Abstract
J. F. Abisambra,* U. Jinwal,† J. C. O'Leary, III,* L. Blair,* L. Wang,* K. Voss,* and C. A. Dickey*
*Department of Molecular Medicine, Byrd Alzheimer's Research Institute, University of South Florida, Tampa, FL, USA
†College of Pharmacy, Byrd Alzheimer's Research Institute, University of South Florida, Tampa, FL, USA
Specificity in the chaperone system is dictated by members of the DnaJ/Hsp40 protein family, which is the largest and most functionally diverse family of chaperones. We have preliminary evidence that select DnaJ/Hsp40 family members can distinguish between normal and abnormal forms of the microtubule associated protein tau. Excessive accumulation of tau that is abnormally hyperphosphorylated and proteolytically cleaved in neurons is a pathological hallmark of Alzheimer's disease, as well as 15 other neurodegenerative diseases termed tauopathies. Accumulation of these abnormal tau species is thought to be one of the key mediators of neuronal dysfunction and death. Thus, the ability to manipulate the fate of tau in the neuron would be a significant step forward in understanding the mechanisms of tau quality control. It is likely that the signaling that leads to the preservation or degradation of tau in the brain is linked to the chaperone network through the DnaJ protein family, which binds specifically and localizes tau to the Hsp70 complex. Therefore, this family of “specialist” chaperones represents an important suite of modulators for better understanding the mechanisms of tau biology. Moreover, there is already evidence that the DnaJ family of proteins does have important effects on neuronal function and pathologic accumulation of proteins. We hypothesize that select DnaJ proteins can serve a protective role in Alzheimer's disease by selectively recognizing and delivering aberrant tau for degradation, and that this activity could be manipulated to tune tau fate. We have identified two DnaJ proteins that appear to specifically destabilize distinct tau species. These proteins can target tau through different routes of degradation and they can regulate distinct aberrant species of tau resulting from posttranslational modifications. They also have distinct requirements of Hsp70 in controlling their efficacy towards tau. We predict that we can control tau triage by manipulating discrete DnaJ proteins. These studies have begun to clarify how the chaperone system selects client subsets, paving the way for future structural investigations.
A. R. Bailey,* H. Hou,* D. F. Oregon,* J. Tian,* Y. Zhu,* Q. Zou,* W. V. Nikolic,† M. Bengtson,* T. Mori,‡ T. Murphy,§ and J. Tan*
*Rashid Laboratory for Developmental Neurobiology, Silver Child Development Center, Department of Psychiatry & Behavioral Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL, USA
†Department of Genetics, Faculty of Medicine, University of Kragujevac, Svetozara, Kragujevac, Serbia
‡Departments of Biomedical Sciences and Pathology, Saitama Medical Center and Saitama Medical University, Kawagoe, Saitama, Japan
§Rothman Center for Neuropsychiatry, Department of Pediatrics, College of Medicine, University of South Florida, All Children's Hospital, St. Petersburg, FL, USA
Our goal with this study was to identify any abnormalities in T-lymphocyte development and function that may be associated with overexpression of secreted amyloid precursor protein-α (sAPP-α). Elevated levels of sAPP-α have recently been observed in autistic patient plasma and there are reports of T-lymphocyte abnormalities in autistic patients as well. We designed and generated transgenic mice that overexpress sAPP-α in the brain and plasma, and other organs. Brain and plasma levels of sAPP-α, as well as splenocyte-secreted cytokine levels, were measured by ELISA. We used flow cytometry to investigate precursor and mature T-cell populations in the thymus and spleen. Lastly, molecular biology and immunohistochemistry techniques were used to observe apoptosis in transgenic mouse thymus and zeta-chain-associated protein kinase 70 (ZAP-70) expression in the spleen. We found that splenocytes from transgenic mice secreted increased levels of proinflammatory cytokines interferon-γ (IFN-γ), interleukin-2 (IL-2), and IL-4 after T-cell mitogen stimulation. The mice exhibit increased CD8+ T-cell populations in the spleen and abnormal precursor cell populations in the thymus. Apoptotic signaling proteins are expressed at greater levels in sAPP-α mouse thymus. Finally, after secondary immune challenge, splenocytes from sAPP-α mice produced reduced levels of proinflammatory cytokines and decreased ZAP-70 expression. Given our findings, we conclude that high expression of sAPP-α is associated with abnormal T-lymphocyte populations, development, and function. This may explain the T-lymphocyte abnormalities seen in autism patients.
This work was supported by the Silver Foundation.
W. A. Banks
Veterans Affairs Puget Sound Health Care System, Division of Gerontology and Geriatric Medicine, Department of Medicine, University of Washington School of Medicine, Seattle, WA, USA
The vascular blood–brain barrier (BBB) exists because the capillary bed of the central nervous system (CNS) is specially modified to prevent the uncontrolled leakage of material from blood to brain. The BBB is also endowed with many characteristics that allow it to function as a regulatory interface between the CNS and blood so that it influences brain nutrition and homeostasis and participates in a humoral-based brain–body communication. Three aspects in which the BBB is relevant to inflammatory–neurodegenerative interactions are explored in this lecture. First, the BBB controls the ability of drugs, including those relevant to the treatment of neurodegenerative diseases, to enter the brain. For example, many small, lipid-soluble molecules penetrate the BBB by way of transmembrane diffusion. Brain-to-blood efflux systems limit the ability of many otherwise promising drug candidates from entering the CNS. One such efflux system is P-glycoprotein. However, P-glycoprotein activity can be decreased with activation of the innate immune system, thus allowing entry of drugs into the brain that would be otherwise excluded. Likewise, tumor necrosis factor (TNF)-mediated upregulation of P-glycoprotein could lead to less drug entering the brain. Second, the BBB itself is an immune tissue in that it secretes immunoactive agents such as cytokines, prostaglandins, and nitric oxide. We have demonstrated that both brain endothelial cells and their companion pericytes respond to lipopolysaccharide by secreting cytokines. Secretion of interleukin-6 (IL-6) and granulocyte macrophage colony stimulating factor (GM-CSF) from brain endothelial cells act in a paracrine fashion to enhance the penetration of the AIDS virus HIV-1 across the BBB. Third, the BBB is a target for the immune system. For example, efflux systems relevant to neurodegenerative diseases such as Alzheimer's disease (AD) are altered by immune activation. Low-density lipoprotein receptor-related protein 1 (LRP-1) at the BBB pumps amyloid beta peptide (ABP) out of the brain. According to the neurovascular hypothesis, LRP-1 activity is deficient in patients with AD and in animal models of AD. Antisense knockdown of LRP-1 leads to decreased efflux of ABP, increased brain levels of ABP, and cognitive impairments. LRP-1 itself is increasingly oxidized in the brains of patients with AD. Lipopolysaccharide (LPS) decreases ABP efflux from brain and the antioxidant N-acetylcysteine reverses this effect. This suggests that neuroinflammation could result in LRP-1 oxidation and increased ABP retention, thus fostering the progression of AD. In these and other ways, the BBB interacts with neuroinflammation to affect the course of neurodegenerative diseases.
P. C. Bickford*†
*James A. Haley Veterans' Administration Hospital Research Service, Tampa, FL, USA
†Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Changes in innate immune function are early and critical events in brain aging. Importantly, neurodegenerative diseases emerge and are exacerbated with this context. Functional changes in microglial phenotype can lead to dysregulation of proinflammatory molecules that initiates neuronal dysfunction and reduced synaptic plasticity in the aged brain. Microglia in young and nondiseased brain are constantly surveying the environment for “danger” signals. In this so-called “resting” phenotype, microglia are not static, but quite active. Neurons produce signaling molecules [e.g., CX3CL1 (fractalkine)] that turn off production of induced nitric oxide synthase (iNOS), interleukin-1β (IL-1β), tumor necrosis factor-α (TNF-α) among other proinflammatory molecules. With aging, we and others have demonstrated that there is a decrease in the production of these “calming” signals, which leads to a disrupted regulation of microglia in the brain. Additional changes with age in the peripheral immune system also influence the brain as serum proteins and peripheral immune cells communicate with the brain, and may also contribute to the disruption of “resting” microglial phenotype. These extrinsic factors have been shown to have a negative impact on the brain and the neurogenic niche. Over the years we have examined a number of ways to modulate the innate immune system including stem cells, nutritional supplements, and chemokine treatment.
L. S. J. Breger,*† S. B. Dunnett,† and E. L. Lane*
*School of Pharmacy & Pharmaceutical Sciences, Cardiff University, Cardiff, UK
†Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK
The dopamine precursor L-dopa can alleviate the motor dysfunction associated with the degenerative condition Parkinson's disease (PD) but unfortunately long-term treatment is associated with major side effects: patients develop abnormal involuntary movements (AIM) called dyskinesia. An alternative approach, currently under investigation in a new clinical trial, is the replacement of the lost striatal dopaminergic innervation by transplantation of fetal ventral mesencephalon (VM) dopaminergic precursor cells. Despite showing benefits in some patients, this technique has been associated with the appearance of motor complications following transplantation in the absence of drug treatment, so-called graft-induced dyskinesia (GID). The causes of this variability and side effects in cell therapy efficacy are still to be determined. Patients entering clinical trials are typically at an advanced stage of the disease; they have been under L-dopa treatment for some time and remain under medication after transplantation. Studies have previously looked at the role of L-dopa on transplanted neuron survival but only in syngeneic models, while patients will receive donor tissue from allogeneic sources. Additionally previous work has not considered the role of prior exposure to L-dopa versus posttransplantation administration only. In this study, we investigated the effect that chronic L-dopa treatment has on the survival and the function of the graft. 6-OHDA-lesioned rats were treated daily with either saline or L-dopa (10 mg/kg, coadministrated with 15 mg/kg benserazide) for the 8 weeks prior to and/or the 8 weeks following transplantation of ventral mesencephalon (either rat e14 or mouse e12). The animals were assessed for functional recovery and dyskinesia at regular intervals. No spontaneous GID was observed “off” drug in any group. The different treatment regimes did not affect functional recovery but influenced the AIM score when the animals were assessed posttransplantation following L-dopa administration. We show that L-dopa treatment is not detrimental to the survival and function of the graft and may even be beneficial. Histological analysis will examine the consequences of L-dopa administration on graft integrity and be related to animals' performances on behavioral assessments. This finding is of significant clinical relevance, as questions remain about the selection of patients for transplantation, L-dopa administration may be key to the anticipated outcome.
M. Brownlow,*† L. Benner,*† D. D'Agostino,*† M. N. Gordon,*† and D. Morgan*†
*Byrd Alzheimer's Research Institute, University of South Florida, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
Interest is emerging in the involvement of dietary manipulations and their pharmacological outcomes in neurodegenerative diseases. Recent studies suggest that Alzheimer's disease (AD) patients display an energy imbalance with brain hypometabolism and/or mitochondrial deficits. Ketogenic diets (KD), widely investigated in the treatment and prevention of seizures, have been suggested to bypass metabolic deficits present in AD brains by providing ketone bodies as an alternative fuel. We investigated the effects of a ketogenic diet on two mouse models of AD. Five-month-old amyloid precursor protein/presenilin 1 (APP/PS1) and Tg4510 mice were kept on either a KD or a control (NIH-31) diet for 3 months, and then submitted to a battery of behavioral tests. Body weight and food intake were monitored throughout the study; blood was collected at 4 weeks and at the end of the study for ketone and glucose assessments. Both APP/PS1 and Tg4510 mice weigh less than nontransgenic mice, despite an increased food intake. Interestingly, the ketogenic diet did not affect these differences in body weight or food consumption. We found that both mouse models of AD presented hyperactivity, compared to nontransgenic, age-matched controls, and that this effect was not prevented by KD. This was measured by both open field and y-maze tests. Mice kept on KD performed significantly better on an endurance trial in the rotarod compared to mice on the control diet. Only a genotype effect was observed in the radial arm water maze test with no significant differences between diets. Brain tissue was collected at the end of behavioral testing, and analysis of amyloid, tau, and microglial markers is ongoing. These initial data suggest that the metabolic rate of the transgenic mice is considerably greater than the nontransgenic mice and that ketogenic diet may play an important role in motor performance in mice.
Research supported by IIRG-10-174448.
S. A. Busch,* M. DePaul,† J. A. Hamilton,* N. Lehman,* R. Cutrone,* R. J. Deans,* A. E. Ting,* R. W. Mays*, and J. Silver†
*Athersys, Inc., Department of Regenerative Medicine, Cleveland, OH, USA
†Case Western Reserve University, Department of Neurosciences, Cleveland, OH, USA
Adult adherent stem cells are known to have immunomodulatory capabilities, but their potential to alter inflammatory processes and promote regeneration after spinal cord injury has yet to be fully elucidated. We have developed an in vitro model in which adult rat dorsal root ganglion (DRG) axons are confronted with a gradient of increasing inhibitory proteoglycan and decreasing laminin. Growth cones in this environment develop a characteristic dystrophic morphology and, when contacted by activated macrophages, undergo dramatic axonal dieback. In this study, we sought to determine if culturing DRG neurons with rat multipotent adult progenitor cells (MAPCs) or MAPC-conditioned media (MAPC-CM) could prevent macrophage-mediated axonal dieback. In the presence of MAPCs or MAPC-CM dystrophic axons became active and extend. Macrophages contacted these axons extensively, but axonal retraction was prevented. We extended these findings to human MAPCs, or MultiStem® cells, and determined that they were also able to prevent macrophage-mediated axonal dieback. We next sought to determine if MAPCs could prevent axonal dieback or promote regrowth of injured axons in vivo in a dorsal column crush model of spinal cord injury. We transplanted MAPCs into the spinal cord immediately following injury and measured axonal position. Seven days postlesion, MAPC-transplanted animals showed a significant increase in the extent of axon extension into the lesion core compared to vehicle controls. We next sought to examine the effects of MultiStem cells in a more translationally relevant contusion model of spinal cord injury. Using an Infinite Horizon impactor we completed a pilot study to determine the behavioral outcome of multiple different force parameters for 6 weeks postinjury and confirmed that we have created a consistent moderate severe spinal cord injury. We monitored urine production and voiding using a metabolic chamber and electrophysiology in which we record the pressure of the bladder (a measure of detrusor activity) and EMG activity of the external urethral sphincter. At the tested force of 225 kDa, we found that bladder dysfunction is still prevalent in injured rats at 6 weeks postinjury. We have since examined bladder function in individual animals receiving intraspinal injections of either rat or human MultiStem cells. In both injured cell-treated groups, bursting was seen in correlation with a void, whereas in injured untreated animals, no bursting was seen. While these recordings of the cell-treated animals are not indicative of completely normal function, they demonstrate promising data suggesting that recovery of bladder function will be stimulated after treatment with MultiStem cells. We are currently examining the effects of MultiStem cells on long-term behavioral and functional outcomes in this model with the aim of accelerating the translation of MultiStem therapy into clinic for treatment of spinal cord injury.
S. A. Busch,* J. A. Hamilton,* B. Yang,† S. I. Savitz,† R. J. Deans,* and R. W. Mays*
*Athersys, Inc., Department of Regenerative Medicine, Cleveland, OH, USA
†University of Texas Medical School at Houston, Houston, TX, USA
Numerous stem and progenitor cell types are currently being evaluated as potential therapeutic treatments following ischemic insult to the central nervous system (CNS). Previous studies have demonstrated varying levels of functional improvement in animal models of ischemia after infusion of adult stem/progenitor cells; however, little is known regarding the mechanisms by which they provide benefit. Early indications that transplanted stem cells might provide true neural replacement and integration have not been supported by the growing body of literature investigating cellular therapies. The presently described studies were designed to examine the mechanistic interaction between the injured tissue environment and transplanted MultiStem® cells. The MultiStem product is derived from the multipotent adult progenitor cell (MAPC) technology, is manufactured under strict specifications and release criteria, and has received allowance from the Food and Drug Administration for testing in humans in the treatment of acute myocardial infarction, graft-versus-host-disease in leukemia patients, inflammatory bowel disease, and ischemic stroke. In the presented preclinical studies, 4 million cells were administered intravenously (IV) to adult rats 24 h after middle cerebral artery occlusion/common carotid artery ligation. Tissue gene expression was surveyed via microarray analysis 3 and 28 days after cell (or vehicle) administration. Comparison of stroked animals to sham-injured animals at the early time point revealed evidence of a substantial immune response to the injury, both within infarcted brain tissue and at the level of peripheral lymphoid tissue (spleen). Examination of gene expression changes induced by MultiStem cell treatment suggested significant attenuation of the immune response to ischemic stroke, both in the CNS and in the periphery. Comparison of gene expression changes at the later time point showed evidence of improved neuronal activity/signaling-related genes in infarcted brain tissue, and significant preservation of splenic immune homeostasis, in animals treated with MultiStem cells. Subsequent studies were designed to examine behavioral deficits, brain infarct volume, changes in splenic mass and splenocyte phenotype, and serum cytokine response after MultiStem cell or vehicle administration in stroked animals. Data from these studies will be presented, along with an overview of the design and progress of our recently initiated Phase II clinical trial.
L. Cui* and L-R. Zhao*†
*Department of Neurology, Louisiana State University Health Sciences Center, Shreveport, LA, USA
†Department of Cellular Biology and Anatomy, Louisiana State University Health Sciences Center, Shreveport, LA, USA
Stroke, which mainly attacks the elderly, is the leading cause of adult permanent disability worldwide. Stroke also presents an enormous public health problem and a serious public financial burden in the US. Chronic stroke is the time period beyond 3-6 months after stroke onset. Currently, there is no pharmaceutical therapy for treatment of chronic stroke. Recently, we have demonstrated that the combination of two hematopoietic growth factors, stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF) (SCF + G-CSF), leads to the long-term therapeutic effects on chronic stroke in animal models. It remains unknown, however, how SCF + G-CSF repairs a permanently damaged brain in the setting of chronic stroke. Here we have determined the effects of SCF + G-CSF on dendritic plasticity of the cortical neurons outside the infarct cavities in the aged brain of chronic stroke. Using live brain imaging, the layer I dendritic spines in the chronic stroke brain was scanned before treatment, 2 and 6 weeks after treatment in aged transgenic mice carrying yellow fluorescent proteins exclusively in layer V pyramidal neurons. We observed that SCF + G-CSF significantly increased the number of mushroom-type spines and prevented stroke-induced synaptic degeneration 2 and 6 weeks after treatment. Immunofluorescence data revealed that SCF + G-CSF treatment in chronic stroke augmented apical dendritic branching and increased the number of functional postsynapses (postsynaptic density-95 positive puncta) in peri-infarct cortex 6 weeks after treatment. These data suggested SCF + G-CSF rebuild up functioning neuronal networks in the aged brain of chronic stroke. This study provides direct evidence supporting the beneficial effects of SCF + G-CSF on neuronal network remodeling in the aged brain of chronic stroke and offers new insights into the contribution of hematopoietic growth factors on brain repair in chronic stroke. These findings would help in developing a new therapeutic strategy to treat chronic stroke.
D. Dai,* J. Bernitz,* D. E. Redmond, Jr.,† E. Y. Snyder,* and M. M. Daadi*‡§
*Program in Stem Cell & Regenerative Biology, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
†Departments of Psychiatry and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
‡Department of Neurosurgery, Stanford Institute for NeuroInnovation and Translational Neurosciences, Stanford University School of Medicine, Stanford, CA, USA
§Molecular Medicine Research Institute, Sunnyvale, CA, USA
Optimal therapeutic human neural stem cells (hNSCs) for Parkinson's disease (PD) should be scalable, express midbrain identity, engraft in animal models of PD, and differentiate into functional dopaminergic neurons, ideally the A9 type, that appropriately reinnervate the striatum in PD. The search for such a cell line continues. In the present study, we have successfully and efficiently directed a fetal hNSC towards the midbrain DA identity. The hNSCs were perpetuated for over 25 passages in defined media containing epidermal growth factor (EGF), fibroblast growth factor (FGF), and leukemia inhibitory factor (LIF). Their midbrain identity was revealed by the expression of the transcription factors Nurr1 and Pitx3. When induced to differentiate by growth factor omission, approximately 5% of the total cells expressed the neuronal marker microtubule-associated protein 2 (MAP2) after 7 days in vitro (DIV). This proportion increased to 60% after 15 DIV. To induce the DA phenotype, single cell dissociated hNSCs were plated in control media or in media containing 20 ng/ml of bFGF and 75% (v/v) of glial conditioned media. After 24 h in culture, 1% of the total cells expressed tyrosine hydroxylase (TH, marker for DA neurons), which represented approximately 30% of the total number of β-tubulin class III immunoreactive neurons. A time course study demonstrated that the number of dopaminergic neurons steadily increased to 3% of the total DAPI-positive cells after 7 DIV. This proportion of TH-ir cells represented approximately 76% of the total neuronal population. To test the ability of this cell line to engraft and differentiate into DA neurons, we transplanted them into the basal ganglia and substantia nigra (SN) of an MPTP-lesioned nonhuman primate (NHP) model of PD. Two months after cell transplantation, the grafted hNSCs differentiated into TH-expressing cells. These data demonstrate for the first time that hNSCs incubated in the DA-inducing conditions maintain the newly induced phenotype in vivo after 2 months. Further studies are necessary to identify the long-term functional integration and dopamine replacement in the denervated striatum.
This work was supported the California Institute of Regenerative Medicine, Axion Research Foundation, St Kitts Biomedical Research Foundation and by the State of Connecticut under the Connecticut Stem Cell Research Grants Program. Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the State of Connecticut.
J. E. Davies,* C. J. Proschel,†‡ S. M. Green,* K. H. Jasper,* M. Noble,†‡ M. Mayer-Proschel,†‡ and S. J. A. Davies*§
*Department of Neurosurgery, University of Colorado School of Medicine, Aurora, CO, USA
†Department of Biomedical Genetics, University of Rochester, Rochester, NY, USA
‡Institute for Stem Cell and Regenerative Medicine, University of Rochester, Rochester, NY, USA
§Gates Center for Regenerative Medicine and Stem Cell Biology, University of Colorado School of Medicine, Aurora, CO, USA
We have previously shown that transplanted astrocytes derived from bone morphogenetic protein-treated embryonic glial restricted precursors (GDAsBMP) can suppress scar formation, support axon regeneration, enhance neuroprotection, and promote robust functional recovery when transplanted to acute spinal cord injuries in adult rats (see http://jbiol.com/content/5/3/7). To test the ability of these cells to promote recovery after long-term chronic spinal cord injury, GDAsBMP or undifferentiated glial restricted precursors (GRPs) were transplanted into the spinal cords of adult rats at time points of 1 or 8 months after unilateral transection injury of the cervical dorsolateral funiculus at the C3/C4 spinal level. This injury caused long-lasting, stable locomotor deficits as measured by the horizontal ladder test (skilled task) and the CatWalk apparatus (unskilled task). Animals that received transplants of GDAsBMP at either 1 or 8 months after spinal cord injury showed robust improvements in locomotor function. In contrast, the horizontal ladder performances of rats that received transplants of undifferentiated GRPs at either 1 or 8 months after injury were not different from those of media-injected injury control animals at all time points posttreatment. Transplantation of GDAsBMP, but not GRPs, also promoted a 67% increase in synapsin-1 immunodensity, an indicator of increased synaptic plasticity, within cervical motor neuron pools. The results of our present study are therefore consistent with our previous comparisons of the effects of transplanted rat and human GDAs versus undifferentiated GRP cells in acute spinal cord-injured rats and provide support for the development of GDAsBMP transplantation as a therapy for the long-term chronically injured human spinal cord.
Funding: Private donations from the SCI community to S.J.A.D.; Carlson Stem Cell Trust to C.J.P. and M.N.; NYS DOH Spinal Cord Injury Research C023691 to M.M.-P.
C. Edwards,* V. Delic,* N. Copes,* O. Phillips,* T-A. Phan,* S. Medrano,* K. Noble,* P. Bickford,†‡ and P. Bradshaw*
*Department of Cell Biology, Microbiology, and Molecular Biology, University of South Florida, Tampa, FL, USA
†Center for Excellence in Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
‡James A. Haley Veterans' Administration Medical Hospital, Tampa, FL, USA
NT-020, a patented proprietary formulation of blueberries, green tea, carnosine, and vitamin D3 available from Natura Therapeutics, Inc., has been shown to improve spatial memory performance, increase progenitor cell proliferation, and decrease inflammation in aged rats. Here, we show that NT-020 can also extend the mean and maximal life span of C. elegans nematode worms and increase their resistance against heat stress. C. elegans given NT-020 also had increased oxygen consumption rates and decreased ATP levels, a sign of uncoupling of mitochondrial ATP synthesis from electron transport chain function. In independent experiments, we have observed that several other metabolites of mitochondrial carbohydrate, fatty acid, and protein catabolism that extended life span in C. elegans also uncoupled mitochondrial oxidative phosphorylation when administered exogenously. Chemical uncouplers of mitochondrial oxidative phosphorylation, such as dinitrophenol and carbonyl cyanide m-chlorophenyl hydrazone (CCCP), when used at low concentrations to induce “mild uncoupling” have been found to increase the mean life span in fruit flies and mice and also the maximal life span in C. elegans. It is hypothesized that these compounds function, in part, through decreasing mitochondrial reactive oxygen species (ROS) production, but also by activating the AMP kinase and protein kinase A signaling pathways. We are currently conducting experiments to determine if NT-020 functions to extend life span and uncouple mitochondria in an aak-2 (AMP kinase) mutant C. elegans strain to test this assertion. We are also testing the effects of NT-020 on mitochondrial function in an N2a neuroblastoma cell model of β-amyloid toxicity and in E1A-transformed murine embryonic fibroblasts from premature aging mitochondrial DNA mutator (exonuclease-deficient DNA polymerase gamma knock-in) mice. Specifically we are determining the effects of NT-020 on oxygen consumption, ATP levels, and ROS production in these cell lines to determine if the bioenergetic effects observed in C. elegans are evolutionarily conserved and may play a role in the neuroprotective effects of NT-020 in mammals.
P.C.B. is a cofounder of Natura Therapeutics, Inc.
J. Ehrhart,*†‡ D. Darlington,* C. D. Sanberg,‡ N. Kuzmin-Nichols,‡ P. R. Sanberg,† and J. X. Tan*†
*Rashid Laboratory for Developmental Neurobiology, Silver Child Development Center, Department of Psychiatry & Behavioral Medicine, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
‡Saneron-CCEL Therapeutics, Tampa, FL, USA
Pathological amyloidosis is a hallmark of Alzheimer's disease (AD), a dementia disorder affecting 20 million worldwide. Most current therapeutic strategies for the symptomatic and disease-modifying treatment of AD have met with disappointment. Consequently, a more effective treatment or prophylaxis is needed. Amyloidβ (Aβ) is a key protein in the regulation of AD. Postmortem and basic research studies, demonstrating glial cell activation in close proximity to amyloid plaques (a recognized inflammation marker), have confirmed that inflammatory processes play a role in the pathology of AD. Previously, we have shown that human umbilical cord blood cells (hUCBCs, U-CORD-CELL™) provide cognitive recovery in animal models of neurodegenerative disease through modulation of immune function. Moreover, we showed that multiple low-dose injections of HUCBCs in AD mouse models caused a reduction in cerebral Aβ levels/β-amyloid deposits, which further resulted in increased serum levels of Aβ1-40,42. In this study, we continued investigation of the safety and efficacy of hUCBCs in a double transgenic presenilin-amyloid precursor protein (PSAPP) mouse model of AD. Results demonstrate that hUCBCs are able to improve memory function and locomotor ability in these PSAPP mice. Furthermore, by using ELISA and Western blotting techniques we were able to observe altered amyloid precursor protein processing, a major hallmark in Alzheimer's disease. This resulted in reductions in both the Aβ-amyloidogenic pathway and overall reductions in the Aβ peptide. Whereas data suggest that hUCBC infusion mitigates AD-like pathology, no optimal dosing and safety data have been elucidated. As part of our safety characterization, the purpose of this study was to determine hUCBC presence and biodistribution in hUCBC-infused PSAPP mice 24 and 72 h postadministration. Longer term incubations of 7 and 30 days in Spraque-Dawley rats were also performed to further reinforce our findings.
Saneron CCEL Therapeutics, Inc. is a biotechnological company that uses umbilical cord blood cells for the treatment of a number of disorders. J.X.T. is a consultant for and P.R.S. is cofounder of Saneron. C.D.S., N.K.-N., P.R.S., and J.X.T. are inventors on patents and/or patent applications for the use of umbilical cord blood in the treatment of a number of disorders.
L. Feng,* A. Sharma,† D. F. Muresanu,‡ and H. Sharma†
*Department of Neurology, Norman Bethune International Peace Hospital Army, ShiJiaZhuang, Hebei Province, China
†Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology & Intensive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden
‡Neurology, University of Medicine & Pharmacy, Cluj-Napoca, Romania
Chronic neuropathic pain is caused by brain or spinal cord injury, nerve lesion, diabetic neuropathy, amputation, and neuromuscular disorders. Neuropathic pain enhances sensitivity to touch and pain perception including phantom pain. So far there are no suitable treatments available. Thus, new treatment options to reduce these symptoms and to enhance pain management are needed. In animal models of chronic neuropathic pain, some of the clinical symptoms can be simulated by constriction, ligation, or transection of sensory and/or motor spinal nerves. Rats develop neuropathic pain slowly and hypersensitivity could be seen up to 2–4 weeks after nerve injury. In a rat model of L4 and L5 nerve ligation, our laboratory was the first to show that breakdown of the blood–spinal cord barrier (BSCB) to albumin and activation of astrocytes over 2–10 weeks were most prominent on the ipsilateral side of the cord. Hypersensitivity to pain lessened after 4 weeks, but neurodegenerative changes progressed over time. This suggests that neuropathic pain could induce neurodegenerative changes in the cord. However, effects of nanoparticles in modifying neuropathic pain syndrome are still not well known. Thus, we examined the role of nanoparticles on the development of neuropathic pain, BSCB dysfunction, astrocytic reactivity, and neural injury after spinal nerve ligation. In addition, the potential role of cerebrolysin, a mixture of several neurotrophic factors and active peptide fragments was also evaluated in this model. Spinal nerve ligation of L4 and L5 was performed and rats were administered Cu, Ag, or Al nanoparticles (50–60 nm; 50 mg/kg, IP) once daily for 10 days. In these animals, albumin immunoreactivity for BSCB dysfunction, glial fibrillary acidic protein (GFAP) reactivity for astrocytic activation, and Nissl staining for neural injuries were examined after 2, 4, 8, and 10 weeks after nerve ligation. Nanoparticle-treated rats exhibited prolonged hypersensitivity to external stimulation (fur touching) up to 8 weeks. Leakage of albumin and activation of astrocytes in the spinal cord segments T10, T12, and L5 were exacerbated by 120% at 4 weeks, 250% at 8 weeks, and 300% at 10 weeks after ligation in the nanoparticle-treated group. This effect was most marked in Cu- and Ag-treated animals. Neuronal injury closely corresponded to albumin leakage in the spinal cord. Cerebrolysin in high doses (5 ml/kg) if coadministered with nanoparticles daily was able to reduce morphological changes in the cord effectively. However, cerebrolysin given after 4–6 days of nanoparticle administration failed to induce sufficient neuroprotection. The drug also reduced hyperalgesia if given as a pretreatment. These observations are the first to show that nanoparticles potentiate the duration of hyperalgesia of neuropathic pain and exacerbate disturbances in spinal cord microfluid environment. Cerebrolysin in high doses is able to thwart these changes indicating a potential role of the drug in pain management. This research is very timely in neuropathic pain and the data are important for our increased understanding of these phenomena in order to find a good cure.
K. D. Fink,*†‡ A. T. Crane,* J. Rossignol,*§ X. Lévêque,†‡ P. Starski,* R. Hearnes,* M. Lu,*§ T. H. Nguyen,¶ L. Lescaudron,†‡ and G. L. Dunbar*§
*Field Neurosciences Institute Laboratory for Restorative Neuroscience, Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
†Université de Nantes, Faculté des Sciences et des Techniques, Nantes, France
‡INSERM U643, ITERT, CHU Hotel Dieu, Nantes cedex, France
§Field Neuroscience Institute, Saginaw, MI, USA
¶INSERM U948, Biothérapies hépatiques, CHU Hotel Dieu, Nantes cedex, France
Over the past two decades, embryonic stem cell (ESC) therapies have emerged as one of the top candidates for treating CNS disorders because these cells can differentiate into neuronal lineages following transplantation. However, the use of ESCs raises many ethical issues, including the limited availability of ESCs and their propensity to form tumors following transplantation. In the last 10 years, the use of adult stem cells, such as bone marrow-derived mesenchymal stem cells (MSCs), has gained significant interest due to the fact that they are readily available, are subject to fewer ethical considerations, and have been shown to survive following transplantation into the brains of animals that model various diseases. However, MSCs lack the ability to differentiate into neuronal lineages following transplantation. Currently, the use of induced pluripotent stem cells (iPSCs) provides considerable promise as an alternative approach. The use of iPSCs circumvents ethical issues and they are more readily available, as they are derived from adult tissue, but the method of producing these cells usually involve pro-oncogenes, such as c-Myc and kruppel-like factor 4 (Klf4), which become permanently integrated into the genome during reprogramming with retroviruses or lentiviruses. The use of a non-integrating adenovirus for cell transfection avoids this problem. However, the extent to which transplanted adenovirus-generated iPSCs can survive and differentiate into functional neurons, without forming tumors, is unknown and is the focus of the present study. In the first part of this study, we found that iPSCs could be derived from both rat bone marrow MSCs and tail-tip fibroblasts (TTF) using a single cassette lentivirus (LN) [octamer binding transcription factor 4 (Oct4), sex determining region Y box 2 (Sox2), Klf4, and c-Myc] or a combination of adenoviruses (AD) (Oct4, Sox2, Klf4, and c-Myc). This was confirmed using flow cytometry and immunocytochemistry for the pluripotent markers stage-specific embryonic antigen 3 (SSEA3), SSEA4, Tra-1-60, Nanog, and Oct4. The iPSCs (MSC-AD, MSC-LN, TTF-AD, and TTF-LN) were analyzed in vitro for integration of the reprogramming genes, neurotrophic factors, proliferation, and pro- and anti-inflammatory molecules using RT-PCR. Following in vitro analysis, 400,000 TTF-AD iPSCs were unilaterally transplanted into the right striatum of 20 healthy adult rats. Brains were analyzed for graft survival, inflammatory response, and neuronal or glial differentiation at 5, 21, 63, and 90 days posttransplantation. Graft survival was observed at all time points, although graft size decreased dramatically between 5 and 21 days and 21 and 63days posttransplantation, indicating either migration of the cells or graft rejection. Immature, developing, and mature neuronal phenotypes were observed within the transplantation sites at 5, 21, 63, and 90 days posttransplantation, respectively. The observed survival, differentiation into mature neuronal phenotypes, and the absence of tumor formation suggests that these adenovirus-created iPSCs may provide a safe and viable alternative to ESCs and MSCs for therapeutic treatment of CNS disorders.
Support for this project was provided by a PUF grant (to K.D.F.) and funding from the John G. Kulhavi Professorship and Field Neurosciences Institute (to G.L.D.).
D. L. Fischer,*†‡ C. J. Kemp,‡ T. J. Collier,†‡ S. L. Wohlgenant,† B. F. Daley,† K. Steece-Collier,†‡ and C. E. Sortwell†‡
*MD/PhD Program, College of Human Medicine, Michigan State University, MI, USA
†Neuroscience Program, Michigan State University, MI, USA
‡Department of Translational Science & Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
Deep brain stimulation (DBS) is the most common neurosurgical treatment for the alleviation of Parkinson's disease (PD) motor symptoms. A recent clinical trial showed equivalent efficacy for motor symptoms for DBS that targeted either the subthalamic nucleus (STN), the traditional target site, or the globus pallidus interna (GPi) (Follett et al., N. Engl. J. Med., 2011). Whereas the GPi has been less commonly targeted for DBS than the STN, the same study suggests that GPi DBS may provide better outcomes for patients with psychiatric comorbidities. Beyond symptomatic efficacy, our laboratory and others have demonstrated that high-frequency DBS of the STN provides neuroprotection for dopaminergic neurons of the substantia nigra pars compacta (SNc) in the 6-hydroxydopamine (6-OHDA) rat model of PD (Spieles-Engemann et al., Neurobiol. Dis., 2010). Clinical evidence also suggests that STN DBS may have disease-modifying effects in PD patients. However, whether GPi DBS has similar neuroprotective potential has not been investigated. In the present study, using identical experimental parameters as our previous work on STN DBS, we investigated whether DBS of the GPi halts ongoing SNc degeneration induced by 6-OHDA. We conducted long-term GPi DBS in male rats that received unilateral, intrastriatal 6-OHDA. Two weeks post-6-OHDA, rats with approximately 50% loss of SNc DA neurons were randomly assigned to receive active or inactive stimulation continuously for 2 weeks. During this 2–4-week post-6-OHDA interval, SNc degeneration typically progresses to 75% in lesioned, untreated rats. In contrast, STN DBS halts SNc degeneration at 50%. Forelimb use asymmetry measured via the cylinder task was used to prescreen for lesion severity and functional efficacy of GPi DBS. Electrode placement in the GPi was verified postmortem via Kluver-Barerra histochemistry. Tyrosine hydroxylase-immunoreactive (TH-ir) neurons of the SNc were quantified using unbiased stereology. All rats exhibited an approximate 75% loss of THir SNc neurons 4 weeks postlesion regardless of treatment group; no difference between active and inactive groups receiving GPi DBS was observed. Our data reveal that long-term GPi DBS does not halt ongoing SNc degeneration induced by 6-OHDA. These results are in sharp contrast to the neuroprotective effect of STN DBS under identical lesion and treatment time course parameters. Despite equivalent symptomatic efficacy of STN and GPi DBS in PD patients, these data suggest that stimulation-associated neuroprotection is specific to STN placement. Studies in our lab are continuing to investigate the mechanism of STN DBS-mediated neuroprotection.
Supported by the Morris K. Udall Center of Excellence for Parkinson's Disease Research at Michigan State University NS058830 (T.J.C.).
M. Fjodorova, E. Torres, S. Dunnett, and A. Rosser
Brain Repair Group, School of Biosciences, Cardiff University, Cardiff, UK
Embryonic ventral mesencephalic grafts contain two dopamine (DA) neuron subtypes: A9 neurons of the substantia nigra pars compacta and A10 neurons of the ventral tegmental area. It has previously been reported that A9 and A10 cells can be identified on the basis of their morphology, location within the graft, and protein expression. We have already characterized DA cell populations in embryonic age 12-day-old (E12) and E14 grafts into the striatum in a unilateral rat model of Parkinson's disease. Expression of a G-protein-gated inwardly rectifying K+ channel subunit (Girk2) and calbindin in tyrosine hydroxylase-positive (TH+) cells allowed for visualization of A9-and A10-type DA neurons, respectively. E12 grafts produced fivefold larger DA cell yields than E14 grafts. In both donor age groups, 83% of TH+ neurons were costained with Girk2. In E12 grafts 15% of TH+ neurons colabeled with calbindin, in E14 grafts 27% of TH+ neurons were calbindin positive. However, in both E12 and E14 grafts, 14% of calbindin-positive neurons colabeled with Girk2. Graft morphology analysis showed that a greater proportion of TH+ cells coexpressed Girk2 in the periphery rather than in the center of the graft, whereas the opposite was true for TH+/calbindin+ cells. These findings suggest that Girk2 and calbindin are good but not perfect markers of A9 and A10 DA neuron subtypes in dopaminergic grafts. Now we aim to address the effect of the host striatum on the development of DA neuron subtypes. This experiment will look at E12 and E14 grafts implanted into different cerebral targets to see if the site of implantation affects the proportions and distribution of DA cell phenotypes seen in the graft. The striatum, nucleus accumbens, and prefrontal cortex all receive midbrain dopamine innervations, whereas the hippocampus receives none and will make a good control site. We also aim to elaborate the overlap in Girk2 and calbindin staining in the graft, and investigate the morphology of TH+/Girk2+ and TH+/calbindin+ neurons in the grafts to confirm that they represent two different subtypes of DA neurons.
S. Garbuzova-Davis,*†‡ S. Mitril,* S. Sallot,* D. Hernandez-Ontiveros,* and P. R. Sanberg*‡§
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology University of South Florida, Morsani College of Medicine, Tampa, FL, USA
‡Department of Pathology and Cell Biology University of South Florida, Morsani College of Medicine, Tampa, FL, USA
§Department of Psychiatry, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
Mucopolysaccharidosis type III (MPS III), or Sanfilippo syndrome, is an autosomal recessive disorder caused by deficiency of a specific enzyme leading to accumulation of heparan sulfate (HS) within cells and to eventual progressive cerebral and systemic organ abnormalities. Different enzyme deficiencies in the HS degradation pathway comprise the four MPS III subcategories (A, B, C, D). Since neuropathological manifestations are common to all MPS III types, determining blood–brain barrier (BBB) condition may be critical to understand potential additional mechanisms of this devastating disorder. Garbuzova-Davis et al. (2011) showed BBB structural and functional impairment of various brain structures in a MPS III B mouse model. Endothelial and pericyte cell damage and astrocyte degeneration compromise the BBB even at the early disease stage, resulting in vascular leakage. Edematous spaces around microvessels and highly vacuolated perivascular macrophages have been noted. Accumulation of GM3 ganglioside, a secondary storage product, was determined in endothelium microvasculature of multiple brain structures. These results indicate severe BBB breakdown, which might accelerate neuronal damage. However, little is known about the BBB competence in MPS III patients. The aim of this study was to determine structural and functional integrity of the BBB in various brain structures of postmortem tissues from patients with MPS III A and MPS III D and age-matched controls without neurological or immunological status (NICHD Brain and Tissue Bank for Development Disorders, University of Maryland, Baltimore, MD). BBB structural characteristics were examined in microvessels from the primary motor cortex, hippocampus, putamen, and cerebellum via electron microscopy. BBB functional integrity was investigated by tests for vascular leakage of endogenous IgG. Western immunoblot for tight junction proteins (occludin and claudin-5) was performed in serial brain sections. Immunohistochemical staining for lysosomal accumulation within endothelial cells was also performed. Major findings of our study were: 1) capillary ultrastructure revealed endothelial and pericyte cell damage; 2) lysosomal zebra bodies were indicated in a majority of endothelial cells and pericytes, rupturing cell membranes; 3) severe edematous space was noted around microvessels; 4) IgG microvascular leakage was clearly indicated in multiple brain structures; 5) reductions of occludin and claudin-5 were noted with variations between MPS III types; 6) extensive lysosomal accumulation was determined in brain microvasculature endothelium. These new findings of BBB structural and functional impairment, although from only two cases, MPS III A and MPS III D, may have implications for disease pathogenesis and should be considered in treatment development for MPS III. Special attention should be given to endothelial cell function in view of possible deterioration of influx and efflux transport systems needed to maintain proper CNS homeostasis.
Supported by the Children's Medical Research Foundation.
L. E. Glover, N. Tajiri, J. Ehrhart, J. Tan, Y. Kaneko, and C. V. Borlongan
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
We recently reported the importance of timing of therapeutic intervention in experimental traumatic brain injury (TBI) (Glover et al., in press), which may be characterized by genetic and histologic neurovascular proteinase alterations (Borlongan and colleagues, 2009), and inflammation and apoptosis (Borlongan and colleagues, 2010) even at the early stage of the disease. Although such an immediate cell death cascade has become established in adult TBI, to date the pathophysiology underlying neonatal TBI is poorly understood. The objective of the present study was to determine the role of cytokine regulation following TBI in neonatal Sprague-Dawley rats. Seven-day-old Sprague-Dawley rats were subjected to TBI using the controlled cortical impact (CCI) injury model. Age-matched littermates that did not receive TBI served as the control for this study. Immediately following TBI (within 15 min), rats were euthanized and the brains were divided into the ipsilateral (left) and contralateral (right) hemispheres and flash frozen in liquid nitrogen. A BioRad 23-Plex panel was used to measure cytokine levels. Data were unexpected and revealed that 18 of 23 cytokines were significantly downregulated in the hemisphere contralateral to the TBI-impacted hemisphere. The three cytokines, interleukin-5 (IL-5), IL-6, and macrophage inflammatory protein-3a (MIP-3a), were significantly suppressed in both ipsilateral and contralateral hemispheres of TBI rats compared to the control rats. The remaining five cytokines did not significantly differ between TBI and control rats, indicating an overwhelming downregulation of the entire panel of cytokines analyzed here. Note that a parallel study processing plasma, instead of brain tissues, for the present cohort of animals revealed neither upregulation nor downregulation of any of these same cytokines between TBI and controls. Contrary to the early inflammatory response markers seen in adult TBI, the present neonatal TBI study demonstrated the reversed cytokine profile of downregulation, suggesting a robust endogenous anti-inflammatory response being mounted surprisingly from a brain area remote from the site of injury (i.e., the contralateral hemisphere). This regenerative process may be unique to the highly plastic neonatal brain, which is shown here as equally capable of cytokine regulation following TBI.
This research was supported by the University of South Florida Department of Neurosurgery and Brain Repair Funds.
S. E. Gombash,*† F. P. Manfredsson,‡ C. J. Kemp,‡ S. L. Wohlgenant,‡ D. L. Fischer,‡ B. F. Daley,‡ T. J. Collier,‡ J. W. Lipton,‡ A. Cole-Strauss,‡ R. J. Mandel,§ S. M. Fleming,*¶ and C. E. Sortwell‡
*Department of Neurology, University of Cincinnati, Cincinnati, OH, USA
†Graduate Program in Neuroscience, University of Cincinnati, Cincinnati, OH, USA
‡Department of Translational Science and Molecular Medicine, Michigan State University Grand Rapids, MI, USA
§Department of Neuroscience, University of Florida, Gainesville, FL, USA
¶Department of Psychology, University of Cincinnati, Cincinnati, OH, USA
Aging is the single most important risk factor for Parkinson's disease (PD). Mutations in the gene encoding α-synuclein (α-syn) have been linked to familial PD and viral vector-mediated α-syn overexpression in the rodent nigrostriatal dopamine (DA) system recapitulates key pathological features of PD. To date, nigrostriatal pathology has been examined in young adult animal models overexpressing α-syn but has not been examined in the aged brain. The aged nigrostriatal system may have increased susceptibility to α-syn-mediated neurotoxicity due to enhanced nigral oxidative stress and neuroinflammation, both linked to α-syn mechanisms of neurotoxicity. To determine whether α-syn-mediated pathology is amplified with aging, we first intentionally developed a model with limited nigrostriatal degeneration in young adult rats. Recombinant adeno-associated virus serotype 2/5 expressing human wild-type α-syn mixed 1:1 with rAAV2/5 expressing green fluorescent protein (GFP) or rAAV2/5-GFP alone was injected intranigrally in rats 3 months of age. Every 4 weeks rats were tested for motor deficits using the cylinder test, bracing test, bilateral tactile stimulation test, and amphetamine-induced rotational asymmetry. Young rats were sacrificed at 2 and 3 months post-vector injection and were analyzed for α-syn and GFP immunolabeling, stereology for surviving nigral tyrosine hydroxylase-ir neurons, and levels of striatal DA and DA metabolites. Modest reductions in striatal DA (~30%) and surviving nigral DA neurons (~20%) were observed in young rats. No differences were detected between groups in behavioral performance, except that α-syn/GFP injected animals displayed deficits in the bracing task at 1 and 2 months. To make direct comparisons in aged rats, 20-month-old rats were injected with identical vectors, analyzed for motor impairments, and sacrificed 3 months post-vector injection. Analysis of toxicity in aged rats is ongoing with the addition of α-syn-ir nigral neuron and striatal aggregate quantification. Results will determine if the aged nigrostriatal system is more susceptible to α-syn-mediated neurotoxicity. If so, α-syn overexpression in the aged rat may be an improved model for development of novel PD therapies and delineating the cause(s) of increased susceptibility may provide insight into the disease process itself.
Supported by NS058682 (C.E.S.) and the Morris K. Udall Center of Excellence for Parkinson's Disease Research at MSU NS058830 (T.J.C).
E. J. Gonzalez-Rothi,* A. Rombola,† L. Fernandez,* M. Alappattu,* A. Daly,† C. A. Rousseau,† R. A. Federico,* B. E. O'Steen,† K. V. Vandenborne,* P. J. Reier,† D. D. Fuller,* and M. A. Lane†
*Department of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
†Department of Neuroscience, College of Medicine University of Florida, Gainesville, FL, USA
Cervical spinal cord injury (cSCI) significantly impairs upper extremity function. Although some spontaneous functional recovery has been demonstrated, the extent of recovery is limited and the underlying mechanisms are poorly defined. Using a well-established rodent model of high cSCI, lateral C2 spinal cord hemisection (C2Hx), the present work examines the muscular and neuroanatomical substrates underlying upper extremity dysfunction and recovery following incomplete cSCI. Gross forelimb motor function was assessed prior to, and at 1 and 8 weeks postinjury using the limb-use asymmetry (cylinder) test. Immunohistochemical techniques were used to assess forelimb muscle fiber cross-sectional area (CSA). The neuroanatomical circuitry of the forelimb was assessed using retrograde transneuronal tracing techniques. Initial results indicate dramatic reductions in ipsilateral forelimb use (p < 0.01) and muscle fiber CSA (p < 0.05) at 1 week post-C2Hx. By 8 weeks postinjury, improvements in ipsilateral forelimb use (p < 0.01) and increased muscle fiber CSA (p < 0.05) were observed. Taken together, these initial results indicate substantial muscular remodeling and modest recovery of forelimb function following cSCI. Preliminary results from tracing studies reveal the location and distribution of forelimb motoneurons and interneurons in the cervical spinal cord and associated neurons within the brain stem and motor cortex of uninjured animals. Ongoing studies are exploring the neuroanatomical circuitry associated with forelimb dysfunction and recovery after injury. These experiments are the first to examine the combined muscular and neuroanatomical mechanisms underlying neuroplasticity and recovery of upper extremity function following cSCI and may aid in identifying potential therapeutic targets for rehabilitation interventions.
B. Hattiangady,*†‡ R. Kuruba,‡ B. Shuai,*†‡ B. Waldau,‡ and A. K. Shetty*†‡
*Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White, Temple, TX, USA
†Research Service, Veterans Affairs Medical Centers of Durham, NC and Temple, TX, USA
‡Division of Neurosurgery, Duke University Medical Center, Durham, NC, USA
Status epilepticus (SE) is an emergency condition typified by prolonged seizure activity, which affects over 150,000 Americans every year. Despite the antiepileptic drug therapy after SE, a sizeable fraction of SE survivors develop temporal lobe epilepsy (TLE), typified by recurrent complex partial seizures and impairments in memory and mood function. Currently, there is no efficient therapy for easing both complex partial seizures and memory and mood dysfunction in TLE. In this context, neural stem cell (NSC) grafting approach has received great interest because of the ability of NSC grafts for mediating functional recovery in other prototypes of brain injury. We examined the efficacy of grafting of subventricular zone (SVZ)-derived NSCs into the hippocampus for easing spontaneous recurrent seizures (SRS), and memory and mood dysfunction after SE. We induced SE in adult F344 rats via graded intraperitoneal injections of the excitotoxin kainic acid (3 mg/kg/h for 3-5 h). Seven days after SE, hippocampi of a cohort of rats received grafts of NSCs expanded in culture from the postnatal SVZ (grafted SE group; 4 grafts/hippocampus; ~90,000 live cells/site). For comparison of the efficacy, additional groups of rats that underwent SE received either dead NSC grafts (sham-grafted SE group) or no grafts (SE-only group). At 3-5 months after SE, rats from all groups were scored for the frequency and intensity of spontaneous recurrent seizures (SRS) by investigators who were unaware of group affiliations of different rats via intermittent direct observations (8 h/week; 32 h/month). At 6 months post-SE, rats in all groups were examined for recognition memory function using a novel object recognition test and depressive-like behavior using a forced swim test. At 8 months post-SE, rats from grafted SE and SE-alone groups were implanted with epidural electrodes and electroencephalographic (EEG) activity was recorded continuously for 5 days (~120 h) using a video-EEG system (AS40; Grass Telefactor). In comparison to rats belonging to sham-grafted SE and SE-alone groups, rats in the grafted SE group exhibited greatly reduced SRS (78-82% reduction in all SRS; 72-82% reduction in stage-V SRS; 30% reduction in the duration of individual SRS; and 65% reduction in SRS activity time for the recording period). Analyses with EEG also revealed much reduced frequency and intensity of SRS in the grafted SE group, in comparison to the SE-alone group. Grafted SE rats also displayed much improved recognition memory and mood function than SE-alone rats (p < 0.01). Histological analyses revealed a yield of graft-derived cells that was equivalent to ~110% of injected cells, and pervasive migration of graft-derived cells into dentate granule cell layer as well as other regions of the hippocampus. Phenotypic analyses revealed differentiation of ~25% of graft-derived cells into GABAergic interneurons and other graft-derived cells into different types of glia. Thus, intrahippocampal grafting of SVZ-derived NSCs early after SE is highly efficacious for easing both SRS and other comorbidities of TLE.
D. G Hernandez-Ontiveros, M. C. O. Rodrigues, A. Frisina-Deyo, C. V. Borlongan, P. R. Sanberg, and S. Garbuzova-Davis
Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by motor neuron degeneration in the brain and spinal cord resulting in progressive muscle weakness, paralysis, and death. Blood–brain barrier (BBB) and blood—spinal cord barrier (BSCB) impairment have been suggested as important factors involving ALS pathogenesis. In the G93A superoxide dismutase 1 (SOD1) rodent model of ALS, structural and functional alterations of the BBB/BSCB have been detected in the presymptomatic disease stage, worsening with disease progression. We hypothesized alterations to the microvascular integrity of the BBB/BSCB in ALS patients. Capillaries in gray and white matter of the medulla, cervical, and lumbar spinal cord postmortem tissues, obtained from tissue banks, from 25 sporadic ALS patients and 18 age-matched control subjects were analyzed for structural (electron microscopy) and functional integrity (immunohistochemistry). Tight junction protein expressions were also evaluated (Western blot). Ultrastructural analysis of capillaries in ALS tissues confirmed endothelial and pericyte cell damage, intra- and extracellular edema, and a large perivascular accumulation of collagen. Western blot analysis revealed downregulation of tight junction proteins in both gray and white matter capillaries in all examined ALS tissues. IgG vascular leakage was detected by immunohistochemistry, indicating BBB/BSCB disruption. Staining for CD31 (PECAM-1) and CD105 (endoglin) revealed ill-defined vessel margins and discontinuities in the endothelial lining accompanied by asymmetrical increases in the capillary wall extensions. These results demonstrate microvascular alterations in ALS leading to substantial BBB/BSCB impairment.
Supported by the Muscular Dystrophy Association (Grant #92452).
M. J. Hooshmand,*† K. Fousek,†‡ J. Lucero,† R. Nishi,†§ K. Huang,† N. Jadhaw,† J. Dhillon,† H. Perez,†¶ N. Uchida,# B. J. Cummings,*†**†† and A. J. Anderson*†‡§**††
*Institute for Memory Impairments and Neurological Disorders, University of California, Irvine, CA, USA
†Sue and Bill Gross Stem Cell Center, University of California, Irvine, CA, USA
‡Masters Program in Biotechnology, University of California, Irvine, CA, USA
§Christopher Dana Reeve Foundation, University of California, Irvine CA, USA
¶CIRM Stem Cell Research Biotechnology Training Program, California State University, Long Beach, CA, USA
#Stem Cells Inc., Newark, CA, USA
**Physical Medicine and Rehabilitation, University of California, Irvine, CA, USA
††Anatomy and Neurobiology, University of California, Irvine, CA, USA
The average age of the clinical population of spinal cord injury (SCI) is 34 years old. Yet biological mechanisms of recovery/repair in most animal models are generally addressed in young rodents between 8 and 12 weeks. Further, while the thoracic contusion paradigm is the experimental model of choice for SCI, the clinical incidence of cervical SCI is greater than thoracic and is frequently accompanied by vertebral compression. Notably, oligodendrocytes are particularly vulnerable to ischemia/compression injuries, and chemical demyelination models demonstrate an age-related failure of remyelination by endogenous oligoprogenitors. Additionally, unilateral injuries, in particular, result in predominant loss of function on the ipsilateral side but may also alter the contralateral niche. In the present study we: 1) characterize pathology in young versus aged animals in a newly developed unilateral cervical injury model that combines contusion and compression of the spinal cord; and 2) test the capacity of human neural stem cells (hCNS-SCns) to promote recovery/repair in this model. Immunodeficient mice (young: 3–4 months old; aged: 16–17 months old) received 30 kDa unilateral contusion injuries with a 5-s compression on the same side as the contusion at the C5 vertebral level. Nine days postinjury, aged mice received either hCNS-SCns or vehicle while young animals only received vehicle. Following terminal behavioral analyses, tissue was collected for histology. Immunostaining using myelin basic protein revealed significantly reduced white matter sparing on the injured versus control sides of the spinal cord (p < 0.01), validating the efficacy of our model to produce a demyelinated niche. No significant differences were found between white matter sparing in vehicle-treated aged versus young animals. In parallel, no significant differences in either ladderbeam or CatWalk of young versus aged mice were detected, suggesting that age did not affect demyelination or spontaneous recovery. CatWalk analysis revealed improved stepping pattern (p = 0.05) and higher maximum contact area in aged animals receiving hCNS-SCns in comparison to vehicle (p = 0.05) and horizontal ladderbeam data showed a significant reduction in the number of forelimb errors made by cell-treated aged animals compared to vehicle (p < 0.05). No evidence of allodynia/altered hindlimb sensitivity was observed in any of the groups. Histological data demonstrated the presence of 82,519 hCNS-SCns in aged mice 12 weeks posttransplant, 1.1 times more than the number of cells originally transplanted, suggesting robust survival. As well, only 5.5% of cells were found at the injury epicenter, suggesting long-distance migration to areas where hCNS-SCns could mediate repair. Further, only 7.8% of engrafted cells expressed astrocytic markers while 72% of hCNS-SCns expressed Olig-2, suggesting predominant oligodendrocytic differentiation. Collectively, these results suggest that the aging injured niche supports oligodendroglial cell fate and may mediate axonal repair. The data extend our previous findings in thoracic SCI, now demonstrating cell-mediated recovery of motor function following cervical SCI in aged animals, and suggest the capacity of hCNS-SCns to improve motor function and promote oligodendroglial differentiation in animals at multiple ages and SCI types.
This work was supported by NIH R01NS049885 (A.J.A.), NIH/NIA T32 (M.J.H.), and Christopher and Dana Reeve Foundation.
S. Hussey,* E. J. Gonzalez-Rothi,† D. Sanchez,* B. E. Osteen,* D. D. Fuller,† M. A. Lane,* and P. J. Reier*
*Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
†Department of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
Cervical spinal cord injury (SCI) compromises phrenic circuitry resulting in diaphragm paresis or paralysis. While research using a lateralized C2 hemisection model has revealed the potential for spontaneous recovery and anatomical plasticity, less is known about respiratory plasticity following more clinically relevant contusion injuries at the level of the phrenic nucleus (C3–C5/6). The goal of the present study was to define the extent of phrenic motoneuron (PhMN) compromise following lateral midcervical contusions and concomitant temporal changes in diaphragm electromyogram (diaEMG) activity and ventilatory function. Telemetric electrodes were bilaterally implanted in the diaphragms of adult female rats. One week later, all animals received a C3/4 contusion (Infinite Horizon, 150 kDa present force). DiaEMG recordings were made simultaneously with ventilatory assessment (using whole-body plethysmography) in awake unanesthetized animals. Measurements were made preinjury, daily during the first week postinjury, and weekly thereafter while animals were breathing room or hypercapnic (7% CO2) air (using whole-body plethysmography). Initial results demonstrate the feasibility of long-term telemetric recordings of diaEMG activity. Within the first two postinjury days, diaphragm activity increases while ventilation is relatively unaffected. Furthermore, diaphragm activity and overall ventilatory responses to respiratory challenge (hypercapnia) are dramatically attenuated compared with that seen preinjury. By 1 week postinjury, the ventilatory response to challenge normalizes. Although there is some recovery of diaEMG responses to challenge, the extent remains significantly reduced relative to what occurs preinjury. This deficit remains persistent at 4 weeks postinjury. Persistent diaphragm dysfunction in the presence of otherwise normal ventilatory patterns suggests significant compensation in other spinal respiratory motor regions (e.g., intercostal circuitry). Ongoing studies are quantifying PhMN primary and secondary loss and examining the relationship between motoneuron loss and diaphragm dysfunction.
Supported by NIH RO1 NS054025, the Anne and Oscar Lackner Chair in Medicine, and University of Florida Research Opportunity Funds.
H. Ishikawa,* K. Sinozuka,* N. Tajiri,* L. E. Glover,* J. Vasconcellos,* A. Mayo-Perez,* C. Metcalf,* Y. Kaneko,* S. U. Kim,† and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Department of Neurology, University British Columbia, Vancouver, BC, Canada
Stem cell therapy has emerged as an experimental treatment for stroke. Despite the reported functional recovery in transplanted stroke animals and limited clinical trials in stroke patients, a major gap in our knowledge is the mechanism of action underlying cell therapy. We examine the fate differentiation of transplanted human cerebral endothelial cells (HEN6s) in a stroke animal model, using markers of neurogenesis and vasculogenesis as outcome parameters, combined with stress and sensorimotor tests using the Rat Grimace Scale (RGS) and modified Neurogical Severity Score (mNSS). Ten-week-old rats underwent a 1-h middle cerebral artery occlusion. Animals were randomly assigned to receive stereotaxic transplantation of vehicle, 1, 2, or 4 million HEN6s 3e h after occlusion. Rats were sacrificed 7 days postreperfusion for immunohistochemistry using antibodies against neuronal, vascular, and specific human transplanted cell markers. 2,3,5-Triphenyltetrazolium chloride staining was conducted in alternate sections to reveal infarct volume. All animals were videotaped for 15 min at day 1, 3, and 7 for the RGS stress test. After recording, subjects' sensorimotor function was assessed by mNSS. Increased expression of host neuronal and vascular markers was detected in the stroke core, and closely adjacent to the transplanted cells. Some transplanted cells differentiated into a microvascular phenotype and juxtaposed to the host vasculature. Neurogenic and vasculogenic upregulation was more pronounced in animals that received the 4 million cell dose, but the other doses also exhibited both regenerative processes. Infarct volume in transplanted stroke animals was significantly lower than vehicle-infused stroke animals. The mNSS revealed significant improvement of sensorimotor functions in 4 million HEN6s in comparison with saline group (p < 0.05). Interestingly, RGS revealed higher stress score in 4 million HEN6s than the other groups (p < 0.05). We found a correlation between vasculogenesis and neurogenesis following transplantation of HEN6s, suggesting a dual pronged regenerative process in stroke animals, which correlated with improved sensorimotor functions. The increased stress behavior in transplanted stroke animals may be related to host immune response to the human xenograft. A better understanding of regenerative processes, as well as potential side effects, will allow a critical assessment of the risk-to-benefit ratio of cell therapy in stroke.
P. Jensen,*† M. Heimberg,† A. D. Ducray,† H. R. Widmer,† and M. Meyer*
*Department of Neurobiology Research, Institute of Molecular Medicine, University of Southern Denmark, Odense C, Denmark
†Department of Neurosurgery, University of Berne, Inselspital, Berne, Switzerland
Trefoil factor 1 (TFF1) belongs to a family of soluble peptides with a characteristic tree-looped trefoil structure. TFFs are particularly expressed in the gastrointestinal tract, where they play critical roles in the function and structural composition of the mucosal barrier. More recently, TFF1 has been suggested to function as a neuropeptide, but only limited information is available on its expression and functional significance in the CNS. Our preliminary studies have revealed that TFF1 is expressed in the ventral mesencephalon of the developing and adult rat brain. This finding is of special interest in relation to Parkinson's disease, a neurodegenerative disease mainly characterized by a progressive loss of dopaminergic neurons in the substantia nigra (SN) of the ventral mesencephalon. In the present study, we investigated the expression pattern of TFF1 in adult 6-hydroxydopamine (6-OHDA)-lesioned hemiparkinsonian rats versus unlesioned controls. In the unlesioned ventral mesencephalon, TFF1-immunoreactive (-ir) cells were predominantly detected in the SN pars compacta (SNc) and the ventral tegmental area. While we found that around 90% of the TFF1-ir cells in the SNc coexpressed tyrosine hydroxylase (TH), only a subpopulation of TH-ir neurons also expressed TFF1. We observed that some TFF1-ir cells in the SNc coexpressed the calcium-binding proteins calbindin or calretinin and NeuN, suggestive of a neuronal phenotype, which was supported by a lack of colocalization with the astroglial marker glial fibrillary acidic protein. Intrastriatal injection of the retrograde tracer Fluorogold resulted in labeling of a number of TFF1-expressing cells in the SNc, showing that a significant fraction of the TFF1-ir cells were projection neurons. This was also reflected by unilateral loss of TFF1-ir cells in the SNc of 6-OHDA-lesioned rats. In unlesioned rats only a few small TFF1-ir cells were scattered over the striatum, but their numbers were significantly increased after 6-OHDA lesion. At present it remains unclear whether TFF1 expression was upregulated in already existing cells or whether the lesion had induced neurogenesis of TFF1-ir cells. In conclusion, our findings demonstrate that distinct subpopulations of rodent midbrain dopaminergic neurons express TFF1 and that this expression pattern is altered in a rat model of Parkinson's disease.
S. Jergova, D. Collante, N. Pathak, S. Jani, S. Gajavelli, and J. Sagen
University of Miami, Miller School of Medicine, Miami Project, Miami, FL, USA
Neuropathic pain following spinal cord injury (SCI) insufficiently responds to current pharmacological treatment. Therefore, it is necessary to identify new therapeutic targets and approaches. Hypothesized mechanisms underlying chronic neuropathic pain following injury to the nervous system include increased hyperexcitability of spinal dorsal horn neurons due to loss or dysfunction of inhibitory γ-aminobutyric acid (GABA)-ergic interneurons, and enhanced excitatory glutamate signaling through NMDA receptors. Targeting GABAergic and glutamatergic signaling at the same time may enhance pain relief. Our previous research showed that intraspinal transplantation of recombinant GABAergic progenitors releasing NMDA receptor antagonist serine histogranin (SHG) attenuates hyperalgesia after SCI. The current experiment aims to increase the amount of released SHG using plasmids with multiple copies of the SHG gene to enhance the analgesic effect of grafted recombinant cells for SCI induced pain. GABAergic neuronal precursor cells (NPCs) were harvested from E14 rat embryos and transduced by lentivirus encoding six copies of SHG cDNA. Spinal cord clip compression was used to induce central neuropathic pain. One week after intraspinal injection of engineered cells, attenuation of tactile allodynia was observed in SCI animals, with further improvement in the following weeks. Over 50% reduction in cold allodynia was observed 2 weeks postgrafting. The analgesic effect of grafting persisted for 6 weeks and was reduced by intrathecal injection of SHG antibody. In order to further enhance antinociceptive effect, we were looking for potential candidates acting synergistically with SHG. It has been shown that SHG enhances the effect of opioid treatment and thus reduces aversive side effects of high-dose opioid use. Endomorphins (EMs), endogenous opioid peptides with high selectivity for μ-opioid receptors, are therefore suitable for gene therapy in combination with SHG. To test the efficiency of such combined therapy, an intraspinal injection of a viral construct encoding EM1 and an intrathecal injection of SHG were evaluated in SCI animals. Lenti-EM1 was intraspinally injected 2 weeks post-SCI injury. Attenuation of tactile and cold allodynia was observed 2 weeks post-lenti-EM1 injection and persisted for several weeks. Intrathecal injection of SHG further reduced SCI-induced hypersensitivity to cold and tactile stimuli. Our results indicate that engineered constructs encoding EM1 and multi-SHG could provide significant pain relief in SCI animals. The use of recombinant neuronal progenitor cells or direct gene therapy could be a promising way to reduce pain following SCI.
Supported by NS51667 and CNF 190926.
N. M. Kanaan,* N. C. Kuhn,* C. S. Sortwell,* C. Jiang,† and F. P. Manfredsson*
*Department of Translational Science & Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids, MI, USA
†Center for Proteomics and Systems Biology, University of Texas, Health Science Center, Houston, TX, USA
Parkinson's disease (PD) is a relatively common progressive neurodegenerative disorder generally affecting the aging population. PD patients typically present with unilateral motor symptoms such as resting tremor, which progresses bilaterally over time with increasing severity. Motor symptoms arise from the progressive degeneration of dopaminergic neurons in the substantia nigra pars compacta (SNc), resulting in the loss of dopaminergic innervation in the terminal fields of the caudate/putamen. Although a majority of PD cases are sporadic, several familial forms of PD have been identified. α-Synuclein (α-syn) was the first such gene to be identified. Alterations in the coding sequence and expression levels of the α-syn gene (SNCA) are causative in, or confer enhanced risk for, developing PD and several other neurodegenerative disorders. Although the exact mechanisms whereby α-syn impacts disease pathogenesis are poorly understood, the prevailing hypothesis states that aggregated forms of α-syn are directly toxic to the cell. However, we previously showed that significant loss of α-syn in postdevelopmental dopamine neurons leads to neurodegeneration, suggesting that maintaining some level of functional α-syn is required for dopamine neuron survival. In contrast to the prevailing hypothesis, we propose that α-syn aggregation leads to the reduced bioavailability of monomeric, functional α-syn, thus leading to neurotoxicity through a loss-of function (LOF) mechanism. To study this hypothesis we utilized a form of α-syn that cannot form aggregates, yet retains its function. Previously, we demonstrated that intramolecular cysteine interactions stabilize a particular conformation of α-syn, preventing oligomerization and aggregation in vitro. Here, we utilized recombinant adeno-associated virus type 2/5 (rAAV2/5) to unilaterally deliver a mutant form of a nonaggregatable α-syn (with enhanced cysteine interactions) to the rat substantia nigra (SN). These vector injections were performed in both naive rats and rats receiving SN injections of rAAV2/5 human wild-type α-syn, a rodent model of α-syn aggregation that exhibits neurodegeneration. In contrast to overexpression of wild-type human α-syn, overexpression of the nonaggregatable form of α-syn did not cause significant loss of neurons in the SN. Furthermore, coexpression of nonaggregatable α-syn together with wild-type α-syn prevented nigrostriatal denervation, indicating that this species of α-syn provides neuroprotection. Quantification of tyrosine hydroxylase-positive (TH+, a dopamine neuron marker) neurites in the striatum and TH+ neurons in the SN is under way. Our results suggest that, at least in part, neurotoxicity due to α-syn overexpression is due to an aggregation-dependent LOF toxicity, which can be rescued by supplementing a form of α-syn that cannot form aggregates. Moreover, these findings are in support of our novel hypothesis that α-syn-mediated disease is the result of loss of available functional α-syn.
L. Kelly,*† P. Carvey,† R. Bakay,*† and J. Kordower‡
*Department of Neurosurgery, The Graduate College, Rush University, Chicago, IL, USA
†Department of Pharmacology, The Graduate College, Rush University, Chicago, IL, USA
‡Department of Neurological Sciences, The Graduate College, Rush University, Chicago, IL, USA
Parkinson's disease (PD) is the second most prevalent neurodegenerative disorder and clinically characterized by a battery of debilitating motor and nonmotor symptoms associated with the loss of dopamine (DA) neurons in the substantia nigra pars compacta (SNpc). Postmortem analyses routinely reveal Lewy bodies (LBs) in the brain, the pathological hallmark of the disease, which are proteinaceous cytoplasmic aggregates comprised largely of the presynaptic terminal protein α-synuclein (α-syn). How, when, and why LBs form are unknown. There is an increasing body of evidence suggesting that peripheral factors may contribute to the central degenerative process. Symptomatically, gastrointestinal (GI) impairment is present in approximately 80% of PD patients who suffer from constipation years to decades before the onset of movement disorders. Pathologically, while LBs distribute throughout the central nervous system (CNS) from the lower brain stem to the cerebral cortex, α-syn aggregates and LBs have also been identified in the enteric nervous system (ENS) of PD patients and appear to be present prior to clinical PD symptom onset. Anatomically, the intestinal ENS is extensively innervated from the duodenum to the transverse colon by vagal nerves that originate in the brain stem. These observations present the intriguing possibility that α-syn may accumulate in the GI tract and be transported to the brain stem where its aggregation can initiate neuronal degeneration. Even if transport to the brain is not the initiating event in PD, the presence of α-syn aggregation in the GI tract presymptom development may provide the means to develop a simple biomarker to identify patients presymptomatically so that therapeutics can be designed to prevent the onset of parkinsonian symptoms. Unfortunately, little is known regarding how these LBs form, when they form, or if α-syn is transported into the brain or vice versa. We hypothesize that systemically administered lipopolysaccharide (LPS) and 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) will compromise GI permeability and promote α-syn overexpression in neurons of the submucosal and myenteric plexuses of the GI system that then spread via vagal networks to the medulla where it can initiate neuropathology. To support this hypothesis, preliminary studies from our laboratory revealed that systemically administered LPS (a common bacterial endotoxin) and MPP+ (a dopamine neurotoxin) causes increased α-syn accumulation in the descending colons of mice in addition to significant SNpc neuron loss (Kelly et al.; poster presentation, ASNTR, 2011). Additional findings from our laboratory revealed similar descending colonic α-syn accumulation in MPTP-lesioned and aged nonhuman primates compared to young primates (Kordower et al.; poster presentation, SFN, 2011). Recently published work from our lab in conjunction with others demonstrated that increased intestinal permeability correlates with sigmoid mucosa α-syn staining and endotoxin exposure markers in early PD (Forsyth et al., 2011). We propose to advance these studies by characterizing intestinal permeability and α-syn accumulation in vagal and nonvagal inputs throughout the ENS and CNS in a novel systemic LPS/MPTP model of PD. Effectively modeling the pathogenic process leading to SNpc cell death in PD is fundamental for developing preventative or disease-modifying therapies. These results are expected to have a positive impact because they will contribute to the use of routine colonic biopsies as potential tools for premortem neuropathological diagnoses of PD, as well as contribute to the relatively understudied peripheral pathology of PD.
C. J. Kemp,* S. E. Gombash,† S. L. Wohlgenant,* F. P. Manfredsson,* D. L. Fischer,‡ B. F. Daley,* K. Steece-Collier,* T. J. Collier,* and C. E. Sortwell*
*Department of Translational Science & Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Neuroscience Program, University of Cincinnati, Cincinnati, OH, USA
‡MD/PhD and Neuroscience Programs, Michigan State University, Grand Rapids, MI, USA
Deep brain stimulation of the subthalamic nucleus (STN DBS) is the most practiced neurosurgical intervention for Parkinson's disease (PD). Our laboratory and others have demonstrated that STN DBS provides neuroprotection for dopamine (DA) neurons of the substantia nigra pars compacta (SNc) in preclinical neurotoxin models of PD; however, the duration of this neuroprotection is unclear. In the present study we investigated the effects of STN DBS in the 6-hydroxydopamine (6-OHDA) rat model of PD during, immediately following, and long after the cessation of stimulation. Rats were unilaterally lesioned with intrastriatal 6-OHDA, and electrodes were implanted in the ipsilateral STN. Two weeks postlesion, rats were randomly assigned to receive either continuous STN stimulation lasting 2 weeks (active, n = 5) or not (inactive, n = 4) for 2 weeks' duration. The effect of STN DBS on contralateral forepaw use was assessed (cylinder task): at baseline; 2 weeks following 6-OHDA insult' and on completion of 2 weeks of continuous STN DBS, both during stimulation and immediately after cessation. Cylinder task was repeated at 5 and 24 h poststimulation and at monthly intervals for 4 months. At the conclusion of the study neuroprotection of the nigrostriatal system was evaluated via quantification of the tyrosine hydroxylase immunoreactive (TH-ir) neurons in the SNc and optical density of TH-ir neurites in the striatum. Active rats demonstrated complete amelioration of 6-OHDA-induced deficits in contralateral forepaw use during stimulation that continued for 24 h following cessation of stimulation. Further, for the duration of the 4-month study, active rats used their contralateral forepaw with greater frequency than inactive rats. Active rats exhibited an attenuated TH-ir neuron cell loss in the SNc. Conversely, inactive rats displayed continuing nigral TH-ir neuron degeneration. No differences in striatal TH-ir neurite density were observed between treatment groups. In conclusion, neuroprotection of DA neurons in the SNc and functional improvements that follow a 2-week interval of STN DBS are long lived, persisting for 4 months after the stimulation period has ended. Further, the full magnitude of STN DBS-mediated improvements in forepaw akinesia can persist for as long as 24 h after the cessation of stimulation. Lastly, our finding that STN DBS provides neuroprotection for SNc DA neurons, but not striatal TH-ir neurites, suggests that long-term improvements in forepaw akinesia during and following STN DBS is not entirely dependent on striatal DA levels.
Supported by the Morris K. Udall Center of Excellence for Parkinson's Disease Research at Michigan State University NS058830 (T.J.C.).
D-K. Kim, J. Watanabe, G. J. Block, and D. J. Prockop
Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine at Scott & White, Temple, TX, USA
Ischemia preconditioning of brain and other tissues increases the levels of stanniocalcin-1 (STC-1), a multifunctional hormone that was recently shown to reduce reactive oxygen species (ROS) by increasing expression of mitochondrial uncoupling protein 2. We recently prepared recombinant human STC-1 in an E. coli system that provides large yields of the protein at low cost. The rhSTC-1 was biologically active as demonstrated by inhibition of apoptosis of mouse macrophages (RAW 264.7) exposed to either lipopolysaccharide (LPS) or hydrogen peroxide (H2O2). In tests of neural cells, the rhSTC-1 reduced NF-κB signaling in a line of rat neuronal cells (B35) in which hypoxia was mimicked by exposure to CoCl2. The rhSTC-1 also reduced cell death in primary cultures of rat cortical neurons exposed to both CoCl2 and interleukin-1β (IL-1β). In addition, the rhSTC-1 reduced upregulation of IL-1β and induced nitric oxide synthase (iNOS) in microglia (BV2) exposed to oxygen and glucose deprivation. The results supported previous indications that upregulation of STC-1 largely explains the protective effects of ischemia preconditioning in the brain. The results also suggested that rhSTC-1 might serve as a therapy for ischemia/reperfusion of the CNS.
Supported in part by NIH/NCRR grant P40 RR 17447.
S. U. Kim*†
*Division of Neurology, Department of Medicine, University of British Columbia, Vancouver, Canada
†Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea
Cell replacement therapy and gene transfer to the diseased or injured brain have provided the basis for the development of potentially powerful new therapeutic strategies for a broad spectrum of human neurological diseases. However, the paucity of suitable cell types for cell replacement therapy in patients suffering from neurological disorders has hampered the development of this promising therapeutic approach. In recent years, neurons and glia have successfully been generated from stem cells such as embryonic stem cells (ESCs), induced pluripotent stem cells (iPSCs), mesenchymal stem cells (MSCs), and neural stem cells (NSCs), and extensive efforts by investigators to develop stem cell-based brain transplantation therapies have been carried out. We have previously generated continuously dividing immortalized cell lines of NSCs by introduction of oncogenes and used these immortalized NSC lines in cell and gene therapy studies in animal models of Parkinson's disease (PD), Huntington's disease (HD), amyotrophic lateral sclerosis (ALS), Alzheimer's disease (AD), stroke, and brain tumors. Recently the FDA approved the City of Hope Medical Center in California to proceed with the first human NSC clinical trial to treat brain tumor and seven patients are receiving this suicide gene therapy. This human NSC line was produced in my laboratory in UBC and the study is the first to use human NSC line to deliver an anticancer therapeutic agent.
S. M. Kousik,*† S. M. Graves,*† T. C. Napier,*† and P. M. Carvey*†‡
*Department of Pharmacology, Rush University Medical Center, Chicago, IL, USA
†Center for Compulsive Behavior and Addiction, Rush University Medical Center, Chicago, IL, USA
‡Department of Neurology, Rush University Medical Center, Chicago, IL, USA
Methamphetamine (meth) causes excess dopamine (DA) release and DA neurotoxicity. With an estimated 26 million users globally, meth addiction is made more alarming by reports revealing that meth abuse is a risk factor for Parkinson's disease (PD) as meth abusers display reduced DAergic function and motor deficits similar to PD patients. Potential mechanisms of meth neurotoxicity include DA-quinone and reactive oxygen species-aided neuroinflammation, blood– brain barrier (BBB) dysfunction, and hyperthermia. Clinical studies also report reduced regional cerebral blood flow (rCBF) in the putamen and cortex of detoxified meth abusers. Our study focused on how meth-induced changes in the BBB, tissue perfusion, and the DA system contribute to the vulnerability of meth abusers to PD. We hypothesized that acute and chronic meth treatment would result in BBB dysfunction, tissue hypoperfusion in brain regions similar to those reported clinically, and nigrostriatal DA damage. Rats were treated with acute saline, 3 or 9 mg/kg meth, or trained to self-administer meth chronically (~2.2 mg/kg/day for 14 days), then perfused with fluorescein isothiocyanate-labeled albumin (FITC-LA), a vascular integrity marker. Additional rats treated with acute 9 mg/kg meth also were perfused with Microfil-MV for microcomputed tomography (μCT). Rats given acute 3 or 9 mg/kg meth displayed FITC-LA leakage in the prefrontal cortex and nucleus accumbens shell, suggesting that BBB dysfunction was region selective. Compared to controls, the dorsal striatum of all meth-treated rats uniquely exhibited a striking absence of FITC-LA [F(3, 106) = 221.119], suggesting that meth induced region-specific hypoperfusion. Selective dorsal striatum hypoxia was further verified through increased immunostaining of hypoxia inducible factor 1α, a hypoxia marker, in all meth-treated rats. μCT revealed that meth treatment lowered striatal vascular volume and vessel thickness compared with saline controls. Sensorimotor regional hypoperfusion and hypoxia may destroy DA terminals in striatal regions affected by PD. Thus, we assessed tyrosine hydroxylase (TH) and observed significant reductions in striatal TH [F(3, 32) = 14.777] and an 18% decrease in TH+ cells in the substantia nigra [F(3, 30) = 10.964] in rats trained to self-administer meth, suggesting that chronic meth may render this system more vulnerable to PD. Our findings concur with clinical reports of selective reduced rCBF in the sensorimotor striatal regions. Reduced rCBF and hypoxia may destroy striatal DA terminals leading to DA cell damage in the nigra, thus contributing to meth abuse as a risk factor for PD. This is the first preclinical study on meth-induced cerebral vascular changes as a new mechanism of neurotoxicity and may provide a novel model for meth-induced PD risk. Our results also suggest that striatal hypoxia may contribute to idiopathic PD.
Supported by NS052414, DA15760, DA024923, and the Kenneth Douglas Foundation.
C. Lopez,* E. J. Gonzalez-Rothi,† L. M. Mercier,* A. M. Rombola,* J. Loftus,* B. E. O'Steen,* D. D. Fuller,† M. A. Lane,* and P. J. Reier*
*Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
†Department of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
Cervical spinal cord injuries frequently result in life-threatening respiratory dysfunction even at chronic postinjury times. Injuries rostral to C3-5 are especially devastating due to loss of bulbospinal inspiratory input to phrenic motoneurons (PhMNs), resulting in severe impairment of diaphragm function. Experimentally, this has been extensively illustrated by a lateralized C2 spinal hemisection (C2Hx) model, which results in paralysis of the ipsilateral hemidiaphragm. While some spontaneous diaphragm recovery has been demonstrated, the extent of recovery remains limited. The present work aims to improve the extent of recovery using a cell transplantation approach. A primary goal of these studies was to also determine whether donor neuronal progenitors can become synaptically integrated with the phrenic circuitry and mediate improved ipsilateral hemidiaphragm function. For these experiments, E13-14 rat fetal spinal cord tissue was grafted into acute C2Hx lesions of adult, female Sprague-Dawley rats. Lesion-only animals served as controls. A retrograde transynaptic tracer—pseudorabies virus (PRV)—was applied to the ipsilateral diaphragm or injected directly into transplanted tissue, to assess synaptic integration between the host neurons and transplanted cells. Terminal diaphragm EMG (diaEMG) recordings were also obtained 24-72 h after PRV delivery. Recordings were made during eupneic breathing (exposed to compressed air) or respiratory challenge (elicited by hypercapnia; 7% CO2 delivered via nose cone). DiaEMG in untreated animals revealed only limited activity ipsilateral to C2Hx. Hypercapnia increased diaphragm activity, but ipsilateral output remained impaired. In contrast, transplant recipients showed improved muscle activity ipsilateral to injury during eupneic and challenged breathing. PRV tracing revealed connectivity between donor neurons and the host phrenic circuitry ipsilateral to injury. PRV injection into transplanted tissue revealed integration between donor neurons and 1) host spinal interneurons in surrounding gray matter and 2) medullary neurons in the ventral respiratory column, raphe, and reticular nuclei. These results provide evidence for graft-mediated enhancement of post-C2Hx diaEMG activity that may involve a host-graft interneuronal relay.
Supported by the Brain and Spinal Cord Injury Research Trust (M.A.L.), NIH RO1 NS054025 (P.J.R.), and the Anne and Oscar Lackner Chair in Medicine (P.J.R.).
L. M. Mercier,* N. L. Arias,* K. Z. Lee,† E. J. Gonzalez-Rothi,† B. J. Dougherty,† B. E. O'Steen,* D. D. Fuller,† P. J. Reier,* and M. A. Lane*
*Department of Neuroscience, College of Medicine, University of Florida, Gainesville, FL, USA
†Department of Physical Therapy, College of Public Health and Health Professions, University of Florida, Gainesville, FL, USA
Respiratory rhythm originates in ventral respiratory column (VRC) neurons in the medulla, which extend axonal projections to spinal neurons controlling inspiratory and expiratory muscle activity. Interruption of those bulbospinal pathways results in altered patterns of ventilation. While various neuroplastic changes have been shown in spinal respiratory centers following cervical spinal cord injury (SCI), it is unknown whether medullary respiratory circuitry is also affected. Since supraspinal functional map changes have been reported in other motor systems following SCI, we tested the hypothesis that a high cervical lateralized hemisection (C2Hx) will alter representation of brain stem respiratory neuronal activity. Adult female rats received C2Hx, and at 2 or 12 weeks postinjury electrophysiological mapping was performed of the medullary neurons active during breathing. Electrodes were inserted into multiple VRC locations (spaced 200 μm, and at depths ~500 μm apart) to determine the respiratory activity, inspiratory or expiratory, at each site ipsilateral to the C2Hx. External intercostal electromyogram (EMG) recordings obtained simultaneously defined the pattern of inspiratory activity. Phasic respiratory activity was confirmed audibly and by correlation with EMG signals. Maps from C2Hx animals were compared to those obtained from spinal-intact rats. In naive rats, inspiratory activity occurs predominantly in the rostral VRC, whereas expiratory activity is mostly present in the caudal VRC with each partitioned by an intermediate zone of activity overlap. By 2 weeks postinjury, a reversal of this pattern had occurred in which inspiratory activity recording sites were more frequently represented caudally and expiratory activity sites increased rostrally. At 12 weeks postinjury, the rostral area of where expiratory activity has been detected had expanded caudally. Furthermore, there was a decrease in the number of sites at which inspiratory activity could be detected. These findings may reflect changes in ventilatory behavior, as well as compensation in spinal respiratory motor function.
Supported by NIH RO1 NS054025 (P.J.R.), the Anne and Oscar Lackner Chair in Medicine (P.J.R.), and the Brain & Spinal Cord Injury Research Trust (BSCIRT) Development Funds (M.A.L.).
J. M. Morganti,*† K. R. Nash,* B. A. Grimmig,† S. Ranjit,‡ B. Small,§ P. C. Bickford,*†¶ and C. Gemma†¶
*Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
†Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA
‡Honors College, University of South Florida, Tampa, FL, USA
§School of Aging Studies, University of South Florida, Tampa, FL, USA
¶James A. Haley Vererans' Administriation Hospital Research Service, Tampa, FL, USA
The exact roles for the two types of endogenous CX3CL1 (soluble and membrane-bound fracktaline) in neurodegenerative pathophysiology remain elusive. Recent literature suggests an anti-inflammatory and thus neuroprotective role for supraphysiological levels of exogenous soluble CX3CL1 peptide. However, it has been difficult to delineate the function of the soluble and membrane-bound forms of CX3CL1, as both are natively present in the brain. As such, we examined each form's ability to regulate neuroinflammation in a mouse model of Parkinson's disease initiated by the neurotoxin 1-methyl-4-phenyl-1,2,3,6- tetrahydropyridine (MPTP). We were able to delineate the function of both CX3CL1 forms by using adeno-associated virus (AAV)-mediated gene therapy to selectively express synthetic variants of CX3CL1 that remain permanently soluble (sFKN) or membrane bound (mFKN). Briefly, three groups of 12–16-week-old male CX3CL1-null mice (n = 30 per group) were injected with mFKN, soluble sFKN, or rAAV-expressing green fluorescent protein (GFP) for a viral and protein control, while C57BL/6 (wild-type; WT) mice were injected with sterile saline (WT, n = 60). Mice from each rAAV group received bilateral direct injections into the substantia nigra pars compacta (SNpc) using a CED 26-gauge needle. The group of WT mice received sterile saline via the same injection parameters. Six weeks following SNpc injections, sFKN, mFKN, GFP-treated CX3CL1-null mice, and WT mice were serially injected IP with MPTP (10 mg/kg body weight, 4 injections, with 1-h interval) or sterile saline as an injection control. Five days after IP injections, all mice were analyzed for motor coordination using a rotarod apparatus. Following behavioral analysis, animals were randomly divided for either perfusions or biochemical analyses. Here we show that the CX3CL1-null mice that received sFKN had reduced impairment of motor coordination, less exacerbation of dopaminergic (DA) neuron loss, as well as ameliorated microglia activation (CD11b and CD68) and proinflammatory cytokine [tumor necrosis factor-α (TNF-α) and interleukin-1B (IL-1B)] release, resulting from MPTP exposure. Conversely, CX3CL1-null mice that received mFKN had a phenotype similar to CX3CL1-null GFP-injected mice. To this point, both groups of mice (mFKN and GFP) had significant impairment of motor coordination, exacerbated loss of DA neurons, as measured by tyrosine hydroxylase (TH) and NeuN immunoreactivity. Furthermore, microglia activation (CD11b and CD68) was significantly upregulated in mFKN and GFP groups compared to sFKN. Lastly, there was a significant upregulation of both TNF-α and IL-1B in mFKN- and GFP-treated mice. Together, mFKN-treated mice phenotypically resembled mice injected with GFP for all measurable outputs, suggesting that the full-length membrane-bound form of CX3CL1 is insufficient in providing the anti-inflammatory effects associated with CX3CL1. As such we conclude that the soluble CX3CL1 is the anti- inflammatory and microglia-calming agent in a MPTP mouse model of PD.
D. F. Muresanu,* A. Sharma,† R. Patnaik,‡ and H. S. Sharma†
*Neurology, University of Medicine & Pharmacy, Cluj-Napoca, Romania
†Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology & Intensive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden
‡Institute of Technology, Banaras Hindu University, Varanasi, India
The possibility that nanowired cerebrolysin, a mixture of different neurotrophic factors [e.g., brain-derived neurotrophic factor (BDNF), glial-derived neurotrophic factor (GDNF), nerve growth factor (NGF), ciliary neurotrophic factors (CNTF)] and other active peptides, will be more efficient in inducing neuroprotection following hyperthermia-induced brain injury compared to normal cerebrolysin was examined in this investigation. Whole body hyperthermia (WBH) was induced in rats by exposing them in a biological oxygen demand incubator (BOD) at 38°C for 4 h. This experimental condition induces profound cellular damage in the brain. Since brain injury depletes endogenous sources of neurotrophins, it is likely that cerebrolysin could replace these stores and attenuate WBH-induced brain damage. Previous studies in our lab showed that pretreatment with cerebrolysin 2.5 ml/kg before the onset of WBH significantly reduced brain pathology, blood–brain barrier (BBB) breakdown, and edema formation. However, postapplication of cerebrolysin after WBH was ineffective in inducing neuroprotection. There are reports that nanowired delivery of drugs potentiates the effect of parent compounds and also the drug remains active within the CNS for a long time due to slow metabolism. It appears that nanowired cerebrolysin might have some better therapeutic efficiency in reducing brain damage even administered at different time intervals after WBH. For this purpose, titanium (TiO2) nanowires were used to tag cerebrolysin and the nanowired drug delivery was done in a dose of 2.5 or 5 ml/kg IV at 30, 60, 90, or 120 min after WBH in rats. In these animals BBB permeability, brain edema, and neuronal injuries were examined. The results show that nanowired delivery of cerebrolysin in low doses induced pronounced neuroprotection if administered 30–60 min after WBH. However, high doses of nanowired cerebrolysin (5 ml/kg) achieved significant neuroprotection when administered even 90 or 120 min after WBH. On the other hand, normal cerebrolysin in identical doses was ineffective in reducing brain damage administered either 30 or 90 min after WBH. These observations suggest that nanowired cerebrolysin is the most effective therapy for protecting hyperthermia-induced brain damage. In addition, nanowired cerebrolysin is also able to reduce brain damage if given at different time periods after WBH. This indicates that cerebrolysin may have promising therapeutic potentials for treating brain dysfunction in heat-related illnesses. This presentation is very timely to show how nanowired drugs could enhance therapeutic strategies to treat CNS injuries, even administered various times after the insult in situations of whole body hyperthermia. Thus, the data may have immense clinical value that could be applied in humans exposed to high ambient temperatures for combat or other stressful situations.
C. Perez, S. Jergova, D. Collante, S. Gajavelli, and J. Sagen
University of Miami, Miller School of Medicine, Miami Project, Miami, FL, USA
Chronic neuropathic pain significantly affects the quality of life of peripheral nerve injury or spinal cord injury patients. With low efficacy of current pharmacotherapy for chronic pain, the identification of alternative approaches and new therapeutic targets is essential.
The venoms of marine snail genus Conus are a natural source of various peptides (conopeptides) with potent analgesic effects, some of them already FDA approved or involved in Phase I clinical trials. However, continuous administration of pharmacological agents is often associated with undesirable side effects. This can be overcome by locally targeted therapy, using transplantable recombinant cells as minipumps. Our previous research showed that transplantation of recombinant stem cells equipped with analgesic molecules such as conopeptides is an eligible strategy to reduce neuropathic pain symptoms. In a search for more effective analgesic agents, cannabinoid (CB) receptor agonists have emerged, as robust antinociceptive effects have been reported in various pain models, including spinal cord injury-induced pain. However, most of the available drugs that interact with CB receptors are derived from cannabis and considered clinically unacceptable for long-term therapy due to psychoactive side effects. Therefore, specific compounds interacting with CB receptors without aversive side effects are of clinical interest. In this study we evaluated the ability of several Conus venom extracts to interact with CB1 receptor. The venom extracts of six Conus species were analyzed in vitro. HEK293 cells expressing CB1 were treated with venom extracts for 30 min, fixed, and immunostained. Internalization of CB1 receptor was evaluated using fluorescence immunocytochemistry and confocal imaging or high content screening. Results showed the highest rate of CB1 internalization in HEK293 cells after treatment with venoms of C. textile and C. miles. HPLC fractions (seven per species) of these venoms were subsequently analyzed using a similar approach. Based on the analysis, two subfractions with the highest CB1 agonist activity were evaluated in a formalin test after their intraperitoneal (IP), intrathecal (IT), or intracerebroventricular (ICV) injection. IT injection of the C. tex fraction reduced flinching/licking behavior during the second phase of a formalin test and ICV injection of the C. mil fraction reduced behavior during the first phase. The results indicate the presence of CB1 agonists within the Conus venom extract and their potential analgesic effects. The identification of the single analgesic agents with CB1 agonist activity within the selected venom fractions is in the progress. The final peptide compounds of screened venoms with CB1 receptor affinity could subsequently be used as a new analgesic agent in recombinant cell or gene therapy approaches.
K. M. Piltti,*†‡§ G. M. Funes,*§ S. N. Avakian,*§ C. S. Carlock,*§ N. Uchida,¶ B. J. Cummings,*†‡§ and A. J. Anderson*†‡§
*Sue & Bill Gross Stem Cell Center, University of California, Irvine, CA, USA
†Physical & Medical Rehabilitation, University of California, Irvine, CA, USA
‡Institute for Memory Impairments & Neurological Disorders, University of California, Irvine, CA, USA
§Anatomy & Neurobiology, University of California, Irvine, CA, USA
¶StemCells Inc., Newark, CA, USA
We have previously shown that human central nervous system-derived stem cells (hCNS-SCs) promote locomotor recovery after spinal cord injury (SCI) by integrating into host tissue, and that the majority of the hCNS-SCs differentiated into oligodendrocytes when 75,000 cells were transplanted into the spinal cord 9 or 30 days post-SCI (Cummings, 2005; Hooshmand, 2009; Salazar, 2010). In these studies, the total number of surviving cells was positively correlated with improvements in functional recovery (Hooshmand, 2009). However, the capacity of the injured spinal cord niche to accommodate donor cells and provide sites and cues for cell differentiation and/or integration may be limited. In this study, we investigated the association between cell dose (i.e., the number of transplanted hCNS-SCs) and engraftment, survival, or differentiation by transplanting 10,000, 100,000, 250,000, or 500,000 cells into the spinal cords of nonobese diabetic severe combined immunodeficient (NOD-scid) mice after moderate (50 kDa) midthoracic (T9) contusion SCI at an early chronic stage 30 days postinjury. At 16 weeks posttransplantation, unbiased stereological quantification of hCNS-SCs using a human-specific cytoplasmic marker (SC121) demonstrated that the number of engrafted cells was positively correlated with the transplanted cell dose in each group (Pearson r = 0.79, two-tailed p < 0.0001); that is, a linear relationship was observed between initial transplant dose and the estimated total number of surviving cells determined at 16 weeks posttransplantation. Engraftment and survival were observed across all dose cohorts; however, the ratio of surviving cells relative to the initial cell dose was noted to be significantly higher in the low-dose group when compared to the other dose cohorts (one-way ANOVA, Tukey's test p < 0.05). hCNS-SCs transplanted at all doses showed normal migration from the injection sites into the parenchyma. Quantification of hCNS-SC differentiation revealed that the majority of cells differentiated into SC121+/Olig2+ oligodendrocytes/oligoprogenitors, regardless of the dose group, and that the total number of SC121+/Olig2+ cells was greater in higher dose than lower dose groups. However, the proportion of SC121+/Olig2+ cells varied from 82 ± 7% in the 10,000 cell dose group to 56 ± 4% in the 500,000 cell dose group when normalized to the total number of surviving SC121+ cells. Statistical analysis revealed that the number of engrafted SC121+ cells and SC121+/Olig2+ cells exhibited a negative correlation (Pearson r = −0.62, two-tailed p = 0.002). In contrast, no correlation was found between the number of engrafted SC121+ cells and number of either SC121+/doublecortin (DCX+) neural progenitors (7 ± 1.2%) or SC123+ human astrocytes (15 ± 1.5), suggesting the effect of cell dose on fate was lineage specific. Additionally, the number of SC121+ cells not labeled by any of these three lineage markers exhibited a positive correlation with the number of engrafted SC121+ cells (Pearson r = 0.6, two-tailed p = 0.003), suggesting increasing cell dose may alter the kinetics of differentiation. No aberrant histological findings were observed in any transplanted spinal cords, regardless of dose. These preliminary data suggest a possible correlation between cell dose and/or fate/differentiation kinetics in the injured spinal cord, but overall, similar patterns of survival, fate, and migration were observed in the injured spinal cord at transplantation doses ranging from 10,000 to 500,000 cells.
A. L. Rachubinski* and K. B. Bjugstad*†
*Neuroscience Program, Colorado Intellectual & Developmental Disabilities Research Center, University of Colorado Denver, Aurora, CO, USA
†Department of Pediatrics, Colorado Intellectual & Developmental Disabilities Research Center, University of Colorado Denver, Aurora, CO, USA
The implantation of neural progenitor cells (NPCs) into the brain can benefit adult models of neurodegeneration, for example by normalizing cell populations in the dopamine-depleted striatum of parkinsonian primates (Bjugstad et al., Cell Transplantation, 14:183; 2005). However, there are many disorders that are present from birth and that continue into adulthood and aging, such as Down syndrome (DS). How NPC-based therapies might also benefit these conditions is unknown. Using a DS mouse model, we sought to answer the question of NPC longevity. Ts65Dn trisomic mice (DS modeling) and their disomic littermates (normal controls) were implanted into the hippocampus on postnatal day 2 (PND2) with NPCs, saline, or remained untreated. Twelve months later, all mice were tested for cognitive function, the long-term survival of NPCs, hippocampal cell numbers, and the presence of extracellular tau accumulation. We previously reported on the cognitive outcomes. Briefly, the implantation of NPCs in trisomic mice improved performance on the conditioned taste aversion, but not on the novel object recognition task. Trisomic mice given saline control injections improved performance on both cognitive tasks. Disomic mice, implanted with either saline or NPCs, appeared impaired in both tasks. The current presentation reports on the survival of the NPCs and long-term changes in the hippocampus of these aging mice. Surviving NPCs were found in the brains of both disomic and trisomic mice, but rarely at the site of implantation, the hippocampus. Most NPCs were found in the subventricular zone (SVZ) and surrounding areas (e.g., striatum and septum). No hypocellularity in CA1, CA3, or the dentate gyrus was found in hippocampus of trisomic mice. Both implantation procedures, though, tended to reduce the number of hippocampal neurons in disomic and trisomic brains. Hippocampal cell numbers were not correlated with cognitive performance. Extracellular tau aggregations were found with greatest frequency in the stratum lacunosum-moleculare in CA1, compared to other areas in the hippocampus or brain. Trisomic brains had significantly more extracellular tau aggregations than the disomic brains. Brains that were implanted with NPCs, regardless of evidence for survival 12 months later, were associated with fewer extracellular tau aggregations. This was found to be true in both disomic and trisomic brains. Saline injections significantly elevated tau presence in both karyotypes. In light of the saline implantation-induced changes, and the lack of a clear benefit of NPCs, we conclude that the effects of NPCs cannot be distinguished from those induced by the implant procedure. Further, these effects, while initiated neonatally, have long-term consequences. An alternative route of NPC administration, which enhances cell survival and minimizes implant injury to the brain, may be advisable to separate out discrete NPC effects.
This work was supported by a Ruth L. Kirschstein National Research Service Award (NRSA) (F31NS060517), The Anna and John J. Sie Foundation, and The Linda Crnic Institute.
I. Rattray,* R. Gale,† E. Smith,* K. Matsumoto,* G. Bates,† and M. Modo*
*Institute of Psychiatry, King's College London, London, UK
†Medical and Molecular Genetics, King's College London, London, UK
The R6/2 transgenic mouse line exhibits a rapid onset of Huntington's disease (HD)-like symptoms. The current study provides a interdisciplinary, longitudinal description of the progressive pathology exhibited by this model. For this purpose, both male and female R6/2 mice along with wild-type littermate controls were repeatedly assessed on a variety of functional measures, including motor performance on an accelerating rotarod, anxiety-like behavior, and locomotor activity in an open field, as well as cognitive performance in the passive avoidance test. Alongside this behavioral characterization, we have implemented serial in vivo MRI for studying age-related changes in brain atrophy and tissue T2 relaxometry. Scanning was conducted at 4, 8, 12, and 14 weeks of age and was separated by the behavioral assays. To support these in vivo measures, postmortem quantification of neuropathological markers including stereological neuronal cell counts and mutant huntingtin protein expression have been taken. Marked abnormalities were observed in the R6/2 mice, including the development of deficits at the rotarod and an escalating hypoactivity in the open field. A progressive atrophy in a variety of brain regions was evident alongside a shortening of T2 relaxation times. We will perform a correlative analysis with the aim of linking changes in behavioral phenotypes with the development of brain abnormalities. This investigation will provide crucial information regarding the neurobiological underpinnings of brain atrophy in the R6/2 mice and how these relate to the emergence of behavioral impairments.
Supported by the Medical Research Council and the CHDI Foundation.
P. R. Sanberg,* S. Garbuzova-Davis,* J. Tan,* D. J. Eve,* C. V. Borlongan,* L. E. Cruz,† N. Kuzmin-Nichols,‡ and C. D. Sanberg‡
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Cryopraxis Criobiologia Ltda, Rio de Janeiro, Brazil
‡Saneron-CCEL Therapeutics, Tampa, FL, USA
Cell therapy has great potential for the treatment of neurodegenerative diseases. The mononuclear cell fraction from umbilical cord blood (HUCBCs) is a heterogenous population, including stem cells, lymphocytes, and monocytes, and provides benefit following a single relatively high intravenous dose in animal models of Alzheimer's disease (AD), amyotrophic lateral sclerosis (ALS), and Sanfilippo syndrome type B (mucopolysaccharidosis III B; MPS III B). Very few (if any) of these injected cells remain inside and outside of the CNS a few months after a single injection. This, in conjunction with the progressive nature of these diseases, suggests that repeated injections of lower cell doses may prove to be more effective than a single dose. Using 7-month-old transgenic PSAPP and Tg2576 mice as models of AD, 100,000 HUCBCs were administered intravenously every 2 weeks for 2 months and then monthly for an additional 4 months. Multiple injections of this low dose of HUCBCs were found to reduce amyloid accumulation and associated astrogliosis as well as attenuate CD40L-induced inflammatory responses to a greater extent than seen in mice treated with either a single higher dose of HUCBCs (5 × 106) or vehicle. A single dose of intravenously administered HUCBCs (25 × 106) into presymptomatic G93A SOD1 mice (an animal model of ALS) was found to be optimal for delaying the disease progression, extending the life span and decreasing proinflammatory cytokines in these mice. Since the equivalent dose in humans is unlikely to be feasible, multiple weekly injections of 106 or 2.5 × 106 HUCBCs were administered to presymptomatic (9 weeks old) and symptomatic (13 weeks old) G93A SOD1 mice. The multiple 106 cell doses in presymptomatic and 2.5 × 106 cell doses in symptomatic mice were found to delay motor functional deterioration, extend life span, and reduce microglial responses compared to vehicle-treated mice. A single intravenous administration of 3 × 106 HUCBCs into 3-month-old α-N-acetylglucosaminidase (Naglu) knockout mice (an animal model of MPS III B caused by deficiency of the enzyme Naglu), modestly improved the behavioral outcomes, reduced heparin sulfate accumulation, and modified inflammation compared to vehicle-treated mice. By comparison, multiple same dose cell injections, given monthly for 6 months, were found to lead to prolonged benefits in behavior, greater cell migration into the brain, and reduced heparin sulfate accumulation versus mice given a single cell dose. These studies show that multiple low doses of HUCBCs administered intravenously are more beneficial than a single high cell dose. The use of multiple injections is likely to provide long-term benefit against the progressive nature of these diseases, by providing sustained replacement of the Naglu enzyme in MPS III B as well as long-term trophic support against the progressive degeneration in AD and ALS. In addition, use of smaller doses will be easier to translate into the clinical setting. These studies therefore provide the groundwork for more comprehensive investigation to optimize the frequency and size of multiple injections in relation to their safety and clinical translatability for these and potentially other neurodegenerative diseases.
P.R.S., S.G.D., J.T., C.V.B., and L.E.C. are founders and/or consultants of Saneron CCEL Therapeutics. L.E.C. is Founder and P.R.S. is consultant of Cryopraxis Criobiologia Ltda. These projects were funded in part by the Children's Medical Research Foundation and the NIH.
D. E. Sanchez, K. Salazar, L. M. Mercier, B. E. O'Steen, P. J. Reier, and M. A. Lane
Department of Neuroscience, McKnight Brain Institute, University of Florida, Gainesville, FL, USA
Injuries in the midcervical spinal cord dramatically impair breathing. This is due primarily to disruption of bulbospinal respiratory fiber pathways and direct loss of phrenic motoneurons (PhMNs) and prephrenic interneurons. A consistent respiratory outcome of C3–C5 contusions in rodents is chronically diminished diaphragm EMG (diaEMG) responses to respiratory challenge (e.g., hypercapnia). Recent experimental studies have shown that this deficit may be due primarily to gray matter damage. Therefore, the present study will test the hypothesis that restitution of gray matter integrity following midcervical contusion injury will improved diaphragm function. Lateralized C3/4 contusions were produced in adult female rats using the Infinite Horizon Impactor (200 kilodyne preset force), and the animals were divided into treated and untreated groups. One week postinjury in treated animals, dissociated E13.5–14 fetal spinal cord (FSC) tissue was injected into the lesion epicenter. Animals were allowed to recover for 1 month posttransplant. At that time, pseudorabies virus (PRV) was applied directly to the diaphragm or injected into the site of transplantation, to establish connectivity between 1) donor neurons and the host phrenic circuit and 2) host and donor neurons. Terminal EMG recordings were made in spontaneously breathing animals to assess ipsi- and contralateral diaphragm activity under baseline (eupneic breathing) and challenge (exposed to hypercapnia) conditions. While diaphragm activity usually increases during respiratory challenge, all injured untreated animals exhibited blunted ipsilateral EMG responses to respiratory challenge 5 weeks postinjury. In contrast, graft recipients showed elevated EMG activity during challenge. PRV delivered to the ipsilateral hemidiaphragm resulted in infection of the PhMN pool and second-order host interneurons, as well as labeling of a contingent of donor neurons. Intragraft injection of PRV resulted in labeling of host interneurons in the vicinity of the grafts. Additional immunohistochemical analyses revealed enlarged astrocytes surrounding the donor tissue and both rostral and caudal to the site of injury/transplant in the host spinal cord. These data demonstrate that restoration of gray matter integrity by spinal interneuronal progenitors can ameliorate an aspect of postcervical SCI respiration, even in the presence of substantial astrocytic accumulation and activation. Our neuroanatomical findings further suggest this functional change is mediated by bidirectional connectivity between donor neurons and host interneurons and PhMNs.
Supported by NIH RO1 NS054025 (P.J.R.), the Anne and Oscar Lackner Chair in Medicine (P.J.R.), and the Brain & Spinal Cord Injury Research Trust Development Funds (M.A.L.).
I. M. Sandoval,* F. Chan,* B. A. Price,* A. K. Gross,† W. W. Hauswirth,‡ T. G. Wensel,* and J. H. Wilson*
*Biochemistry and Molecular Biology, Baylor College of Medicine, Houston, TX, USA
†Vision Sciences, University of Alabama at Birmingham, Birmingham, AL, USA
‡Ophthalmology and Molecular Genetics, University of Florida, Gainesville, FL, USA
Targeted gene editing is a promising potential therapy to treat many devastating genetic diseases caused by dominant mutations. The strategy requires the introduction of a double strand break at a target site in the genome. Repair of the break by the error-prone nonhomologous end joining (NHEJ) pathway can introduce mutations and block expression of the toxic gene, whereas gene correction can be achieved if repair occurs by homologous recombination (HR). To test and develop this approach as a therapy for retinal and other neurodegenerative disorders we have built three mouse models for detection of gene correction in retinal photoreceptor cells. The knocked-in alleles code for mutant forms of human rhodopsin, the most abundant protein in rod cells, fused to enhanced green fluorescent protein (GFP) at the C-terminus; but expression of full length hRho-GFP is only achieved upon successful gene repair, allowing easy quantification of such rare events. In retinas containing an allele with a duplication of one exon (ID2) that causes a frame shift, treatment with recombinant adeno-associated virus (rAAV) expressing endonuclease Isce1 led to a dramatic increase in GFP-positive cells, about 15% of all cells in the injected area. Sequence analysis revealed that double strand breaks (DSBs) in these cells were repaired by HR, while those in the nonfluo-rescent cells were repaired by NHEJ. Mice bearing nonsense mutations that cause retinitis pigmentosa in humans, Q64X and Q344X, had low, and strikingly different, frequencies of spontaneous correction, 10−7 and 10, respectively. The overwhelming majority of these occurred in individual cells rather than pairs or clusters, indicating the mutation happened after the last mitosis, and the frequencies did not increase significantly with age. Since the 5′ untranslated regions of the transcription units were engineered for low translation levels, the retinas do not undergo degeneration, so they are a powerful tool for investigating the ability of treatments, such as rAAV encoding zinc-finger endonucleases designed to cut near the mutation sites, to effect gene repair.
S. Schroeder,*† M. N. Gordon,*† and D. Morgan*†
*USF Health Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
†Department of Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
Immunotherapy, as a potential for treating, or maybe even preventing, Alzheimer's disease (AD) has been extensively studied, primarily with A-β as the target protein, leading to a number of clinical studies. Recently, tau pathology has been receiving more attention as a therapeutic target for immunotherapy, especially due to the stronger correlation between tau pathology and cognitive deficits. In this series of experiments we have tested multiple antibodies against tau for their effects on pathology, seeking to find the most effective agent to pursue further, ideally to clinical trial. In these experiments, we examined the acute results of intracranial administration of antibodies to three different “classes” of tau pathology: PHF-1, which recognizes hyperphosphorylation at Ser396/404; MC-1, an antibody that detects conformational changes of tau protein; and DA 9, a pan-tau antibody that may detect and/or interact with both normal and pathological forms of tau. All of these were compared to anti-GFP control antibody administration, an identical isotype that is not known to be biologically active. We used the rTg4510 mouse model, as it is known for inducing robust tau pathology in cortical and hippocampal areas relevant to AD and other dementias; 30 mice were divided into four groups of 7–8. Each group received unilateral intracranial injection of one of the antibodies listed above, into the right anterior cortex and hippocampus. The left hemisphere was left untreated as an internal control for extent of tau deposition. Four days postinjection tissue was collected for immunohistochemical analysis and future biochemistry. The following histochemical techniques were conducted to assess the effect of the antibodies: Gallyas silver stain, Nissl stain, and immunocytochemical staining for H150, pSer199/202, pSer396, and CD45. Both MC-1 and DA 9 reduced pathology as indicated by some of the markers tested, whereas PHF-1 did not appear to significantly reduce tau pathology. Based on the results presented here, we are inclined to pursue MC-1 further as a treatment of choice.
Supported by NS076308.
M-L. B. Selenica,* D. C. Lee,† B. Manchec,* G. Pena,* K. Nash,* J. Alvarez,* P. Reid,* D. Morgan,* and M. N. Gordon*
*Department of Molecular Pharmacology & Physiology, Byrd Alzheimer's Institute, University of South Florida, Tampa, FL, USA
†College of Pharmacy, University of South Florida, Tampa, FL, USA
Bone marrow-derived cells (BMDCs) of the monocyte/macrophage lineage include two subtypes: “resident” (expressing CCR2neg, CX3CR1hi) and “inflammatory” (CCR2hi Ly6C+). Macrophages and microglia become activated through two activation states: M1, which incites tissue damage and promotes proinflammatory cytokine profiles, and M2, which promotes wound repair and dampens proinflammatory pathways. We examined whether cytokine- induced M1 versus M2 activation promote recruitment and infiltration of peripheral monocytes into the CNS, and which subtype(s) of monocytes respond to M1 versus M2 stimulator cocktails. To do so, we transduced endogenous BMDCs with green fluorescent protein (GFP) by intraosseous injections of recombinant adeno-associated virus (serotype 9; rAAV9). Mice received injections of rAAV9-GFP bilaterally into both femurs. Three weeks later, mice received an intracranial injection of either an M1 cytokine cocktail [tumor necrosis factor-α (TNF-α), 667 ng; interleukin-12 (IL-12), 26 ng; and IL-1β, 26 ng], an M2 cytokine cocktail (IL-4, 800 ng and IL-13, 240 ng) or vehicle (PBS). Brain and bone marrow were analyzed by flow cytometry for GFP expression, microglia/macrophage markers, and “inflammatory” markers (CCR2hi, Ly6C+) 3 days later. In bone marrow tissue [after red blood cell (RBC) lyses] rAAV9-GFP transduced 4% of the total BMDC population. Forty percent of the BMDCs expressed Ly6C, 5% were labeled as CCR2hi, 10% versus 3% were labeled as CD45+ and CD11b+, respectively. In brain tissue, M1 stimuli significantly induced recruitment of GFP+ cells into the brain compared to M2- and vehicle-injected animals. M1 cytokine cocktail, but not M2 cocktail, increased Ly6C+, CD11b+, and CD45+ expression in brain microglia. M1 activation induced recruitment and infiltration of an “inflammatory” subset of peripheral monocytes because the GFP+/Ly6C+ population was increased only in this group. However, both M1 and M2 activation of microglia induced increases in CCR2 expression within brain microglia, indicating activation of brain microglia due to M1/M2 stimuli. Next, we will use immunohistochemistry to determine the functional state the transduced GFP+ cells that homed into CNS by staining for markers of M1 (IL-1β, Marco) versus M2 (YM1, Arg-1) activation. Overall, these experiments will identify specific inflammatory stimuli that enhance recruitment and infiltration of endogenous inflammatory monocyte subsets into CNS.
Supported by NIH grant AG015490.
M. Shahaduzzaman,* J. McAleer,* J. Grieco,* J. Golden,* J. Glover,* J. Hall,* M. Cortes-Salva,† J. Antilla,† J. Cuevas,‡ K. R. Pennypacker,‡ and A. E. Willing*‡
*Center for Excellence in Aging & Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA
†Department of Chemistry, University of South Florida, Tampa, FL, USA
‡Molecular Pharmacology and Physiology, University of South Florida, Tampa, FL, USA
In humans, white matter comprises about 50% of brain volume and is adversely affected in most cases of stroke. In rodents, white matter constitutes only 14% of brain volume and most animal stroke models have largely neglected the study of white matter injury after stroke. Oligodendrocytes are more sensitive to excitotoxicity and oxidative stress than neurons, so a drug targeted only to gray matter will not necessarily be efficient in minimizing damage or enhancing recovery after stroke. In our previous study, we report that Sigma1 and 2 receptor (σ1/σ2Rs) agonist [1,3 di-o-tolylguanidine (DTG)] treatment decreased short-term infarct volume when administered 24 h poststroke, but the effect was not maintained over the long term and therefore did not improve behavioral outcomes (Leonardo et al., 2010). In the present study we administered a newly synthesized mixed σ1/σ2Rs agonist, N′N-di-NAPH-HCL (NAPH), which is based on the structure of DTG. The present study examines long-term therapeutic effects of this compound on infarct volume and white matter as well as neurobehavioral outcomes. Male Sprague-Dawley rats were divided into two groups (n = 16/group) receiving either daily subcutaneous injections of NAPH or vehicle for 3 days starting at 24 h poststroke. Our results demonstrated that infarct volume in the stroke group receiving vehicle only (n = 10) was 30.49 ± 8.71%, whereas animals receiving NAPH (n = 12) showed infarcts involving only 13.93 ± 3.79% of the hemisphere 30 days poststroke. We also observed that NAPH protects white matter from stroke-induced lesions, suggesting that NAPH exerts its neuroprotective effects via preservation of both gray matter and white matter tracts. Future studies will examine in vitro effects of NAPH on oligodendrocyte (OL) apoptosis. This study demonstrates that NAPH reduces infarct volume and protects white matter from stroke-induced lesion, and σRs may prove to be a valuable therapeutic target for stroke.
This study was supported in part by James and Esther King Florida Biomedical Team Science Program #09KT-02.
H. S. Sharma,* D. F. Muresanu,† R. Patnaik,‡ and A. Sharma*
*Laboratory of Cerebrovascular Research, Department of Surgical Sciences, Anesthesiology & Intensive Care Medicine, University Hospital, Uppsala University, Uppsala, Sweden
†Neurology, University of Medicine & Pharmacy, Cluj-Napoca, Romania
‡Institute of technology, Banaras Hindu University, Varanasi, India
The possibility that exposure of silica dust to hypertensive individuals may exacerbate brain pathology and sensory motor dysfunctions, particularly at high environmental temperature, was examined in this investigation. Since military combat operation is often associated with both psychological and physical stress resulting in hypertensive episodes, it is quite likely that additional exposure of silica at high environmental temperature will further deteriorate hypertension induced neurological dysfunctions. Hypertension was produced in rats (200-250 g) by 2-kidney one clip (2K1C) method causing elevation of mean arterial blood pressure (MABP) by 20 ± 6 mmHg after 1 week and 40 ± 5 mmHg after 4 weeks. In these rats, SiO2 nanoparticles (50-60 nm) were administered 50 mg/kg, IP, once daily for 1 week and then subjected to partial restraint in a Perspex box restricting their movement for 4 h either at room temperature (21°C) or at 33°C in a biological oxygen demand incubator (BOD, wind velocity 2.6 cm/s, relative humidity 65-67%). In these animals, behavioral functions, blood-brain barrier (BBB) permeability to Evans blue albumin (EBA) and radioiodine (iodine), brain water content, and neuronal injuries were determined. Hypertensive rats subjected to 4-h restraint at room temperature did not exhibit BBB dysfunction, brain edema, neural injury, or alterations in Rotarod or inclined plane angle performances. However, when these hypertensive rats were subjected to restraint at 33°C, breakdown of the cortical BBB (EBA +38%, radioiodine +56%), brain water (+0.88%), neuronal damages (+18%), and behavioral impairment were much more exacerbated. Interestingly, SiO2 exposure to these hypertensive rats further exacerbated breakdown of the BBB (EBA 280%, radioiodine 350%), brain edema (4%), and neural injury (30%) after restraint stress depending on the ambient temperature. SiO2 treatment also induced brain pathology and alteration in behavioral functions in normotensive rats after restraint at high temperature, a feature not seen in saline-treated normotensive or hypertensive rats. These observations clearly show that hypertension significantly enhances restraint-induced brain pathology and behavioral anomalies, particularly at high ambient temperature and SiO2 intoxication further exacerbate these brain damage and cognitive dysfunctions.
H. Shen, Y. Luo, H-S. Liu, S. J. Yu, Y. Yang, B. J. Hoffer, and Y. Wang
Neural Protection and Regeneration Section, National Institute on Drug Abuse, Baltimore, MD, USA
Current pharmacological treatments for stroke are limited by a narrow therapeutic time window. We here describe a delayed, noninvasive poststroke therapeutic approach involving functional recovery after stroke. Rats were treated with cocaine- and amphetamine-regulated transcript (CART), delivered into the nostrils, from day 3 after middle cerebral artery occlusion. CART treatment improved behavioral recovery and reduced neurological scores. In the subventricular zone (SVZ), CART enhanced immunolabeling of bromodeoxyuridine, the neuroprogenitor cell (NPC) marker Musashi-1, and the proliferating cell nuclear antigen, as well as upregulated brain-derived neurotrophic factor (BDNF) mRNA. Overexpression of BDNF by local administration of AAV-BDNF in SVZ enhanced migration of SVZ cells toward the ischemic cortex and induced behavioral improvement. In SVZ culture, CART increased neurosphere formation and cell migration. CART-mediated cell migration was significantly antagonized by anti-BDNF blocking antibody, suggesting this response is mediated through BDNF. Diffusion-tensor imaging and 1H-magnetic resonance spectroscopy were used to examine reinnervation in the lesioned cortex. Significant increases in the fractional anisotropy ratio and N-acetylaspartate levels were found in the lesioned cortex after CART treatment. Western analysis indicated that CART significantly increased the expression of growth-associated protein 43 in the lesioned cortex. Taken together, these data suggest that CART enhances reinnervation in the lesioned cortex. In conclusion, poststroke intranasal treatment with CART facilitates behavioral recovery, which is associated with proliferation and migration of NPCs from SVZ and an increase in neural innervation in the lesioned cortex. Our results may provide a new treatment strategy for stroke patients, enabling a noninvasive and longer treatment window of days after stroke occurrence.
K. Shinozuka, N. Tajiri, N. Weinbren, Y. Kaneko, and C. V. Borlongan
Center of Excellence for Aging & Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
Stress has been implicated as a major exacerbating factor of stroke pathological manifestations, including immune response. The present study was designed to investigate the interaction between stroke and stress, with the latter characterized behaviorally by a novel pain perception scale, and cellularly by DNA damage in the thymus, an important organ for immune defense system (i.e., T-lymphocyte maturation). Fifteen Sprague-Dawley rats received middle cerebral artery occlusion (MCAo) to induce experimental stroke. Thirteen rats served as intact control subjects. Routine behavioral and histological assays were performed to determine stroke symptoms. For characterization of stress, the subjects were videotaped for 15 min at 1 day before and 0, 1, and 3 days after MCAo. After video recording at day 3, all animals were sacrificed and their thymus collected. Subjects' pain perception, which is considered as one of the poststroke stressors, was analyzed from the video in accordance with Rodent Grimace Scale (Longford et al., 2010; Sotocinal et al., 2011). DNA damage in the thymus was analyzed by immunostaining with gamma-histone 2a (H2AX) antibody, which is known as a DNA double strand break marker. Stroke animals displayed the characteristic motor asymmetry and cerebral infarction, whereas intact control subjects exhibited normal behaviors and nondetectable pathological damage to the brain. Pain scores before MCAo revealed no significant differences across groups. Following MCAo, significant treatment effects on pain scores were detected. Stroke subjects showed significantly higher pain score than those in intact subjects (p < 0.05). Gamma-H2AX-immunoreactive cells were localized at the boundary between medulla and cortex of the thymus, and were highly expressed in the stroke group compared to the control group. This is the first report extending the utility of the rodent grimace scale in a neurological condition. Here we show that the typical stroke neurobehavioral symptoms produced by MCAo were accompanied by a pain response. Moreover, we demonstrate that this stroke-induced behavioral stress response coincided with an upregulation of a cellular stress marker involving DNA damage in the thymus. These results suggest that care and management of stroke patients, at least in the acute setting, may benefit from a careful consideration of treating stressors. Ongoing studies are utilizing these unique and sensitive stress assays as surrogate markers of therapeutic benefits of cell therapy in experimental stroke.
J. A. Shires,*† S. Ohshima-Hosoyama,* N. Goecks,‡ V. Joers,*† C. R. Swanson,*† V. Bondarenko,* K. Cumminsford,* R. Velotta,* H. A. Simmons,* K. Brunner,* A. Alexander,‡ B. Blits,§ M. Sonnemans,§ S. Hermening,§ and M. E. Emborg*†‡
*Wisconsin National Primate Research Center, University of Wisconsin-Madison, Madison, WI, USA
†Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI, USA
‡Department of Medical Physics, University of Wisconsin-Madison, Madison, WI, USA
§Amsterdam Molecular Therapeutics. Amsterdam, The Netherlands
Application of convection enhanced delivery (CED) to gene therapy is proposed as a method to optimize treatment delivery by maximizing intracerebral distribution of viral vector per inoculation, minimizing the risks associated to multiple injections sites. Several adeno-associated viral vector (AAV) serotypes are currently being evaluated to deliver therapeutic transgenes to the brain. While AAV2 is widely used for preclinical and clinical research, other serotypes are emerging for their different cellular affinity and transfection efficacy. Here we report our analysis in nonhuman primates of the feasibility of applying CED technology for the delivery of AAV5 encoding for glial-derived neurotrophic factor (GDNF) or green fluorescent protein (GFP). Adult rhesus monkeys were screened for the presence of antibodies against AAV5. Four seronegative animals were selected, and underwent baseline MRI and angiography for evaluation of anatomy and targeting purposes. Two animals received a high dose (1E11 gc per injection) and the other two a low dose (1E10 gc per injection) of AAV5 encoding for GDNF into the right ventral postcommisural putamen and for GFP into the contralateral hemisphere. Each vector inoculation (one per hemisphere) had a volume of 30 μl, and it was performed under intraoperative MRI monitoring using CED methods (rate of infusion: 1 μl/min). Six weeks after surgery the animals were necropsied and the brains removed and processed. Evaluation of imaging obtained during surgery, as well as postmortem analysis of intracerebral expression of GDNF and GFP, confirmed accurate and replicable targeting of the postcommissural putamen nucleus in all monkeys. Distribution beyond the target was titer dependent. Retrograde transport of both recombinant proteins into the substantia nigra was observed at the two titer levels. Quantification of dopaminergic markers revealed increased striatal and nigral TH optical density immunoreactivity in the side ipsilateral to GDNF treatment, suggesting its biological activity. Although stereological cell count of TH-positive nigral neurons did not reveal differences between treatments and titers, nigral neuronal volume was greater in the side ipsilateral to GDNF compared to GFP, hinting at a biological effect of GDNF. Our results suggest that application of intraoperative MRI-guided CED for gene transfer is an accurate and replicable method of targeting that facilitates viral vector distribution and efficacy to deliver a therapeutic transgene. Additional molecular biology analysis of GDNF and GFP expression is currently under way.
Supported by P51 RR000167 (NIH-NCRR), to the Wisconsin National Primate Research Center, University of Wisconsin-Madison, NIH-NIHMS P32 GM007507 (J.A.S.). This research was conducted in part at a facility constructed with support grants RR15459-01 and RR020141-01.
R. D. Shytle,* A. J. Smith,* P. Kavuru,† L. Wojtas,† and M. J. Zaworotko†
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Health Morsani College of Medicine, Tampa, FL, USA
†Department of Chemistry, College of Arts and Sciences, University of South Florida, Tampa, FL, USA
Flavonoids have been studied extensively due to the observation that diets rich in these compounds are associated with lower incidences of neurodegenerative diseases, such as Alzheimer's disease. One of the most studied flavonoids, quercetin, is also the most abundant of these compounds in the plant kingdom. While numerous therapeutic bioactivities have been identified in vitro, quercetin's in vivo efficacy in pure form is limited by poor bioavailability, primarily due to its low solubility and consequent low absorption in the gut. Cocrystallization has gained attention recently as a means for improving the physicochemical characteristics of a compound. Here, we synthesized and evaluated four new cocrystals of quercetin (QUE): quercetin/caffeine (QUECAF), quercetin/caffeine/methanol (QUECAF·MeOH), quercetin/isonicotinamide (QUEINM), and quercetin/theobromine dihydrate (QUETBR·2H2O). Each of these cocrystals exhibited pharmacokinetic properties that are vastly superior to quercetin alone. Cocrystallization was able to overcome the water insolubility of quercetin, with all four cocrystals exhibiting some degree of solubility. The QUECAF and QUECAF·MeOH cocrystals increased the solubility of QUE by 14-and 8-fold when compared to QUE dihydrate. We hypothesized that this improved solubility would translate into enhanced systemic absorption of QUE. This hypothesis was supported in our pharmacokinetic study. The cocrystals outperformed QUE dihydrate with increases in bioavailability up to nearly 10-fold. Studies are now under way to determine if these quercetin cocrystals provide better access to the brain, and thus superior neuroprotective efficacy over quercetin alone.
The authors are inventors on patents covering the composition of matter and use of nutraceutical cocrystals.
R. D. Shytle,* A. J. Smith,* P. Kavuru,† and M. J. Zaworotko†
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Health Morsani College of Medicine, Tampa, FL, USA
†Department of Chemistry, College of Arts and Sciences, University of South Florida, Tampa, FL, USA
New research suggests that lithium is an essential trace element in higher animals and may be an important therapeutic tool for developing novel stem cell therapies as well as treating neurodegenerative disease. Whereas lithium is the only medication that consistently reduces suicide in vulnerable populations and is still considered the gold standard for treating bipolar disorder, the lithium formulations currently available, such as lithium chloride and lithium carbonate, provide a narrow therapeutic window due, in part, to their poor physicochemical properties and thus require routine blood monitoring to prevent systemic lithium toxicity. In an effort to develop new lithium compounds with improved oral bioavailability as well as selected uptake by the brain over other organs, we have recently evaluated the pharmacokinetics of some new ionic cocrystals of lithium developed by our interdisciplinary team at USF. We will present preliminary data from one of our compounds exhibiting vastly superior oral absorption and blood–brain barrier penetration in rats. We believe that these new lithium cocrystals could potentially lower the dose of lithium needed to achieve therapeutic effects in the CNS without the toxic peripheral side effects that arise from conventional lithium medications.
Y. A. Sidorova,* M. M. Bespalov,* M. Karelson,†‡ and M. Saarma*
*Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
†Tallinn University of Technology, Tallinn, Estonia
‡GeneCode Ltd., Tallinn, Estonia
Chronic or neuropathic pain is caused by damage of sensory neurons and affects up to one sixth of the Western population at various times of an individual's life. This condition may develop as a result of physical trauma, disease processes (e.g., diabetes), exposure to drugs and chemical compounds, or infections (e.g., HIV) (Ossipov et al., 2011). Current treatments poorly control chronic pain and bring only symptomatic relief to suffering people. They do not eliminate the cause of the condition or, in other words, they do not promote regeneration or rescue of damaged neurons. In addition, existing neuropathic pain treatment strategies are accompanied by the drug tolerance development. Thus, the necessity to develop novel medications supporting injured neurons in patients with chronic pain is clear. It is well established that neuronal cells require constant support for survival and proper functioning that is provided by small secretory proteins called neurotrophic factors. One of such proteins called artemin (ARTN) supports sensory neurons and, as was recently shown, is able to relieve symptoms and repair damaged neurons in animal models of neuropathic pain (Gardell et al., 2003; Wang et al., 2008). Thus, ARTN can pave the way to the development of a totally new approach for neuropathic pain management. ARTN belongs to the glial cell line-derived neurotrophic factor (GDNF) family ligands (GFLs) and signals via a receptor complex consisting of transmembrane rearranged during transfection (RET) receptor tyrosine kinase (RTK) and a co-receptor-GDNF family receptor α3 (GFRα3). Ligand binding to GFRα3 leads to phosphorylation of RET, followed by subsequent activation of intracellular signaling cascades including MAPK (mitogen activated protein kinases), Src, and AKT (protein kinase B), which support the survival and stimulate the regeneration of affected neurons. In spite of promising data from animal models of neuropathic pain, ARTN itself is not a good pharmacological agent: it is expensive to produce and store, it does not penetrate tissue barriers, and does not diffuse much from the site of application (Bespalov et al., 2011). These problems can be overcome by the development of small molecules acting similarly to ARTN, but with significantly improved pharmacological properties. We established a panel of assays to facilitate the selection of small molecular weight ARTN mimetics and screened chemical library of 30,000 drug-like compounds. We identified several molecules activating RET and its downstream targets in the cells expressing ARTN receptors. On the basis of active scaffolds discovered in high-throughput screening, a focused library of chemicals was generated and tested for the ability to activate RET, MAPK, and Akt. The most potent molecules from the focused library were tested for their specificity towards RET, cytoxicity and ability to support survival and sprouting of sensory dorsal root ganglion (DRG) neurons in vitro. Our results show that the selected compounds do not activate another receptor tyrosine kinase called TrkB (that transmits signals from ARTN-unrelated neurotrophic factors) and do not significantly stimulate TrkB-dependent intracellular signalling. Preliminary data demonstrate that one molecule efficiently promotes survival and induces neuritogenesis from DRG in vitro. Selected compounds are also neither toxic nor survival-promoting towards non-neuronal cells (fibroblastic origin) in the concentrations active on DRG neurons. Selected compounds have also demonstrated efficacy in the Chung and Bennett rat models of neuropathic pain. Currently, these compounds are being tested for advanced ADME-Tox (absorption, distribution, metabolism, excretion-toxicity) properties.
G. A. Smith,* A. Heuer,† S. V. Precious,† A. Klein,† I. Jaeger,‡ C. M. Kelly,† E. L. Lane,§ M. Li,‡ and S. B. Dunnett†
*Mclean Hospital/Harvard Medical School, Boston, MA, USA
†Brain Repair Group, Cardiff University, Cardiff, UK
‡Imperial College London, London, UK
§Welsh School of Pharmacy, Cardiff University, Cardiff, UK
Parkinson's disease is currently treated by L-dopa; however, long-term treatment causes debilitating L-dopa-induced dyskinesia (LID) and other therapeutic strategies are much needed. The transplantation of ventral mesencephalon (VM) tissue has shown encouraging results at clinical trial, yet a proportion of patients developed graft-induced dyskinesia (GID). The mechanism of this remains unclear and needs to be elucidated to aid both the continuation of VM clinical trials and the initiation of new stem cell clinical trials. Deficits on behavioral tests, graft-mediated recovery, LID, and GID can be tested experimentally in hemiparkinsonian rat models. However, there has been little translation to the mouse. Mice were unilaterally lesioned with 6-OHDA directed at the medial forebrain bundle and assessed on: rotarod, cylinder, corridor, balance beam, locomotor activity, psychostimulant, and spontaneous rotational behavior to determine levels of dopamine depletion and to predict those that would develop LID, as seen by a dose escalation (2–25 mg/kg). 6-OHDA-lesioned mouse models that displayed LID from two strains (C57/Bl6 and CD1) were then used for the transplantation of dissociated E14 VM tissue (corresponding strain and crossover strains) and mouse epiblast-derived stem cells (EpiSCs) differentiated toward a dopaminergic phenotype, with the aim of modeling GID driven by amphetamine (2.5 mg/kg). Amphetamine-mediated GID has so far only been demonstrated in the rat using rat primary tissue. Rotarod, apomorphine-induced rotations, and locomotor activity were significantly correlated with the development of LID, yet many more tests were correlated to dopamine depletion. GID was induced in mice using primary tissue from the same strain, different from those that were not strain matched or that received EpiSCs. LID and GID mouse models may have wide implications for combined use with the transplantation of different mouse cell lines and transgenic models in future experiments.
V. M. Spruance, E. J. Gonzalez-Rothi, B. E. O'Steen, D. D. Fuller, M. A. Lane, and P. J. Reier
Department of Neuroscience, College of Medicine and McKnight Brain Institute, University of Florida, Gainesville, FL, USA
Injury in cervical spinal cord frequently impairs respiratory function with the greatest deficits arising from direct damage to the phrenic circuit and diaphragm paresis/paralysis. It is known from experimental studies using a lateral C2 hemisection (C2Hx) in the adult rat, however, that spontaneous recovery of diaphragm function can occur. Recent experiments have identified cervical interneurons that could contribute to respiratory neuroplasticity following lateralized hemisection lesions at C2 (i.e., C2Hx). However, prephrenic interneuron activity following C2Hx is not well defined. In fact, little is known about the change in activity within spinal neurons following injuries at this level. The present study aims to address this gap in knowledge. Activity of cervical interneurons and motoneurons in the normal (naive) adult rat spinal cord was examined using immunocytochemical assessment of c-fos expression. These results were compared against i) naive animals exposed to a respiratory challenge (10 min of hypoxia) and ii) animals 2 weeks following C2Hx. All animals were euthanized at the same time of day under the same conditions. Transynaptic tracing with pseudorabies virus (PRV) applied to the ipsilateral hemidiaphragm was used in a subset of animals to label phrenic motoneurons and prephrenic interneurons ipsi- or contralateral to injury. Very limited c-fos expression was seen in the cervical cords of naive animals. Following exposure to hypoxia, however, there was a slight increase in motoneuron c-fos staining in the region of the phrenic nucleus, as well as interneuronal labeling in laminae VII and X. This pattern was consistent with PRV first- and second-order labeling. An even greater increase in c-fos-positive motoneurons and interneurons was observed 2 weeks post-C2Hx. While there was a moderate increase in c-fos in laminae VII and X, there was an even greater increase in the dorsal horns both above and below the injury level. Immunocytochemistry for neuronal and astrocytic markers confirmed that c-fos expression was limited to neurons. Studies are under way to electrophysiologically record neuronal activity from identified regions in naive and injured animals. These results are consistent with initial electrophysiological recordings showing cervical interneurons that are responsive to hypoxia and may thus be responsive to conditions requiring increased respiratory drive and phrenic motoneuron output.
Supported by NIH RO1 NS054025 (P.J.R.), the Anne and Oscar Lackner Chair in Medicine (P.J.R.), and the Paralyzed Veterans of America (M.A.L.).
L. Stone,*† C. Nan,* M. Ritchie,* F. Xiao,* M. Donatelle,*‡ G. Larson,* E. Leathly,*† S. Greimel,*† and W. Low*†
*Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
†Graduate Program in Neuroscience, University of Minnesota, Minneapolis, MN, USA
‡College of St. Catherine, University of Minnesota, Minneapolis, MN, USA
Despite the high prevalence and devastating outcome of ischemic stroke, there remain few options for treatment following stroke onset. The treatments that are currently available remain limited in the time window following stroke in which they are effective and in their success in ameliorating stroke injury in the brain as well as functional outcome. Current therapies for stroke, such as tissue plasminogen activator (tPA), are only effective up to 18 h following stroke onset. We have tested a novel stroke therapy that has shown a significant reduction in stroke infarct volume as well as improved functional recovery following stroke in the rat when administered 48 h following stroke onset. In the present study we compared high versus low passage nhUCBSCs to determine whether highly expanded nhUCBSCs are as therapeutically effective as low passaged cells. Using the middle cerebral arterial occlusion (MCAO) model of stroke in Sprague-Dawley rats, we administered nhUCBSCs by intravenous administration 2 days following stroke induction. These human cells were injected into rats without any immune suppression, but no adverse reactions were detected. Both behavioral and histological analyses have shown that the administration of these cells reduces the infarct volume by 50% as well as improves the functional outcome of these rats following stroke for both high and low passaged nhUCBSCs. Staining for donor cells using antibodies that recognize human nuclei in the rat brains have shown that the injected cells are not present 5 days following injection. We believe the therapeutic effect of these cells is not due to any engraftment in the host brain, but rather that these cells provide a profile of secreted cytokines/growth factors that help combat a chronic inflammatory response that follows stroke. Our results demonstrate that high passaged nhUCBSCs are as effective therapeutically as low passaged cells. As a consequence high passaged nhUCBSCs can serve as an abundant source of cells for the treatment of ischemic brain injury. Moreover, the therapeutic benefits can be achieved without the need of immunosuppressive agents. nhUCBSCs have great potential to provide a novel therapy for stroke with no known ill-effects, and are effective at later time points following stroke than methods that are currently available in the clinic.
N. Tajiri,* L. E. Glover,* T. Shimizu,† G. W. Arendash,‡ and C. V. Borlongan*
*Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†Department of Psychology, University of South Florida, Tampa, FL, USA
‡Department of Cell Biology, Microbiology, & Molecular Biology, College of Arts and Science, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) has become a signature wound of the war in Iraq and Afghanistan. Many American soldiers, even those undiagnosed but likely suffering from mild TBI, display Alzheimer's disease (AD)-like cognitive impairments, suggesting a pathological overlap between TBI and AD. This study examined the possible mechanistic link between TBI and AD by characterizing the histological effects of TBI in presymptomatic amyloid precursor protein/presenilin 1 (APP/PS1) AD-transgenic (tg) mice. These AD-tg mice display AD-like behavioral deficits by 15-17 months of age and AD-like neuropathology as early as 6 months of age. To reveal the effects of TBI on the AD-tg mice, the present study began at age 3 months and ended at age 5 months. Nontransgenic (non-tg) mice served as the control group. There were four treatment groups: AD transgenic mice + TBI (n = 8), AD transgenic mice + sham surgery (n = 8), nontransgenic mice + TBI (n = 7), and nontransgenic mice + sham surgery (n = 7). AD-tg mice and non-tg mice received an experimental TBI at the right parietal cortex using the controlled cortical impact (CCI) model. β-Amyloid (Aβ) was significantly increased by at least eight times in the cortex of AD-tg mice that received TBI compared to the non-tg mice that received TBI and the AD-tg and non-tg mice that underwent sham surgery. A significant decrease in microtubule associated protein 2 (MAP2)-positive cells was observed in the cortex of both the AD-tg and non-tg mice that received TBI compared to the AD-tg and non-tg mice subjected to sham surgery. Similarly, there was a significant increase in Aβ accumulations by at least 10 times in the hippocampus of the AD-tg mice that received TBI compared to all other groups. Conversely, there was significant decrease in MAP2-positive cells in the hippocampus of the AD-tg mice that underwent TBI surgery compared to the non-tg mice that received sham surgery. These results suggest that TBI may precipitate AD-like pathology in presymptomatic AD-tg mice. A recent article (Tran et al., J. Neurosci., 2011) reported that controlled cortical impact TBI model promoted 3xTg-AD mice to display intra-axonal Aβ accumulations and increased phospho-tau immunoreactivity at 24 h and up to 7 days after TBI. The present study replicated the accelerated Aβ aggregations in the APP/PS1 tg mice, but also extended such AD pathology up to 6 weeks post-TBI. Our observed histological data should allow correlative analyses between Aβ accumulations and TBI, providing support for a causal role of TBI in AD-related pathological manifestations. The recognition of this direct link between TBI and AD should aid in developing novel treatments, such as cell-based therapies, directed at abrogating cellular injury and Aβ deposition in both cortex and hippocampus.
C. C. Tate, M. A. Dao, M. McGrogan, and C. C. Case
SanBio, Inc., Mountain View, Ca, USA
SB623 cells are genetically modified human bone marrow stromal cells (MSCs) that are currently being tested in a Phase I/IIa clinical trial to treat stroke disability in the US. SB623 cells have been shown to promote recovery following experimental brain injury and neurodegenerative disorders (e.g., stroke, Parkinson's disease, retinal degeneration). SB623 cells secrete many beneficial soluble and insoluble factors that support host neural cell survival and regeneration and have immunomodulatory functions. In the current study, we examined in vitro the ability of SB623 cells and their parent MSCs to promote angiogenesis through secreted factors. Human umbilical vein endothelial cells (HUVECs) were cultured in either MSC- or SB623 cell-conditioned medium or unconditioned medium. After 16 h, there was enhanced tube formation for HUVECs cultured in conditioned medium compared to unconditioned medium. HUVECs that were “starved” of supplements for 24 h were rescued by MSC- and SB623 cell-conditioned medium. Specifically, there was a significant increase in HUVEC survival and proliferation when the endothelial cells were cultured for 7 days with MSC- or SB623 cell-conditioned medium compared to unconditioned medium (p < 0.05). Adult rat aortic rings were cultured with either MSC- or SB623 cell-conditioned medium or unconditioned medium for 7 days. While there were no significant differences across the groups in the number of new vessels sprouting from rings, there were significantly more branches for the rings in conditioned medium from SB623 cells compared to either MSC-conditioned medium or unconditioned medium (p < 0.05). These data indicate that SB623 cell-secreted soluble factors promote angiogenesis, which likely contributes to promoting recovery in the injured and diseased brain.
Y. D. Teng,*†‡ S. C. Benn,§ S. N. Kalkanis,*§ J. M. Shefner,¶ R. C. Onario,*†‡ B. Cheng,# M. B. Lachyankar,‡ M. Marconi,‡** J. Li,** N. J. Maragakis,†† J. Lládo,†† K. Erkmen,*‡ D. E. Redmond, Jr.,‡‡ R. L. Sidman,*,** S. Przedborski,§§ J. D. Rothstein,†† R. H. Brown, Jr.,§¶¶ and E. Y. Snyder*‡**##
*Department of Neurosurgery, Harvard Medical School, Boston, MA, USA
†SCI Research, Veterans Affairs Boston Healthcare System, Boston, MA, USA
‡Department of Neurology, Children's Hospital-Boston, Harvard Medical School, Boston, MA, USA
§Department of Neurology, Massachusetts General Hospital, Harvard Medical School, Boston, MA, USA
¶Department of Neurology, State University of New York, Syracuse, NY, USA
#Department of Biostatistics, Mailman School of Public Health, Columbia University, New York, NY, USA
**Department of Neurology, Beth Israel-Deconess Medical Center, Harvard medical School, Boston, MA, USA
††Department of Neurology, Johns Hopkins University, Baltimore, MD, USA
‡‡Departments of Psychiatry and Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
§§Movement Disorder Division, Neurological Institute, Center for Neurobiology & Behavior, College of Physicians & Surgeons, Columbia University, New York, NY, USA
¶¶Department of Neurology, University of Massachusetts Medical School, Worcester, MA, USA
##Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
Amyotrophic lateral sclerosis (ALS) is a lethal disease characterized by unremitting motor neuron (MN) degeneration. Multiple pathophysiological processes involving MNs and other cell types have been implicated. Stem cells within the central nervous system have also been increasingly viewed as mediating multiple actions in fulfillment of their primary biological functions of organogenesis and maintenance of homeostasis. We tested the hypothesis that implanted nonpre-differentiated multipotent neural stem cells (NSCs) may be broadly beneficial against this array of pathological mechanisms in the superoxide dismutase 1 (SOD1)G93A transgenic ALS mouse model. Based on meta-analysis of 11 independent studies performed by a consortium of ALS investigators, we observed that, via multiple inherent processes, migratory multipotent NSCs (including of human origin) can forestall the onset and progression of symptoms and substantially prolong survival in these ALS mice (by 300–400% in some cases), particularly if spinal regions that mediate life-sustaining functions (e.g., respiration and hindlimb function) are rendered chimeric. While a very small proportion of donor-derived cells appear to begin pursuing a neuronal phenotype (in this nonneurogenic albeit “degenerating” region), the likely beneficial mechanisms included: 1) preservation of host MNs and their functional neuromuscular connections (affirmed by electrophysiological measures of motor unit number and activity, by plethysmography, and by cell counts); 2) creation of an anti-inflammatory local milieu; 3) inhibition of mutant astrogliosis; 4) mitigation of signs of intraneuronal pathology (e.g., filamentary tangles, aggregates, axonal debris), pathologic findings in both familial and sporadic ALS. NSCs did not repress mutant SOD1G93A expression, but rather its clinical manifestations, suggesting that such interventions may be effective for a broad range of etiologies. We conclude that transplanted multipotent NSCs exert profound widespread, beneficial pleiotropic “chaperone” influences on degenerating host neurons and disease-permissive host glia. It is this homeostatic, modulatory action that may represent the most accessible (hence near-term) therapeutic application of stem cell biology against heretofore untreatable degenerative diseases like ALS.
F. Vahidy, S. Kar, I. Aisiku, H. Juneja, D. Lee, J. Garret, S. Alderman, and S. I. Savitz
Department of Neurology, University of Texas Medical School at Houston, UT-Health, Houston, TX, USA
Cell therapy is a new, investigational approach for stroke. Mononuclear cells (MNCs) from the bone marrow reduce neurological deficits in animal stroke models and permit autologous administration. In March 2009, we launched the first study in the US to test the safety and feasibility of a bone marrow harvest followed by intravenous readministration of autologous MNCs in 10 patients with acute ischemic stroke, the results of which have recently been published. We then expanded the study to 25 patients and present our findings for the entire cohort up to 3 months after enrollment. Patients presenting to our stroke center with acute ischemic stroke were eligible for enrollment. Inclusion criteria were 18–83 years of age, ischemic stroke, and an NIHSS 6–18 for left hemisphere and 6–15 for right hemisphere infarcts. Initial infarct size had to be ≤145 cc on brain imaging. Using conscious sedation, bone marrow (up to 2 ml/kg) was aspirated from the iliac crest and MNCs were separated by density gradient at a GMP facility. MNCs (a maximum of 10 million cells/kg) were administered IV on average 5 h after harvest. The window for both harvest and infusion had to occur between 24 and 72 h after stroke. Patients were monitored during the harvest, infusion, and hospitalization and are currently being followed for up to 5 years. The time window and dose were chosen based on animal studies. We compared the study patients against 107 historical controls derived from our stroke database and matched for age and NIHSS stroke severity during the eligible time window for enrollment. Twenty-five patients were enrolled with an average age of 61 ± 13 years old. Bone marrow aspiration was successfully completed in all patients (average total procedure time: 50 min). MNC viability was greater than 96%. MNCs were successfully infused without study related complications. In two patients, transaminases had transiently increased from 1 to 3 times upper limit of normal and were attributed to statin drugs. There was one death at 40 days due to a pulmonary embolism related to the stroke. There have been no study-related severe adverse events. Using historical controls, we created a linear regression model of stroke recovery using the 90-day modified rankin scale (mRS) as an outcome plotted against the 7-day mRS score. An average trajectory of recovery was generated along with the 95% confidence interval (CI). Superimposed on this model, we plotted the mRS scores of the study patients. Nearly all the patients fell within or below the range at 90 days. Eight out of the 25 patients were below the 95% CI. This study suggests that bone marrow harvest and reinfusion of MNCs were safe and feasible in acute stroke patients. The comparison with historical controls reveals the possibility of a signal that some of the study patients improved better than the predicted model of recovery.
Supported by the NIH R21HD060978.
D. R. Wakeman,* S. Kriks,† H. B. Dodiya,* L. Studer,† and J. H. Kordower*
*Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
†Center for Stem Cell Biology, Memorial Sloan-Kettering Cancer Center, New York, NY, USA
Human pluripotent stem cells are a promising alternative substrate for cellular transplantation in Parkinson's disease. Efficient directed differentiation of embryonic and induced pluripotent stem cells into functional midbrain dopamine (DA) neurons was recently demonstrated using a unique floor-plate induction strategy (Kriks et al., Nature, 2011). In vivo survival and function were shown in 6-hydroxydopamine-lesioned mice and rats verifying robust survival of midbrain DA neurons derived from human embryonic stem cells (hESCs), including restoration of amphetamine-induced rotation behavior and improvements in tests of akinesia and forelimb use. While these results were extremely promising, the clinical translational value remained untested. Therefore, we sought to test the scalability and short-term potential of hESC-derived midbrain dopaminergic neurons in parkinsonian nonhuman primates. Midbrain floor-plate precursors were derived from WA-09 hESC (H9) utilizing dual SMAD inhibition in combination with small molecule activators of sonic hedgehog and canonical WNT signaling. Engraftable midbrain DA neuroblasts were obtained by day 25 and transplanted bilaterally into the striatum of aged, MPTP-lesioned Rhesus monkeys (10–12 kg). After 1 month (n = 2) and 3 months (n = 2) posttransplantation, robust donor-derived grafts were located within the graft zone. Immunohistochemical analysis using a human cytoplasm specific antibody (STEM-121) revealed extensive target-specific axonal outgrowth from grafted cells, specifically along endogenous white matter tracks. Maintenance of the midbrain dopaminergic lineage was confirmed by coexpression of FoxA2 and tyrosine hydroxylase (TH) in combination with human cytoplasm. By 3 months, grafted cells matured into highly arborized, mature TH-ir dopaminergic neurons reminiscent of host nigral neurons. The results demonstrate excellent graft survival, maintenance of the dopaminergic phenotype, and lack of neural overgrowth in parkinsonian monkeys and indicate considerable promise for the development of pluripotent cell-based therapies in Parkinson's disease.
C. Yang,* B-F. Ma,† L-R. Zhao,†‡ and W-M. Duan*
*Department of Anatomy, Capital Medical University, Beijing, China
†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
We have recently demonstrated that adeno-associated virus vector 9 (AAV9)-mediated erythropoietin (EPO) gene delivery into the brain protects dopaminergic neurons in the substantia nigra in a rat model of Parkinson's disease. However, immune responses against AAV9-EPO vectors in rat brain have not yet been determined. In the present study, we planned to examine whether preexposure to AAV9-EPO vectors with an intramuscular or intrastriatal injection would reduce AAV9-mediated EPO transgene expression in rat brain. We first characterized transgene expression and immune responses against AAV9-EPO vectors in rat striatum at three time points (4 days, 3 weeks, and 3 months) and with doses ranging from 109 to 1013 viral genomes. To sensitize the immune system, rats received an injection of AAV9-EPO either into the muscle or into the left striatum, and then received an injection of AAV9-EPO into the right striatum 3 weeks later. We observed that transgene expression and immune responses exhibited in a time course and dose-dependent manner. The levels of EPO transgene expression in the right striatum of the muscle-EPO/striatum-EPO rats were lower than the other two groups of the muscle buffer/striatum-EPO rats and the striatum buffer/striatum-EPO rats, but not lower than the striatum-EPO/striatum-EPO rats 3 weeks after readministration. In contrast, the expression of major histocompatibility complex class I and II antigens in the muscle-EPO/striatum-EPO rats was at greater levels than in the other three groups. Infiltration of CD4 and CD8 lymphocytes and accumulation of microglia/macrophage and astrocytes was observed in the right striatum. We also observed that the sera from the muscle-EPO/striatum-EPO rats contained greater levels of antibodies against AAV9 capsid protein than the other three groups. There were detectable levels of antibodies against EPO protein in the sera from the muscle-EPO/striatum-EPO and the striatum-EPO/striatum-EPO rats. Interestingly, EPO gene expression was negatively correlated with the levels of circulating antibodies against AAV9 capsid protein. Our results suggest that preimmunization with an intramuscular injection can efficiently immunize animals, leading to the reduction of transgene expression in the striatal readministration. Although an intrastriatal injection of virus vectors can immunize animals, it appears not to harm EPO transgene expression in the following application in the striatum. It therefore highlights that preimmunization may jeopardize AAV vector-mediated gene transduction.
Y. Yu,*† J. Chao,‡ A. E. Ropper,*† H. Wang,‡ S. Schachter,§ R. Langer,¶ and Y. D. Teng*†
*Neurosurgery and PM&R, Harvard Medical School/Brigham and Women's Hospital/Spaulding Rehabilitation Hospital, Boston, MA, USA
†Division of SCI Research, Veteran Affairs Boston Healthcare System, West Roxbury, MA, USA
‡Department of Chemistry, Chemical Biology & Biomedical Engineering, Schaefer School of Engineering & Science, Stevens Institute of Technology, Hoboken, NJ, USA
§Neurotechnology Program, Center for Integration of Medicine and Innovative Technology (CIMIT), Boston, MA, USA
¶Department of Chemical Engineering, MIT, Cambridge, MA, USA
One of the central processes of post-spinal cord injury (SCI) pathology is inflammation caused by reactive nitrogen or oxygen species (RNS/ROS) that are generated by activation of infiltrating white blood cells and residential microglia around the injury epicenter. Among these radicals, peroxynitrite (ONOO-) leads secondary insult to the neural cells primarily through lipid peroxidation, protein nitration, and DNA breakage. Although antioxidant drugs were used to impede RNS/ROS, they showed no definitive benefit in clinical trials despite promising data of in vitro studies. Such outcomes may result from poor bioavailability of systemically administered antioxidants to the injury zone. Hence, we hypothesize that the true efficacy of ONOO- scavengers could be realized if they are administered directly to the lesion site via controlled local release. To test this hypothesis, MnTBAP, a new superoxide dismutase mimetic, is embedded in thermoreversible poly-(NiPAAm-co-AAc) hydrogel that gelates inside the injury epicenter following injection (30 nmol/μl; total: 4 μl in 3 loci/rat) at 1 h post-T10 contusion SCI (n = 7/group), and releases MnTBAP under a predetermined rate. In two consecutive studies, we found that hydrogel-based delivery of MnTBAP significantly reduces hindlimb deficits in coordinated motor functions, compared with saline-injected or lesion alone controls (p lt 0.05; n = 7/group). Mechanistic analyses show that the recovery is mediated by mitigation of RNS-triggered protein nitration, gliosis, neural apoptosis, and overall scales of lesion volume. The effectiveness observed in the current regimen suggests that hydrogel-controlled in situ administration of RNS/ROS scavengers may provide even wider therapeutic windows to ameliorate deleterious consequences of SCI.
Supported by CIMIT under U.S. Army Medical Research Acquisition Activity Cooperative Agreement DAMD-17-02-2-0006; VA RRD.
R. Zhang,* J-P. Lee,† M-C. Yan,‡ S. Duggineni,§ D. R. Wakeman,¶ W. Niles,* Y. Feng,* Z. Huang,*§ and E. Y. Snyder*
*Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
†Tulane University School of Medicine, New Orleans, LA, USA
‡The University of Southern California, Los Angeles, CA, USA
§SUNY Upstate Cancer Research Institute, Department of Pharmacology, State University of New York, Syracuse, NY, USA
¶Rush University, Chicago, IL USA
Interactions between the chemokine receptor CXCR4 and its natural agonist ligand stromal cell derived factor-1 (SDF-1α) are recently identified to be essential for adult neurogenesis inside the central nervous system. Based on the existing information regarding the structure–functional relationship between CXCR4 and its natural and unnatural ligands, in this study we have hypothesized a novel design strategy for synthetic bifunctional peptide agonists, which are capable of activating CXCR4-mediated signaling inside human neural stem cells, particularly for a guided cell migration. Among a set of thus generated peptides, one was identified to be ideal as such a candidate, with highly competent CXCR4 binding, and also activates multiple downstream signaling events in supporting an effective chemotaxis function. In recipient healthy adult mouse brain, the novel agonist peptide is found to be highly stable and widely distributed, and leads to extensive migration of human neural stem cells engrafted into the contralateral hemisphere. Compared to the engraftment of exogenous recombinant human SDF-1α, the peptide elicited significantly less immunogenicity to microglia. These discoveries shed new light on an effective guidance of stem cell engraftment, which is among the key questions in cell therapies for neurodegenerative diseases.
Y. Zhang,* A-C. Granholm,† L. Shan,* O. Diaz,* N. Malik,* L. Olson,‡ K. Huh,* B. J. Hoffer,* C. R. Lupica,§ A. F. Hoffman,§ and C. M. Bäckman*
*Integrative Neuroscience Research Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA
†Department of Neurosciences and the Center on Aging, Medical University of South Carolina, Charleston, SC, USA
‡Cellular Neurobiology Research Branch, National Institute on Drug Abuse, National Institutes of Health, Baltimore, MD, USA
§Department of Neuroscience, Karolinska Institutet, Stockholm, Sweden
Clinical trials in patients with Parkinson's disease (PD) have shown that transplants of embryonic mesencephalic dopamine (DA) neurons form new functional connections within the host striatum, but the clinical benefits have been highly variable. A major obstacle preventing cell replacement from becoming a viable treatment for PD is the poor survival rate and integration of grafted DA neurons. Recent evidence has shown that activation of Akt, a serine/threonine kinase that promotes cell survival and growth, and is inhibited by the lipid phosphatase and tensin homolog (PTEN), increases the ability of neurons to survive after injury and to regenerate lost neuronal connections. We have generated a conditional mouse knockout model that constitutively expresses Akt in DA neurons following cell-specific deletion of PTEN (DA-PTEN KO). Ventral mesencephalic (VM) tissue from DA-PTEN KO or control animals was transplanted bilaterally into the DA-depleted striata of MitoPark mice that express a parkinsonian phenotype because of impairment of mitochondrial function. After transplantation into MitoPark mice, PTEN-deficient DA neurons were less susceptible to cell death, and exhibited robust and more extensive fiber outgrowth when compared to control grafts. Voltametric measurements demonstrated that DA release and reuptake were significantly increased in the striata of animals receiving DA-PTEN KO transplants. These animals also displayed enhanced locomotor and circadian rhythm activity, relative to control transplanted MitoPark mice. Together, these results indicate that upregulation of the Akt signaling pathway during neuronal transplantation may provide a valuable strategy to significantly enhance the survival and function of DA neurons with the host striatum, thereby providing an improved system for cell transplantation.
Y. Zhang,* G. E. Meredithm,*† N. Mendoza-Elias,*† D. J. Rademacher,‡ and K. Steece-Collier‡
*Department of Cellular & Molecular Pharmacology, Rosalind Franklin University of Medicine and Science, Chicago, IL, USA
†Department of Pharmaceutical Sciences, Rosalind Franklin University of Medicine and Science, Chicago, IL, USA
‡Department of Translational Science & Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
Levodopa (L-dopa)-induced dyskinesias (LIDs) are among the most common and disabling complications in the treatment of Parkinson's disease. Previous studies have suggested that changes in glutamate function lead to an aberrant synaptic plasticity in dyskinetic animal models. While changes in glutamate function are supported by clinical and preclinical data, the source and manner of this change is not understood. To our knowledge, this is the first study to examine ultrastructural changes in the corticostriatal and thalamostriatal glutamate input in a model of LIDs. We used stereology (physical disector) to count synapses in the striatum of adult male rats that received a sham (two groups, n = 5-6/group) or 6-hydroxydopamine (6-OHDA; three groups, n = 5-6/group) lesion, and were treated with L-dopa (L-dopa and benserazide, each at 12.5 mg/kg, IP) or saline for 3 weeks. Abnormal involuntary movements (AIMs) were rated following the L-dopa injection, 3 times/week, and rats were categorized into severe or mild AIMs groups. Rats were anesthetized and perfused; brains were cut, immunoreacted with VGlut1 (corticostriatal terminals) or VGlut2 (thalamostriatal terminals) antisera, and prepared for stereology. We sampled synapses in a striatal volume of 150-190 μm2 over four serial sections in 15 planes (60 sections total) per animal with the electron microscope. Reference volumes were compared. The 6-OHDA lesion significantly reduced the total number of asymmetrical (excitatory) inputs from the cortex (p < 0.05) but not the thalamus. Rats with severe AIMs showed a significant increase in the number of VGlut1 (corticostriatal) synapses (p < 0.01) and a decrease in VGlut2 (thalamostriatal) synapses (p < 0.05) versus controls. Rats with severe AIMs also showed a significant increase in VGlut1 (p < 0.01) and decrease in VGlut2 (p < 0.05) axospinous synapses when compared to controls. Interestingly, there was a significant decrease for both VGlut1 and VGlut2 multisynaptic boutons (MSBs) in rats with severe AIMs versus controls (p < 0.05). These data show that corticostriatal contacts are increased but thalamostriatal synapses are decreased in the dyskinetic striatum, and that these changes are due to sprouting or loss rather than remodeling of existing boutons, as demonstrated by the significant decrease in MSBs. This study identifies novel potential mechanisms underlying the development of LIDs, data that could lead to new targets for therapeutic development.
G. Cisbani,* M. Saint-Pierre,* D. Soulet,*† T. Freeman,‡ and F. Cicchetti*†
*Centre de Recherche du CHUL (CHUQ), Québec, Canada
†Département de Psychiatrie et Neurosciences, Université Laval, Québec, Canada
‡Center of Excellence for Aging and Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida, Tampa, FL, USA
Neuronal transplantation has been proposed as a potential strategy to replace lost neurons in neurodegenerative diseases, such as Huntington's disease (HD). Host brain ability to integrate and provide trophic support, as well as yielding proper vascularization to the graft has been suggested to be crucial for graft survival. Methodology of transplantation, and especially cell preparation (solid vs. suspension cells), may also play a critical role in graft survival. We recently reported that grafts undergo disease-like degeneration 10 years postsurgery in HD. In the present study, we analyzed the brain of an HD patient who received solid grafts, and evaluated the adequacy of vascular integration of these grafts as potential mechanism for suboptimal survival. The grafted brain of an HD patient 10 years postsurgery has been evaluated immunohistochemically for markers of blood vessels (van Willebrand factor; vWF), vascular endothelial growth factor (VEGF), pericytes, astrocytes (glial fibrillary acidic protein; GFAP), gap junctions (connexin 43; cx-43). In parallel, cell suspensions were transplanted into an HD mouse model (Yac128 mice) and their control (NC), and the grafts were analyzed 1 and 3 months posttransplantation. Grafts found in the HD patient presented a reduced expression of VEGF and a lower density of capillaries than the host brain. Moreover, we observed a reduced number of astrocytes within the graft and limited interaction of these cells with blood vessels, suggesting a lack of functional blood brain barrier (BBB) components. The lack of astrocytes was accompanied by the absence of connexin 43 in the grafted tissue, important for cell communication and buffering of harmful signals. Interestingly, when dissociated cells were transplanted in the striatum of Yac128 or NC mice, the graft survival was excellent and neither the graft vascularization nor the interaction between astrocytes and capillaries were altered. Our study demonstrates the importance of an adequate vascularization for graft survival and integration into the host brain. In particular, the methods of cell preparation are essential to insure a proper integration.
B. Grimmig,* L. Le,* S. Acosta,* Y. Kaneko,* C. Hudson†, C. Borlongan,* C. D. Sanberg,‡ and P. C. Bickford*†
*Center of Excellence for Aging Brain Repair, Department of Neurosurgery and Brain Repair, University of South Florida Morsani College of Medicine, Tampa, FL, USA
†James A. Haley Veteran's Administration Hospital, Tampa, FL, USA
‡NaturaTherapeutics, Inc., Tampa, FL, USA
Neural stem cells have the ability to self-renew and replenish damaged neurons; these cells can be used in cell replacement therapy for neurodegenerative diseases. Aging reduces the regenerative potential of stem cells in many niches within the body in both a cell autonomous as well as a cell nonautonomous manner. The cell nonautonomous impact of aging on stem cell niches has been studied with the technique of parabiosis where the circulation of a young and an aged rat are combined together. Under these circumstances it has been observed that aged progenitor cells in liver, muscle, brain, and many other tissues when exposed to the circulation of a young rat increased proliferation and regeneration index, and vice versa, serum from an old rat reduces the regenerative potential of young cells. We have examined the effect of nutritional supplementation to impact the aged niche. Young and aged F344 rats were treated with NT-020 (a patented proprietary formulation of blueberries, green tea, carnosine, and vitamin D3 available from NaturaTherapeutics, Inc.) for 30 days (0.5% NT-020 in the diet) and then the serum of these animals was used in cell culture of rat mesenchymal stem cells (MSCs) and rat hippocampal neural stem cells (NSCs). We have demonstrated that the aged serum decreases stem cell proliferation while serum from aged rats treated with NT-020 is similar to young in that there is little impact on NSC proliferation in vitro. We now show that this effect is also observed with MSCs. Furthermore, recent studies have demonstrated that one mechanism involved in the effect of aged serum to reduce MSC proliferation is via WNT/β-catenin activation of senescence genes p53 and p16INK4A. We report here that aged rat serum also increases β-catenin in NSCs and that NT-020 treatment of aged rats reduces β-catenin when compared to that observed in NSCs treated with aged rat serum.
P.C.B. is a cofounder of NaturaTherapeutics, Inc. C.V.B. is a consultant of NaturaTherapeutics, Inc.