic potential for Parkinson's disease (PD). Our laboratory and others have found 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. In previous studies we have demonstrated that striatal overexpression of PTN can protect the nigrostriatal system of rats from 6-hydroxydopamine (6-OHDA). PTN appears to function in the nigrostriatal system in a manner similar to glial cell line-derived neurotrophic factor (GDNF) (i.e., expression peaks during nigrostriatal development and is downregulated in adulthood). It has been reported that overexpression of GDNF in the striatum can be detrimental, causing decreases in dopamine (DA) production, downregulation of tyrosine hydroxylase immunoreactivity, and aberrant sprouting in downstream targets. It is possible that supraphysiological levels of GDNF participated in these detrimental side effects. The present series of experiments were conducted to determine, for the first time, the level of peak striatal PTN expression during rat development in order to provide a reference point for future PTN gene transfer studies. We used Western blot analysis to examine the time course of PTN expression in the rat striatum at multiple pre- and postnatal time points (E15, E18, E20, P1, P2, P14, P17, and P35). Striatal tissue was collected from embryonic (saline rinsed) and postnatal (saline perfused) Sprague-Dawley rats from multiple litters and separated by gender when possible. Initial experiments examined PTN expression between gender-identified samples of identical ages, followed by examination of PTN expression across developmental ages. Western blot results revealed no gender differences in PTN levels at P1, P2, P14, and P35. Peak protein expression occurred during P1–P2 with mean peak PTN expression estimated by digital densitometry (n = 4) to be 1171.46 ± 312.14 ng/mg protein. Retrospective comparisons to striatal PTN levels produced in our previous experiment utilizing recombinant adeno-associated virus 2 serotype 1 (rAAV2/1)-mediated gene transfer revealed that neuroprotection was observed when striatal PTN levels were approximately double that of peak developmental levels at P1–P2. Ongoing experiments using rAAV2/1 PTN will examine if a critical threshold of PTN expression is required to provide neuroprotection from 6-OHDA. Results from these studies will provide the optimal transduction parameters for effective PTN gene transfer as a therapeutic strategy for PD.
Supported by NS058682 and the University of Cincinnati Neuroscience Graduate Program.
N. Gorenkova*, C. Franco†, Z. Hassani*, J. Price*, J. West†, and M. Modo*
*Centre for Cellular Basis of Behaviour, King's College London, UK
†Department of Bioengineering, Rice University, Houston, TX, USA
Stroke causes extensive cellular loss that leads to a disintegration of the afflicted brain tissue. Although transplanted neural stem cells can recover some of the function lost after stroke, recovery is incomplete and restoration of lost tissue is minimal. Restoration and regeneration of tissue in the stroke cavity is likely to require not only neural stem cells (NSCs), but also endothelial cells and a structural support in the early phase after implantation. We demonstrated here that NSCs and endothelial cells can be incorporated into PEG-based hydrogel beads. Under MRI guidance, these beads were injected into the stroke cavity. After 7 days, only remnants of the hydrogel beads were present with transplanted bromodeoxyuridine (BrdU)-labeled cells having migrated out of these support structures. Transplanted endothelial and neural stem cells can be found within the lesion cavity. Some integration with host tissue can be observed as well as some primitive organization of the endothelial cells into tubular structures. This tissue engineering approach could therefore be a novel way to enhance brain repair that provides a substantial step forward in the technology development required for clinical translation.
A.-Ch. Granholm*‡, A. Fortress*, J. Lockrow*, A. Moore*, B. Williams*, K. Helke†, and M. Buhusil*‡
*Department of Neurosciences, Medical University of South Carolina, Charleston, SC, USA
†Department of Laboratory Animal Research, Medical University of South Carolina, Charleston, SC, USA
‡Center on Aging, Medical University of South Carolina, Charleston, SC, USA
Age-related memory loss is a common clinical complaint that will increase exponentially with the aging of the baby boomer generation. The most common form of dementia is Alzheimer's disease, and patients with this devastating neurological disorder exhibit significant loss of basal forebrain cholinergic neurons, which correlates closely with cognitive impairment. Recent studies suggest that age-related neurodegenerative diseases may, at least in part, be caused by altered growth factor production, release, or uptake. Our previous studies have shown that nerve growth factor (NGF) levels are elevated in the hippocampus of aged rats but reduced in the basal forebrain, where cholinergic neurons are degenerating as a result of this imbalance, similar to what has been found in the Alzheimer patient. We then proceeded to investigate whether this imbalance was due to reduced release of NGF, or transport to the basal forebrain via cholinergic axons. Interestingly, we found that release mechanisms were not altered, but the response of high-affinity NGF TrkA receptors to the released NGF appeared to be hampered by the aging process. In addition, it was found that the ability to respond to NGF injected into the hippocampus with TrkA and extracellular signal-regulated kinase (ERK) phosphorylation in cholinergic neurons directly correlated with performance in a spatial memory task. Our work now continues to explore whether cleavage of pro-NGF into mature NGF is altered with aging in rats, as has been demonstrated in patients with Alzheimer's disease. Initial work suggests that this is the case, and that the low-affinity p75 receptors and their coreceptor sortilin are also altered with aging. The data suggest that the aging process results in an abnormal response to exogenous NGF, as well as abnormal cleavage of endogenous pro-NGF into the mature form; and thus, targeting the NGF cleavage mechanisms may represent a possible treatment avenue for cognitive impairment with aging.
This work was supported by a grant from the National Institutes on Aging (AG10755).
P. J. Hallett*, A. Vinuela*, P. Licznerski*, T. D. Sotnikova†, M. G. Caron†, R. R. Gainetdinov†, and O. Isacson*
*Udall Parkinson Disease Research Center of Excellence, Center for Neuroregeneration Research, McLean Hospital/Harvard Medical School, Belmont, MA, USA
†Department of Cell Biology, Duke University Medical Center, Durham, NC, USA
The replacement of functional living synapses and nerve terminals by new neurons is a unique feature of neural transplantation to Parkinson's disease (PD) models. We have investigated how functional and connective differences between types of dopaminergic (DA) donor neurons can influence transplantation outcomes. We transplanted fetal DA neurons from mice lacking the DA transporter (DATKO) or from wild-type (WT) mice, into a rat model of PD with L-dopa induced dyskinesia (LID), to address how excessive and unregulated DA stimulation influences dyskinesias. Grafts containing WT DA neurons improved parkinsonian signs to a similar extent to DATKO grafts, but provided a more complete reduction of LID. Interestingly, OFF-L-dopa-induced dyskinesias were not observed with either DATKO or WT grafts. Both WT and DATKO grafts reinnervated the host striatum to a similar extent, but DATKO grafts produced a greater (threefold) and more diffuse increase in extracellular striatal DA levels as determined by microdialysis. DATKO grafts induced a more complete normalization of striatal DA receptor supersensitivity than WT grafts, as assessed by behavioral and receptor autoradiography analyses, and both graft types induced a similar downregulation of proenkephalin (PENK) and fosb/Δfosb in striatal neurons. In summary, DATKO grafts causing high and diffuse extracellular DA levels provide functional recovery even in the presence of abnormally high extracellular DA levels. WT DA neurons appear the most effective neuronal type to restore function and reduce LID. These findings suggest that it is the presence of DA terminals, and not the DA levels per se, that is important for restoration of function following transplantation in PD. As a continuation of these studies we are now examining factors that modify the host striatal environment (overexpression of α-synuclein and neuroinflammation) that hypothetically can influence the morphology of synaptic connections formed between graft–host, and subsequently influence graft function and behavioral recovery.
B. K. Harvey*, J. Chou*, H. Shen*, Y. H. Chiang†, B. J. Hoffer*, and Y. Wang*
*National Institute on Drug Abuse, Baltimore, MD, USA
†Taipei Medical University, Taipei, Taiwan
Diadenosine tetraphosphate (AP4A), two adenosine moieties bridged by four phosphates, is an endogenous purinergic ligand found in the brain. Previous studies have shown that AP4A reduced neurodegeneration caused by the dopaminergic neurotoxin 6-hydroxydopamine in rat striatum and substantia nigra. The purpose of this study was to determine whether AP4A is protective against methamphetamine (MA)-mediated toxicity. Primary neuronal cultures were prepared from rat embryonic (E15) ventral mesencephalic tissue. Cultures treated with 2 mM MA exhibited decreased tyrosine hydroxylase (TH) immunoreactivity, increased cleaved caspase-3 immunoreactivity, and TUNEL labeling. All these changes were lessened by pretreatment with AP4A. The protective effect of AP4A was also found in vivo. Adult Sprague-Dawley rats were injected with AP4A (25 μg/20 μl) or vehicle intracerebroventricularly followed by four doses of MA (5 or 10 mg/kg), given subcutaneously every 2 h. Administration of MA reduced locomotor activity 1 day after injection, which was significantly antagonized by pretreatment with AP4A. Using immunohistochemical analysis, TH fiber density in substantia nigra pars reticulata was found reduced while cleaved caspase-3 immunoreactivity in striatum was increased after MA treatment; these responses were also significantly antagonized by AP4A. Taken together, our data show that AP4A has protective effects against MA-mediated toxicity both in vitro and in vivo. The mechanism of action involves suppression of MA-induced apoptosis.
Supported by the National Institute on Drug Abuse, Intramural Research Program, NIH and Taipei Medical University, Taiwan.
T. Hayashi*, Y. Kaneko*, S. J. Yu*, O. Parolini†, and C. V. Borlongan*
*Department of Neurosurgery, Center for Aging and Brain Repair, University of South Florida, Tampa, FL, USA
†Centro di Ricerca E. Menni, Fondazione Poliambulanza Istituto Ospedaliero, Brescia, Italy
Recent studies have demonstrated that the human placenta is a good source of stem cells. We have provided laboratory evidence that transplantation of these human placenta-derived cells in an in vivo stroke model promotes functional recovery. However, the mechanisms underlying these observed therapeutic benefits of human placenta-derived cells remain poorly understood. Here, we examined the expression of two discrete types of melatonin receptors and their role in proliferation and differentiation of cultured human amniotic epithelial cell (AECs). Human AECs were obtained from the amnion, which was provided by Dr. Parolini under approved institutional guidelines. Immunocytochemical studies were performed to reveal: (1) melatonin receptor expression in cultured AECs, and (2) proliferation and differentiation of cultured AECs with or without melatonin supplementation in the growth media. Furthermore, melatonin dose-dependently suppressed proliferation, but enhanced neural differentiation (TuJ1 and GFAP) of melatonin receptor 1-expressing AECs. These results suggest a novel role for melatonin in modulating neural differentiation of human placenta-derived AECs as donor cells for transplantation in neurological disorders. That melatonin receptor 1 rather than melatonin receptor 2 was detected in AECs implicates melatonin receptor 1 as principally mediating these physiological effects of melatonin.
D. C. Hess*, R. J. Deans†, and R. W. Mays†
*Medical College of Georgia, Augusta, GA, USA
†Athersys, Inc., Cleveland, OH, USA
Stroke is the third leading cause of death and the leading cause of disability in the US. In 2008, approximately 780,000 Americans suffered a stroke with total associated costs estimated at $65 billion. The mean lifetime cost following ischemic stroke of a single patient in the US is projected as ~$150,000 including inpatient treatment, rehabilitation, and follow-up care necessary for lasting defects. Current therapy for stroke is severely limited; other than one protein therapy, tissue plasminogen activator (tPA), no approved specific treatment is available. Only ~5% of Americans suffering ischemic stroke who would benefit actually receive tPA due to delayed recognition of the symptoms coupled with the limited window for receiving treatment. This means ~610,000 Americans suffered an ischemic stroke in 2008 that could have benefited from an effective alternative cellular therapy. The number of affected individuals, the costs necessary to facilitate care and rehabilitation, and the lack of current therapies reiterate that stroke represents a current significant unmet medical need. Athersys has developed MultiStem®, a proprietary adult adherent stem cell clinical product, under strict specifications and release criteria approved by the FDA. Athersys has established the efficacy of MultiStem® in multiple preclinical animal disease models including ischemic stroke, hypoxic-ischemic (HI) injury, acute myocardial infarct (AMI), and graft versus host disease (GVHD). Subsequently, Athersys has undertaken safety profiling of MultiStem® in multiple preclinical animal studies, including GLP tumorigenicity studies in NOD-SCID and nude mice, GLP safety studies in stroke-injured rats, and long-term safety (>1 year) studies in stroke-injured and HI-injured rats. As a result, the FDA has approved the manufacturing and basic safety profile of MultiStem® for use in humans in the treatment of ischemic stroke, AMI, and GVHD. Athersys has successfully initiated patient enrollment and treatment using MultiStem® in AMI and GVHD patients. Our phase I clinical stroke design is a randomized, double-blind, placebo controlled, multicenter, dose escalation study. The primary objective is to determine the maximum tolerated dose of MultiStem® from administration of a single intravenous dose. Secondary endpoints include determining the long-term safety of the cells, gathering preliminary data on functional outcomes including exploratory MRI to correlate outcome with imaging in MultiStem®-treated patients. This study represents an initial step in developing a new class of therapy for treatment of ischemic stroke.
A. Jain, H. Henao, R. Puentes, D. Bleicher, D. Maggio, and D. D. Pearse
The Miami Project to Cure Paralysis, The Department of Neurological Surgery, The University of Miami Miller School of Medicine, Miami, FL, USA
Physical injury to the spinal cord often results in permanent functional loss. Following spinal cord injury (SCI), an ensuing cascade of molecular and cellular events occurs to create an inhibitory environment for the endogenous regeneration of axons, and thus severed connections and locomotor function are not restored. Cellular transplantation has been demonstrated to be a promising therapeutic strategy to promote axon regeneration and functionality. Schwann cells, which have been of interest due to their regenerative roles in the peripheral nervous system, supporting axon growth through the secretion of extracellular matrix molecules and growth factors, as well as the capacity for remyelination, and repair severed axons, have significant preclinical research associated with them. However, concerns and questions have been raised regarding whether this therapy may enhance sensory plasticity and thus contribute to and enhance neuropathic pain, an already major problem associated with SCI that is largely refractory to treatment. This study sought to identify whether Schwann cell transplantation would enhance neuropathic pain beyond that observed following SCI. For this study, the Multicenter Animal Spinal Cord Injury Study (MASCIS) Impactor was used to produce a thoracic (T8) contusion (25.0 mm weight) in female Fisher rats. Two million Schwann cells were transplanted at the injury epicenter at 8 weeks post-SCI. Prior to injury and every week for 18 weeks after injury, thermal hyperalgesia and cutaneous allodynia upon the hindpaws and back (above, at, and below the injury level of the animals) were performed for three different experimental conditions: sham injury, injury only, and injury with Schwann cell transplantation. Biochemical and histochemical analyses were performed to determine production of inhibitory neurotransmitters and sprouting of serotonergic fibers. We found that there were no significant changes in thermal hyperalgesia and cutanous allodynia responses between injury only and injury with Schwann cell transplant groups in the chronically injured spinal cords. This suggests that as a clinical therapeutic treatment, the transplantation of Schwann cells, although altering sensory plasticity at the anatomical level, does not enhance behavioral hypersensitivity after chronic, contusive SCI.
P. Jensen*†, M. Meyer*, A. D. Ducray†, and H. R. Widmer†
*Department of Anatomy and Neurobiology, University of Southern Denmark, Odense, Denmark
†Department of Neurosurgery, University of Bern, Bern, Switzerland
Parkinson's disease (PD) is a neurodegenerative disorder mainly characterized by a progressive loss of dopaminergic neurons in the substantia nigra. Cell replacement therapy in PD is still at an experimental stage and the limited availability of suitable donor tissue presents a major complication for a successful clinical application of this approach. Effective numerical expansion of fetal dopaminergic precursor cells hence might offer a strategy to minimize this problem. In this context, the role of oxygen tension may be of critical importance. While physiological oxygen tension in the CNS is in the range of 1–5%, traditionally cell culturing is performed at high nonphysiological oxygen tension. In line with this notion, positive effects on both neural precursor cell proliferation and dopaminergic differentiation by culturing cells at a more physiological oxygen tension have been reported. In the present study, we investigated the influence of fibroblast growth factor 2 (FGF2) and FGF8 on expansion of precursor cells cultured at high (20%) and low (3%) oxygen tension. For that purpose tissue from embryonic day 12 rat ventral mesencephalon was mechanically dissociated and cultured for 4 days in serum-free medium in the absence (controls) or the presence of FGF2 or FGF8. After mitogen withdrawal and addition of serum, cells were differentiated for 6 days. We first observed that significantly more cells incorporated bromodeoxyuridine (BrdU) in cultures exposed to FGF2 (51-fold) and FGF8 (13-fold) than in control cultures. Notably, expansion at low oxygen tension significantly increased the number of BrdU-expressing cells in both FGF2 (1.7-fold) and FGF8 (3.0-fold) exposed cultures compared to high oxygen tension. After differentiation the number of beta-III-tubulin expressing cells was significantly higher in low as compared to high oxygen tension cultures. Importantly, the number of tyrosine hydroxylase-immunoreactive (TH-ir) cells was significantly higher in FGF2-expanded and FGF8-expanded cultures compared to controls. Low oxygen tension in combination with FGF2 expansion resulted in the most pronounced increase in TH-ir cell numbers, which was significantly higher compared to high oxygen tension, while no such effect was detected for HPLC measured dopamine levels released into the culture medium. However, switching FGF2-expanded cultures from low to high oxygen tension during the last 2 days of differentiation resulted in both a significantly higher number of TH-ir cells and release of dopamine as compared to all other treatment groups. In contrast, such effects were not observed for FGF8-expanded cultures. Taken together, the combination of FGF2 treatment at low oxygen and exposed to high oxygen tension during differentiation hence provided the most effective numerical expansion of fetal mesencephalic precursor cells. Our findings demonstrate that low oxygen tension serves a critical role for the induction and differentiation of TH expressing neurons in culture.
S. Jergova*, L. Collado*, M. Collado*, M. Varghese*, O. Furmanski*†, A. Hama*, S. Gajavelli*, and J. Sagen*†
*The Miami Project to Cure Paralysis, University of Miami, Miller School of Medicine, Miami, FL, USA
†Neuroscience Program, University of Miami, Miller School of Medicine, Miami, FL, USA
Chronic neuropathic pain is a common symptom in clinical practice with inconsistent response to a drug therapy. Loss of endogenous spinal inhibitory neurons, which normally serve to limit pain, has been implicated in pathological pain states. Intrathecal administration of GABA receptor agonists or transplantation of GABA-producing cell lines can alleviate injury-induced pain symptoms in rodent models. However, intraparenchymal grafts of GABAergic neuronal precursor cells have the potential to integrate and restore spinal inhibitory neural circuitry for long-term pain alleviation. Previous studies in our lab have shown primary neurospheres generated from E14 rat telencephalic neural precursor cells (NPCs) contain numerous GABAergic neurons and can attenuate spinal cord injury-induced neuropathic pain in rats. To amplify the GABAergic fate of NPCs following transplantation and to inhibit gliogenesis, we combined environmental and molecular manipulation of cultured NPCs. Recent findings in our lab showed that differentiation under FGF-2 withdrawal resulted in higher yields of GABA-immunoreactive cells and low level of glial markers. In addition, transformation with a lentiviral vector driving constitutive expression of MASH1, a proneuronal basic HLH transcription factor, was used to promote GABAergic neurogenesis. Neuropathic pain was induced in rats by unilateral chronic constriction injury (CCI) of the sciatic nerve. Nociceptive responses were quantified using a paw pressure test for mechanical hyperalgesia at weekly intervals following CCI. At 1 week following CCI, animals exhibiting hyperalgesia received either: 1) predifferentiated GABAergic NPCs from primary cultures, 2) MASH1-transduced FGF-2-deprived neurospheres, or 3) PBS injected into the dorsal horn at the lumbar enlargement ipsilateral to the peripheral nerve injury. Control animals that received PBS only continued to develop progressive decrease in withdrawal threshold to mechanical stimulation indicative of ongoing neuropathic pain. In contrast, mechanical hyperalgesia was significantly attenuated, with approximately 50% increase in withdrawal thresholds, in both groups of transplanted animals compared with controls. Immunocytochemical evaluation revealed numerous GABAergic neurons in the spinal gray matter of transplanted animals. Results of these studies suggest that inhibitory neuronal replacement therapy could be an alternative strategy in chronic neuropathic pain management.
Supported by NS51667.
U. K. Jinwal*†, Y. Miyata§, J. Koren, III*†, J. R. Jones*†, J. H. Trotter†, L. Chang§, L. O'Leary*, D. Morgan†, D. C. Lee†, C. L. Shults*†, A. Rousaki‡, E. J. Weeber†, E. R. P. Zuiderweg‡, J. E. Gestwicki‡§, and C. A. Dickey*†
*Department of Molecular Medicine, Byrd Alzheimer's Institute University of South Florida, Tampa, FL, USA
†Department of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute University of South Florida, Tampa, FL, USA
‡Department of Biological Chemistry, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
§Department of Pathology, Life Sciences Institute, University of Michigan, Ann Arbor, MI, USA
Tau is a microtubule-associated protein that abnormally accumulates in a group of neurodegenerative diseases collectively termed tauopathies. Posttranslational modifications subvert the removal of tau through an unknown mechanism, allowing it to form pathogenic aggregates. Recently we found that aging increases levels of heat shock proteins in the brain, and this was accelerated by tau overexpression. Here, we speculated that pharmacological manipulation of heat shock protein 70 (Hsp70), a component of the cellular chaperone machinery, might impact tau processing and regulate disease pathology. To test this model, we employed a high-throughput chemical screen to identify both inhibitors and activators of Hsp70's ATPase activity. Using this battery of chemical probes to tune chaperone function, we found that inhibitors dramatically decreased both tau and phosphotau levels by approximately 90%, while activators led to their accumulation (up to 300%). Degradation of tau in response to Hsp70 inhibitors was rapid (~5 min) and mediated by the ubiquitinproteasome system (UPS). Moreover, artificially increasing the levels of Hsp70, when concurrent with pharmacological inhibition of its ATPase activity, expedited tau degradation. Finally, we found that tau levels were reduced in models of tauopathy, including in cells expressing clinically relevant tau mutants, organotypic brain slices, and transgenic mice. These findings reveal an unexpected path towards therapeutic intervention into tauopathies via targeting the ATPase activity of Hsp70.
S. Johansson, A. C. Vernon, J. Price, and M. Modo
Centre for Cellular Basis of Behaviour, King's College London, Institute of Psychiatry, London, UK
Immunological rejection is one of the major obstacles to the clinical implementation of transplanted cells for brain repair. The expression of major histocompatibility complex (MHC) antigens is generally thought to be the main factor in graft rejection. We here demonstrate that two human neural stem cell (hNSC) lines (HPC0A07 and STROC05 cells) that differ in their MHC expression when stimulated using rat inflammatory cytokines in vitro exhibit substantial differences in graft survival. STROC05 cells survived well, whereas only a few HPC0A07 cells remained by 90 days in immunocompetent hosts. However, neither line expressed MHC antigens or complement (CD40, CD80, CD86) in vivo. Immunosuppression (methylprednisolone and cyclosporine A) increased graft survival, suggesting the involvement of T cells in the immune response. Indeed, HPC0A07 cells elicited a twice as strong infiltration of lymphocyte compared to STROC05 cells. This increase in lymphocytes is due to a fourfold higher expression of FasL (in the absence of TGF-β) on HPC0A07 that induced a proliferation of CD45+ cells. Differential graft survival in these two hNSC lines therefore is unlikely to be a consequence of a difference in MHC expression, but is mainly due to a difference in their propensity to induce a lymphocyte proliferation through the Fas system. The advantage of these cells lines over mixed cell sources is that these cells can be thoroughly characterized in vitro and hence improve the clinical implementation and robustness of cell therapy.
L. Kelly*†, M. Newman*†, A. Smith*, 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
This is the first study indicating human umbilical cord blood (hUCB) cells, both the heterogeneous mononuclear fraction (MNF) and neural-induced CD-133 (nCD133) stem cells (a subclass of hUCB cells), yield significant behavioral improvements over neural-induced human embryonic stem cells (nhESC) in Parkinson's disease. 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 in treating various nonmalignant and malignant diseases. The present series of studies determined the viability and potential of nhESCs, MNF and nCD133s to repair, replace, or regenerate the loss of DA in the striatum of parkinsonian-like rats. Rats (n = 28) were lesioned twice in the right medial forebrain bundle with 6-hydroxydopamine (6-OHDA) or sham lesioned (n = 8). Baseline rotational behavioral testing was performed 1–2 days before lesion surgery and 3 weeks after for inclusion criteria. In this model of PD, at least 60% of DA neurons in the SNpc are lost, with ~80% depletion of DA levels in the striatum, and full PD-like motor deficits are observed by 3 weeks postlesion. Unilateral cell transplantations were performed into the striatum with either nhESCs, MNF, or nCD133s. Animals were sacrificed and brains were processed for immunohistochemical dependent measures. In each group of rats, the brains, spleens, bone marrow, and peripheral blood were taken for immunohistochemistry. Tissue sections were stained for tyrosine hydroxylase (TH), neuronal nuclei (NeuN), human nuclei (HuN), or double-labeled for NeuN and HuN for cell counts. The specific aim of our study was to determine whether the nCD133 stem cells could yield behavioral improvements that have been previously reported for nhESC transplantations. The most fundamental finding was that both the MNF and nCD133s produced significant improvements in the rotational, swing, and forelimb behavioral tests to nearly complete recovery. This indicated an increase in dopamine, which led to the improved motor function. The nhESC stem cells, however, only showed significant improvements in the swing and forelimb behavioral tests, and these were markedly less robust than both hUCB groups. In addition, histologically, the transplanted hUCB cells appeared to be more numerous and widely distributed than the nhESCs. Interestingly, both hUCB groups were distributed bilaterally and heavily localized throughout blood vessels. If part of the profound hUCB improvement is due to a neurotrophic factor cascade, their migratory capacity could account for this observation. Furthermore, while hESCs carry a substantial ethical burden, restriction, and cost, hUCB cells are a superior alternative without ethical implications, are easily attainable from every live birth at low costs, are rich in immature stem cells, and have proven benefit in other current clinical diseases.
Z. Z. Khaing, S. K. Seidlits, R. R. Rosenberger, J. E. Vanscoy, and C. E. Schmidt
Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA
Our current research aims to optimize the properties of photo-crosslinkable, hyaluronic acid-based hydrogels to encourage differentiation of ventral midbrain progenitors (E13.5) into dopamine neurons. Hyaluronic acid (HA) is a major component of the extracellular matrix with marked effects on cellular proliferation, cell migration, and scar-free wound healing processes. Moreover, HA is present in large quantities during brain development and downregulated in adults. Recently, it has also become apparent that mechanical properties of the matrix that surrounds the cells can have profound effects on the behavior of these cells. Here we used HA (high molecular weight form, 1–2 × 106 Da) and modified it with methacrylic anhydride (MA), resulting in a photo-crosslinkable form of HA. By varying the molar ratio of MA to HA, we have obtained a wider range of degrees of methacrylation, and thus mechanical properties of hydrogels, than we have achieved previously using glycidyl methacrylate. We confirmed the degrees of methacrylate group per HA disaccharide using H+-NMR and found them to range from 39% to 160%. We then evaluated the swelling ratios of the hydrogels in physiologically relevant buffers as well as their degradation when exposed to the HA-specific enzyme hyaluronidase. As expected, a higher degree of methacrylation resulted in decreased swelling ratios and degradation rates, indicating more tightly crosslinked hydrogel networks. We quantified the bulk compressive modulus of each type of hydrogel, and evaluated the cellular response to differences in hydrogel modulus. Dissociated ventral midbrain progenitors from mice were mixed with the hydrogel solution in the presence of the water-soluble photoinitiator Irgacure 2959 (I2959), and exposed to UV light, creating a three-dimensional culture environment. Initial analysis by phase contrast microscopy indicated that neural progenitor cells preferentially extend processes in hydrogels with lower compressive moduli (39% methacrylated) over hydrogels with higher compressive moduli (93% methacrylated). These processes within the “softer hydrogels” were later identified as positive for β-III tubulin, a marker for differentiated neurons. In addition, few glial fibrillary acidic protein (GFAP) positive profiles, indicative of differentiated astrocytes, and nestin, a marker for immature neuronal progenitors, were observed. Progenitor cells within the “harder hydrogels” (93% methacrylated) were positive for all three markers, but the majority of cells were present in rounded “clumps.” The ability to control the mechanical properties of a 3D culture environment within HA-based hydrogels may allow for the optimization of 3D culture of various cell types to achieve specific responses. Currently, we are investigating this hydrogel system to preferentially encourage differentiation of midbrain progenitors into dopamine neurons.
Z. Z. Khaing*, J. E. Vanscoy*, S. K. Seidlits*, R. J. Grill†, and C. E. Schmidt*
*Department of Biomedical Engineering, University of Texas at Austin, Austin, TX, USA
†Department of Neurosurgery, University of Texas Health Science Center at Houston, Houston, TX, USA
Although many exciting advances are being made in the field of spinal cord injury (SCI) repair, significant functional recovery is still not a reality. A major hurdle for regeneration after SCI is the ability of the axons to penetrate and grow through scar tissue to reach distal targets. It is well established that extracellular matrix (ECM) molecules play significant roles by either promoting or inhibiting regenerating axons. In the spinal cord, degradation of intact high molecular weight (MW) hyaluronic acid (HA), a major component of native ECM in the central nervous system (CNS), can induce proliferation of astrocytes after injury to the adult rat spinal cord. Moreover, the addition of soluble high MW HA into cultures can inhibit astrocyte proliferation. Here we studied the effects of high MW HA in the activation of astroglial cells, and the production of astroglial scar component, chondriotin sulphate proteoglycan (CSPG), using in vitro cultures of spinal cord astrocytes. Our results, using immunocytochemistry, indicate that the presence of high MW HA did not significantly decrease the number of proliferating astrocytes (BrdU-positive cells), but it decreased the amount of CSPG (CS-56-positive profiles) present in the culture. Moreover, to study the effects of high MW HA after injury to the CNS, HA hydrogels were implanted into hemisected adult rat spinal cords. Grafts and graft/host interfaces were assayed at 1, 3, and 7 days following implantation. Immunohistochemical staining for markers of macrophage/microglia (ED1) and activated astrocytes (GFAP) revealed that both markers were decreased in and around the injury site in HA-implanted spinal cords compared to the controls. In addition, our data indicate that CSPG levels in areas directly adjacent to the injury were also attenuated at 3 and 7 days after SCI compared to spinal cords without any implants. Our combined in vitro and in vivo data suggest that the breakdown of a major component of the normal ECM component, HA, can result in the activation of astrocytes after SCI and subsequently contribute to the formation of glial scar. Understanding basic mechanisms that induce scar formation after SCI will greatly enhance our ability to design repair therapies to overcome scar-induced inhibition of regenerating axons.
R. Kuruba*†, B. Hattiangady*†, B. Shuai*†, and A. K. Shetty*†
*Division of Neurosurgery, Duke University Medical Center, Durham, NC, USA
†Medical Research and Surgery Services, VA Medical Center, Durham, NC, USA
Status epilepticus (SE)-induced hippocampal and extrahippocampal neurodegeneration typically leads to several epileptogenic changes and culminates in the occurrence of chronic epilepsy characterized by spontaneous recurrent motor seizures. Epilepsy affects ~2 million Americans and ~50 million people worldwide and temporal lobe epilepsy is one of the most prevailing types of chronic epilepsy. As recurrent seizures in a significant fraction of TLE patients are resistant to antiepileptic drugs, development of alternative therapies that prevent or restrain chronic epilepsy after SE or brain injury has immense value. In this study, we tested the effects of grafting of hippocampal stem/progenitor cells (NSCs) into hippocampi of rats shortly after SE for restraining chronic epilepsy development. We expanded NSCs from embryonic day 19 hippocampi in culture as neurospheres in the presence of epidermal growth factor and fibroblast growth factor. The neurospheres cells were labeled with chlorodeoxyuridine (CldU) and triturated to obtain a suspension of hippocampal NSCs for grafting. Four grafts of CldU-labeled hippocampal NSCs were placed into each hippocampus of adult F344 rats (SE rats with grafts; n = 7) at 6 days after kainic acid (KA)-induced SE. Each transplant contained 80,000 live hippocampal NSCs at the time of grafting. A second group of rats received sham-grafting surgery at 6 days post-SE for comparison of the effects of HSC grafting (SE rats with sham surgery; n = 7). We measured both frequency and intensity of SRMS in both groups at 4–6 months postgrafting. SE rats with sham surgery exhibited robust chronic epilepsy at 4–6 months post-SE. In this group, the average frequency of SRMS varied from 0.2 to 0.4/h, the average duration of individual SRMS was 30–40 s, and average frequency of stage V seizures (bilateral forelimb clonus with rearing and falling) was 0.05–0.1/h. In contrast, SE rats with grafts exhibited much reduced frequency and intensity of SRMS at 4–6 months post-SE. In these rats, the average frequency of SRMS was 0.05–0.15/h, the average duration of individual SRMS was 10–20 s, and the average frequency of stage V seizures was 0.02–0.05/h. Overall, SE rats with grafts exhibited 50–62% reductions in seizure frequency and intensity, in comparison to SE rats with sham surgery. Histological analyses of hippocampi revealed neurodegeneration in all groups. Analyses of transplants using CldU immunostaining revealed the presence of grafts. Although a small core of the graft could be seen at the injected site, a vast majority of graft-derived cells migrated into different regions of the hippocampus. Stereological quantification of graft-derived cells using the optical fractionator method (StereoInvestigator, Microbrightfield Inc.) revealed that the yield of surviving cells is equivalent to 31% of grafted cells. Phenotypic analyses revealed the presence of neurons, astrocytes, and oligodendrocytes among the graft-derived cells. Additional phenotypical analyses and quantification are in process. Thus, grafting of hippocampal NSCs shortly after SE is efficacious for restraining spontaneous recurrent motor seizures during the chronic phase of epilepsy. Furthermore, it appears that the effects of grafting are mediated by graft-derived cells. This was evidenced by the presence of graft-derived cells in different regions of the hippocampus and by no effects of sham-grafting surgery on the evolution of chronic epilepsy after SE.
Supported by grants from the National Institute of Neurological Disorders (RO1 NS 054780 to A.K.S.) and Department of Veterans Affairs (VA Merit Review Award to A.K.S.).
C.-T. Lee, J. Chen, S. Errico, L. T. Worden, R. Amable, and W. J. Freed
Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, DHHS, Baltimore, MD, USA
Cocaine adversely affects brain development in animals, and contributes to impaired cognitive development in humans. Although the molecular mechanisms responsible are unknown, the effects of cocaine on development of subhuman primate brain include interference with the proliferation of neural progenitor cells. Mechanisms involved in the effect of cocaine on development were explored using a neural progenitor cell line (AF5), primary human brain cell cultures, and developing fetal rat brains. In AF5 cells, cocaine inhibited cell proliferation by interfering with the cell cycle at the G1-to-S transition, but did not cause cell death. Using microarray analysis and real-time RT-PCR, cyclin A2 was identified as being the cell cycle regulator altered by cocaine exposure. Cocaine was also found to downregulate cyclin A2 in human neural progenitor cells, A2B5+ progenitor cells, and fetal rat brains exposed to cocaine in utero. Compensating for cyclin A downregulation by transfection of cells with a plasmid to express cyclin A2 reversed the inhibitory effect of cocaine on cell proliferation. Transcriptional regulators of cyclin A2 were screened, resulting in identification of ATF4, which responds to endoplasmic reticulum (ER) stress, as being the mediator of cocaine-induced cyclin A2 repression. It was determined that cocaine causes AF5 cells to accumulate reactive oxygen species (ROS) due to N-oxidation of cocaine via cytochrome P450. Cocaine-induced ROS accumulation causes ER stress, as evidenced by increased phosphorylation of eukaryotic initiation factor 2 (EIF2α) and increased expression of ATF4. In developing fetal rat brains, cocaine inhibited neural precursor proliferation in the ventricular proliferative zone. It was determined that inhibitors of oxidative cocaine metabolism, such as cimetidine, prevent the inhibition of cell proliferation and cyclin A downregulation by cocaine, both in vitro and in developing rat brain. In developing rat brain, cocaine was found to disturb neural progenitor proliferation as well as cell migration. In addition, cocaine differentially affects gene expression in neural progenitor cells, neurons, astrocytes, A2B5+ progenitors, and microglia, suggesting that the overall effect of cocaine on brain development involves a complex interaction between multiple cell types. Downregulation of cyclin A2 mediates the inhibition of neural progenitor proliferation by cocaine, and may comprise a principal mechanism through which cocaine interferes with development of the brain. Oxidative metabolism of cocaine via cytochrome P450 was found to be responsible for at least this effect of cocaine. These findings could lead to the strategies to prevent adverse effects of cocaine on brain development.
D. C. Lee, L. Lebson, J. Rizer, C. Ruiz, R. Rojiani, M. N. Gordon, and D. Morgan
Alzheimer's Research Laboratory and Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
Aging and neuroinflammation have been considered to promote neurodegenerative disorders such as Alzheimer's (AD) and Parkinson's diseases. Activation of microglia/macrophages may contribute to disease pathology as well as to therapeutic relief. Classical activation couples with proinflammatory cytokine profiles referred to as M1 activation, whereas alternative (M2) activation associates with dampened proinflammatory cytokine signaling and healing responses. Several reports argue for the existence of both M1 and M2 states during chronic neuroinflammatory diseases such as AD. However, the effects of aging on M1 and M2 activation are less well understood. We performed intraparenchymal injections into hippocampus (HPC) with an M1-activating cocktail (TNF-α/IL-1β/IL-12) or an M2-activating cocktail (IL-4/IL-13) designed to elicit either an M1 or an M2 bias into 6-, 12-, or 24-month-old mice. Three days postinjection, several mouse brain gene products were selectively identified from the M1 or M2 activating cocktails via microarray. Several genes that were selectively expressed by the M1 cocktail included CXCL13, MARCO, TIMP1, calgranulin A and B, and RANTES, while another subset of genes were that were selectively induced by the M2 cocktail included FIZZ1, IGF-1, and EAR 11. In general, aging resulted in smaller inductions after M2 cocktail injections, while M1 cocktail inductions tended to increase. This suggests aging will bias microglial activation towards an M1 state. Furthermore, several purported M2 markers (i.e., argi-nase1 and YM1) were upregulated paradoxically by both M1- and M2-stimulating cocktails. However double-labeling with MARCO (M1 selective) and YM1 (M1 and M2 responsive) revealed two separate populations of cells, with no colocalization. In addition, double-labeling with the myeloid cell marker calgranulin A and microglial markers showed cell populations consisting of calgranulin A+/CD11b+ or calgranulin A+/IBA-1-, further suggesting the presence of multiple subtypes of myeloid-derived cells. These data show that M1 or M2 cytokine cocktails selectively induce different gene products and phenotypic profiles for microglia in the mouse CNS, and that age influences the activation responses.
This work was supported by P01 AG 04418, R01 AG 15490, R01 AG18478, and P50 AG 225711.
C. A. Lieu, N. I. Marupudi, M. A. Berk, R. Ramachandra, and T. Subramanian
*Departments of Neurology and Neural & Behavioral Sciences, Penn State Hershey College of Medicine, Hershey, PA, USA
Parkinson's disease (PD) has been characterized by gradually progressive significant loss of nigrostriatal neurons, unilateral symptoms at the onset of disease, and motor fluctuations including drug-induced dyskinesias (DID) in advanced disease. Recent studies confirm that a purely hemiparkinsonian animal or patient does not exhibit DID until a threshold of >95% of the nigrostriatal pathway is degenerated in one hemisphere or there is bilateral clinically apparent parkinsonism. Interhemispheric (IH) nigrostriatal connections have been reported to constitute 2–10% of the nigrostriatal pathway in mammals. The role of the IH nigrostriatal pathway in the genesis of DID has not been investigated. In the present study, we used the retrograde tracer Fluorogold (FG) to better understand the IH connections in normal rats, striatal partial lesion model (Sauer and Oertel), and the complete lesion model (Ungerstedt) of PD in the rat. A 2% solution of FG was injected unilaterally at one, two, or three sites into the striatum. A subset of the rats were rendered parkinsonian by unilateral injection of 6-hydroxydopamine into the striatum to produce a striatal partial lesion or into the medial forebrain bundle/substantia nigra (MFB) to produce a complete lesion. The stepping and cylinder behavioral tests were used to confirm parkinsonism. Exposure to levodopa caused marked dyskinesias in all MFB-lesioned animals, while striatal partial lesions showed no apparent dyskinesias. Brain sections were analyzed for FG tracer using unbiased design-based stereological counts. The extent of striatal labeling with FG was similar between rats that received single injection of 0.5 μl FG versus rats that received two or three separate 0.2-μl FG injections, and allowed coverage of approximately 75% of the neostriatum. Unilateral striatal FG labeling demonstrated the presence of 3.0 ± 1.6 (n = 8) IH nigrostriatal neurons in normal rats and in the partially lesioned (Sauer and Oertel) hemiparkinsonian rats. The morphology and distribution within the substantia nigra pars compacta of these IH-FG-labeled neurons were identical to the ipsilateral FG-labeled nigrostriatal neurons. There were no IH-FG-labeled nigrostriatal neurons in the MFB-lesioned (Ungerstedt) animals. Preliminary tyrosine hydroxylase immunohistochemistry suggest that IH-FG-labeled nigrostriatal neurons are dopaminergic. Our data demonstrate that the IH nigrostriatal neurons are preserved in the striatal partial lesion model of PD and that such animals do not develop DID. We hypothesize that the preservation of the IH nigrostriatal neurons may be important to prevent the genesis of DID. Additional studies in other animal models, such as the overlesioned primate model of PD, are warranted to determine the presence of these IH nigrostriatal connections and how they modulate DID.
Funded in part by the NIH NINDS RO1NS42402, HRSA DIBTH0632, PA Tobacco Settlement Funds Biomedical Research Grant, PSUHMC Movement Disorders Brain Repair Fund, and NCCAM R21 AT001607.
Q. Li*, M. N. Gordon*, B. Chackerian‡, J. Alamed*, K. E. Ugen†, and D. Morgan*
*Alzheimer's Research Laboratory, Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
†Department of Molecular Medicine, University of South Florida, Tampa, FL, USA
‡Department of Molecular Genetics and Microbiology, University of New Mexico, Albuquerque, NM, USA
It has been demonstrated that vaccine targeting amyloid-β (Aβ) peptide lowers cerebral Aβ and improves cognition in mouse models of Alzheimer's disease. However, the clinical trial was halted due to the fact 6% of the patients developed aseptic meningoencephalitis related to T-cell infiltration. In this study, we investigated the possible use of a novel vaccine designed to avoid the problems encountered in the human clinical trial. We immunized amyloid precursor protein (APP) transgenic mice (Tg2576) at the age of 9 months with either N-terminal Aβ 1–9 (10 μg) or C-terminal Aβ 28–40 (10 μg) peptide fragments conjugated to QB bacteriophage without using adjuvant. It was hoped that this would elicit a strong immune response, with large amounts of antibodies produced, but very little T-cell activation (to minimize the risk of inflammation). We also wished to compare whether the N-terminal or the C-terminal of the amyloid molecule might make the best vaccine. Our data showed the anti-Aβ antibodies response induced by N-terminal vaccine was earlier and greater than the C-terminal after two inoculations. However, after the fourth boost, the antibody titers raised against the A β 28–40 domain got a maximal level that was much higher than the A 1–9 domain. Both vaccines predominately induced IgG1 and IgG2b, Th2-type, immune response in all six immunized APP transgenic mice. Intriguingly, the N-terminal vaccine also induced a robust anti-Aβ IgM response but not Aβ C-terminal domain. There was no anti-Aβ antibody response in mice treated with PBS vehicle control. Both IgG and IgM induced by the vaccines recognized their immunogens and synthetic human full length Aβ as well as bound the endogenous Aβ plaques in the brain in unvaccinated APP Tg mice. Both vaccines reduced amyloid burden in the brain in APP Tg mice and the C-terminal vaccine also significantly enhanced circulating total Aβ levels. CD45, Fcy receptor, and YM-1 immunostaining showed that both N-terminal and C-terminal Aβ peptide treatment decreased CD45 and Fcy receptor expression and did not induce a YM-1 response. Prussian blue staining showed no micro-hemorrhages induced by the immunizations of either N-terminal or C-terminal domain in these middle-aged mice. Thus, both vaccines induced a sufficiently robust antibody response to reduce Aβ deposits in the brain of APP transgenic mice with neither microglia activation leading to an inflammatory response nor microhemorrhage. This novel Aβ vaccination approach could be more effective with a greater safety profile than traditional vaccines in Alzheimer's disease therapy.
Y. Luo, C.-C. Kuo, H. Shen, J. Chou, N. H. Greig, B. J. Hoffer, and Y. Wang
National Institute on Drug Abuse and National Institute of Aging, NIH, Baltimore, MD, USA
Cerebral ischemia can activate endogenous repair processes, such as the proliferation of progenitor cells in the subventricular zone (SVZ). Most of these new cells, however, die shortly after injury. We demonstrate here a novel therapeutic strategy for treatment of stroke 1 week after injury through enhancement of the survival of endogenous progenitor cells from the SVZ. The p53 inhibitor pifithrin-α (PFT-α) was administered to stroke rats from days 6 to 9 after a 90-min middle cerebral artery occlusion (MCAo). PFT-α enhanced functional recovery as assessed by a significant increase in multiple behavioral measurements. Delayed PFT-α treatment had no effect on the cell death processes in the lesioned cortical region. However, it enhanced the survival of SVZ progenitor cells and promoted their proliferation and migration. PFT-α inhibited the expression of a p53-dependent proapoptotic gene, termed PUMA (p53-upregulated modulator of apoptosis), within the SVZ of stroke animals. The enhancement of survival/proliferation of NPCs was further found in SVZ neurospheres in tissue culture. PFT-α dose-dependently increased the number and size of new neurosphere formation. In conclusion, our study demonstrates that delayed treatment with a p53 inhibitor PFT-α is able to modify stroke-induced endogenous neurogenesis and improves the functional recovery in stroke animals.
Supported by NIDA & NIA, NIH.
B.-F. Ma*, Y.-Q. Xue*, L.-R. Zhao*†‡, and W.-M. Duan*‡
*Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA
†Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
‡Gene Therapy Program, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Accumulating evidence shows that the adult brain contains endogenous neural stem cells (NSCs) and possesses some ability for self-repair. The mobilization and neuronal differentiation of endogenous NSCs may provide a new therapeutic strategy for brain repair, in particular for treating neurodegenerative disorders such as Parkinson's disease (PD). However, the regulation of mobilization and neuronal differentiation of endogenous NSCs is challenging. The objectives of the present study were to examine whether hematopoietic cytokines, granulocyte colony-stimulating factor (G-CSF), and stem cell factor (SCF) could enhance mobilization of NSCs in the subventricular zone (SVZ) to the 6-hydroxydopamine (6-OHDA)-lesioned striatum, and whether ectopic expression of the transcription factors Nurr1 and Mash1 could induce mobilized endogenous NSCs from the SVZ to differentiate into dopaminergic (DA) neurons in a mouse model of PD. Young female C57/BL6 mice received either Nurr1/Mash1 lentiviral vectors or GFP lentiviral vectors into the right SVZ. Three weeks after viral injections, mice received subcutaneous injections of G-CSF (50 μg/kg/day) and SCF (200 μg/kg/day) for 7 days. Intrastriatal injections of 6-OHDA were made 3 days after G-CSF and SCF treatment into the right striatum. D-Amphetamine-induced rotational asymmetry was examined 2 weeks after 6-OHDA lesioning. Mice received intraperitonal injections of bromodeoxyuridine (BrdU, 50 mg/kg) twice a day during week 1 after 6-OHDA lesioning. Mice were perfused after the behavioral test, and brain sections were prepared for immunocytochemistry. To determine whether gene transduction of Nurr1/Mash1 lentiviral vectors was functional, we prepared NSCs from the SVZ of neonatal rats and transduced NSCs with lentiviral vectors carrying both Nurr1 and Mash1 genes. We observed that Nurr1- and Mash1-transduced NSCs gave rise to numerous DA cells that were double labeled for tyrosine hydroxylase (TH) and β-III-tubulin in the cultures 7 days after cell differentiation. By injecting GFP lentiviral vectors into the right SVZ of mice, we demonstrated that numerous GFP-labeled cells were colocalized with doublecortin (Dcx), suggesting that GFP-labeled cells are NSCs. G-CSF and SCF treatment significantly increased the number of BrdU-positive cells in the injection sites in the striatum. There were numerous cells double labeled for BrdU and Dcx. In addition, G-CSF and SCF treatment significantly attenuated rotational asymmetry 2 weeks after 6-OHDA lesioning. Interestingly, a number of TH-positive cells were present in 6-OHDA-lesioned striatum of Nurr1/Mash1 lentiviral vector-injected mice. These results suggest that systemic administration of G-CSF and SCF can enhance mobilization of endogenous NSCs in the SVZ to 6-OHDA-lesioned striatum, and that ectopic expression of Nurr1 and Mash1 can induce mobilized endogenous NSCs from the SVZ to differentiate into DA neurons in a mouse model of PD, facilitating behavioral recovery. The control of mobilization and differentiation of endogenous NSCs will likely afford a new opportunity for using induced neuronal replacement as a therapeutic strategy for neurodegenerative diseases, such as PD.
J. D. Macklis,
Harvard University, Harvard Medical School, Massachusetts General Hospital, Boston, MA, USA
Given the heterogeneity of central nervous system (CNS) neuronal subtypes, and the complexity of their connections, detailed understanding of molecular controls over differentiation, connectivity, and survival of specific neuronal lineages will contribute not only to 1) understanding of the development, evolution, organization, and function of CNS circuitry, but also to 2) support or regeneration of vulnerable populations in neurodegenerative [e.g., amyotrophic lateral sclerosis (ALS), hereditary spastic paraplesia/primary lateral sclerosis (HSP/PLS), Huntington's disease (HD), Parkinson's disease (PD)] or acquired disease [e.g., spinal cord injury (SCI)], 3) enabling accurate models of neuron type-specific disease, 4) identification of disease genes, and 5) attempts to functionally repair CNS circuitry. For example, data from our lab demonstrate that new neurons can be added to adult neocortical circuitry via manipulation of transplanted or endogenous precursors in situ (including induction of limited neurogenesis of clinically important corticospinal motor neurons—CSMN—in adult mice), indicating that cellular repair of cortical and cortical output circuitry is possible, if controls over specific lineage differentiation are understood. Using FACS-purified CSMN and other projection neuron populations at critical stages of development in vivo, we have identified both developmentally regulated transcriptional programs of novel and largely uncharacterized genes, and cell-extrinsic controls, that are instructive for development of specific neuron lineages as they develop in vivo (in particular, for CSMN and other projection neuron populations); these control key developmental processes from arealization to subtype-specific differentiation and axonal outgrowth. Loss-of-function and gain-of-function analysis for multiple identified genes and molecules revealed combinatorial molecular-genetic controls over the precise development of key forebrain projection neuron populations that may allow directed control of neural precursors/progenitors/stem cells [embryonic stem/induced pluripotent (ES/iPS) cells] toward accurate disease models, neuronal support or regeneration, or functional CNS repair.
L. Madhavan, B. F. Daley, K. L. Paumier, and T. J. Collier
Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
Stimulation of endogenous (host) neural precursor cells (NPCs) and exogenous NPC transplantation provide alternate promising options for Parkinson's disease (PD) therapy. So far, therapeutic strategies in PD precursor cell research have been divided into studying cell transplantation or endogenous cell stimulation, separately. Because future cell therapies for PD will likely involve a combination of both stimulation of endogenous cells and transplantation, we study these two modes of precursor cell therapy jointly to determine whether and how communication between these two cell types may contribute to neural protection and repair in a rat PD model. Human placental alkaline phosphatase (hPAP)-labeled NPCs were transplanted unilaterally into host rats, which were subsequently infused ipsilaterally with 6-hydroxydopamine (6-OHDA). The reaction of host NPCs to the transplantation and 6-OHDA was tracked by bromodeoxyuridine labeling. Two weeks after transplantation, in animals transplanted with NPCs, we found evidence of elevated host subventricular zone NPC proliferation, neurogenesis, and migration to the graft site. In these animals, we also observed a significant preservation of striatal tyrosine hydroxylase (TH) expression and substantia nigra TH cell number. At 5 weeks after transplantation the striatal and nigral neuroprotection were still observed pointing to the persistence of the neuroprotection phenomenon. Behaviorally, rats receiving NPC transplants before the 6-OHDA infusion showed significantly improved symmetry of spontaneous forepaw placement in the cylinder task as compared to rats receiving 6-OHDA alone. In terms of mechanisms underlying the neuroprotection and the endogenous NPC response to transplantation, we have seen no evidence of DA neuron replacement by NPC-derived cells; however, the NPCs expressed glial cell line-derived neurotrophic factor (GDNF), sonic hedgehog (Shh), and stromal cell derived factor 1α (SDF1α), providing a molecular basis for the observed phenomena. These data together suggest plausible synergy between exogenous and endogenous NPC actions leading to neuroprotection in our rat PD model. Current studies are examining further this plausible synergism, by determining whether endogenous NPCs actually play a role in the observed neuroprotection by inhibiting host NPC proliferation and neurogenesis using cytosine-D-arabinofuranoside (Ara-C). In parallel, the roles of the graft expressed GDNF, SDF1α, and SHH in the observed endogenous NPC response to transplantation and neuroprotection are also being determined by using RNA interference (RNAi) techniques.
Supported by NIH grant NS055295.
Y. A. Maksoud
Your Biology, NFP, Hickory Hills, IL, USA
About 90 years ago, Ramon Cajal (a Nobel Prize laureate) concluded that it is not possible for the central nervous system (CNS) to regenerate after injury. Scores of years elapsed until the early 1980s when Aguayo demonstrated that retinal ganglionic cells could regenerate through a permissive environment. Since then, compelling evidence has accumulated indicating that regenerating the cells and tissues of the CNS is possible. Strategies to enhance the regenerative capacity of the CNS include molecular, cellular, and axonal guidance approaches that enhance one or more of the following cellular and histological processes: neurogenesis, synaptogenesis, angiogenesis, and axonal and dendritic outgrowth. Cellular strategies include the use of stem cells, activated macrophages, Schwann cells, olfactory-ensheathing cells, and genetically modified cells. Molecular approaches include neurotrophic factors (NGF, BDNF, GDNF, FGFs, CNTF, NT-3, and NT-4/5), anti-Rho materials (cAMP and statins), antiapoptotic factors, biomolecules that antagonize the local inhibitory molecules produced by the injury (anti-NOGO and anti-MAG), and enzymatic degradation of chondroitin sulfate proteoglycans by chondroitinase ABC.
F. P. Manfredsson*§, A. Rising*§, C. Burger‡, K. Hasona*§, N. Muzyczka†§, and R. J. Mandel*§
*Department of Neuroscience, University of Florida College of Medicine, Gainesville, FL, USA
†Department of Molecular Genetics and Microbiology, University of Florida College of Medicine, Gainesville, FL, USA
‡Department of Neurology, University of Wisconsin-Madison, Clinical Science Center, Madison, WI, USA
§Powell Gene Therapy Center, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL, USA
Recombinant adeno-associated virus (rAAV)-mediated overexpression of glial cell line-derived neurotrophic factor (GDNF) in the nigrostriatal tract has been shown to lead to loss of body weight in rodents. In this study we utilized this phenomenon in order to study the in vivo activity of a bicistronic tetracycline (tet)-regulated rAAV vector containing both the ptTA2 tet-off transactivator and GDNF under the expression control of a tetracycline response element/CMV hybrid promoter. Animals were bilaterally injected in the substantia nigra (SN) with rAAV expressing GDNF either under the control of the tet-regulated expression cassette or a constitutive chicken β-actin (CBA) promoter. Animals receiving rAAV-CBA-GDNF displayed an immediate loss of body weight following viral injections as did animals in the rAAV-tet-GDNF group receiving normal rat food (“ON”). Over a period of 6 months the animals in the two rAAV-tet-GDNF groups were studied in a cross-over fashion where the food was switched between normal and 3 g/kg doxycycline (dox, “OFF”) every 6 weeks. At the apex of each food cycle the “ON” animals displayed a significantly lower body weight than that of the “OFF” group and a rAAV-GFP-injected control, whereas the “OFF” group returned to body weights indistinguishable from that of the GFP control. GDNF levels in “OFF” rats were undetectable either via ELISA or immunostaining. Striatal GDNF levels in the “ON” group were roughly ninefold lower than that of the CBA-GDNF group. An additional study was designed to evaluate whether there was a dynamic dox dose-response for striatal GDNF levels. Five groups of animals received bilateral nigral injections of the same regulated vector used in the previous study and was thereafter placed on dox doses ranging from 0 to 500 mg/kg food. Surprisingly, all dox doses blocked weight loss induced in “ON” animals (normal food). A clear dox dose-response of striatal GDNF levels was apparent with total shut off occurring at 500 mg/kg diet. Measurements of corresponding serum levels of dox levels showed that serum levels below 0.8 μg/ml were required for GDNF expression shut-down whereas 5 μg/ml is the level required for antibiotic activity. Finally, because the striatum has been a preferred target in clinical trials aimed at reducing neural degeneration in Parkinson's disease, we also wanted to investigate whether direct injections of rAAV-GDNF to the striatum would lead to any measurable weight loss. However, even after achieving striatal levels of GDNF similar to those resulting in significant weight loss in the nigral injection paradigm, no reduction in body weight due to intrastriatal injections was observed.
F. P. Manfredsson*, N. Tumer†‡, B. Erdos†‡, T. Landa†‡, C. S. Broxson†‡, L. F. Sullivan*, A. C. Rising*, K. D. Foust*, Y. Zhang†‡, N. Muzyczka§, O. S. Gorbatyuk§, P. J. Scarpace†‡, and R. J. Mandel*
*Department of Neuroscience, Powell Gene Therapy Center, McKnight Brain Institute, University of Florida College of Medicine, Gainesville, FL, USA
†Geriatric Research, Education and Clinical Center, Department of Veterans Affairs Medical Center, Gainesville, FL, USA
‡Department of Pharmacology and Therapeutics, College of Medicine, University of Florida, Gainesville, FL, USA
§Department of Molecular Genetics and Microbiology, Powell Gene Therapy Center, Genetics Institute, College of Medicine, University of Florida, Gainesville, FL, USA
Glial cell line-derived neurotrophic factor (GDNF), a member of the transforming growth factor-β family, has previously been shown to reduce body weight when delivered intracerebroventricularly in Parkinson disease patients. Additionally, our lab has shown overexpression of GDNF by recombinant adeno-associated virus (rAAV) delivered to the hypothalamus (HYP) produced weight loss in rats. These observations, combined with fact that the nigrostriatal tract is known to effect feeding and weight loss, led us to examine the affects of injections of rAAV-GDNF into the HYP and the substantial nigra (SN) in aged F344 × BN rats. The rats showed significant weight loss from the injections into both HYP (HYP-GDNF) and the SN (SN-GDNF) compared to rAAV-GFP control injections. However, the extent of weight loss was much greater in the SN-GDNF animals compared to the HYP-GDNF animals. SN-GDNF rats showed a significant decrease in food consumptions out to 5 weeks after injection. There was no difference in activity between the groups. Immunohistochemistry stains and ELISA assays showed extensive and significant GDNF levels in the HYP from both injections sites over GFP controls. We found a known downstream protein, phosphorylated extracellular signal-regulated kinase (p-ERK), of GDNF to be expressed in the paraventricular hypothalamic area (PVH). In order to gleam a better understanding of the pattern and specific nuclei in the PVH that was being targeted by GDNF to elicit the p-ERK expression, we decided to stain the PVH for oxytocin. Oxytocin is limited to the parvocellullar PVH and would thus give a clear anatomical delineation of the PVH to help localize the p-ERK expression pattern. This hypothalamic double staining of oxytocin and p-ERK from SN-GDNF rats showed little colocalization. Oxytocin staining was seen surrounding the area of ERK activation and indicates ERK is activated in the medial parvocellullar division (MPD) of the PVH. The MPD contains almost exclusively corticotrophin releasing hormone neuroendrocrine neurons, which are activated by p-ERK. GDNF overexpression in the SN may act on the noradrenergic efferrents projecting to the hypothalamus causing phosphorylation of ERK in the MPD and lead to the weight loss observed in the rats.
F. Marschinke, P.-A. Oldenborg, and I. Strömberg
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
Transplants of fetal ventral mesencephalon (VM) are used as a treatment strategy in Parkinson's disease patients. Cultures of organotypic fetal VM have been utilized to find guiding cues for dopamine nerve fiber formation to enhance the outgrowth from grafted dopamine neurons. In VM cultures two timely separated sequences of dopamine nerve fiber growth have been observed. The first appearing one is a long-distance outgrowth in absence of glial cells and disappears at longer time points. With the guidance of glial cells a later long-term dopamine nerve fiber outgrowth has been observed from 5 to 7 days in vitro (DIV). The integrin associated protein CD47 is expressed in all tissues and serves as a ligand for the signal regulatory protein (SIRP)a, which is, for example, a promoter for migration and synaptogenesis. Furthermore, CD47 acts as a receptor for the extracellular matrix glycoprotein thrombospondin 1 (TSP-1). Embryonic day 14 fetal VM tissues from CD47+/+ and CD47-/- mice were used to study astrocytic migration and the outgrowth of the different dopamine nerve fiber populations. The cultures were performed either at days 7 or 14 and stained for tyrosine hydroxylase (TH), an indirect marker for dopamine neurons and the astrocytic marker vimentin. TH immunohistochemistry demonstrated that the non-glial-guided dopamine nerve fiber outgrowth in CD47-/- mice reached significantly longer distances from the tissue slices and had a massive appearance compared to CD47+/+ mice after 14 DIV. No difference was seen after 7 DIV. A similar effect appeared when adding antibodies against CD47 or Sirpα to the medium of CD47+/+ cultures. Addition of TSP-1 antibodies to the medium of CD47+/+ cultures did not affect the non-glial-guided TH-positive outgrowth. The astrocytic migration and the glial-guided dopamine nerve fiber outgrowth did not differ between CD47+/+ and CD47-/- mice at both time points. For the first time, it has been observed that the non-glial-guided dopamine nerve fiber outgrowth do not degenerate after 14 DIV. These findings can be used in further transplant experiments.
N. I. Marupudi, C. Lieu, J. Tombran-Tink, R. Ramachandra, and T. Subramanian
Department of Neurology, Penn State University, Hershey Medical Center, Hershey, PA, USA
Human retinal pigment epithelial cell (hRPEC) xenotransplants into the striatum of nonimmunosuppressed parkinsonian animals and hRPEC allografts in Parkinson's disease (PD) patients have shown significant amelioration of parkinsonism in open label trials with no evidence of a host immune response or graft rejection. Blinded placebo controlled phase 2 studies, completed recently on 71 patients, did not meet primary end points but did not cause any safety concerns. hRPECs appear to provide antiparkinsonian benefits by secreting small amounts of L-dopa and produce growth factors that provide survival advantage to the adjacent microenvironment, such as pigment epithelium-derived growth factor (PEDF). In this study we examined whether biodegradable poly(lactic-co-glycolic acid) (PLGA) nanospheres containing PEDF whole peptide could potentially provide neuroprotection in a chronic slowly progressive rat model of unilateral PD. Sixteen female rats received stereotactic intrastriatal deposits of the retrograde tracer substance Fluorogold® (FG) (0.5 ill of 2% solution) unilaterally on day 0 and 6-OHDA on day 7 at the same coordinates as the FG injections. On day 8, animals were randomized to receive either intrastriatal injections of empty nanoparticles (group 1) or PEDF-PLGA (4 itg PEDF protein/animal, group 2), and were administered in two sites adjacent to the site of FG and 6-OHDA injections. All animals had baseline and weekly behavioral testing and were sacrificed on day 30. Brains were fixed with paraformaldehyde/lysine/periodate (PLP) and coronally sectioned at 60-μm thickness. Adjacent sections were histologically evaluated for FG labeling and tyrosine hydroxylase immunoreactivity using design-based stereological assessments. General histology, accuracy of targeting, and host response to striatal injections were evaluated using cresyl violet staining. Appropriate depletion of nigrostriatal dopaminergic neurons in the substantia nigra pars compacta was verified in all control animals treated with empty nanoparticles. Evaluators of behavioral testing and cell counts were blinded to the nature of treatments in the two groups of animals. Animals treated with PEDF-PLGA (group 2 animals) had an average difference of 2.5 (n = 8, σ2 = 0.3) steps between both forepaws on the stepping behavioral task. Animals treated with nanoparticles alone had an average difference of 4.2 steps (n = 6, σ2 = 1.3). Furthermore, representative stereological counts on animals receiving nanoparticles alone (n = 3; average raw score = 4649; σ = 721; CE = 0.33) had about 70% fewer number of FG-positive neurons in the substantia nigra compared to animals treated with PEDF-PLGA (n = 3; average raw score = 15,376; σ = 6631; CE = 0.20).
This study was funded in part by the American Parkinson Disease Association (APDA) summer student fellowship to NIM, NIH NINDS RO1NS42402, HRSA DIBTH0632, PA Tobacco Settlement Funds Biomedical Research Grant to TS and PSUHMC Movement Disorders Brain Repair Fund.
R. F. Mervis*†, J. Kotick†‡, M. Shah†‡, A. Winkler§, K. Kasimos‡, S. Joshi‡, S. Scheff¶, and E. J. Mufson#
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University South Florida College of Medicine, Tampa, FL, USA
†NeuroStructural Research Labs, Inc., Tampa, FL, USA
‡Honors College, University of South Florida, Tampa, FL, USA
§University of South Florida College of Medicine, Tampa, FL, USA
¶Sanders-Brown Center for Aging, University of Kentucky, Lexington, KY, USA
#Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
Cortical dendritic morphology (i.e., branching and spine configuration) may play an important role in Alzheimer's disease (AD)-related cognitive decline. This study assessed dendritic morphology in three clinical groups: Individuals with no cognitive impairment (NCI), mild cognitive impairment (MCI), and mild to moderate AD. Formalin-fixed tissue blocks from the frontal and (area 9) and the parietal cortex (area 39–40) were stained with the Golgi method, and layer II–III pyramidal neurons were randomly selected for analysis by an investigator blinded to clinical status using coded slides. Dendritic analysis in the parietal cortex revealed a significant progressive loss of branching that paralleled increasing cognitive deficits: e.g., NCI > MCI > AD. In frontal cortex, MCI pyramidal neurons manifested increased dendritic branching and spine number compared to NCI with a subsequent significant reduction of these parameters in AD. This transient increase in branching and spines may represent a compensatory neuroplastic response that may assist in maintaining cortical circuitry and delay more severe cognitive dysfunction in MCI. Although the basis for this increase in MCI is unknown, mutations in amyloid-β (Aβ) and/or tau genes may enhance dendritic pruning (microtubule depolymerization in the shafts of dendrites) by promoting enhanced calcium influx from the endoplasmic reticulum, or Aβ could promote spine formation via actin polymerization.
Supported by AG14449.
C. Miller*, P. R. Sanberg*†‡§¶, N. Kuzmin-Nichols#, C. D. Sanberg#, and S. Garbuzova-Davis*†‡§
*Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery, 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 College of Medicine, 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 astrocytes contribute to this damage by promoting inflammation through the release of various proinflammatory cytokines. Current treatments for ALS are only palliative. Cell therapy shows promise as a treatment for ALS. Garbuzova-Davis et al. (2003) demonstrated that a low dose (106) of mononuclear human umbilical cord blood (MNC hUCB) cells administered intravenously to G93A mice delayed symptom progression and modestly prolonged life span, possibly through neuroprotection. Recently, an optimization study by Garbuzova-Davis et al. (2008) was performed comparing three different doses (10 × 106, 25 × 106, and 50 × 106) of intravenously injected MNC hUCB cells. 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 dosage effects of MNC hUCB cells upon spinal cord microglia in ALS mice. After behavioral tests were conducted for the optimization study, mice were sacrificed and cervical/lumbar spinal cords were removed and cut into coronal sections. Immunohistochemical staining of microglia was performed on the spinal cord tissue using antibody IBA1 and microglia density (MD) was measured in the gray matter of the ventral horn region of spinal cord cross sections. The spinal cord MD was compared between cell-treated, nontreated (media injected), nontreated [cyclosporine (CsA) injected] G93A, and C57BL/6J control mice. Consistently reduced MDs were noted in mice receiving 25 × 106 cells in both the cervical (p < 0.01) and lumbar (p < 0.05) spinal cords. ALS mice injected with 10 × 106 or 50 × 106 cells showed no significant differences from nontreated G93A mice. Interestingly, mice receiving 50 × 106 cells demonstrated higher MDs, similar to those of nontreated G93A mice. These results confirm that the 25 × 106 cell dose, previously shown most beneficial in regards to mouse life span and disease progression, also provides the most anti-inflammatory benefit through microglia inhibition. Further investigation is needed to establish MNC hUCB cell effects on activated microglia.
Supported in part by NIH STTR (Phase I) grant IR41NS46870–01A1.
A. J. Monahan, Z. Ling, and P. M. Carvey
Rush University, Chicago, IL, USA
Numerous studies have approached nonfamilial Parkinson's disease from the standpoint of adult exposure. Data from our lab, however, implicate prenatal exposure as being an important consideration. When gravid female rats were exposed to bacterotoxin lipopolysaccaharide (LPS), offspring exhibited reduced numbers of dopamine (DA) neurons, less striatal DA, an increase in DA activity, and an increase in proinflammatory cytokine release. The mechanism through which LPS produces this effect is unknown. Using in vitro techniques, we hypothesize that prenatal LPS exposure alters the early stages of DA process development by reducing the trophic environment, leading to underdevelopment and loss of DA neurons. Female rats at E10.5 were injected with 100,000 endotoxin units of LPS or Hank's balanced salt solution (SAL). At E14.5, the mesencephalic region [substantia nigra, (SN)] and lateral ganglionic eminence [striatum (ST)] were dissected from each fetus and pooled per animal. Single cells were plated as SN-ST cocultures according to the following scheme: SAL-SN/SAL-ST; SAL-SN/LPS-ST; LPS-SN/SAL-ST; LPS-SN/LPS-ST. The cocultures were incubated for 7 days at 37°C undisturbed, followed by fixation and analysis of numbers of tyrosine hydroxylase-immunoreactive neurons (TH-ir) and average length of neurite extensions per condition. In addition, supernatant was also collected from primary cultures of the SN, ST, or parietal cortex (PC), which had been exposed to LPS or SAL prenatally. The supernatant was then exposed to untreated SN primary cultures and cultures were assessed for numbers of TH-ir neurons and average length of neurite extension. The results from these experiments demonstrated three overt effects. First, cocultures established from fetuses prenatally exposed to LPS had reduced process extensions morphologically and a reduced average length of process extension compared with control cultures. Second, there were fewer TH-ir neurons in the LPS-SN/LPS-ST condition compared with the SAL-SN/SAL-ST and SAL-SN/LPS-ST conditions [F(3, 26) = 19.5, p < 0.001]. In addition, SN monocultures established from LPS prenatally exposed fetuses exhibited drastic reductions in TH-ir cell counts when compared with controls [F(5, 32) =31.69, p < 0.001]. Third, supernatant collected from ST primary cultures exposed to LPS prenatally resulted in a decrease in TH-ir neurons compared with control cultures [F(7, 31) = 3.300, p < 0.01]. In conclusion, given the reduced process extension and reduced TH-ir cell counts observed in the LPS-SN/SAL-ST, LPS-SN/LPS-ST, and SN monoculture conditions, LPS may mediate its effect by altering levels of neurotrophic factors essential for proper development of the nigro-striatal pathway. With an alteration in DA neuron process extension, access to target-derived trophic factors would also be altered, leading to further reductions of DA neurons. The data presented here are consistent with our previous observations in vivo. Further studies will access whether neurotrophic factors associated with the SN or ST are responsible for the prenatal LPS effects on developing DA neurons.
Supported by DOD and the Richard Nopar Foundation.
S. Moradian*, C. G. Pick†, V. Rubovitch†, J. Kaminsky‡, and R. F. Mervis‡§
*The Honors College, The University of South Florida, Tampa, FL, USA
†Department of Anatomy and Anthropology, Tel-Aviv University, Sackler School of Medicine, Tel-Aviv 69978, Israel
‡Neurostructural Research Labs, Inc., Tampa, FL, USA
§Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL, USA
Single and multiple mild concussions are a frequent occurrence in sports. Clear morphological deficits are difficult to discern but there may be long-lasting behavioral and learning disturbances, the basis of which is poorly understood. Here, we investigated the effects of concussion on brain circuitry. We used the Golgi impregnation method, which randomly stains the dendritic branches and spines of about 6–8% of all neurons. The vast majority of synapses occur on the dendritic spines. We evaluated dendritic parameters using a closed-head model of minimal traumatic brain injury (mTBI). Mice were exposed to a weight drop of 10 g (to the temporal right hemisphere) either only once, or for 2 or 3 times (with a week interval between mTBI episodes). An additional group was used to compare the effects a single 30 g weight drop. Animals were sacrificed 72 h after the last TBI event. All slides were coded and layer V pyramids (apical and basilar trees) from the injured hemisphere were randomly selected for spine analysis. Most notable in these early findings are: (1) a single mTBI episode appeared not to affect dendritic spines density; (2) two mTBI episodes resulted in reduced spine density in both apical and basilar trees; however, and surprisingly, (3) three 10 g mTBI episodes or a single 30 g mTBI episode was not associated with spine loss, but by an increase in spines. Although the time frames for these two groups are different, both of these latter findings may be attributed to a compensatory dendritic hypertrophic response associated with neuronal dropout. Additional assessment of dendritic branching and stereological evaluation of neuronal numbers will further clarify the roles of these parameters in modifying cortical circuitry following multiple concussion-related traumatic brain injury.
J. Musso, III*, D. J. Eve*, D.-H. Park*†, C. Oliveria‡, K. Pollick‡, A. Hope‡, M.-O. Baradez‡, J. D. Sinden‡, and P. R. Sanberg*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery, Korea University Medical Center, Korea University College of Medicine, Seoul, Korea
‡ReNeuron Ltd., Guildford, UK
During the middle cerebral artery occlusion in the rat model of stroke, cerebral blood flow is impaired. The long-term effects of the occlusion on cerebral blood flow have not been investigated to any real extent. It is likely that any treatment for stroke would need to influence the effects on cerebral blood flow. This was tested using a neural stem cell line known to release angiogenic factors such as vascular endothelial growth factor (VEGF). A cohort of male Sprague-Dawley rats were given a middle cerebral artery occlusion, and the hemispheric cerebral blood flow was measured by laser doppler at the time of occlusion, as well as 4, 10, and 16 weeks after occlusion. Under the present experimental conditions, there was a significant right hemisphere bias (p < 0.01; t-test) before surgery, and this difference between the left and right hemisphere cerebral blood flow was observed to appear to switch in direction in a visually oscillatory fashion over time in the sham-treated group, whereas the stroke animals showed left hemisphere dominance throughout the study. One group of rats was intraparenchymally transplanted at the 4 weeks point (after the measurement of cerebral blood flow) with a human neural stem cell line (CTX0E03 cells) known to have some benefit in stroke models. Cerebral blood flow in the lesioned hemisphere of the cell-treated group was significantly improved (p < 0.05; t-test) compared to the untreated rats and also demonstrated a similar oscillatory nature to that observed in sham-treated animals. These findings suggest that multiple bilateral monitoring of cerebral blood flow over time can show the effects of stem cell transplantation efficiently as well as functional tests in an animal stroke model.
This study was funded by ReNeuron Ltd, Guildford, UK and PRS is on the Scientific Advisory Board of ReNeuron.
N. D. Neckel,
Department of Neuroscience, Georgetown University, Washington, DC, USA
The CatWalk (Noldus Inc., Wageningen, NE) is quickly becoming the tool of choice for researchers who wish to quantitatively assess footfalls and gait in rodent models. The device works by recording the time and location of an animal's footprints as it transverses an illuminated glass walkway. From these data a wide variety of parameters can be calculated, but researchers should be aware of exactly how these parameters are calculated by the software, and if those calculations suit their needs. Our research with rats that have received a cervical over-hemisection injury has found that custom calculations are more appropriate. When calculating stride length and base of support the CatWalk software assumes that rodents travel in a straight line parallel to the coordinate frame dictated by the software. In our experience, following a spinal cord injury rats do not walk in straight lines but tend to list and wobble as they travel down the walkway. Stride length is the distance between two successive footprints, and the Catwalk uses forward displacement as this measure. Because our rats did not walk in a straight line, we used the resultant of the forward displacement and lateral displacement as our stride length. This correction resulted in a 7.8% increase in stride length compared to the Catwalk value. Base of support is a measure of how far from the midline of the body that the forelimbs or hindlimbs are. The CatWalk takes the difference of the average lateral position of the right limb and the left limb to calculate base of support. Because our animals varied the lateral position of their footprints, we deemed that an average of the instantaneous base of support would be more appropriate. For each set of four footprints a body axis was calculated and the distance between the right limb and left limb from this body axis was measured. This ensured that the calculation of base of support was independent of the animal's orientation in the coordinate frame of the software. Our custom base of support calculation resulted in a 10% decrease in forelimb base of support and a 9.7% increase in hindlimb base of support compared to the catwalk values. In addition to parameter calculations, we have found that researchers who use the CatWalk should be aware of how footprints are identified. Following a cervical hemisection injury, we have found that rats reluctantly put weight on their more injured limb, and often begin steps with a “double-tap.” This results in the CatWalk recording two separate steps that do not change position. It is up to the researcher to either discard the data from the first step of the “double-tap,” or merge the data into one step as we have done with custom software. We believe that the CatWalk is a very effective system for quantifying rodent locomotion, but urge fellow researchers to make sure that the CatWalk software is calculating gait parameters in a manner that suits the researcher.
N. Nevalainen, S. af Bjerkén, G. Gerhardt, and I. Strömberg
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
Long-term treatment with levodopa (L-dopa) in patients with Parkinson's disease causes side effects known as levodopa-induced dyskinesia. It has not been established why this phenomenon occurs, although many studies indicate the involvement of serotonergic nerve fibers to convert L-dopa into dopamine (DA), and thereby causing the involuntary movements. In the present study, L-dopa was locally applied into the striatum of anesthetized rats and the conversion into DA was observed with electrochemical methods. Nerve terminals were stimulated with potassium chloride before and after L-dopa administration in the DA- and DA/serotonin-denervated rat brain. In DA-denervated striatum, an increase in extracellular DA concentration was seen when stimulating terminals with potassium chloride after local L-dopa administration. Interestingly, no such increase was detected in the DA/serotonin-denervated striatum, thereby supporting the statement that serotonergic nerve fibers convert L-dopa into DA in the absence of dopaminergic nerve fibers. Furthermore, an instant conversion of L-dopa into DA was observed independent of the lesions, resulting in significantly higher and prolonged extracellular concentration than when stimulating nerve fibers with potassium chloride. To conclude, serotonin nerve fibers convert L-dopa into DA in the DA-depleted striatum; meanwhile, they appear not to be responsible for the instant conversion of L-dopa. Hence, an unidentified mechanism that gives rise to several-folds higher levels of DA in the striatum than serotonergic nerve fibers do is yet to be established, and may be responsible for the fluctuations in motor control seen in Parkinson's disease patients.
M. B. Newman, A. P. Smith, L. P. Kelly, and R. A. E. Bakay
Department of Neurosurgery, Rush University Medical Center, Chicago, IL, USA
Hematopoietic human umbilical cord blood (hUCB) cells are a well-established and characterized reliable source of cells that are rich in stem and progenitor cells. These cells have been used successfully for over 20 years to treat children with malignant and nonmalignant diseases. While hUCB cells possess several attractive properties for use in cell transplantation, the most advantageous is their immune immaturity. The cells' immune immaturity directly relates to a low incidence of graft versus host disease and, therefore, a low rejection rate of the transplanted cells. This is one of the major reasons we chose to investigate hUCB cells and their subpopulation of CD133 stem cell potential, once transplanted, to repair or to induce regeneration of the damaged tissue and/or pathway in adult rats lesioned in the middle forebrain bundle with a dopamine (DA) neurotoxin, 6-hydroxydopamine, which is a well-utilized animal model of Parkinson's disease (PD). This presentation will discuss our methods and the results of this study, and our current direction. All cells were cultured in proliferation media, supplemented with the human recombinant proteins stem cell factor, thrombopoietin, and interleukin-3 for 5 to 45 days in vitro (DIV). CD133 stem cells were further cultured for neural induction with the addition of epidermal and basic fibroblast growth factors for 3–4 DIV, and then switched to neural media supplemented with glial cell-derived neurotrophic factor. CD133 cells were not allowed to fully differentiate and kept as neurospheres. The mononuclear fraction of hUCB cells (hUCB MNF) and neural-induced CD133 stem cells [now neural progenitor cells, (CD133 NPC)] were transplanted into the striatum of rats 4 weeks after 6-hydroxydopamine lesions at a concentration of 1 × 106 cells. Apomorphine-induced rotation, forelimb placement, and elevated body swing behavioral tests were preformed on shams and cell transplanted rats at: prelesion (inclusion criteria), 3 weeks postlesion (baseline and exclusion criteria), and post-cell transplant 1 and 2 months. Rats that received either the hUCB MNF or the CD133 NPC cells showed significant behavioral motor function improvements in all behavioral tests compared to sham rats and almost complete recovery compare to their own baseline measures. Most interestingly, the forelimb and swing tests, which do not involve a drug-mediated response, clearly demonstrated improvements in motor deficits for rats receiving either the MNF or NPC cells. While there were cells present and viable at the site of injection, the majority of transplanted cells had migrated into other brain regions and into peripheral organs. Currently, we are examining the blood, spleen, and bone marrow from these rats for transplanted cells and are investigating other mechanism, beside an increase in DA, to account for the remarkable motor improvements. In addition, we have transplanted the CD133 NPC into nonhuman primates and will discuss our findings thus far.
T. Nguyen*, N. Hupalo*, J. Jimenez*, D. Morgan†, and N. Alcantar*
*Chemical Engineering Department, University of South Florida, Tampa, FL, USA
†Molecular Pharmacology and Physiology Department, University of South Florida, Tampa, FL, USA
Alzheimer's disease (AD) is a common degenerative disease with an ever-growing number of victims, unknown cause, and no effective treatment available. Among patients with AD three main hallmarks are common: presence of extracellular amyloid β (Aβ) plaques, formation of neurofibrillary tangles, and finally neuronal death. Extracellular plaques are mainly composed by Aβ140 and Aβ142. Our study is focused on understanding the process of plaque formation and possible clearance. By controlling media conditions we are able to monitor and study the aggregation pathways of these peptides by the mean of Atomic Force Microscopy (AFM), hence imaging their topological feature changes (monomeric, oligomeric, and fibrillar species) over time. The data extracted from the AFM images become a useful tool to estimate the rate of aggregation, as well as the species assemble mechanisms. This analytical technique in combination with others is currently been used by our group to study and monitor the effects of monoclonal antibodies on peptide aggregates, such as the breakdown of large peptide aggregates (fibrils) and/or the decrease of aggregation.
J. B. Nikas, and W. C. Low
Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
The striatal concentrations of 17 metabolites of R6/2 transgenic mice with Huntington's disease, as well as those of wild-type (WT) mice, were obtained via in vivo 1H proton nuclear magnetic resonance spectroscopy (NMRS) at 8 and 12 weeks of age. The 1H NMR scans were conducted with a magnetic field of 9.4 Tesla. The time-dependent variable was collapsed in order to develop a single diagnostic biomarker that would be applicable from 8 to 12 weeks of age. Receiver operating characteristic (ROC) curve analysis was performed in order to ascertain those metabolites that are most influential and have the highest predictive accuracy. Analysis of the following three probabilities—sensitivity, 1 – specificity, and area under the curve—revealed that creatine (Cr), creatine + phosphocreatine (Cr + PCr), N-acetyl-aspartate (NAA), and glutamine (Gln) are by far the four most important metabolites in the differentiation between R6/2 and WT. Discriminant analysis was employed to derive the discriminant function whereby unknown mice can be diagnosed as either R6/2 or WT based on their individual discriminant function score (R6/2 mice have negative scores, whereas WT ones have positive scores). Analysis of variance in the discriminant analysis showed that only 8 of the 17 metabolites are significant variables. Those eight were used to derive the discriminant function, and they are in descending order of significance: Cr + PCr, Gln, Cr, NAA, glutathione (GSH), glycerophosphorylcholine + phosphorylcholine (GPC + PC), myoinositol (mIns), and phosphorylethanolamine (PE). Of those eight, the first four were shown to be by far the most important metabolites in the differentiation between R6/2 and WT, thus corroborating the findings from ROC curve analysis. The diagnostic biomarker developed based on the aforementioned discriminant function was tested with 11 unknown mice, and its prediction results were compared with genotyping—the gold standard. The discriminant function correctly diagnosed all of the 11 unknown mice, with a positive likelihood ratio approximating infinity [1/0(→ ∞)], and with a negative likelihood ratio equal to zero (0/1 = 0). Moreover, also based on the discriminant function, a computer program was developed to assess the percent deficit of the R6/2 mice with respect to the normal (WT). Transcending the animal model, and from a clinical perspective, this approach could have great utility in patients with Huntington's disease for assessing the efficacy of new treatments before and after therapy, thus yielding an objective and accurate evaluation of the treatment itself. Clustering analyses (Fuzzy, Medoid, and K-means) performed on the 17 metabolites revealed that the Cr + PCr variable by itself is by far the most significant metabolite in the differentiation between R6/2 and WT (average silhouette value = 0.833). Because the Cr and PCr reaction, and more importantly its equilibrium state (Cr + PCr), is of exclusive importance to the mitochondria of the cells, these results also lend further weight to the proposed hypothesis that Huntington's disease is characterized by mitochondrial dysfunction.
M. M. Pabón*, C. E. Hudson*‡, J. Jernberg*, A. Bachstetter*, S. A. Acosta*†, C. Gemma†‡, R. Klein§, and P. C. Bickford*†‡
*Center for Excellence in Aging and Brain Repair, Department of Neurosurgery, University of South Florida, Tampa, FL, USA
†Molecular Pharmacology and Physiology, University South Florida, Tampa, FL, USA
‡James A. Haley VA Hospital, Tampa, FL, USA
§Department Pharmacology Toxicology and Neuroscience, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Parkinson's disease (PD) is a neurodegenerative disorder affecting the motor system, including motor coordination and speed as well as producing rigidity and tremor. The symptoms of PD are mainly due to a progressive loss of dopaminergic neurons within the pars compacta of the substantia nigra (SN). This degeneration decreases the levels of the neurotransmitter dopamine in the nigrostriatal system. A recent article has demonstrated that using an adeno-associated virus (AAV) expression vector with WT a-synuclein placed into the SN of rats leads to a 20% loss of dopamine (DA) neurons at 4 weeks after transduction, but that loss of DA neurons progresses over time and at 6 months after the insult there is a 50–60% loss of DA neurons (Gorbatyuk et al., 2008). In the present study, we used a PD model involving an AAV9 vector encoding wild-type human α-synuclein treatment to induce loss of DA neurons in the SN. It is our hypothesis that inflammation via microglial activation may play a role in the ongoing degeneration of DA neurons following α-synuclein. Spirulina, a blue-green algae, has been shown to have antioxidant and anti-inflammatory properties. For example it has been shown that when rats were fed a spirulina-enriched diet for 4 weeks, there was a significant increase in the regeneration of DA terminals into the TH-negative zone of the striatum 1 month following an intrastriatal injection of 6-OHDA. This decrease was accompanied by a reduction in microglia activation as determined by immunohistochemical major histocompatibility complex II (MHC-II) expression (OX-6), suggesting that decreases in microglia activation modulate the beneficial effects of spirulina on 6-OHDA-induced striatal dopaminergic lesion (Strömberg et al., 2005). In the present study we examined the effect of a spirulina-enriched diet on AAV-9 α-synuclein-induced loss of dopaminergic neurons in the substantia nigra. Rats were fed either a spirulina (0.1%)-enriched diet or control NIH31 diet for 1 month prior to treatment with either AAV-9 synuclein, AAV9-GFP, or Ringer's solution. One month following the treatment rats were euthanized and prepared for immunohistochemical measurement of TH and Ox-6. The AAV-9 synuclein treatment decreased the numbers of TH-positive cells counted using unbiased stereological techniques in the SN pars compact. Treatment with the spirulina diet attenuated the loss of dopaminergic neurons when compared to the contralateral side. Furthermore, there was an increase in microglia activation in AAV-9 α-synuclein group, which was partially reversed by the spirulina diet. We observed a correlation between the loss of TH+ cells and an increase in OX-6+ microglia, suggesting that spirulina's antioxidant/anti-inflammatory actions may allow for neuroprotection in an inflammatory environment.
V. K. Parihar*†, B. Shuai*†, B. Hattiangady*†, R. Kuruba*†, and A. K. Shetty*†
*Medical Research and Surgery Services, VA Medical Center, Durham, NC, USA
†Division of Neurosurgery, Duke University Medical Center, Durham, NC, USA
Neural stem cells (NSCs) in the adult mammalian hippocampus produce neurons throughout life. Hippocampal neurogenesis is very sensitive to stress. Particularly, it is known that unpredictable chronic mild stress has negative impact on neurogenesis. However, the effects of predictable chronic mild stress (PCMS) such as routine stress experienced in day-to-day life are unknown. We hypothesize that PCMS enhances neurogenesis in the adult hippocampus because of the adaptation of the organism to predictable nature of the mild stress. To address this, we quantified hippocampal neurogenesis in adult rats that underwent 28 days of exposure to PCMS. A group of 3-month-old male Sprague-Dawley rats (n = 6) were exposed daily to 5 min of restraint stress for 28 days. Another group of age-matched male rats (n = 6) served as controls. At the end of the 28-day period, rats in both groups received four intraperitoneal injections of 5′-bromo-2′-deoxyuridine (BrdU; one injection every 6 h at a dose of 50 mg/kg body weight). Animals were euthanized at 6 h after the last BrdU injection and 30-μm-thick serial sections (every 15th) through the entire hippocampus were processed for BrdU immunostaining. Another series of sections was processed for doublecortin (DCX; a marker of newly born neurons) immunostaining. The production of newly born cells per day from NSCs was quantified by counting the numbers of BrdU+ cells in the subgranular zone-granule cell layer (SGZ-GCL) using the optical fractionator method (StereoInvestigator; Microbrightfield). Furthermore, the status of neurogenesis was quantified through stereological counting of DCX+ neurons in the SGZ-GCL. Additionally, the overall growth of individual DCX+ newly generated neurons that are relatively mature (i.e., neurons with vertical dendrites) was assessed through morphometry using Neurolucida (Microbrightfield). Animals exposed to 5 min of restraint stress for 28 days exhibited 51% increase in the production of new cells/day in the SGZ-GCL (mean ± SEM = 6,974 ± 396), in comparison to age-matched naive rats (4,616 ± 206; p < 0.01). Analyses of DCX+ neurons revealed 53% increase in the numbers of newly born neurons in the SGZ-GCL of rats exposed to PCMS (17,732 ± 644), in comparison to age-matched naive rats (11,585 ± 833; p < 0.01). Characterization of individual DCX+ neurons demonstrated increased total dendritic length, and increased numbers of dendritic segments and nodes in DCX+ neurons of rats exposed to PCMS. Increased production of new cells in the SGZ-GCL suggests that NSCs in the adult hippocampus respond positively to PCMS through increased proliferation. On the other hand, increased numbers of DCX+ neurons in rats exposed to PCMS implies that increased production of new cells translates into an enhanced neurogenesis. Enhanced dendritic growth suggests that the milieu of the hippocampus in rats exposed to PCMS is conducive for rapid growth of newly born neurons. The results provide novel evidence that predictable chronic mild stress enhances both production and growth of newly born neurons in the adult hippocampus. Considering the significance of neurogenesis to learning, memory, and mood, it is plausible that predictable chronic mild stress has beneficial effects, which may include better cognitive function and improved mood.
Supported by a Gulf War Research Grant from the Department of Veterans Affairs (A.K.S.).
D.-H. Park*†, D. J. Eve*, J. Musso, III*, C. Gemma*, A. D. Bachstetter*, A. Wolfson*, A. Schlunk*, M.-O. Baradez‡, J. D. Sinden‡, and P. R. Sanberg*
*Center of Excellence for Aging and Brain Repair, and Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery, Korea University Medical Center, Korea University College of Medicine, Seoul, Korea
‡ReNeuron Ltd., Guildford, UK
It is now well accepted that the brain is able to generate new neurons from a population of resident multipotential neural stem cells (NSCs) located in two discrete regions of the brain: subventricular zone and the subgranular zone (SGZ) of the dentate gyrus (DG). However, the capacity for neurogenesis appears to diminish over the life span of an organism. Methods to potentiate the proliferation of new neuronal or glial cells within the central nervous system from resident NSCs could have therapeutic potential following an insult, such as stroke, or to replace lost cells as a result of a neurodegenerative disease. Previously, we have demonstrated increased neurogenesis following intravenous human umbilical cord blood implantation. Here, we implanted human neural stem cells (CTX0E03), originally derived from fetal cortical tissue, directly into the ventricles of aged Fisher rats (22 months old). The following day, the rats were injected twice intraperitoneally with 50 mg/kg BrdU and were transcardially perfused 1 day later. The brains were then removed and cryopreserved before being cut into 40-μm sagittal sections. Immunohistochemical staining for BrdU (marker of proliferation) and doublecortin (DCX; immature neurons), and immunofluorescent staining for IBA-1 (microglia), GFAP (astrocyte) and HuNu (transplanted human fetal cells) was performed on free-floating sections. Quantification and imaging of labeled cells within the SGZ region was performed using the optical fractionator method of unbiased stereological cell counting. Defining the SGZ of the DG as a two-cell-diameter band on both sides of the granular cell layer (GCL), the number of BrdU+ cells within the SGZ was counted. DCX+ cell counts were made in the SGZ/GCL, due to possible cell migration. BrdU staining demonstrated significantly increased proliferation in the SGZ of the cell-treated rats [t(4) = 8.75, p = 0.0009; n = 3], whereas the number of DCX+ cells was also significantly increased in the cell-treated animals compared with the vehicle [t(8) = 4.29, p = 0.002; n = 5]. Immunofluorescence studies showed that colocalization of BrdU and IBA-1 was very limited, and BrdU and GFAP coexpression was not found within the SGZ. Absence of double labeling for human nuclear antigen suggested that the increased proliferation was from endogenous neural progenitor cells rather than the implanted cells. The present study demonstrates that implanted exogenous NSCs may stimulate endogenous neurogenesis in aged animals. The cells are believed to be exerting their influence either directly by inducing neurogenesis or indirectly by reducing inflammation to stimulate neurogenesis from the injection site, possibly due to the rapid secretion of neurotrophic factors such as vascular endothelial grown factor (VEGF), which have been known to encourage angiogenesis and/or neurogenesis.
This study was funded by ReNeuron Ltd., Guildford, UK and P.R.S. is on the Scientific Advisory Board of ReNeuron.
A. V. Pendharkar*, A. De†, H. Wang†, X. Gaeta*, N. Wang*, R. H. Andres*, X. Chen†, S. S. Gambhir†, and R. Guzman*
*Department of Neurosurgery, Stanford University School of Medicine, Stanford, CA USA
†Molecular Imaging Program, Stanford University School of Medicine, Stanford, CA, USA
Stem cell transplantation represents a promising experimental therapeutic avenue for CNS disorders. Before these therapies can come to fruition, however, in-depth studies must be conducted to assess stem cell survival and biodistribution, especially in developing minimally invasive intravascular delivery techniques. In the present study, we have designed a multimodality approach with which intravascular transplants can be studied for their biodistribution and survival. We have created a mouse neural progenitor cell line harboring a triple-fusion reporter gene encoding a luciferase, red fluorescent protein, and thymidine kinase PET reporter gene. To combine high sensitivity and spatial resolution, we have also included super paramagnetic iron oxide (SPIO) labeling for magnetic resonance (MR) imaging to create a clinically applicable system to monitor neural stem cells after transplantation. C17.2 mouse neural progenitor cells were transduced by a lentivirus harboring a trifusion reporter gene containing a synthetic Renilla luciferase, monomeric red fluorescent protein (RFP), and a truncated version of sr39 thymidine kinase (TK). Cells were also transfected with SPIO particles. Following transduction, RFP-positive cells were selected using fluorescent-activated cell sorting (FACS). Cells were analyzed for TK and luciferase activity 48 h after FACS selection. Negative controls for both assays were performed using the parental C17.2 neural progenitor cells. Adult nude mice were subsequently injected with either FACS isolated C17.2 neural progenitor cells positive for the triple-fusion reporter gene or parental C17.2 neural progenitor cells. FACS-sorted or wild-type cells (5 × 105) were transplanted into the right or left striatum, respectively. Control injections of the respective cells were also performed subcutaneously into the shoulders. In vivo bioluminescence imaging for luciferase and T2*-weighted MRI was performed 3 days following transplantation. Fluoro-3-[(hydroxymethyl)butyl]guanine-positron emission tomography (FHBG-PET) imaging was also performed. FACS analysis confirmed the presence of RFP, and thus the triple-fusion reporter gene in the C17.2 mouse neural progenitor cell line. In vitro assays also demonstrated luciferase and thymidine kinase activity in the RFP sorted cell line. Wild-type cells did not exhibit any thymidine kinase and luciferase activity. The in vivo data validated the activity of the triple-fusion reporter gene after transplant, both in subcutaneous and intracerebral locations. Specific luciferase signal emission from the bolus of transduced cells from the shoulder, as well as the brain after intracerebral transplantation was observed. Wild-type cells transplanted on the contralateral shoulder and striatum did not emit any signal above background levels. PET confirmed the presence of the transduced cells and activity of the triple-fusion gene in vivo. SPIO transfection resulted in a typical susceptibility artifact on T2 and T2*-weighted MRI. In conclusion, we describe a novel method for tracking cells after transplant. With this approach we obtain high spatial resolution from MRI and high sensitivity with PET. This method will enable studies of biodistribution and cell migration in both short- and long-term studies of CNS disorders.
C.-S. Piao*, and L.-R. Zhao*†
*Department of Neurology, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA
†Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center-Shreveport, Shreveport, LA, USA
Stem cell factor (SCF) and granulocyte colony-stimulating factor (GCSF) are known as growth factors for hematopoietic stem cells/hematopoietic progenitor cells (HSCs/HPCs), and SCF and GCSF contribute to regulation of lineage commitment for HSCs/HPCs. In previous studies, we have demonstrated that receptors for SCF and G-CSF are expressed by neural stem cells (NSCs) and that SCF and GCSF have therapeutic effects on brain ischemia in rats. However, the biological function of SCF and GCSF on NSCs and how SCF and GCSF repair the brain after experimental stroke remain unclear. In this study we determined whether SCF and GCSF regulate NSC differentiation. NSCs were dissected from the ventricular zone of rat embryonic brains. SCF and GCSF were added to NSCs during NSC differentiation. We observed that the number of TuJ1-positive neurons was increased and the number of GFAP-positive astrocytes was decreased by SCF and GCSF. This immunohistochemistry finding was further confirmed by Western blot. Additionally, using quantitative RT-PCR, we found that the expression of the proneuronal gene, Ngn 1, was upregulated and Notch 1 was suppressed by SCF and GCSF treatment. These data suggest that SCF and GCSF control lineage commitment for NSCs and that SCF and GCSF promote neuronal fate determination and differentiation of NSCs. Our study, for the first time, demonstrates that hematopoietic growth factors can control NSC differentiation. The effects of SCF and GCSF on neurogenesis may contribute to brain repair after experimental 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.
C. Priest, A. Davies, E. Wirth, A. Conta, Y. Polonskaya, and J. Polonskowski
Geron Corporation, Menlo Park, CA, USA
Cellular therapeutics require multidisciplinary development efforts to enable reliable production and characterization of the intended cell types, rigorous testing in preclinical models, and the design of clinical trials to assess the safety and benefit of the therapy in appropriate patient populations. We have developed processes to produce and cryopreserve a population of differentiated cells (GRNOPC1) that contains human embryonic stem cell-derived oligodendrocyte progenitors from characterized and dedicated stem cell banks. GRNOPC1 is characterized for cell composition, sterility, viability, and the lack of adventitious agents. GRNOPC1 produces numerous neurotrophic factors and can also induce myelination of axons in rats with spinal cord injuries and in shiverer mice that lack compact myelin. GRNOPC1 has been tested for the ability to repair damaged tissue and demyelinated axons following spinal cord contusion injuries. Administration of GRNOPC1 into the contusion site 7 days after a thoracic spinal cord injury in rats improved hindlimb locomotor activity and reduced parenchymal cavitation at the injury site 9 months posttransplantation. Fascicles of myelinated neural fibers were observed crossing the injury epicenter after GRNOPC1 transplantation, in contrast to the cavities that developed at the injury epicenter in animals injected with vehicle alone. Extensive preclinical studies were performed to assess the biodistribution, safety, toxicology, and tumorigenicity of GRNOPC1 following transplantation into the rodent spinal cord. Histological and quantitative PCR-based analyses at multiple time points up to 12 months posttransplantation were performed to ascertain the survival and distribution of GRNOPC1 in both central and peripheral tissues. In addition, clinical, cellular, and biochemical assessments for systemic toxicity were made at 2, 6, and 9 months posttransplantation. Assessments of allodynia were performed in control and GRNOPC1-transplanted animals up to 9 months posttransplantation. All animals that received GRNOPC1 were monitored for the development of ectopic tissue or teratoma formation for up to 12 months postadministration. The results of these studies were submitted as part of an Investigational New Drug (IND) application to the FDA. Preparations are under way to initiate a Phase 1 clinical trial to assess the safety of GRNOPC1 in patients with subacute, complete American Spinal Injury Association Classification (ASIA) A, thoracic injuries whose last fully preserved neurological level is T3 to T10. The results of these preclinical studies and the clinical trial design will be presented.
A. Rachubinski, S. K. Cornelius, K. N. Maclean, and K. B. Bjugstad
Department Pediatrics, University Colorado Denver, Aurora, CO, USA
Currently, treatments for Down syndrome (DS) are directed at restoring already impaired neurological functions in children and adults with DS. However, a treatment used earlier in development might postpone or prevent the later cognitive delays. Neural progenitor cells (NPCs) have been used in a wide range of adult neurodegenerative disorders with promising behavioral and neuroanatomical results. The present study sought to determine if NPCs could be used as a long-term treatment for DS when implanted shortly after birth, before the DS brain is subjected to postnatal neurodegenerative changes. This study used the Ts65Dn mouse model of DS, which has delayed postnatal development, low birth weight, and age-associated cognitive impairment. Trisomic and disomic Ts65Dn littermates were bilaterally implanted in the hippocampus with 100,000 C17.2 murine neural progenitor cells (mNPC), saline, or not transplanted, on postnatal day 2 (PND 2). As some of the first developmental delays observed in infants with DS are in motor skills, such as rolling over, crawling, and walking, gross motor skill acquisition was used as an initial assessment of mNPC effects in these mice. All pups were tested daily for developmental milestones: righting, cliff avoidance, negative geotaxis, eye and ear opening. Compared with their disomic littermates, trisomic pups were delayed in the righting response, cliff avoidance, eye and ear opening by ~24 h, consistent with previously reported results. No karyotype differences were seen in negative geotaxis. Trisomic/mNPC pups improved their righting response to the same level as disomic/saline controls (p < 0.01). Treatment with mNPC had no effect on cliff avoidance or negative geotaxis in trisomic mice, although disomic pups may have been delayed. mNPC significantly shortened the time to eye opening (p < 0.01) in disomic and trisomic pups. Disomic/mNPC reached eye opening ~12 h before disomic/saline controls. Both trisomic/mNPC and trisomic/saline groups showed a 1.5 day improvement compared to trisomic/no treatment controls, although they did not improve to the level of disomic/saline control (p < 0.05). Ear opening was not affected by the implantation of mNPC. The current study demonstrates that mNPC can be implanted into the neonatal brain without negative side effects and for some milestones, the mNPC may be inducing immediate benefits. Because many milestones emerge within days of PND 2, the transplanted mNPC may not have been in place long enough to induce measurable effects for all milestones. Subtle differences, like the time to eye opening, suggest that the mNPC could be causing changes in the brain that may not be evident until additional testing of the mice is done. Current studies are focusing on mNPC effects at these later ages. These same litters of mice are behavior tested as they reach early adulthood, with subsequent neuroanatomical analyses to determine the fate of the mNPC and changes in the host brain.
Supported by: NIH/NINDS 1F31NS060517-01A1.
A. Rehnmark*, P. C. Bickford†, and I. Strömberg*
*Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
†Department of Neurosurgery, University of South Florida, Tampa, FL, USA
Diets enriched in antioxidants are known to be beneficial for health. Antioxidants have a modulatory effect on the immune response and oxidative stress, which are believed to be involved in degeneration of dopamine neurons observed in the substantia nigra in patients suffering from Parkinson's disease. Blueberries are rich in antioxidants and, according to previous studies, have a positive effect on regeneration of the nigrostriatal dopaminergic neurons in rats after partly 6-OHDA depletion. This recovery seems to be associated with an early microglia response, which is observed 1 week after 6-OHDA depletion in rats fed with blueberry-enriched diet but not in controlfed animals. However, no regeneration is observed in the globus pallidus despite an early microglia response. This study was focused on measuring factors that might be involved in the regenerative process such as glial cell line-derived neurotrophic factor (GDNF) and the proinflammatory cytokine tumor necrosis factor-α (TNF-α). Additionally, the extracellular matrix molecule tenascin-C and the phenotype of the microglia were examined. Therefore, rats treated with blueberry-enriched or control diets were subjected to a striatal injection of 6-OHDA. In both blueberry- and controlfed animals, GDNF levels were significantly higher in the 6-OHDA-lesioned striatum compared to the control side, while no difference was observed in the lesioned side 1 week after the lesion independent of diet. GDNF levels were significantly higher in the globus pallidus compared to the striatum, suggesting that GDNF is not involved in the recovery of the striatal dopaminergic neurons because no regeneration was observed in the globus pallidus. TNF-α measurements revealed neither difference when comparing lesioned or control striata, nor when comparing between the diets. However, tenascin immunoreactivity was more intense in 6-OHDA-lesioned striata of blueberryfed compared to control animals, indicating that tenascin might be involved in the recovery process of dopamine neurons. Immunohistochemistry for ED-1-positive microglia, a marker for phygocytotic cells, revealed a faster inflow of ED-1-positive cells after blueberry treatment, suggesting that an early clearance of debris in the lesioned tissue was found after treatment with antioxidants. In conclusion, early phagocytocis and enhanced tenascin-c levels after striatal 6-OHDA injection might be important for recovery of dopamine neurons in rats fed with blueberry-enriched diet.
J. M. Ross*†, S. Brené‡, J. Öberg§, R. Sitnikov§, K. Pernold†, M. Terzioglu¶, A. Trifunovic¶, J. Kehr#, N.-G. Larsson¶**, B. J. Hoffer*, and L. Olson†
*The National Institutes of Health, National Institute on Drug Abuse, Baltimore, MD, USA
†Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
‡Department of Neurobiology, Health Sciences and Society, Karolinska Institutet, Stockholm, Sweden
§Department of Clintec, Karolinska Institutet, Stockholm, Sweden
¶Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
#Department of Physiology and Pharmacology, Karolinska Institutet, Stockholm, Sweden
**Max Planck Institute for Biology of Ageing, Cologne, Germany
Mitochondrial dysfunction may underlie aging-related alterations in neuronal function and has been implicated in neurodegenerative diseases, such as Alzheimer's and Parkinson's diseases, as well as in stroke. A homozygous knock-in mouse expressing a proof-reading-deficient version of the nucleus-encoded catalytic subunit (PolgA) of mitochondrial DNA (mtDNA) polymerase has been developed to study the effects of progressive mitochondrial dysfunction, and shows many signs of premature aging (Trifunovic et al., 2004). The mtDNA mutator mouse has a three- to fivefold increase in levels of mtDNA point mutations as well as increased levels of mtDNA deletions, with an approximately linear increase in mutation levels from midgestation to late adult life. The mouse has a reduced life span, reduced fertility, weight loss, reduced subcutaneous fat, enlarged heart, anemia, alopecia, kyphosis, osteoporosis, and sarcopenia. The aging phenotype appears not to be due to increased reactive oxygen species (Trifunovic et al., 2005). Furthermore, the prematurely aging mouse has hearing loss with apoptotic neurons in both the spiral ganglion and the cochlear nucleus (Niu et al., 2007). 1H Magnetic resonance spectroscopy (1H-MRS) is being used to monitor the in vivo metabolism of these animals. 1H-MRS demonstrates a twofold increase in brain lactate levels as early as 6–9 weeks of age to at least 35–38 weeks of age (oldest age group studied; average life span is 46 weeks). High-performance liquid chromatography (HPLC) confirms the 1H-MRS finding of high lactate in brain tissue in mtDNA mutator mice, and also reveals a significant increase in brain lactate levels in aged (86 weeks) wild-type controls. A double staining for the activities of cytochrome coxidase (COX) and succinate dehydrogenase (SDH) shows mitochondrial respiratory dysfunction in cerebral cortex, hippocampus, striatum, and thalamus as early as 9 weeks of age in mtDNA mutator mice and in aged (2.5 years) wild-type controls. Additionally, HPLC measures a threefold increase in plasma lactate levels in mtDNA mutator mice, and a twofold increase in 86-week-old wild-type controls. Interestingly, lactate levels are also elevated in cardiac muscle from 25–29-week-old mtDNA mutator mice and 86-week-old wild-type controls. Additional data indicate higher activity in lactate dehydrogenase (LDH) in brain and liver, and LDH mRNA expression differences in brain.
Supported by the National Institute on Aging, the National Institutes of Health, the Swedish Research Council, and Swedish Brain Power.
J. Rossignol*†, C. Boyer*, X. Lévêque*, G. L. Dunbar†, and L. Lescaudron*‡
*INSERM U643, ITERT, CHU Hotel Dieu, Nantes, France
†Field Neurosciences Institutes, Department of Psychology and Program in Neuroscience, Central Michigan University, MI, USA
‡Faculté des Sciences et des Techniques, Université de Nantes, France
Adult bone marrow-derived mesenchymal stem cells (MSCs) can elevate levels of trophic factors and have the potential to differentiate into multiple cell derivatives (from mesodermic and neuro-ectodermic lineages), as well as the ability to transdifferentiate into neurons (albeit in only a few studies). Because they lack major histocompatibility complex II (MHC-II) expression and have a low-level MHC-I expression, transplanted MSCs have less inflammatory responses from host tissue than what is observed following xenogeneic transplants of neuroblasts. Given these advantages, MSCs provide a potential means for treating brain dysfunction, such as what occurs in Huntington's disease (HD). We have previously observed beneficial effects of bone marrow transplants in the quinolinic acid rat model of HD. In the present study, we investigated the potential efficacy of MSC transplants in the 3-nitropropionic acid (3NP) rat model of HD. Male Sprague-Dawley rats were randomly assigned to one of three groups: sham (injected with PBS and treated with or without vehicle); 3-NP (with or without vehicle); 3-NP + TP (with MSC transplants). All animal received IP injections of 3NP or PBS twice daily. During 3NP intoxication, animals received intrastriatal injections of 200,000 or 400,000 MSCs and underwent several weeks of behavioral testing followed by an anatomical investigation of the transplanted site at 71 days posttransplantation. Modest recovery was observed on the rotarod and stepping tests and, at sacrifice, MSCs were still viable and metabolically active, albeit with undifferentiated morphology (oval/round shape). Only a very weak inflammatory response from the host brain was observed. Because very few MSCs showed transdifferentiation, the observed recovery of function was attributed to the release of trophic factors and/or extracellular matrix components of the MSCs, although recovery was also observed in vehicle-treated 3NP rats, indicating factors in the medium (DMEM) may confer beneficial effects in this model of HD. Our results provide further evidence that the beneficial effects of MSC transplants in rodent models of HD come primarily from their ability to produce trophic or growth factors, such as brain-derived neurotrophic factor (BDNF) or nerve growth factor (NGF), although factors in DMEM need to be further investigated for their potential therapeutic effects.
Acknowledgments: Association Huntington France (L.L., J.R.), INSERM (L.L.), French Ministry of Research and Education (J.R.), Field Neurosciences Institute and funds from the John G. Kulhavi Professorship at Central Michigan University (G.L.D.).
J. Sanchez-Ramos*§, S. Song*§, T. Mori‡, N. Patel§, C. Cao†, and G. Arendash†§
*Department of Neurology, University of South Florida, Tampa, FL, USA
†Florida AD Research Center, Tampa, FL, USA
‡Saitama Medical Center/University, Japan
§James A. Haley VA Hospital, Tampa, FL, USA
Filgastrim (granulocyte colony-stimulating factor; G-CSF) is a multimodal hematopoietic growth factor, which also has profound effects on the diseased central nervous system. Recent work in our laboratory has demonstrated that administration of G-CSF to cognitively impaired Alzheimer's disease (AD) transgenic mice (APP + PS1) reversed cognitive performance and significantly decreased amyloid-β (Aβ) deposition in hippocampus and entorhinal cortex. Additional studies have revealed that G-CSF-treated tg AD mice exhibit a significant increase in total microgliosis and increased synaptophysin immunostaining in hippocamal CA1 and CA3 regions. We also have found that trafficking of bone marrow-derived cells from blood to brain plays a role in decreasing Aβ deposition. To test the hypothesis that the procognitive effects of G-CSF are mediated by direct actions on neural stem/progenitor cells, we measured the extent to which G-CSF promotes neurogenesis both in vitro and in vivo. The functional significance of increased synaptophsin expression in CA1 and CA3 regions of hippocampus associated with the reduction of amyloid was assessed electrophysiologically in hippocampal slice preparations. At an intracellular level, we have elucidated a novel signaling pathway triggered by G-CSF that involves switching to an alternatively spliced variant of protein kinase C (PKC-δ2) resulting in a) increased hippocampal stem/progenitor cell proliferation and/or decrease in cell death. To summarize, we will review our current understanding of the mechanisms by which G-CSF reduces amyloid deposition/levels, induces cellular antiapoptotic programs, stimulates hippocampal neurogenesis, and restores synaptic functional activity.
M.-L. B. Selenica, C. Dickey, M. Gordon, and D. Morgan
Department of Molecular Pharmacology and Physiology, University of South Florida College of Medicine, Tampa, FL, USA
It has been proposed that Aβ1–42 and its derived oligomers may contribute toward changes in the levels of hyperphosphorylated tau and thus linking both pathologies observed in AD. Furthermore, the correlation of soluble Aβ-derived oligomers with the level of synapto-toxicity, activated microglia and neurotoxicity has been shown in several studies. Tg 4510 mouse model was developed to generate a model mimicking the taupathy observed in the frontotemporal dementia with parkinsonism. In this model the CAMKIIα promoter is driven by tetracycline operator to overexpress human P301L tau in the forebrain. These inducible transgenic mice develop tangle pathology, cognitive deficits, and neuronal loss at age-dependent mode and start showing tau hyperphosphorylation as early as the age of 3 months. In our study we attempt to investigate the impact of Aβ1–42-derived oligomers on tau hyperphosphorylation by utilizing these models. We also investigate other disease-relevant markers such as neuronal loss and microglia activation when ex vitro oligomers are introduced to brain of the tau mice. We used osmotic minipump system to infuse 0.0045 mg/ml soluble Aβ1–42-derived oligomers in the hippocampus of 3-month-old Tg4510 mice for 2 months until reaching an age of 5 months. Previously, a robust increase of phosphorylated tau levels in Tg4510 mouse has been shown at this age. Also, Aβ1–42-derived oligomers were incubated in vitro for 2 months and analyzed biochemically in order to monitor the kinetic change of the oligomeric sample with time. Coronal sections of the brain from all groups were immunohistochemically analyzed by using different phosphotau-specific antibodies in order to measure the levels of phosphorylated tau when compare to the nontransgenic cohort. Markers of activated microglia such as CD45+ and immunoreactivity of Floro-Jade dye were used for further analyses on all animals.
Research supported by the following NIH grants: AG 15490, AG 18478 AG 04418, AG 25509.
S. D. Shahaduzzaman*†, J. E. Golden*‡, S. M. Green*†, T. Womble*†, P. R. Sanberg*†, K. R. Pennypacker*‡, and A. E. Willing*†‡
*Center of Excellence for Aging & Brain Repair, University of South Florida, Tampa, FL, USA
†Department of Neurosurgery, University of South Florida, Tampa, FL, USA
‡Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
During stroke there is an abrupt oxygen and glucose deprivation that results in irreversible neuronal damage. In this study we examined the neuroprotective effect of human umbilical cord blood (hUCB) cells in an oxygen-glucose-deprived (OGD) environment, and the gene expression induced in the neurons by OGD and hUCB cells. Neuronal cells were isolated from E17 Sprague-Dawley rats and cultured in media for 6 days. Four experimental conditions were applied; neuronal cells were cultured alone or cocultured with hUCB cells under either normoxic or OGD conditions. After 20 h of culture, the viability of neuronal cells was determined using fluorescein diacetate/propidium iodide (FDA/PI) labeling. RNA samples were extracted from cell cultures and loaded onto an Affymetrix rat U34 genome chip for microarray analysis. Microarray results were quantified by polymerase chain reaction (PCR) (RT-PCR). Under OGD conditions, hUCB cells cocultured with neuronal cells significantly increased neuronal cell viability (52 ± 6%) when compared to the neuronal cells cultured alone (28 ± 4%) (p < 0.001). The microarray data indicated that 64 genes were induced in the neuronal cells by the presence of hUCB under normoxic conditions, including genes known to be important in the detoxification, immunogenic, cell trafficking, and signaling pathways. Under OGD, 84 genes were induced in the presence of hUCB cells. The gene expression profile of the OGD group differed significantly from the normoxia group, with only one gene, myotropin, common to both groups. Based on the results of these microarray analyses, 16 genes of interest were selected and the expression profiles were quantitatively confirmed by RT-PCR. In particular, ATF1, Akt3, chemokine-2, chemokine-20, cytochrome P450, doublecortin, glycine receptor, glutaredoxin, myotropin, protein tyrosine phosphatase, peroxirederoxin, and Vcam1 are being examined. The FDA/PI stain indicated an increase in the viability of neurons cocultured with hUCB and exposed to OGD. The results of these experiments suggest that hUCB alters the gene expression profile to enhance neuronal survival signaling following OGD exposure.
Study supported by NIH Grant #R01NS52839 (A.E.W.). Disclosure: A.E.W. is a consultant and P.R.S. is a cofounder of Saneron CCEL Therapeutics, Inc. A.E.W. and P.R.S. are inventors on cord blood patents licensed to Saneron.
J. Sinden
ReNeuron Limited, Surrey, UK
The UK Medicines and Healthcare Regulatory Authority has recently approved a Phase I clinical trial for stable disabled patients 6–24 months after ischemic stroke. The trial is to be undertaken at Glasgow Southern General Hospital under the leadership of Dr. Keith Muir, of the Department of Neurology, University of Glasgow. This is the first approved trial for a stem cell line in the UK and, along with Geron's recent approval, represents a significant breakthrough in the field. The stem cell line, CTX0E03, was developed from fetal cortex and is a clonal neural stem cell line, expanded using conventional cGMP manufacturing made possible by the c-mycERTAM technology, permitting all clinical trials and approved licensed product to be from the same cell banks. In this talk the steps towards clinical development of this unique stem cell product and the next steps into the clinic will be outlined.
I. Singec, and E. Y. Snyder
Stem Cell & Regeneration Program, Burnham Institute for Medical Research, La Jolla, CA, USA
The work with human embryonic stem cells (hESCs) has demonstrated that pluripotent cell lines can be grown indefinitely and are capable of generating large numbers of defined neural cell types. The ethical problems surrounding the derivation of hESCs and the heterogeneity of currently available cell lines have turned out to be major obstacles in the stem cell field. The recent discovery that human somatic cells such as fibroblasts and keratinocytes can be reprogrammed to a pluripotent state by retroviral delivery of the transcription factors Oct4, Sox2, Klf4, and c-Myc holds great promise to advance cell therapy, drug discovery, and in vitro disease modeling without the problems surrounding hESCs. Taking advantage of this new method, we have generated induced pluripotent stem cells (iPS cells) from human fibroblasts derived from normal individuals and from various patients with genetic disorders. These established iPS cell lines are indistinguishable from classical hESCs regarding morphology, growth requirements, gene expression, and developmental potential. This presentation will summarize our experience with generation of iPS cells and their differentiation into specific neural lineages such as dopamine neurons and motor neurons.
J. R. Sladek, Jr.*, J. D. Elsworth§, R. H. Roth†§, C. Leranth¶, T. J. Collier#, K. B. Bjugstad*, B. C. Blanchard*, R. J. Samulski**, P. Aebischer††, and D. E. Redmond, Jr.†‡
*Department of Pediatrics, University of Colorado School of Medicine, Aurora, CO, USA
†Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA
‡Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
§Department of Pharmacology, Yale University School of Medicine, New Haven, CT, USA
¶Department of Obstetrics and Gynecology, Yale University School of Medicine, New Haven, CT, USA
#Department of Neurology, University of Cincinnati School of Medicine, Cincinnati, OH, USA
**Department of Pharmacology, University of North Carolina, Chapel Hill, NC, USA
††École Polytechnique Fédérale de Lausanne, Switzerland
Transplantation of dopamine neurons from human fetal ventral mesencephalon (VM) has resulted in variable degrees of functional change in humans with Parkinson's disease. In some instances dyskinesias have resulted, which suggests an excess of and/or unregulated release of dopamine from grafted neurons. We speculate that grafts placed into the substantia nigra would offer a better opportunity for afferent control than those placed directly into the striatum. Thus, we have attempted to stimulate neuritic growth from such grafts to the host neostriatum. Previously, we demonstrated that such outgrowth can be achieved with “bridge or helper” grafts of embryonic lateral ganglionic eminence placed at progressively more rostral distances to the VM grafts. The current study attempted to determine whether injection of a growth-promoting factor [i.e., glial cell line-derived neurotrophic factor (GDNF)] into the striatum or placement of fetal striatal cografts in the nigrostriatal pathway might elicit neuritic outgrowth to the caudate nucleus. Four St. Kitts vervets received embryonic DA grafts into the rostral mesencephalon near the host substantia nigra and also received injections of adeno-associated virus 2 (AAV2)/GDNF or equine infectious anemia virus (EIAV)/GDNF into the caudate nucleus. They were injected in the same area with the retrograde tracer fluorogold (FG) at 6 months after grafting and just prior to sacrifice. Three adult monkeys were cografted with fetal VM tissue near the substantia nigra and fetal striatal grafts 2.5 mm more rostral along the nigrostriatal pathway. These animals also received FG injection prior to sacrifice. The FG label was found in both grafted and host TH-positive neurons following dual label immunohistochemical analysis. FG label was found in dopamine neurons in VM grafts only in animals that received AAV2/GDNF vector injections in the ipsilateral, but not contralateral, striatum. All monkeys showed FG labeling in the ipsilateral host substantia nigra to the FG injection. These findings demonstrate that grafted dopamine neurons can extend neurites to a distant target that releases an elevated concentration of GDNF, and suggest further that grafted VM neurons can be placed into more appropriate neuroanatomical sites that may facilitate nigrostriatal tract reconstruction and better neurolgical control of dopamine release.
Supported by NINDS Grants PO1-NS044281, UO1-NS046028, Axion Research Foundation, and the Michael J. Fox Foundation for Parkinson's Research.
A. J. Smith*, R. D. Shytle*†‡¶, J. Tan*†‡¶, P. C. Bickford*§¶, K. Rezai-zadeh†, L. Hou†, J. Zeng†, P. R. Sanberg*‡¶, C. D. Sanberg¶, B. Roschek, Jr.#, R. C. Fink#, and R. S. Albert#
*Center for Excellence in Aging and Brain Repair, Department of Neurosurgery, University of South Florida College of Medicine, Tampa, FL, USA
†Silver Child Development Center, Department of Psychiatry and Behavioral Medicine, University of South Florida College of Medicine, Tampa, FL, USA
‡Neuroscience Program, University of South Florida College of Medicine, Tampa, FL, USA
§Research Service, Veterans Administration Hospital, Tampa, FL, USA
¶Natura Therapeutics, Inc., Tampa, FL, USA
#HerbalScience Group LLC, Naples, FL, USA
While previous studies have shown antiamyloidogenic effects of curcumin, no research has systemically examined optimized turmeric extracts enriched in curcuminoids or turmerones. In a previous in vitro study, the standardized turmeric extract, HSS-888, showed strong inhibition of Aβ aggregation and secretion, indicating the presence in this extract of bioactive molecules that might be therapeutically important. Therefore, in the present study HSS-888 was evaluated in vivo using transgenic “Alzheimer” mice (Tg2576) that overexpress Aβ protein. Following a 6-month prevention period where mice received extract HSS-888 (~5 mg/kg/day), tetrahydrocurcumin (TC), or a control through ingestion of customized animal feed pellets, HSS-888 significantly reduced brain levels of soluble (~40%) and insoluble (~20%) Aβ as well as phosphorylated tau protein (~80%). In addition, primary cultures of splenocytes from these mice exhibited enhanced cellular immunity (increased IL-4 to IL-2 ratio). In contrast, TC treatment only weakly reduced phosphorylated tau protein and failed to alter other therapeutic endpoints. These findings indicate that optimized turmeric extract HSS-888 is enriched in key functional bioactives that may represent an important therapeutic agent for future clinical development for the treatment of Alzheimer's disease.
A. L. Spieles-Engemann*‡, M. M. Behbehani†, T. J. Collier*, K. Steece-Collier*, S. L. Wohlgenant*, V. B. Thompson‡§, J. W. Lipton§, and C. E. Sortwell*
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH, USA
‡Graduate Program in Neuroscience, University of Cincinnati, Cincinnati, OH, USA
§Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA
Deep brain stimulation (DBS) of the subthalamic nucleus (STN) has become a popular neurosurgical treatment strategy for Parkinson's disease. However, the mechanism by which DBS exerts its symptomatic benefits is unknown. In addition, preclinical studies in our laboratory and others have demonstrated that STN DBS can protect substantia nigra (SN) dopamine neurons from experimental toxins and therefore may have additional disease-modifying effects. Our goal in the present study was to investigate whether long-term, high-frequency STN DBS impacts the levels of trophic factors involved in nigrostriatal plasticity. Specifically we examined glial cell line-derived neurotrophic factor (GDNF) and brain-derived neurotrophic factor (BDNF) in the STN and its target structures: SN, globus pallidus interna (GPi), globus pallidus externa (GPe), striatum (STR), and frontal cortex (FC). In the first experiment, unlesioned rats (no 6-OHDA) were implanted unilaterally in the STN with bipolar concentric stimulating electrodes. Stimulation was initiated 2 weeks postsurgery for a period of 2 weeks (130 Hz, 60 μs, 30 μA). Rats in which STN electrodes were never activated served as controls. Enzyme-Linked ImmunoSorbent Assay (ELISA) was used to determine GDNF and BDNF protein levels and real-time reverse transcriptase polymerase chain reaction (qPCR) was used to determine mRNA levels. Results revealed no changes in GDNF protein associated with STN DBS. However, STN DBS was associated with a bilateral threefold increase of BDNF protein in the STR. Additionally, a ninefold increase in BDNF mRNA was observed in the GPe ipsilateral to the stimulated STN. In the second experiment, rats lesioned unilaterally with intrastriatal 6-OHDA were utilized with the lesion and STN electrode implantation occurring during the same surgical session. Rats were assessed for contralateral forelimb akinesia prior to the initiation of stimulation and following the 2-week stimulation interval. The STN and its target structures were microdissected and analyzed via ELISA to determine BDNF protein levels. Behavioral analysis revealed that STN DBS provided functional improvements in lesion-induced contralateral forepaw akinesia, improvements that were not observed when stimulation was turned off, indicating functionally effective stimulation. Analysis of BDNF protein levels in the 6-OHDA-lesioned rats is ongoing. These studies are the first to report alterations in trophic factors after long-term STN DBS. Our results to date suggest that the upregulation of BDNF may play an important role in the therapeutic efficacy and neuroprotective effects of STN DBS.
Supported by the Davis Phinney Foundation and the University of Cincinnati.
A. M. Spieles-Engemann¶, M. M. Behbehani†, T. J. Collier*, S. L. Wohlgenant*, G. T. Mandybur‡, J. W. Lipton§¶, B. T. Terpstra¶, and C. E. Sortwell*
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Department of Molecular and Cellular Physiology, University of Cincinnati, Cincinnati, OH, USA
‡Department of Neurosurgery, University of Cincinnati, Cincinnati, OH, USA
§Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA
¶Neuroscience Graduate Program, University of Cincinnati, Cincinnati, OH, USA
Preclinical studies demonstrate that deep brain stimulation of the subthalamic nucleus (STN DBS) can provide neuroprotection of nigral dopamine (DA) neurons when initiated prior to or soon after experimental toxins. However, the ability of STN DBS to provide neuroprotection for nigral DA neurons once progressive degeneration has been initiated and the toxin is no longer present has never been examined. Further, the ability of STN DBS to protect striatal DA terminals has yet to be determined. To examine neuroprotection, we unilaterally lesioned rats with intrastriatal 6-hydroxydopamine (6-OHDA) and implanted stimulators in the ipsilateral STN. Following a 2-week recovery period, at which point animals had lost approximately 50% of their substantia nigra (SN) DA neurons, STN stimulation began and was administered continuously for 2 weeks. Lesioned rats in which stimulators were implanted in the STN but never activated served as controls. Functional neuroprotection of contralateral forepaw use and general motor activity was assessed using the cylinder task. Morphological neuroprotection of the nigrostriatal system was evaluated via stereological counts of the tyrosine hydroxylase-immunoreactive (TH-ir) neurons in the SN and TH-ir neurites in the striatum as well as analysis of striatal dopamine and dopamine metabolites. Placement of stimulators was confirmed histologically. STN DBS halted ongoing SN DA neuron cell loss. In contrast, rats implanted with inactive electrodes displayed significant nigral DA neuron degeneration over the 2-week stimulation interval. Despite this protection observed at the level of the cell body, stereological analysis of striatal TH-ir neurite density and striatal DA levels revealed that the neuroprotective effects of HF STN DBS did not extend to the striatum. Behavioral analysis revealed that STN DBS provided functional improvements in generalized motor activity (rearing); these improvements were not observed when stimulation was turned off. These studies demonstrate that STN DBS initiated 2 weeks after 6-OHDA injection can halt ongoing, protracted SN DA neuron degeneration but does not restore previously depleted dopaminergic terminals in the striatum. Further studies are warranted utilizing alternate models of Parkinson's disease in which protracted striatal terminal loss occurs in order to determine whether the neuroprotection of nigral cell bodies can be extended to neuroprotection of striatal dopaminergic terminals.
Supported by the Davis Phinney Foundation and the University of Cincinnati.
K. Steece-Collier*, K. Soderstrom†, J. O'Malley*, J. Stancati*, N. Levine*, C. Sortwell*, J. Lipton*, and T. Collier*
*Department of Neurology, Movement Disorders Division, University of Cincinnati, Cincinnati, OH, USA
†Department of Neurology, Rush University, Chicago, IL, USA
Dopamine (DA) denervation in the parkinsonian brain results in numerous changes within the striatum. One such change involves the primary target neuron of afferent input from nigral DA and cortical glutamate neurons, specifically the striatal medium spiny neurons (MSN). Dendritic spines found on MSNs are critical sites for integration of DA and glutamate signaling, and are essential for normal motor behavior. In advanced Parkinson's disease (PD) there is marked atrophy of dendrites and spines on MSNs. Similar pathology is observed in mice and rats with severe DA depletion. The impact of altered MSN morphology on therapeutics such as DA cell replacement is unknown. Indeed, while grafting of embryonic DA neurons has provided significant benefit in some individuals, the overall efficacy has been variable and less than would be predicted from the degree of DA replacement provided in some PD patients. Similarly, while DA grafts in parkinsonian rats can completely reverse rotational abnormalities, more complex behaviors are often not significantly improved. Thus, it is possible that DA terminal replacement, regardless of the approach (stem cells, embryonic neurons, vectored trophic factors) into an environment complicated by severe alterations in morphology of postsynaptic neurons will produce only limited overall benefit. To test this hypothesis, Sprague-Dawley rats with a unilateral nigrostriatal DA lesion received a standard, suboptimal embryonic DA neuron graft (one capable of reversing amphetamine rotations but not more complex sensorimotor deficits). Half the animals received slow-release pellets containing the Cav1.2/1.3 calcium channel blocker nimodipine to prevent dendritic spine loss; the other half received control pellets. Behavioral tests were performed in nongrafted, nonpelleted parkinsonian rats to confirm that acute nimodipine administration over doses spanning three log units had no impact on test behaviors. Data from our studies demonstrated that DA grafts placed into severely parkinsonian rats with a normal compliment of dendritic spines protected by nimodipine (confirmed with Golgi staining) showed enhanced behavioral efficacy measured with the vibrissae sensorimotor test and levodopa-induced rotational asymmetry and dyskinesias. The enhanced behavioral response in nimodipine + DA-grafted compared to vehicle + DA-grafted animals occurred despite no significant difference in graft volume (0.41 ± 0.07 mm3 Veh + DA graft; 0.50 ± 0.06 mm3 Nimod + DA graft) or number of surviving grafted tyrosine hydroxylase positive (TH+) cells (3837 ± 1049 Veh + DA graft; 5368 ± 620 Nimod + DA graft). Severely parkinsonian rats with a normal complement of striatal dendritic spines did however have nearly double the TH+ fiber density compared to DA-grafted rats with dendritic spine loss (11.6 ± 1.56 Nimod + DA graft; 6.18 ± 1.30 Veh + DA graft; 0.27 ± 0.13 Sham + Sham; 0.30 ± 0.03 Nimod + Sham; Spaceballs® adjusted volume/fiber length ± SEM). Ultrastructural analyses of TH+ innervation of striatal targets and correlational analyses with behavior are pending. In conclusion, it is interesting to speculate that the enhanced behavioral efficacy and degree of neurite outgrowth derived from the same number of grafted cells in rats with a normal complement of striatal dendritic spines is related to the increase in physiological input sights for TH+ fiber reinnervation. This will be systematically investigated as this study progresses.
Supported by NIH/NINDS and MJFox Foundation.
S. Stoner*, P. R. Sanberg*†‡§¶, and S. Garbuzova-Davis*†‡§
*Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery, 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 College of Medicine, Tampa, FL, USA
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease involving selective premature motor neuron death in the spinal cord and brain. Recently, it has been shown that the blood-brain/spinal cord barrier (BBSCB) is compromised in G93A SOD1 mice (Garbuzova-Davis et al., 2007). The BBSCB dysfunction might be involved in disease mechanism and disease progression. Vascular problems could also be a potential cause of the premature motor neuron death in ALS. Insufficient supplies of nutrients and oxygen would adversely affect neuron metabolism, possibly leading to an increasingly excitotoxic microenvironment. The aim of this study was to determine microvascular density (MVD) in the gray matter of spinal cord of G93A mice modeling ALS at different stages of disease. Cross sections of cervical and lumbar spinal cords of early and late symptomatic G93A mice and of control C57BL/6 mice were cut on a cryostat, mounted onto slides, and stained with 1% toluidine blue. The spinal cord sections were photographed under a light microscope with 20× magnification. MVD was measured in vessels per micrometer by objectively counting the number of blood vessels present in the gray matter of the ventral horn and dividing this sum by the applicable area. The results show a significant decrease in cervical MVD between control (2.84 ± 0.34) and early (1.58 ±0.36) (p < 0.015) or late symptomatic mice (1.32 ± 0.11) (p < 0.001). Similarly, there were also significant decreases in lumbar MVD in early (1.40 ± 0.09) (p < 0.002) and late symptomatic mice (1.43 ± 0.11) (p < 0.002) versus controls (3.28 ± 0.44). Overall, the decreased microvascular densities observed in the ventral horn of cervical and lumbar spinal cords of G93A mice might account for ALS motor neuron degeneration as a result of an accumulation of metabolite waste and byproducts, probably leading to an increasingly excitotoxic microenvironment for motor neurons. Because vascular insufficiency was found in early symptomatic G93A mice, it is likely that motor neuron degeneration is exacerbated by the deficient blood supply. However, it is uncertain whether the observed vascular deficiencies are primary or secondary effectors in ALS pathogenesis. Further studies in this area are needed.
Supported in part by The Muscular Dystrophy Association.
I. Strömberg, F. Marscinke, N. Nevalainen, M. Chermenina, A. Rehnmark, E. Berglöf, and S. af Bjerkén
Department of Integrative Medical Biology, Umeå University, Umeå, Sweden
Attempts in understanding the guiding cues for nerve fiber formation from ventral mesencepahlic dopamine neurons to enhance graft reinnervation of host brain in Parkinson's disease have been made utilizing organotypic slice cultures. Organotypic slice cultures from E14 ventral mesencephalon result in neurite formation occurring in two temporally separated sequences, where the initial nerve fibers are generated in the absence of astroglial cells, reach for long distances, and degenerates when the second wave of nerve fibers are formed attached to migrating astrocytes, is persistent but terminate to grow after 2 weeks in vitro. The growth factor glial cell line-derived neurotrophic factor (GDNF) affects the two separate nerve fiber formations such that in cultures from E14 gdnf gene-deleted tissue, the glial-associated nerve fibers are hampered in their growth due to reduction in proliferation and thereby migration of astrocytes, while no effects are found on the non-glial-associated growth. Treating the cultures with tumor necrosis factor-α (TNF-α), which is supposed to be toxic for the dopamine neurons, results in enhanced nerve fiber growth and astrocytic migration if the treatment is made during an early phase of the culture. The effect is due to enhanced migration of astrocytes and is mediated through both TNF-α receptors 1 and 2. Inhibiting the astrocytic proteoglycan synthesis affects the glial-associated growth while inactivating the proteoglycans using chondroitinase ABC has no effect on nerve fiber growth. In cultures from CD47 knockout tissue, where CD47 binds to signal regulatory protein alpha (Sirpα) and participates in synapse formation, both the long-distance growing non-glial-associated as well as the glial-associated nerve fibers survive and grow for long time periods and reach long distances. Thus, these data demonstrate that astrocytes are the main key players when guiding nerve fiber growth and that synaptogenesis is an important factor to modulate the length of the nerve fibers.
Y. D. Teng*†‡, D. Yu*†, D. E. Benedict*†, U. Wilhelmsson§, and M. Pekny§
*Department of Neurosurgery, Harvard Medical School, the Brigham and Women's Hospital and Children's Hospital Boston, Boston, MA, USA
†Department of Physical Medicine and Rehabilitation, Harvard Medical School, Spaulding Rehabilitation Hospital, Boston, MA, USA
‡Division of SCI Research, VA Boston Healthcare System, Boston, MA, USA
§Center for Brain Repair and Rehabilitation, Department of Clinical Neuroscience and Rehabilitation, Institute for Neuroscience and Physiology at Sahlgrenska Academy Göteborg University, Göteborg, Sweden
Traumatic spinal cord injury (SCI) leads to severe functional deficits with devastating impacts. To date, effective therapy for SCI is still unavailable. The discovery of neural stem cells (NSCs) has offered new hope in using cell-based therapies to treat neurotrauma. However, the complex postinjury pathology in the spinal cord tissue imposes a major obstacle to the effective engraftment of donor NSCs. Among the factors that impede the biological integration of the NSCs, the quick development of post-SCI reactive astrogliosis, as manifested in the upregulation of hallmark intermediate filament proteins such as glial fibrillary acidic protein (GFAP) and vimentin (Vim), is by far the most notable barrier to interfere the donor–host interaction. While activation of the astrocytes may benefit wound healing process, it also inhibits neural regeneration. To test whether the therapeutic potential of donor human NSC (hNSC) could be enhanced in an experimental SCI model through augmenting donor engraftment, we created a T9–T10 segmental hemisection in both GFAP-/-Vim-/- and wild-type mice, and implanted poly-lactic-co-glycolic (PLGA) polymer scaffold seeded with hNSC immediately following injury. Control mice of either type received injury procedure only. We performed our routine batteries of the behavioral tests 1 day before and post-SCI, as well as weekly thereafter for up to 4 weeks. Neurite tracing for the CNS motor pathways were carried out after the behavioral evaluation, followed by histopathological analyses. Our initial assessment showed significant behavioral improvements in the animals treated with hNSC-seeded polymer implant, relative to the injury alone controls. The levels of neurobehavioral recovery were significantly higher in the treated GFAP-/-Vim-/- mice compared with those of the wild-type mice that underwent the same quality hNSC-polymer placement. These early stage findings indicate that limiting the degrees of post-SCI reactive astrogliosis may enhance the efficacy of NSC-based neural repair for SCI. Further experimental analysis will shed light on more specific mechanisms that might underlie the interaction of the hNSC and the GFAP-/-Vim-/- spinal cord.
V. B. Thompson*, C. E. Sortwell†, T. J. Collier†, K. Steece-Collier†, and J. W. Lipton‡
*Neuroscience Graduate Program, University of Cincinnati, Cincinnati, OH, USA
†Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
‡Department of Psychiatry, University of Cincinnati, Cincinnati, OH, USA
We have previously demonstrated that prenatal exposure to MDMA (ecstasy) from embryonic days 14 to 20 results in a significant increase in dopaminergic fiber innervation of the striatum and the prefrontal cortex in the rat at postnatal day 21. We have also shown that MDMA enhances the survival of primary cultured dopaminergic (DA) neurons, but does not increase fiber density. We therefore hypothesized that factors present in “target” structures may be required to induce the increased neurite branching observed in vivo. The current study sought to determine whether target-derived factors facilitate MDMA-mediated neurite branching. To examine the influence of MDMA and target tissue on DA neuron survival and outgrowth, primary mesencephalic neurons (embryonic day 14, E14) were cocultured with cells derived from the lateral ganglionic eminence (embryonic striatum), and incubated in the presence or absence of MDMA for 96 h. The density of dopaminergic fibers was determined using StereoInvestigator Software employing the petrimetric probe. Cocultures exposed to MDMA (37.5 μM), demonstrated a 20% increase (p < 0.05) in neurites per neuron compared to control cocultures. This finding is consistent with the hypothesis that target-derived factors are critical in recapitulating the increased DA neurite density observed in vivo. A parallel in vivo proteomic survey of control and MDMA-exposed fetuses demonstrated that transgelin-3, a protein implicated in neuronal cytoskeletal dynamics, was reduced by 38% (p < 0.05) immediately after the cessation of MDMA treatment on E20. The expression of its gene, TAGLN3, was similarly reduced by 41% (p < 0.05) as measured by qPCR. In rats, transgelin-3 is normally upregulated throughout the brain during early postnatal development, and has been shown to colocalize with important elements of the cytoskeleton, including tau, α-tubulin, F-actin, and microtubule-associated protein 2 (MAP2). The spatial and temporal pattern of its expression suggests that transgelin-3 may play an important role in neurite extension and path finding during development such that reductions in its expression may result in aberrant connectivity and/or sprouting. In humans, alterations in transgelin-3 expression have been reported in the frontal cortex during alcohol withdrawal and in individuals suffering from schizophrenia, suggesting that it may continue to play an important role in neuronal plasticity in adults. These results suggest that MDMA-mediated DA neurite hyperinnervation is a target driven phenomenon. In addition, alterations in transgelin-3 expression implicate it as a potential mediating candidate for MDMA-induced DA neurite outgrowth observed in the prefrontal cortex (PFC). The mechanism behind MDMA-mediated DA neurite outgrowth may provide important insight into the normal development of the DA neuron, which could be critical in understanding the pathology of disorders as divergent as schizophrenia and Parkinson's disease.
M. Timmer*†, C. Winkler‡, A. Klein§, J. Sauter¶, W. Oertel¶, and G. Nikkhah†
*Hospital Grosshadern, Neurosurgical Clinic, Ludwig-Maximilians-University Munich, Munich, Germany
†Neurosurgical Clinic, Department of Stereotactical and Functional Neurosurgery, Albert-Ludwigs-University Freiburg, Freiburg, Germany
‡Neurological Clinic, Albert-Ludwigs-University Freiburg, Freiburg, Germany
§The Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK
¶Neurological Clinic, University Hospital Giessen-Marburg, Marburg, Germany
Different animal models of Parkinson's disease (PD) are used in order to develop and evaluate new therapies including, for example, deep brain stimulation and stem cell transplantation. Terminal 6-OHDA lesions of the rat striatum represent more closely early and middle stages of human idiopathic PD, whereas the medial forebrain bundle (MFB) lesion corresponds to a more complete destruction of the dopaminergic nigrostriatal pathway seen in late PD stages. In the present study both 6-OHDA lesion models were compared. We investigated the impact of motor training prior to the lesion on functional recovery and morphological pathway destruction after unilateral MFB-lesion versus striatal lesions in a rat model of Parkinson's disease. The experiment comprised seven groups (n = 12 rats each): MFB- and terminal-lesioned rats with or without prelesion training and respective control groups. Complex behavioral performance was investigated immediately after and 1, 2, and 4 months postlesion. Finally, tyrosine hydroxylase (TH) immunohistochemistry and dopamine measurements using HPLC were performed to confirm the extent of the lesion and to analyze the dependency on behavior. Rats with MFB lesion display both apomorphine-induced [–6.8 turns per minute (tpm)] and amphetamine-induced (+7.8 tpm) rotations while the partial lesioned groups elicited amphetamine-induced (+9.4 tpm) rotations only 4 months postlesion. The table lift test showed that trained animals with MFB lesions show better behavioral levels 4 months postlesion compared to untrained animals (p < 0.01), whereas the training levels had no influence in partial lesioned animals. The outcome in the latter behavioral test was dependent on the amount of dopamine measured in the medial caudate putamen unit. Other statistically significant differences included a more pronounced deficit in skilled forelimb use for the untrained rats (2.9 pellets eaten) compared to the pretrained rats (16 pellets eaten) in the groups with MFB lesions (p < 0.001), as well as generally more pronounced deficits in the MFB-lesioned groups compared to the terminal lesioned groups. The degree of sensorimotor deficits is highly correlated with the extent of nigrostriatal pathway destruction. In addition, prelesion motor training can significantly improve postlesion recovery. These data may provide further insights into the functional organization of the basal ganglia and for the optimization of neuroprotective and restorative treatment strategies.
T. Vazin*, K. G. Becker†, J. Chen*, Y. Zhang†, L. Worden*, and W. J. Freed*
*Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, DHHS, Baltimore, MD, USA
†Gene Expression and Genomics Unit, Intramural Research Program, National Institute on Aging, National Institutes of Health, DHHS, Baltimore, MD, USA
Stromal-derived inducing activity (SDIA) refers to the property of mouse stromal cell lines, such as PA6 or MS5, of inducing embryonic stem cells (ESC) to differentiate into dopaminergic (DA) neurons. The potential for clinical application of SDIA is, however, limited due to the use of animal cells, and the molecular nature of SDIA is unknown. We recently reported that PA6 cell surface activity promotes cell survival and general neurogenesis from human ESC (hESC), whereas secreted factors provide lineage-specific instructions and DA-promoting activity (Vazin et al., 2008). In order to identify the chemical factors responsible for SDIA, we carried out gene expression profiling of PA6 cells, and compared the potent PA6 cell line (PA6-DA) to subtypes of PA6 cells (PA6-X1, PA6-X), and to three other cell lines that lack SDIA. Among the factors differentially expressed by PA6 cells were stromal cell-derived factor 1 (SDF-1/CXCL12), pleiotrophin (PTN), insulin-like growth factor 2 (IGF2), and ephrin B1 (EFNB1). The combination of these factors was termed SPIE. In vitro functional analysis of candidate molecules on DA differentiation of BG01V2, BG02, and BG03 hESC lines was carried out following a brief (2–4 day) embryoid body (EB) formation phase. After three additional days of culture under adherent conditions in the presence of SPIE, EBs developed into neuroectodermal cells with a clear neuronal morphology arranged in rosette-like structures. After 10 days, the majority of colonies differentiated from BG01V2- and BG03-derived EBs contained large numbers of cells expressing the midbrain neural progenitor cell (NPC) marker Msx1, and tyrosine hydroxylase (TH). β-III-Tubulin and TH colabeling also revealed TH expression in the majority of neuronal cells. In contrast, when BG02 cells were differentiated under the same conditions, they did not develop into Msx1+ NPC. TH+ neurons were, however, present in the BG02-derived cultures. Characterization of the separate role of each individual factor in DA induction indicated that IGF2-enhanced survival of the proliferating NPC, whereas SDF-1 and EFNB-1 were required for DA induction of hESC. PTN increased the yield of TH+ neurons. In summary, identification of a novel combination of four factors, SDF-1, PTN, IGF2, and EFNB1, which we have termed SPIE, provides a new and efficient approach to generation of DA neurons from hESC under chemically defined conditions, free of xenogenic cells or material. Future investigations of the biological effect and mechanism of action of these factors is likely to provide insights into novel pathways controlling DA development from hESC, ultimately contributing to the advancement of cellular replacement strategies for treatment of PD.
Research supported by the IRPs of NIDA and NIA, NIH, DHHS.
T. M. Vorobyeva, O. G. Berchenko, V. V. Gejko, E. O. Storchak, A. M. Titkova, and S. P. Kolyadko
Department of Neurophysiology and Immunology, Institute of Neurology, Psychiatry, and Narcology, Ukrainian Academy of Medical Science, Kharkiv, Ukraine
In the present study, the effects of transplanting human-derived bone marrow stromal cells or rat-derived embryonic neuronal tissues into rats modeling multiple sclerosis were investigated. Human bone marrow stromal cells, with or without previous retinoic treatment, were transplanted into the periventricular area of the rat brain. Rat embryonic neuronal tissues (16–17 ED) were implanted subcutaneously or into the cerebral sensorimotor cortex. Results showed that bone marrow stromal cells without retinoic treatment stimulate movement activity in the short term, failing to reestablish muscle tone. In contrast, retinoic-treated bone marrow stromal cells restore muscle function in accordance with the absence of proliferative processes in immunocompetent organs. Additionally, activated humoral immunity, normalized phospholipid levels correlating with high levels of myelin in blood plasma, and inflammatory reactions in demyelinated white matter areas of the brain were noted. Transplantation of embryonic neuronal tissues into the sensorimotor cortex restored movement activity in 50% of treated animals. Mechanisms of nonspecific immune resistance were intensified with decreased autoimmune processes. Levels of phospholipids and myelin in plasma were normalized. Decreased inflammatory reactions were accompanied by remyelinization in white matter areas of the brain and spinal cord. When embryonic neuronal tissue was implanted subcutaneously, animal movement activity was also improved. This effect was characterized by normalized phagocitic and humoral mechanisms, decreased lymphocytes and eosinophils, and restored levels of phospholipids, cholesterol, myelin, and TNF-α. Interestingly, myelin was restored in various brain structures such as the white matter of the neocortex, limbic cortex, hippocampus, and striatum of treated animals. Thus, our results show specific effects of different transplant routes and types of administered cells or tissues into a rat model of multiple sclerosis. Implantation of embryonic neuronal tissue was most beneficial in regards to the normalized immune system response and restored myelin.
D. R. Wakeman*†, D. E. Redmond, Jr.‡, J. R. Sladek, Jr.§, and E. Y. Snyder*†
*Biomedical Sciences, University of California, San Diego, La Jolla, CA, USA
†Burnham Institute for Medical Research, La Jolla, CA, USA
‡Psychiatry and Neurosurgery, Yale University, New Haven, CT, USA
§Pediatrics, University of Colorado, Aurora, CO, USA
We and others have shown that increased glial cell-derived neurotrophic factor (GDNF) expression increases the survival of fetal ventral mesencephalon (VM) grafts by several fold as well as eliciting highly directional axonal outgrowth. Furthermore, recent data indicate that GDNF can act as a chemoattractant in stem cell migration in the brain as well as being absolutely essential for the survival of DA neurons. Previously, we demonstrated that human neural stem cells engraft, migrate, and provide functional improvements in behavioral deficits when transplanted into the MPTP-lesioned African green monkey. A very small portion of engrafted cells differentiated into tyrosine hydroxylase (TH+) cells with appropriate morphology within the substantia nigra (SN), but there was no evidence that these cells made any connection with their targets in the striatum. The aim of the present study was to determine whether overexpression of GDNF, delivered by a viral vector into the striatum and human neural stem cells (hNSCs) injected into the SN, could enhance survival, integration, differentiation, and elicit directional neuritic outgrowth of donor-derived cells from the SN to the caudate nucleus and provide evidence of these projections using a retrograde tracer, fluorogold. Ten adult male St. Kitts green monkeys received AAV-5 GDNF unilaterally into two target areas in the striatum (anterior caudate and putamen) and hNSC grafts unilaterally into the ipsilateral SN. Six animals received hNSC expressing enhanced-GFP (hNSCeGFP), two received unmodified wild-type hNSC (hNSCWT), and two received killed hNSCeGFP controls (hNSCKill). Animals were sacrificed after 1.5 months (short term) (1 hNSCWT, 1 hNSCeGFP) or 10 months (long term) after grafting (1 hNSCWT, 5 hNSCeGFP, 2 hNSCKill). Ten days prior to sacrifice, previous striatal targets were injected bilaterally with FluorGold retrograde tracer to determine the extent of donor-derived striatal integration in long-term 10-month animals (the remaining 1 hNSCWT animal was not injected as a control for fluorogold toxicity). After 1.5 months (short term), we found engrafted, bromodeoxyuridine-positive (BrdU+) donor cells distributed throughout the ipsilateral SN, confirming our previous studies. In addition, we also confirmed the expression of AAV-5 GDNF throughout the striatum, with highest expression within the lower needle tract, emanating and diffusing outward from the injection points throughout the striatum. Host striatal cells highly expressing GDNF had the characteristic morphology of stellate astrocytes, the site of endogenous GDNF production, suggesting a more “natural” site of production, processing, and secretion. In addition, GDNF staining was also apparent within the SN, suggesting retrograde transport of GDNF from the striatum to the SN. These data provide evidence that grafted hNSC can engraft and survive in the MPTP-lesioned SN while their distant striatal target is releasing an elevated concentration of GDNF. Data from the remaining animals will be presented at the meeting.
P. Walczak*†, M. Gorelik†‡, M. Levy†‡, N. Rumpal†‡, R. Rifkin†‡, N. Muja*†, H. Kim*†, D. A. Kerr†‡, and J. W. M. Bulte*†
*Radiology, Johns Hopkins University, Baltimore, MD, USA
†Institute for Cell Engineering, Johns Hopkins University, Baltimore, MD, USA
‡Neurology, Johns Hopkins University, Baltimore, MD, USA
Therapeutic intervention for neurodegeneration remains one of the major challenges for modern medicine. One promising approach to replace dead or defective endogenous neural cells is the administration of exogenous progenitors. In the last decades, we have witnessed unprecedented effort by scientists and clinicians to harness the potential of stem cells for the treatment of neurological diseases. Due to the complexity of the central nervous system, as well as limited methodological capabilities, progress in this field was not as rapid as was hoped. Cell therapy, particularly in adult recipients, introduces the challenge of providing guidance for the graft to appropriately migrate, differentiate, and functionally integrate. Before such guidance can be considered, there is a need for the tools that will enable in vivo, realtime monitoring of the status of transplanted cells. Taking advantage of recent developments in molecular biology and imaging, we have developed a system for monitoring the delivery and status of transplanted cells noninvasively. We are centering our studies on the preclinical evaluation of the therapeutic potential of glial-restricted precursor cells (GRPs) for the treatment of myelin diseases. We use high-resolution magnetic resonance cellular imaging to monitor the delivery and acquire information about the position of grafted cells. Reporter gene-based bioluminescent imaging allows us to monitor survival and differentiation. Here, we report on two different approaches and applications for the in vivo imaging of GRPs transplanted into the rodent brain. Approach I: Noninvasive monitoring of the targeted, intravascular cerebral delivery of GRPs. Cells were engineered to express α4 and β1 subunits of VLA-4 integrin (to enhance vascular adhesion) and were labeled with the magnetic resonance (MR) contrast agent Feridex. Recipient rats were injected IP with lipopolysaccharide (LPS), a known inducer of endothelial VCAM-1 expression, and the cells were infused into the carotid artery. MR imaging demonstrated extensive hypointense regions, in the brain, indicating successful targeting. Approach II: Noninvasive monitoring of the survival and differentiation of intracerebrally injected GRPs. Cells were engineered to express the bioluminescent reporter, luciferase, under the control of a constitutive cytomegalovirus (CMV) promoter or the cell type-specific promoters, glial fibrillary acidic protein (GFAP) and cyclic neucleotide phosphodiesterase (CNPase). Cells were stereotactically injected into the brain of immunodeficient (Rag2) or immunocompetent mice. Bioluminescence imaging demonstrated that transplanted GRPs survived for extended periods of time in immunodeficient animals, while, in immunocompetent animals, rejection was initiated 2 weeks after grafting. With cell type-specific promoters, we were able to visualize the process of cell differentiation without the need to sacrifice the animals. In conclusion, we have demonstrated that noninvasive cellular imaging can be used to monitor and guide cell transplantation-based therapies for myelin disorders and can be adapted for other brain disease models.
Supported by: NMSS-PP1491, MSCRF-104062, MSCRF-07062901 and RO1DA026299.
L. Wang*¶, J. Shi†¶, F. W. van Ginkel*§, L. Lan†, G. Niemeyer*, D. R. Martin*, E. Y. Snyder‡, and N. R. Cox*
*Scott-Ritchey Research Center, College of Veterinary Medicine, Auburn University, Auburn, AL, USA
†Department of Anatomy, Physiology & Pharmacology, College of Veterinary Medicine, Auburn University, Auburn, AL, USA
‡Burnham Institute for Medical Research, La Jolla, CA, USA
§Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, AL, USA
¶Department of Anatomy and Physiology, Kansas State University, Manhattan, KS, USA
We and others have reported that neural stem/progenitor cells (NSCs) may exert direct anti-inflammatory activity. This action has been attributed, in part, to T-cell suppression. However, how T cells become suppressed by NSCs remains unresolved. In this study, we explored one of these mechanisms and challenged some previously advanced hypotheses regarding underlying NSC-mediated T-cell suppression. We employed an easily observable and manipulatable system in which activated and nonactivated T cells were cocultured with a stable well-characterized clone of lacZ-expressing murine NSCs. As in previous reports, NSCs were found to inhibit T-cell proliferation. However, this inhibition by NSCs was not due to suppression of T-cell activation or induction of apoptosis of T cells during the early activation stage. High levels of nitric oxide (NO) and prostaglandin E2 (PGE2) were induced in the T cells when cocultured with NSCs. In addition, inducible NOS (iNOS) and microsomal type 1 PGES (mPGES-1) were readily detected in NSCs in coculture with T cells, but not at all in NSCs cultured alone or in activated T cells cultured with or without NSCs. This finding suggested that activated T cells induced NO and PGE2 production in the NSCs. Furthermore, T-cell proliferation inhibited by coculture with the NSCs was significantly restored by inhibitors of NO and PGE2 production. Therefore, NSCs appear to suppress T cells, at least in part, by NO and PGE2 production, which, in turn, would account for the well-documented reduction of central nervous system immunopathology by transplanted NSCs.
T. A. Womble*†, S. M. Green*†, A. P. Nelson*†, M. D. Shahaduzzaman*†, J. E. Golden*‡, P. R. Sanberg*†‡, K. R. Pennypacker*‡, and A. E. Willing*†‡
*Center of Excellence for Aging and Brain Repair, University of South Florida, Tampa, FL, USA
†Department of Neurosurgery, University of South Florida, Tampa, FL, USA
‡Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
When human umbilical cord blood (HUCB) cells are administered following middle cerebral artery occlusion (MCAO) in the rat, they produce a reduction in infarct volume leading to motor function recovery. The HUCB cell preparation is a mixed population composed of immature T cells (CD2), B cells (CD19), monocytes, macrophage (CD14), and stem cells (CD133). In this study we depleted or enriched CD14, CD133, CD2, or CD19 from the mononuclear fraction of HUCB to examine whether the beneficial effects of HUCB injection following MCAO can be attributed to a specific cell population, or a combination of populations. Male Sprague-Dawley rats underwent permanent MCAO followed 48 h later by IV administration of HUCB preparations from which selected cell populations had been depleted or enriched. Thirty days poststroke, a battery of behavioral tests was performed to assess functional recovery. In the depletion phase of the study we observed in the step test for motor asymmetry that the whole mononuclear fraction of cord blood, T cells, B cells, monocytes/macrophage, and stem cells, improved functional recovery of the impaired left forelimb when compared to the control MCAO-only animals. Animals that received the monocyte/macrophage-depleted HUCB preparation performed more poorly than those that received the other depleted HUCB fractions. In the behavioral test of spontaneous activity, HUCB significantly reduced activity compared to the hyperactive MCAO-lesioned animals. There was also a decrease in infarct volume in the HUCB cell treatment compared to the MCAO only. We also observed that depletion of stem cells (CD133) and monocytes and macrophages (CD14) resulted in infarct volumes and motor function comparable to that of the MCAO controls. Immunocytochemistry revealed an increase in the ipsilateral expression of microglia within the striatum area, compared to the MCAO only. In the enrichment phase of the study, we expect that animals receiving CD14+ and CD133+ HUCB cells should have smaller infarcts and better recovery than the MCAO-only animals, although an interaction of the two cell types may be necessary for optimal recovery. Assessment of the enriched fraction of HUCB in MCAO is currently under way.
Supported by NINDS RO1NS52839 (A.E.W.). A.E.W. is a consultant and P.R.S. a cofounder of Saneron CCEL Therapeutics, Inc. (SCT); A.E.W. and P.R.S. are inventors on cord blood patents licensed to SCT.
R. L. Woods, III*, M. K. Louis*, T. Zesiewicz#, Y. Xie*, K. L. Sullivan#, A. M. Miller#, N. Kuzmin-Nichols**, P. R. Sanberg*†‡§¶, and S. Garbuzova-Davis*†‡§
*Center of Excellence for Aging and Brain Repair, University of South Florida College of Medicine, Tampa, FL, USA
†Department of Neurosurgery, 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 College of Medicine, Tampa, FL, USA
#Department of Neurology, University of South Florida College of Medicine, Tampa, FL, USA
**Saneron CCEL Therapeutics, Inc., Tampa, FL, USA
Amyotrophic lateral sclerosis (ALS) is a fatal neurodegenerative disease characterized by motor neuron degeneration and death in the brain and spinal cord. Recent studies (Garbuzova-Davis et al., 2007, 2008) showed disruption of the blood-brain/spinal cord barrier (BBSCB) in the G93A mouse model of ALS might occur due to degenerating capillary endothelial cells followed by vascular leakage. Although BBSCB damage in ALS patients is still unconfirmed, it is possible to suggest endothelium impairment and/or dysfunction in endothelial cell replacement could be taking place in ALS. The aim of this study was to determine levels of circulating endothelial cells (CECs) in the peripheral blood of ALS patients at different stages of disease. Immunohistochemical analysis using CD146 identified CECs in blood smears from ALS patients (n = 11) and healthy controls (n = 5). CEC percentages were determined from the ratio of counted CECs to total mononuclear cells in each blood smear. Our results showed a significant (p < 0.05) reduction of CECs in peripheral blood of ALS patients with moderate (2.9 ± 1.2%) or severe (1.5 ± 0.4%) disease compared to controls (7.4 ± 1.1%). There was a strong positive correlation (r = 0.84) between measured CECs and patient ALSFRS-R (ALS Functional Rating Scale Revised) scores. Thus, the reduction of CECs in the peripheral blood of ALS patients suggests impairment of endothelial cell replacement during the course of disease, possibly leading to BBSCB dysfunction in ALS.
Supported in part by the Muscular Dystrophy Association. S.G.-D. is a consultant and P.R.S. is a cofounder of Saneron CCEL Therapeutics, Inc.
L. T. Worden, C.-T. Lee, and W. J. Freed
Cellular Neurobiology Research Branch, Intramural Research Program, National Institute on Drug Abuse, National Institutes of Health, DHHS, Baltimore, MD, USA
Human embryonic stem cells (hESC) have great potential as a therapeutic and scientific tool; however, chromosomal abnormalities often arise when hESC are cultured for extended periods. We have observed that BG01V2, a hESC line with a karyotype abnormality (trisomy 17), has a heightened ability to differentiate to mature dopaminergic cells using a stromal cell coculture method, compared to other karyotypically normal lines (unpublished data). Here we examine the differences between three normal lines, BG01, BG02, and BG03, and the abnormal line BG01V2, in their ability to differentiate to midbrain dopamine neurons using a chemically defined, feeder-free system. The differentiation method was modified from that reported by Yan et al. (2005). In brief, isolated undifferentiated hESC colonies were cultured in the presence of fibroblast growth factor-2 (FGF2) and fibroblast growth factor-8 (FGF8) to yield neuroepithelial (NE) cells exhibiting neural tube-like rosettes after 14 days. Rosettes were then isolated and grown in the presence of FGF8 and sonic hedgehog (SHH) to yield neural progenitor (NP) cells, and finally mature dopaminergic neurons after a total of 45 days. At the NE cell stage, nestin+ rosettes developed in a ring encircling the center of the colonies in BG01 and BG02 lines. In contrast, BG03 and BG01V2 cells differentiated and formed a monolayer of nestin+ neuroepithelial cells mixed with randomly distributed small rosettes. The speed of differentiation for BG03 and BG01V2 was also faster than the other lines, as there was evidence of MSX1+ and TH+ cells at the NE cell stage. BG01 and BG02 NP cells reformed MSX1+ rosettes when seeded for the final differentiation after 24 days. Rosettes did not re-form in the BG03 or BG01V2 lines, but MSX1+ cells were present, as well as TuJ1+ neurons. The yield of mature TuJ1+/TH+ dopamine neurons was similar for the BG01 and BG02 lines, greatest in the abnormal BG01V2 line, and intermediate in the BG03 line. Characterization of NE, NP, and mature TH+ cells showed few differences between the BG01 and BG02 stem cell lines in terms of speed of progression, number of rosettes formed, or yield of TH+ neurons. The abnormal line BG01V2 displayed an enhanced degree and speed of differentiation relative to all the normal lines, while the speed and degree of differentiation of the BG03 line was intermediate between the other normal cell lines and BG01V2. Therefore, the ability of stem cells to differentiate is influenced by karyotypic abnormality, as well as by other genetic differences between hESC lines not manifest in karyotype analysis.
Research supported by the IRP of NIDA, NIH, DHHS.
S. Yu*, T. Hayashi*, Y. Kaneko*, E. Bae*, C. E. Stahl†, Y. Wang‡, and C. V. Borlongan*
*Center for Aging and Brain Repair, Department of Neurosurgery, University of South Florida, Tampa, FL, USA
†Department of Internal Medicine, Dwight D. Eisenhower Army Medical Center, Augusta, USA
‡Neural Protection and Regeneration Section, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA
The clinical presentation of traumatic brain injury (TBI) involves either mild, moderate, or severe injury to the head resulting in long-term and even permanent disability. The recapitulation of this clinical scenario in animal models should allow examination of the pathophysiology of the disease and its treatment. To date, only a few studies have demonstrated TBI animal models encompassing the three levels of disease severity, and all these previous reports have focused on the resulting brain damage without characterization of behavioral deficits. Thus, in the present study we investigated in rats and mice both brain histopathologic and behavioral alterations arising from mild, moderate, and severe TBI produced by controlled cortical impact injury technique. Here, we replicated the previously observed TBI severity-dependent brain damage as revealed by 2,3,5-triphenyltetrazolium chloride staining (severe > moderate > mild) in rats, but also extended this pattern of histopathologic changes in mice. Moreover, for the first time we showed severity-dependent abnormalities in locomotor and cognitive behaviors in TBI-exposed rats and mice. Next, we used the severe TBI rat model as an initial step to examine the potential of cell therapy. Immediately after TBI surgery, 4 million rat bone marrow stromal cells (BMSCs), lentivirally labeled with green fluorescent protein, were intravenously (i.e., jugular vein) administered. Fluorescent microscopy revealed BMSCs migrated to, but did not express a neural phenotype [using microtubule-associated protein 2 (MAP2), glial fibrillary acidic protein (GFAP), oligodendrocyte (O4) antibodies] within the lesion boundary of the TBI-damaged cortex at 3 days posttransplantation. At the present route, dose, and timing of BMSC transplantation, no improvement in behavioral recovery was detected. Taken together, these results support the use of rodent models of TBI as a sensitive platform for investigations of the disease-induced neurostructural and behavioral deficits, which should serve as key outcome parameters for testing experimental therapeutics, such as cell therapy.
Y. Zhu*, D. Oregon*, H. Hou*, D. Luo*, T. Mori*, B. Giunta*†, Y. Zhao‡, 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
‡Department of Pathology and Laboratory Medicine, School of Medicine, University of Pennsylvania, Philadelphia, PA, USA
§Departments 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
Mutations in the presenilin-1 (PS1) gene are causally linked to familial early-onset Alzheimer's disease (AD). Previous studies have shown alterations in immune function in AD patients, raising the possibility that PS1 plays a role in regulation of immunity. In support of this, mutant human PS1 (both M146V and PSEN1dE9) mice exhibit abnormal T- and B-cell immune responses. To test whether PS1-associated abnormality in immune function could modify AD-like pathology, we reconstituted the immune system with bone marrow cells (BMC) in two different AD mouse models: PSAPP [bearing both mutant human APP (K670N, M671L) and mutant human PS1 (PSEN1dE9) transgenes] and Tg2576 [carrying mutant APP (K670N, M671L) alone]. Here, we report a marked reduction of Aβ levels/β-amyloid plaques and associated inflammation in PSAPP mice following strain-matched wild-type bone marrow infusion. Strikingly, Tg2576 mice showed accelerated AD-like pathology following PS1 mutant mouse-derived BMC infusion. Most importantly, these effects were associated with altered markers of immune function. These data show that hematopoetic cells bearing the mutant human PS1 transgene, an independent genetic cause of familial AD, exacerbate AD-like pathology in AD mouse models, suggesting a novel immunomodulatory therapeutic strategy for AD.