Abstract
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
I give tribute to the scientists and physicians who serve as leaders of the field of stem cell therapy, noting the milestone achievements of many ASTNR members. In my journey with the doctors and students of stem cell therapy, I learned the many challenges they faced in the laboratory and in the clinic in their quest to conquer the potentials and limitations of stem cells, but equally impressive I witnessed the “human” meaning of being a scientist and a physician. Here, I want to share with you glimpses of the triumphs and tribulations of many a nation of stem cell researchers. In doing so, we may be able to appreciate a better perspective of stem cells as a product of science and of the urgent need to translate these biologics to the clinic, as well as a reflection of men and women in search of a meaning of human life and dignity.
S. A. Acosta, N. Tajiri, Y. Kaneko, 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
Long-lasting histological consequences have been suggested as accompanying the chronic stage of traumatic brain injury (TBI) and this presents as a risk factor for the development of dementia and neurodegenerative diseases such as Alzheimer's disease (AD). The region-specific secondary damage after TBI, specifically at the cellular level to cortical and subcortical regions, is not well characterized. In the present in vivo study, the neuronal degeneration was evaluated in proximal and distal areas in a late stage TBI model. Recognizing the contribution of AD-like pathology to TBI, we focused on AD pathological markers in brain regions implicated in the disease process, including neuronal cell loss, inflammation, cell proliferation, neurogenesis, and aberrant intraneuronal β-amyloid precursor protein (β-APP) expression, to link TBI and AD pathologies. Sprague-Dawley male rats were subjected to the moderate controlled cortical impact (CCI) injury model of TBI, and 6 months later were euthanized and their brain tissues harvested. Results from hematoxylin and eosin (H&E) staining revealed a significant 33% and 10% reduction on the ipsilateral and contralateral side, respectively of the number of hippocampal CA3 interneurons of TBI animals compared to sham animals (p < 0.001). Meanwhile, significant increments in major histocompatibility complex II (MHC II)-activated inflammatory cells were detected in many gray matter (8–20-fold increase) and white matter (6–30-fold increase) regions of both the ipsilateral and contralateral hemispheres of TBI animals compared to sham animals (p < 0.001). A cell cycle regulating protein marker revealed significant 1.6- and 1-fold reductions in the subventricular zone (SVZ) and 2.3- and 1.5-fold reductions in the dentate gyrus (DG) of the ipsilateral and contralateral side, respectively, of TBI animals compared to sham animals (p < 0.001). Moreover, immature neuronal markers demonstrated a significant two- and onefold decrements in both the SVZ and in the dentate gyrus ipsilateral and contralateral side, respectively, of TBI animals compared to sham animals (p < 0.001). Additionally, increased β-APP distribution volumes in white matter including corpus callosum, fornix, and internal capsule (4–38-fold increase), as well as in the cortical gray matter, such as the striatum hilus, SVZ, and dentate gyrus (6–40-fold increase) (p < 0.001), except the thalamus (p > 0.05), were detected. These findings indicate AD-like pathological features in brain regions of late stage TBI rats. The observed inflammation, impaired neurogenesis, and β-APP reminiscent of AD may prove to be valuable markers and therapeutic targets of chronic TBI.
Research supported by Department of Defense W81XWH-11-1-0634.
A. E-E. Aly*, O. Sesenoglu-Laird†, L. Padegimas†, M. J. Cooper†, B. L. Waszczak*
*Department of Pharmaceutical Sciences, Northeastern University, Boston, MA, USA
†Copernicus Therapeutics, Inc., Cleveland, OH, USA
The therapeutic potential of glial cell line-derived neurotrophic factor (GDNF) has been limited thus far by its inability to cross the blood–brain barrier. We are investigating the intranasal route of administration of PEGylated lysine 30-mer (PEG-CK30) DNA nanoparticles (NPs) encoding GDNF. These NPs, developed by Copernicus Therapeutics, Inc., compact a single molecule of expression plasmid, have a minimum diameter of 10 nm, and provide a nonimmunogenic, nonviral vector for CNS gene therapy. We have previously demonstrated that DNA NPs expressing either enhanced green fluorescent protein (eGFP) alone, or eGFP linked with hGDNF (pUGG, which incorporates the polyubiquitin C promoter), transfect cells in vitro and brain cells in vivo. We further showed that intranasal administration of Copernicus' pGDNF NPs provides neuroprotection of substantia nigra (SN) dopamine neurons in the rat 6-hydroxydopamine model of Parkinson's disease (PD). Previous studies have determined that intranasal administration of pUGG NPs results in widespread transfection of cells throughout the rat brain at 7 days, with the highest number of eGFP+ cells in the midbrain. Additionally, doublelabel immunohistochemical analysis carried out for eGFP and different cell specific markers [i.e., RECA-1 for rat endothelial cell antigen, glial fibrillary acidic protein (GFAP), the neuronal nuclei marker NeuN, or the dopamine neuronal marker tyrosine hydroxylase (TH)] demonstrated that most of the eGFP+ cells found in the brain 7 days after intranasal administration of pUGG NPs were cells lining the vasculature, most likely pericytes. This is consistent with the distribution of intranasally administered agents by perivascular transport. The goal of the current study was to establish the duration of eGFP expression after intranasal administration of these pUGG NPs. Rats were sacrificed 1 week, 3 months, or 6 months after intranasal administration. Analysis of eGFP expression at the three time points revealed that eGFP expression (above background in saline-treated controls) was highest at 1 week, and persisted at ~40% of maximal expression at both the 3- and 6-month time points. These results provide the first evidence that intranasal delivery of Copernicus' pDNA NPs provides prolonged transgene expression in rat brain, with highest levels at 1 week and continued expression over at least 6 months. These studies will help to establish dosing regimens for intranasal delivery of these NPs as a noninvasive, long-term means of gene therapy for PD and other CNS disorders.
J. D. Anderson*, J. Rossignol†, P. Lein‡, S. EL-Andaloussi§¶, J. Lehtio#, J. A. Nolta*
*Stem Cell Program, Department of Internal Medicine, University of California Davis Health Systems, Sacramento, CA, USA
†College of Medicine, Central Michigan University, Mount Pleasant MI, USA
‡Department of Molecular Biosciences, School of Veterinary Medicine, University of California Davis, Davis CA, USA
§Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden
¶Department of Physiology, Anatomy and Genetics, University of Oxford, Oxford, UK
#Clinical Proteomics, Department of Oncology-Pathology, Karolinska Institute, Stockholm, Sweden
Mesenchymal stem cells (MSCs) are known to facilitate healing of neurological diseases through release of prosurvival factors, including small lipid-bound secreted vesicles called exosomes. Recent studies demonstrate that MSC-derived exosomes function as paracrine effectors of neurovascular remodeling and promote functional recovery in mouse models of ischemic stroke. However, the identity of which components of the exosome proteome is responsible for this effect remains elusive. To address this we used high-resolution isoelectric focusing coupled liquid chromatography tandem mass spectrometry (HiRIEF LC-MS/MS), a novel, unbiased high throughput proteomics approach to comprehensively characterize the proteinaceous contents of MSC exosomes derived from in vivo like conditions (hypoxia, serum starvation). In total, 1,927 proteins were identified and quantified in MSC exosomes, representing to our knowledge the first time their proteome has been comprehensively probed and assessed. Multilayered analyses identified several putative effectors that modulate neurite outgrowth, axonal guidance and oxidative stress. This study also examined the functional role of MSC exosomes using in vitro assays of neurite outgrowth and oxidative stress-induced neuronal apoptosis. This study also examined the functional role of MSC exosomes in vivo in two models of neurologically related diseases: ischemic stroke and retinal degeneration. Taken together these studies provide functional and mechanistic insight into the use of MSC exosomes as a putative therapeutic for neurological indications.
B. Barboni*†, V. Russo*† P, Berardinelli*†, V Gatta,†‡, A. Muttini,*†, L. Stuppia†‡, O. Parolini§, M. Mattioli*†
*University of Teramo, Teramo, Italy
†StemTeCh Group, Chieta, Italy
‡University of Chieti-Pescara, Chieti, Italy
§Fondazione Poliambulanza-Istituto Ospedaliero, Brescia, Italy
The lack of a consistent therapeutic approach to tendon injury represents a great worldwide medical, social, and economic challenge. In fact, after tendon injury the spontaneous healing results in a fibrotic scar formation that is biomechanically inferior to normal tendons. None of the surgical techniques provide long-term repair; thus, cell-based therapy seems to be a promising alternative. In our previous research (Barboni et al.; Muttini et al.), it was evident that ovine amniotic cells were able to stimulate tendon healing by conjugating their strong paracrine role with a direct contribution in the synthesis of new tendon matrix. On the basis of these premises, in this study, taking advantage of the chimerism, the cross-talk between transplanted human amniotic cells and the host tissue of a preclinical ovine tendon defect model has been investigated. In particular, it was analyzed to see if these cells undergo integration by modulating gene expression and influencing the molecular/cellular mechanisms of the host tissue involved in regeneration. PKH26-labeled human amniotic epithelial (80%) and mesenchymal (20%) stem cells (hAECs/AMCs) were transplanted in an ovine preclinical tendon model, explanted after 28 days, and analyzed (Barboni et al.; Muttini et al.). hAEC/AMC gene expression profile was assessed using a Human Whole Genome OneArray™ Microarray (30,968 total probe). Hierarchical clustering analysis was carried out using Cluster 3.0 and Treeview software. Differentially expressed genes were evaluated by t-test (p < 0.05). The genes' biological functions were analyzed by Ingenuity Pathways Analysis software (Qiagen, Redwood City, CA, USA). hAECs/AMCs accelerated the recovery of the macroarchitecture and biomechanical properties of treated tendons. hAEC/AMCs: PKH26 and the human-specific probe COT1 revealed the persistence of the cells within the host tissue where they upregulated 48 human-specific genes involved in epithelial–mesenchymal transition [lysine (K)-specific demethylase 6B (KDM6B), nuclear receptor subfamily 2, group F, member 2 (NR2F2)], protein matrix synthesis [collagen type 1 alpha 1 (COL1A1)], and the inflammatory response [chemokine (C-C motif) receptor-like 2 (CCRL2)]. Host tissue: tendon regeneration was accelerated by an higher expression of crucial growth factors, such as transforming growth factor-β (Tgfβ) and vascular endothelial growth factor (Vegf), that influenced the deposition and alignment of collagen type I fibers and angiogenesis, respectively. This regeneration was also supported by a reduced infiltration of inflammatory cells together with anti-inflammatory M2 macrophage recruitment, as proved by an upregulation of chitinase-like protein 3 (Ym-1), interleukin-10 (Il-10), and cluster of differentiation 206 (Cd206) associated genes in the host tissue. The human amniotic cells in the host tissue actively modulated tendon regeneration by direct differentiation as demonstrated by the modification of their gene profile. The repairing regenerative process of the host tissue was improved by proper extracellular matrix deposition and the inflammatory response recruiting reparative tissue M2 macrophages. These findings demonstrate that human amniotic cells may represent a successful strategy to regenerate biomechanically functional tendons.
This research was granted by Tercas and PRIN and the authors have nothing to disclose.
M. J. Benskey*, N. C. Kuhn*, R. Sellnow*, F. P. Manfredsson*†
*Department of Translational Science and Molecular Medicine, Michigan State University College of Human Medicine, Grand Rapids MI, USA
†Mercy Health Hauenstein Neuroscience Center, Grand Rapids, MI, USA
α-Synuclein (α-syn) has been widely implicated in the pathogenesis of Parkinson's disease (PD). Mutations or multiplications of the α-syn (SNCA) gene result in familial PD, while aggregated α-syn is a primary component of Lewy bodies, the pathological hallmark of sporadic PD. Further, recombinant adeno-associated virus (rAAV)-mediated overexpression of human α-syn in the rat substantia nigra pars compacta (SNc) produces a predictable pattern of α-syn aggregation and neurodegeneration and is a commonly used model to study PD pathology. The strong correlation between aggregated α-syn and the development of PD pathology has led to the theory that α-syn-mediated pathology arises due to a toxic gain-of-function, and many current therapeutic strategies center on eliminating α-syn from midbrain dopamine neurons of the SNc. However, we have shown that removal of endogenous α-syn from SNc neurons of adult rodents and nonhuman primates results in neurodegeneration in vivo. Importantly, this cell loss is rescued by supplementation of α-syn. These results indicate that α-syn is essential for neuronal survival, and has led us to develop the novel hypothesis, that α-syn and associated aggregates are not directly toxic; rather α-syn aggregation produces toxicity by reducing levels of functional monomeric α-syn through sequestration into intracellular aggregates. To test this hypothesis, we aimed to induce aggregation in dopamine neurons of the SNc by overexpressing human wild-type (WT) α-syn in the rat SNc, while also maintaining functional levels of monomeric α-syn through the concomitant expression of a nonaggregatable α-syn (NAS) isoform. In the current study, rAAV was used to deliver human WT α-syn either alone or in combination with the NAS isoform to the rat SNc. One month postsurgery, animals that received rAAV-human WT α-syn displayed a significant increase in forepaw akinesia, indicative of degeneration of the injected SNc. In agreement with the motor behavior, overexpression of human WT α-syn resulted in a significant loss of tyrosine hydroxylase (TH) neurons in the SNc, compared to rAAV-green fluorescent protein (GFP)-injected control animals. In contrast, animals that received the NAS isoform either alone or in combination with human WT α-syn showed no forepaw akinesia or loss of TH neurons in the SNc, compared to rAAV-GFP control animals. These results demonstrate that the toxicity produced by α-syn aggregation can be obviated by maintaining soluble levels of α-syn. Further, these data support the overarching hypothesis that aggregation of α-syn results in toxicity via a de facto loss-of-function, following the removal of physiological forms of the protein from their subcellular compartment.
T. R. Brickler, B. Okeyere, X. Wang, M. H. Theus
Department of Biomedical Sciences and Pathobiology, Virginia–Maryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
Arteriogenesis is the most important adaptive processes known to occur following vascular injury throughout the body. Unlike angiogenesis (the long-term sprouting of new capillary networks), arteriogenesis refers to the immediate growth/enlargement of preexisting arterioles, which are a principal delivery route for oxygen, nutrients, and potential therapeutic agents. Recent studies have established that pial arteriole anastomosis or collaterals develop during three distinct phases: early embryonic proliferation of the vascular plexus at E15.5–E18.5; postnatal pruning at P1–P21; followed by maturation of the pruned collaterals from P21 into adulthood. While having greater numbers of collaterals that form during development can prevent progressive tissue damage in adulthood, tissue reperfusion can also be enhanced by the postinjury process of arteriogenesis. Analysis of the mechanisms regulating arteriole collateralization has important implications for therapies aimed at promoting neurovascular repair following traumatic brain injury (TBI). Murine studies have provided significant insight into collateral development, including strain differences, and its acute hemodynamics in response to environmental stimuli. However, limited methods exist to visualize the morphological development and remodeling in postnatal and juvenile mice that are fast, efficient, and economically feasible. Although changes in the collateral circulation have been extensively studied in cerebral ischemia, our understanding of collateral formation and remodeling is limited in the context of brain trauma. Consistent with previous findings, vessel painting shows postnatal collateral vessel numbers are reduced in CD1 mice from P1 to P21 while adult numbers remain at P21 levels, indicating that collateral pruning is complete by 21 days postnatal. Surprisingly, cortical trauma using the moderate controlled cortical impact (CCI) injury model in juvenile P21 mice caused a significant increase in the number of pial arterioles contralateral to the injury site at 14 days post-CCI injury. This effect was abolished in adult mice, suggesting the potential to re-form collateral arteriole branches remains after pruning is complete in the juvenile cerebral collateral circulation, the mechanism(s) of which is lost during the maturation stage. Future studies will address the role of different vascular factors in the enhancement of arteriogenesis following juvenile TBI. These data highlight a novel healing response that occurs following TBI in juvenile mice that is not exhibited in adult mice.
C. Case*, N. Manley*, E. Wirth*, J. Lebkowski*, I. Llorente†, T. Carmichael†
*Asterias Biotherapeutics, Fremont, CA, USA
†Department of Neurology, Brain Research Institute, David Geffen School of Medicine at UCLA, Los Angeles, CA, USA
AST-OPC1 is a population of early stage oligodendrocyte progenitor cells (OPCs) that is differentiated from human embryonic stem cells (hESCs) using the H1 cell line. The AST-OPC1 differentiation process is commercially scalable, compatible with current good manufacturing practices (cGMP), and has been approved for early phase clinical testing by the US FDA (Geron, Menlo Park, CA, USA). There are multiple pathologies observed in both spinal cord injury (SCI) and white matter stroke (WMS) due to the injury itself and subsequent secondary effects due to edema, hemorrhage, and inflammation. These pathologies include the severing of axons, demyelination, and parenchymal cavitation. Oligodendrocytes, which provide both neurotrophic factor and myelination support for axons, are susceptible to cell death following CNS injury and thus are an important therapeutic target. Replacement of the oligodendrocyte population could support the remaining and damaged axons and also remyelinate axons to help restore electrical conduction and function. Studies of AST-OPC1 in rodent models of SCI provide evidence of the therapeutic benefit of these cells when transplanted directly into the injured spinal cord approximately 1 week after injury. These and other studies have supported the initiation of human clinical trials in both thoracic and cervical SCI. Observations and plans pertaining to an ongoing clinical trial will be presented. AST-OPC1 has also recently been tested in a subcortical WMS model in nonobese diabetic, severe combined immunodeficient, interleukin-2 receptor gamma (NSG) mice with a large infarct area that mimics the larger white matter lesions seen in moderate to advanced human white matter ischemia or vascular dementia. Tissue outcomes in myelination patterns, reactive astrocytosis, microglial/macrophage responses, and the optimum injection site for transplantation were determined. Transplant of AST-OPC1 at subacute time points (7 days after stroke) outside of the ischemic stroke produced widespread migration of these cells throughout subcortical white matter. This resulted in increased myelination within the damaged white matter, reduced measures of reactive astrocytosis, and inflammation. Preliminary behavioral evaluation of recovery will be presented. These studies suggest that AST-OPC1 transplantation in moderate to advanced white matter stroke, as seen in vascular dementia, may provide a therapy to promote white matter repair and recovery.
Y-H. Chiang*†‡, K-Y. Chen‡, C-C. Wu*†, J-C. Ou§, C-F. Chang*†, K-H. Liao¶
*Department of Neurosurgery, Taipei Medical University Hospital, Taipei, Taiwan
†Department of Surgery, College of Medicine, Taipei Medical University, Taipei, Taiwan
‡Graduate Institute of Neural Regenerative Medicine, College of Medical Science and Technology, Taipei Medical University, Taipei, Taiwan
§Department of Emergency Medicine, Shuang Ho Hospital, Taipei Medical University, New Taipei City, Taiwan
¶Department of Neurosurgery, Wan Fang Hospital, Taipei Medical University, Taipei, Taiwan
Mild traumatic brain injury (mTBI) is a major public problem that results in several psychiatric problems. The insulin-like growth factor 1 (IGF-1) system is involved in growth and survival signaling in the central nervous system. In our observational study, 155 mTBI patients and 116 healthy participants (control group) were evaluated for four psychiatric symptoms and problems: anxiety (Beck's anxiety inventory), depression (Beck's depression inventory), daytime sleepiness (Epworth sleepiness scale), and sleep quality (Pittsburgh sleep quality index). The results show a significant relationship between IGF-1 and the participants' age but it is not correlated with the weight. The anxiety, depression, and daytime sleepiness levels were significant between participants with and without brain injury when adjusting their corresponding gender and age. The IGF-1 level did correlate significantly with these continuous scores, but it was also significant when comparing participants with and without anxiety (according to the clinical cutpoint). Our findings suggest that IGF-1 was related with the anxiety problem after mTBI, supporting further investigation of its potential as a biomarker.
C-H. Chou*†‡, M. Modo*§
*McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
†Department of Neuroscience, Institute of Psychiatry, King's College London, London, UK
‡Department of Neurology, Tri-Service General Hospital, Taipei, Taiwan
§Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
Neural stem cell (NSC) transplantation is emerging as a promising therapeutic strategy in neurological disorders, such as stroke, in which neurovascular restoration is at the core of cell-based therapies aiming for tissue regeneration. The concept of a “neurovascular unit” (NVU) includes vasculature as an interdependent functional component, in addition to a purely neurocentric focus, in order to interpret the mechanisms of neurovascular diseases and the therapeutic efficacy of cell-based therapies. To further the application of NSCs in cell transplantation and regenerative medicine, the clinical-grade human NSCs (STROC05 line; provided by ReNeuron Ltd., Guildford, Surrey, UK) were cultured with human cerebral microvascular endothelial cells (hCMEC/D3 line; Pierre-Olivier Couraud, Institut Cochin, Paris, France) with direct physical contact to form a three-dimensional neurovascular cytoarchitecture. We demonstrated that hNSCs facilitate angiogenic morphogenesis of hCMECs, depending on the ratio of hNSCs to hCMECs. This neurovascular environment reciprocally influences both neural and endothelial differentiation. A particular ratio of NSCs to ECs may therefore be favorable to replace lost brain tissues. Furthermore, signaling mechanisms underlying angiogenic morphogenesis in EC/NSC coculture were illustrated with mechanistic interventions against specific soluble factors and contact-mediated neurovascular interactions.
B. Combs*, C. Hamel*, N. M. Kanaan*†‡
*Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
†Neuroscience Program, Michigan State University, Grand Rapids, MI, USA
‡Hauenstein Neuroscience Center, Mercy Health Saint Mary's, Grand Rapids, MI, USA
Conformational changes involving the amino-terminus of tau are associated with pathological forms of the protein and are among the first detectable alterations in Alzheimer's disease (AD) and other tauopathies. A phosphatase-activating domain (PAD), located within this region, is aberrantly exposed in these pathological forms. This exposure initiates a signaling cascade that leads to disruption of anterograde fast axonal transport, a critical process for the maintenance and function of neurons. We have characterized four antibodies that identify the amino-terminus of tau, TNT1, TNT2 (a novel antibody), Tau12, and Tau13, in order to further study this region and its role in neurodegeneration. We refined the epitopes of these antibodies using scanning alanine mutations, determined the relative abilities of the antibodies to identify pathological tau using native conformation and denaturing assays, and examined the patterns of pathology labeled by each of the antibodies in human postmortem AD brain tissue at various Braak stages as well as in other tauopathies. The antibodies could be classified into two basic groups. The TNT1 and TNT2 antibodies bound to tau within amino acids 7–12. They identified aggregated recombinant tau as well as soluble and insoluble forms of tau in AD brain lysates but were unable to detect monomeric recombinant tau or tau extracted from age-matched control brains in their native conformations. However, when normal and pathological forms of tau were analyzed under denaturing conditions, TNT1 and TNT2 did not distinguish between them, indicating that the differences in reactivity observed under native conditions are conformation dependent. These antibodies also specifically labeled tau pathology in all Braak stages and in various non-AD tauopathies. These antibodies preferentially identified early pretangle neurons and reactivity was lost in late stage neurofibrillary tangles (NFTs). In contrast, Tau12 and Tau13 identified discontinuous epitopes in tau's amino-terminus and, despite the close proximity to the TNT1 and TNT2 epitopes, lacked the ability to differentiate between normal and pathological forms of the protein in native or denaturing conditions. These differences suggest that conformational changes in tau, associated with PAD exposure in pathological forms of the protein, are not detected by all amino-terminal antibodies. TNT1 and TNT2 are useful for their highly specific abilities to identify early pathological tau alterations in nondenaturing biochemical assays as well as in postmortem human brain tissue and may help to elucidate toxic mechanisms involving aberrant PAD exposure in AD and other tauopathies.
M. J. Corenblum*, S. Ray†, M. Long‡, B. Harder‡, D. D. Zhang‡, C. A. Barnes*§¶#, L. Madhavan*#
*Department of Neurology, University of Arizona, Tucson, AZ, USA
†Undergraduate Biology Research Program, University of Arizona, Tucson, AZ, USA
‡Pharmacology and Toxicology, University of Arizona, Tucson, AZ, USA
§Department of Psychology, University of Arizona, Tucson, AZ, USA
¶Department of Neuroscience, University of Arizona, Tucson, AZ, USA
#Evelyn F McKnight Brain Institute, University of Arizona, Tucson, AZ, USA
Although it is known that the regenerative function of neural stem/progenitor cells (NSPCs) declines with age, causative mechanisms underlying this phenomenon are not understood. In this context, we have systematically examined subventricular zone NSPCs in various cohorts of rats across the aging spectrum, using in vitro, in vivo, and behavioral techniques. These studies indicate that although NSPC function continuously declines with advancing age, there is a critical time period during middle-age (13–15 months) when a striking reduction in NSPC survival and regeneration (proliferation and neuronal differentiation) occurs. We also find that this specific temporal pattern of NSPC deterioration correlates with the decreasing expression of the redoxsensitive transcription factor nuclear factor (erythroid-derived 2)-like 2 (Nrf2) in the NSPCs. When Nrf2 expression was suppressed in “young” NSPCs, using short interfering RNAs, the survival and regeneration of the NSPCs was significantly compromised and their behavior mirrored “old” NSPCs. Conversely, increasing Nrf2 expression in “old” NSPCs rendered their behavior similar to “young” NSPCs and they showed greater survival and regeneration. Furthermore, analysis of NSPCs in young Nrf2 knock-out (Nrf2-/-) mice revealed a lower number of stem and progenitor cells in the subventricular zones of these animals, when compared to age-matched wild-type controls. In addition, the proliferative capacity of the NSPCs, and their ability to produce new neurons, was also notably compromised in the Nrf2-/- mice. These results identify a novel regulatory role for Nrf2 in NSPC function during aging. The results also have important implications with respect to developing NSPC-based strategies to support healthy aging and to treat age-related neurodegenerative disorders.
G. P. Cortese*, R. Hullinger†, K. O'Riordan‡, C. Burger*
*Department of Neurology, University of Wisconsin-Madison, Madison, WI, USA
†Neuroscience Training Program, University of Wisconsin-Madison, Madison, WI, USA
‡Department of Pharmacology & Therapeutics Biotechnology, Trinity College, Dublin, Ireland
Disorders that span from age-related mild cognitive impairment (MCI) and early stage dementia to later-stage clinical Alzheimer's disease (AD) all have a common link to aging-associated cognitive decline, contributing to the clinical symptomology of memory loss and learning impairments. Aging-associated cognitive decline correlates with specific deficits in hippocampal function, resulting in a reduced ability for learning and memory. There has been significant progress in identifying both behavioral and biological mechanisms of hippocampal dysfunction resulting from aging and memory-related diseases. To date, the most relevant and translational findings known to reverse the cognitive decline associated with aging has been performed in human and rodent models of environmental enrichment (EE), which consist of environmental changes promoting social activity, healthy diet, frequent exercise, quality living conditions, and limited stress. In order to understand the mechanism by which enrichment promotes successful cognitive aging we utilized a rodent model of aging-associated enrichment using 20-month-old Fischer 344 rats that have been exposed to 1 month of social enrichment (SE) and EE. In the work presented here, we have begun to explore the behavioral, molecular, and electrophysiological benefits of enrichment in aged rodents. We find that EE improves hippocampus-dependent learning and memory as measured by performance on the Morris water maze and novel object recognition tasks. Additionally, we find that increases in learning and memory are mirrored by increases in hippocampal long-term potentiation (LTP) following 0.5 theta-burst stimulation (0.5 TBS). Extending these findings, we show that EE-associated increases in LTP are dependent on metabotropic glutamate receptor 5 (mGluR5) activity, as antagonists to mGluR5, but not mGluR1, block enhanced LTP. In addition, we find that this EE-dependent enhancement of LTP requires mGluR5–Homer1c signaling, as addition of the phospholipase C (PLC) antagonist (U73122) prevents LTP enhancement following EE. Furthermore, we show that levels of mGluR5 and Homer1c protein increase in postsynaptic densities prepared from 0.5 TBS hippocampi from aged animals following EE. These findings are important in understanding the electrophysiological and biochemical mechanisms by which environmental enrichment can be used as a behavioral therapeutic strategy to curb the cognitive decline that is strongly associated with normal aging.
S. E. Counts*†
*Department of Translational Science and Molecular Medicine, Department of Family Medicine, Michigan State University, Grand Rapids, MI, USA
†Hauenstein Neuroscience Center, Mercy Health Saint Mary's Hospital, Grand Rapids, MI, USA
The widespread influence of small, noncoding microRNA (miRNA) regulation on neuronal physiology suggests that perturbations in miRNA function might be involved in the pathogenesis of complex neurodegenerative disorders such as Alzheimer's disease (AD). Indeed, AD brains display altered expression of several miRNAs that regulate the β-secretase BACE1, a key enzyme involved in the generation of β-amyloid plaque pathology. However, whether miRNA networks are dysregulated in the brains of people in the prodromal stages of AD such as amnestic mild cognitive impairment (aMCI) and the extent to which these changes have physiologic consequences for the onset of AD remain underexplored. To begin to address these knowledge gaps, we performed large-scale microarray and quantitative PCR (qPCR) studies to compare the levels of miRNAs in postmortem frontal cortex tissue from Rush Religious Orders Study participants who died with an antemortem clinical diagnosis of no cognitive impairment (NCI, n = 12), aMCI (n = 10), or early stage AD (n = 10). Of note, two functional miRNA families, miR-212/132 and miR-23a/b, were significantly decreased ~40–50% in frontal cortex of aMCI and AD subjects compared to NCI (p < 0.01). Surprisingly, human miRNA databases revealed that downregulation of either miR-212/132 or miR-23a/b was predicted to upregulate the deacetylase sirtuin 1 (SIRT1), which mediates neuroplasticity and neuroprotective cell stress responses. To this end, qPCR studies using the same frontal cortex samples revealed that SIRT1 mRNA levels were ~50% higher in aMCI compared to NCI subjects (p < 0.01). Given the relatively delayed involvement of frontal cortex in AD pathogenesis and the ability of this region to respond to the onset of dementia by neuronal reorganization, these data suggest that miRNA-mediated upregulation of SIRT1 might represent a compensatory response to mounting disease processes. To explore this hypothesis mechanistically, in vitro studies using human teratocarcinoma-derived (hNT) neuronal cells showed that the coordinated experimental downregulation of miR-212 and miR-23a increased SIRT1 protein expression by ~100% (p < 0.01). Moreover, downregulation of miR-212 and miR-23a provided ~80% neuroprotection when the cultures were challenged with toxic β-amyloid (p < 0.01), and this effect was reversed in the presence of the SIRT1 inhibitor EX527. Taken together, these data suggest that we have uncovered a novel miRNA-mediated neuroprotective pathway activated during prodromal AD. These findings may reveal new mechanistic insights into gene regulation pathways in AD, leading to innovative therapeutic avenues for modifying disease progression.
Support: NIH AG042146, AG014449, Brinson Foundation.
A. T. Crane*†, P. Swaminathan*, H. Hewitt*‡, F. Xiao*‡, V. Savanur*, J. Voth*, Z. Schultz*, D. Carlson§, S. Fahrenkrug*§, D. Garry*¶, N. Koyanon*¶, J. Dutton*, W. C. Low*‡
*Stem Cell Institute, University of Minnesota, Minneapolis, MN, USA
†Minnesota Craniofacial Research Training Program, University of Minnesota, Minneapolis, MN, USA
‡Department of Neurosurgery, University of Minnesota, Minneapolis, MN, USA
§Recombinetics Inc., St. Paul, MN, USA
¶Department of Medicine, University of Minnesota, Minneapolis, MN, USA
Ethical considerations and limited tissue availability represent a significant hurdle in transplantation of fetal midbrain dopamine (mDA) progenitors for patients with Parkinson's disease (PD). Blastocyst complementation can provide an alternative source of tissue, a process in which pluripotent stem cells (PSCs) from one organism are introduced into genetically engineered organogenesis-disabled blastocysts. Through normal development, injected PSCs can potentially fill the niche and develop into an organ of interest. In an effort to obtain genuine human fetal mDA progenitors, human stem cells were injected into embryos lacking paired-like homeodomain transcription factor 3 (PITX3), a gene involved in the development of the lens, retina, and ventral mesencephalon, a source of mDA progenitors. In the current study, our first experiment represents a proof-of-concept for human–porcine chimerism. Human-induced pluripotent stem cells (hiPSCs) and human umbilical cord blood stem cells (hUCBSCs) were introduced into wild-type porcine blastocysts. In vitro observations indicate human cells can incorporate and proliferate within the porcine inner cell mass. Furthermore, human cells were widely present in E30 porcine fetuses derived from hUCBSC-injected blastocysts, following transfer to surrogate sows. Our second experiment examined the ability for human stem cells to incorporate within the substantia nigra of porcine fetuses. Porcine fibroblasts were genetically engineered to disrupt the function of PITX3 through transcription activator-like effector nuclease (TALEN) technology. PITX3 knockout clones were used for nuclear cloning to produce PITX3-null embryos, which were then injected with hUCBSCs or hiPSCs, transferred to surrogate sows, and examined at E62. PITX3-null fetuses exhibited a closed eye phenotype that was attenuated through injection of hUCBSCs. Interrogation of the fetal eye failed to identify any contribution of human cells within the lens or retina. PITX3-null fetuses also exhibit a reduction in tyrosine hydroxylase (TH) immunoreactivity within the developing substantia nigra. Interrogation of fetuses complemented with either hiPSCs or hUCBSCs also failed to identify human cells within the substantia nigra. In a subset of complemented animals, however, a nontypical PITX3-null phenotype was observed in the morphology of the retina or an increase in TH immunoreactivity within the substantia nigra. These results may suggest a compensatory mechanism provided by the human cells earlier during development. Results from these studies indicate the potential for the use of blastocyst complementation as an alternate source for transplantable tissue. As no human cells were observed in the target tissue, further studies require the mutation of additional genes earlier in the development of the ventral mesencephalon for generating exogenic human mDA progenitors in swine.
I. dela Peña*, W.-X. Shi*†
*Department of Pharmaceutical and Administrative Sciences, School of Pharmacy, Loma Linda University, Loma Linda, CA, USA
†Department of Basic Sciences, School of Medicine, Loma Linda University, Loma Linda, CA, USA
Methylphenidate, the most widely prescribed psychostimulant to treat many neuropsychiatric conditions, has been reported to enhance attention and cognition in survivors of traumatic brain injury. The neural mechanism of this efficacy remains unclear. The prefrontal cortex (PFC), which interacts with cortical sensory systems, motor systems, and subcortical structures, has been thought to play a key role in diverse cognitive functions such as working memory and attention. PFC neurons fluctuate spontaneously between a depolarized UP state and a hyperpolarized DOWN states (in anesthetized animals), which may be crucial for information processing. Using in vivo electrophysiological recordings, we investigated the influence of methylphenidate on activity of the PFC by measuring local field potential responses. We also tested effects of methylphenidate on activity of the dopamine neurons in the ventral teg-mental area (VTA), which has also been suggested to subserve several functions, including cognition and attention. Methylphenidate (0.1–3 mg/kg, IV) altered spontaneous transitions of PFC activity between UP and DOWN states and drove PFC activity to the UP state. This effect was partially reversed by the dopamine receptor D2 antagonist raclopride and by the noradrenergic α1 receptor antagonist prazosin. Since UP states are generated by the synchronous depolarization of large ensembles of cortical neurons, these results suggest that methylphenidate produces excitatory effect on cortical networks in vivo, which may involve activation of both dopamine receptor D2 (DRD2) and α1 receptors. Furthermore, methylphenidate dose-dependently reduced the firing rate of dopamine neurons, an effect reversed by raclopride. Although further studies are required, methylphenidate-induced transition of PFC activity to the UP state may underlie its efficacy to enhance attention and cognition. Its effect on dopamine neurons coincides with the reported effects of methylphenidate and other psychostimulants to inhibit dopamine neuron firing through stimulation of dopamine autoreceptors secondary to its blockade of dopamine transporters (DATs) and an increase in dopamine release, which may ultimately lead to enhancement of signal-to-noise ratio and improvement in attention and/or cognitive performance.
P. Deng*†, J. R. Gutierrez*, A. Torrest*, A. Komarla*†, J. A. Aprile*, S. Kalomoiris†, W. Gruenloh*, G. Annett*, T. Tempkin‡, V. Wheelock‡, D. J. Segal†, J. A. Nolta*, K. D. Fink*
*Stem Cell Program and Institute for Regenerative Cures, University of California Davis Health Systems, Sacramento, CA, USA
†Genome Center, MIND Institute, and Biochemistry and Molecular Medicine, University of California, Davis, CA, USA
‡Department of Neurology, University of California Davis Health Systems, Sacramento, CA, USA
Huntington's disease (HD) is an autosomal dominant neurodegenerative disorder caused by an abnormal expansion of CAG repeats encoding a polyglutamine sequence in the N-terminal region of the HD gene. It has been suggested that postnatal reduction of mutant huntingtin through protein interference or conditional gene knockout could prove to be an effective therapy for patients suffering from HD. Our previous work established allele-specific targeting and silencing by designing transcription activator-like effectors (TALE) to target singlenucleotide polymorphisms (SNPs) in the mutant allele with a Krüppel associated box (KRAB) domain to promote transcriptional repression. We have developed multiple TALEs that are able to significantly reduce mutant huntingtin at the RNA and protein level without affecting the healthy allele in patient-derived HD fibroblasts. Up to a 75% reduction of the mutant allele was observed following purification of only cells that received the TALE. The current study focuses on the delivery of our lead TALE, T3γ, in a panel of adult and juvenile HD fibroblasts, human HD neural precursors, and in the YAC128 transgenic mouse model of HD. T3γ was selected as our lead candidate TALE based on high global minor allele frequency score, robust gene silencing, allele specificity, and the presence of its target SNP in the YAC128 HD mouse model. Mutant allele knockdown was observed across multiple adult and juvenile HD fibroblasts following T3γ treatment, demonstrating consistent gene silencing. Presently, we examine the packaging of T3γ-KRAB into a lipid nanoparticle (LNP)-based delivery system for in vivo use. LNPs have been previously shown to have strong potential as a delivery vehicle for biopharmaceuticals by complexing with apolipoprotein secreted by astrocytes, allowing it to be taken up by neurons and glia via the low-density lipoprotein (LDL) receptor with minimal in vivo toxicity and immunogenicity. We tested the LNP-T3y-KRAB in human HD fibroblasts, neural precursors, and the transgenic YAC128 to evaluate toxicity, biodistribution, and reduction of the mutant allele at the protein and RNA level. This study builds on our previous work of the potential of TALE as a powerful, personalized gene therapy for individuals suffering from Huntington's disease and seeks to identify a robust in vivo delivery mechanism.
Support for this project was provided by a NIH NIGMS Predoctoral Fellowship T32GM099608 (P. Deng), NIH NRSA Postdoctoral Fellowship F32NS090722 (K. D. Fink), a NIH Director's transformative award 1R01GM099688 (J. A. Nolta), The Dake Foundation (K. D. Fink), California Institute for Regenerative Medicine (CIRM) DR2-05415 (V. Wheelock/J. A. Nolta), and philanthropic donors from the HD community, including the Roberson family and TeamKJ.
M. Dezawa
Department Stem Cell Biology and Histology, Tohoku University Graduate School of Medicine, Sendai, Japan
In a rat stroke model treated with a newly discovered human skin-derived “multilineage-differentiating stress enduring (Muse) cells,” neuronal regeneration resulted in significant improvements in neurological and motor functions compared to control groups that were not transplanted with Muse cells. They are unique stem cells that are pluripotent-like because they can generate cells representative of all three germ layers from a single cell, express pluripotency markers, are collectable as pluripotency surface marker stage-specific embryonic antigen 3-positive (SSEA-3+) cells, are able to self-renew, and in vivo they also display high efficiency for differentiating into cells compatible with the tissue they homed to. Human skin-derived Muse cells after engraftment into the stroke tissue were found to possess functional characteristics of neurons as they attain the attributes of the host microenvironment at 3 months. Not only did they spontaneously differentiate into neuronal cells after engraftment, they reconstructed the pyramidal tract and sensory circuit. The latter was shown by recovery of somatosensory evoked potentials in the sensory cortex at 3 months. Muse cells reside in a variety of tissues, including bone marrow, skin, and fat tissues, are readily accessed commercially and can also be easily collected from patient skin biopsies. Since these cells can be derived from dermal fibroblasts, they can be obtained with relative ease, without the need for invasive procedures required for obtaining other kinds of stem cells. Furthermore, Muse cells do not have tumorigenic properties and exhibit exceptional tissue repair potential when introduced into the blood stream. Muse cells do not have to be “induced,” or genetically manipulated, to be pluripotent or be neuronal cells before transplantation as required with some other cell varieties; they already display inherent pluripotent-like properties after isolation and, with their acquired neuronal properties, Muse cells spontaneously repair stroke-damaged sites. Results show that Muse cells are a feasible and promising source for cell-based approaches to ischemic stroke therapy.
M. Dominici
Department of Medical and Surgical Sciences for Children & Adults, University Hospital of Modena and Reggio Emilia, Modena, Italy
Cellular therapies are progressively becoming solid players in cancer treatment. We have been working on projects aimed at modifying cells to insert or increase anticancer properties. In this presentation, I will present data related to the use of modified mesenchymal stromal/stem cells (MSCs) and natural killer T cells (NK/T) for the treatment of neuroectodermal-derived tumors, such as neuroblastoma and high-grade glioma. These strategies rely on viral construct engineering, cell modification, ex vivo expansion, in vitro cytotoxicity, and in vivo models. All these aspects will be dissected with an eye towards the possible clinical translations and the intent to share both encouraging results and ongoing challenges. Based on these experiences we presume that empowering cells by ex vivo manipulations could generate novel therapeutic options for cancer patients still with an unacceptable prognosis.
This work was made possible by the pivotal work of Malvina Prapa, Ph.D., Grisendi Giulia, Ph.D., and Carlotta Spano, Ph.D., and in parts by grants from the Italian Ministry of Health, by AIRC, by ASEOP, and by “Fondazione Guido Berlucchi” on Cancer Research.
C. Drolen*, S. Tamir*, N. Tajiri†, S. A. Acosta†, M. Lee*, S. Shacham*, C. V. Borlongan†
*Karyopharm Therapeutics Inc, Newton, MA, 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
This study evaluated the effects of the selective inhibitor of nuclear export (SINE) compound KPT-350 on inflammatory and neuroprotective endpoints in rodent models of traumatic brain injury (TBI). We investigated the effects of KPT-350 in a rat controlled cortical impact (CCI) model of TBI. KPT-350 or vehicle was administered to CCI rats once daily for 5 days, starting 2 h postinjury. Homogenized tissues from the CCI-impacted area were sampled for cytokine levels. In addition, histological assays, rotarod performance tests, and elevated body swing tests were conducted. Overall, KPT-350 demonstrated potent anti-inflammatory and neuroprotective capabilities, as evidenced by specific alterations in cytokine levels. KPT-350 treatment significantly reduced the TBI-induced lesion volume in the core impact area and decreased damage to the peri-impact area. Behavioral functions were also improved. KPT-350 treatment improves both the acute and subacute phases of TBI by protecting intact brain cells that remain after the primary injury and mitigating the damage caused by secondary inflammation. Together, these data suggest that KPT-350 acts by a dual mechanism of action, modulating key inflammatory mediators and neuroprotective pathways while simultaneously protecting blood–brain barrier (BBB) integrity following TBI. These studies reveal that KPT-350 treatment affords therapeutic benefits in paradigms of TBI and warrants further clinical investigation.
M. F. Duffy*, T. J. Collier*†, K. C. Luk‡, K. L. Paumier*†, D. L. Fischer*, N. K. Polinski*, C. J. Kemp*, J. Q. Trojanowski‡, V. M. Lee‡, C. E. Sortwell*†
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Mercy Health Hauenstein Neuroscience Medical Center, Grand Rapids, MI, USA
‡Center for Neurodegenerative Disease Research, Department of Pathology and Laboratory Medicine, University of Pennsylvania Perelman School of Medicine, Philadelphia, PA, USA
Increased expression of inflammatory markers associated with microglia is observed in postmortem Parkinson's disease (PD) brains, with longitudinal positron emission tomography (PET) imaging studies revealing early and sustained microglial activation in the basal ganglia. α-Synuclein (a-syn) released from neurons has been shown to activate toll-like receptor 2 (TLR2) in microglial cultures, suggesting that α-syn may participate in PD neuroinflammation. However, it remains unclear whether inflammation contributes to nigral degeneration or is merely a secondary consequence of degenerating neurons. Historically, one difficulty in delineating this sequence of events is the lack of an animal model that displays α-syn aggregation and nigral degeneration in a protracted time course. Our lab recently characterized the accumulation of phosphorylated α-syn intraneuronal inclusions and bilateral nigrostriatal degeneration following intrastriatal injection of α-syn preformed fibrils (PFFs) into rats (PMID: 26093169). α-syn PFFs are taken up by striatal terminals and seed endogenous α-syn conversion into a pathological hyperphosphorylated form. Specifically, in our α-syn PFF rat model, we observe widespread Lewy body-like pathology in interconnected regions and ~40% nigral dopamine neuron degeneration over 6 months, providing an optimal platform for studying the time course of α-syn aggregation, neuroinflammation, and nigral degeneration. The present study examined the neuroinflammatory signature resulting from intrastriatal α-syn PFFs. Young adult male Fischer344 rats received unilateral intrastriatal injections (2 × 2 μl) of mouse α-syn PFFs or saline. Specific cohorts of rats (total n = 96) were euthanized at various times postinjection (0.5, 0.75, 1, 2, 3, 4, 5, and 6 months). To quantify the magnitude of nigrostriatal degeneration and number of intraneuronal phosphorylated α-syn inclusions, the substantia nigra was immunostained for a) tyrosine hydroxylase (TH) and cresyl violet or b) phosphorylated α-syn (pS129) and cresyl violet. To analyze striatal denervation, TH immunoreactivity was quantified in striatal sections. To characterize microglia-mediated neuroinflammation, sections were stained with a) pan-microglial marker ionized calcium binding adaptor molecule-1 (iba-1) to quantify microglia density and b) major histocompatibility complex II (MHC II) to assess the number of antigen-presenting microglia. While stereological assessments are still ongoing, preliminary results suggest that a) inflammation is associated with PFF but not saline injection, b) neuroinflammation occurs early in the pathological timeline (2 months; n = 12), and c) neuroinflammation is associated with high levels of phosphorylated α-syn intraneuronal inclusions. Results from this study will provide new insight into neuroinflammatory dynamics and mechanisms in relation to α-syn aggregation and nigral degeneration. These findings may establish the use of the α-syn PFF rat model as a platform for testing anti-inflammatory therapeutics.
Supported by the Morris K. Udall Centers of Excellence for Parkinson's Disease Research at Michigan State University (NS 058830), the University of Pennsylvania (NS 053488), and the Michigan State University Neuroscience Program (T32 NS44928).
N. Egawa*, A. Shindo*, C. Xing*, A. C. Liang*, R. Sudo†, E. H. Lo*, K. Arai*
*Neuroprotection Research Laboratory, Departments of Radiology and Neurology, Massachusetts General Hospital and Harvard Medical School, Charlestown, MA, USA
†Department of System Design Engineering, Keio University, Yokohama, Japan
Oligodendrocyte precursor cells (OPCs) differentiate mostly into oligodendrocytes under both in vitro and in vivo conditions. Mechanisms of OPC differentiation have been extensively examined with in vitro cell culture models. However, those experiments were conducted in two-dimensional culture systems. Here, we developed a novel three-dimensional OPC culture system using gelatin/hyaluronan-mixed gels, wherein cultured OPCs can proliferate and differentiate into oligodendrocytes. Importantly, accessibility to neighboring cells, which we defined based on the concept of hydrokinetic modeling, was a useful index to predict cell viability in the three-dimensional gels. Decreasing the accessibility by changing cell density or hydrogel porosity decreased the number of OPCs and oligodendrocytes thereafter in our three-dimensional culture system. Notch signaling by cell–cell contact may be essential for OPC function in our system because a Notch inhibitor DAPT suppressed the OPC proliferation and differentiation. Taken together, our novel three-dimensional OPC culture system would be useful to examine the mechanisms of OPC function in vitro, and the index of accessibility can be utilized to optimize the conditions of three-dimensional OPC cultures.
D. J. Eve*, M. R. Steele†, P. R. Sanberg*, 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
†Veterans Reintegration Steering Committee, Veterans Research, University of South Florida, Tampa, FL, USA
Traumatic brain injury (TBI) arises from physical damage to the brain as the consequence of an insult and is reflected by psychological and neurological symptoms depending on the severity of the injury. The increase in the use of improvised exploding devices (IEDs) during the recent excursions in Iraq and Afghanistan mean that the US's returning soldiers frequently suffer from some degree of TBI. The primary phase is directly injury related, while a secondary phase arises due to hypoxia, increased oxidative stress, and inflammation. In addition, exposure to the consequences of this warfare mean that a proportion of the returning soldiers are also suffering from posttraumatic stress disorder (PTSD). There is some evidence that PTSD may be a consequence of TBI in some instances. The search for an effective treatment is ongoing and one possible therapeutic candidate is hyperbaric oxygen therapy (HBOT). Some clinical trials suggest benefit with regards to survival and disease severity, while others do not see any improvement compared to a sham, though several of these studies are poorly controlled. Since HBOT has been shown to reduce apoptosis, upregulate growth factors, promote antioxidant levels, and inhibit inflammatory cytokines, there is likely to be an advantageous effect of HBOT in treating at least the secondary phase of TBI and PTSD. The precise timing(s) of HBOT exposure still need to be determined along with the consideration of a putative role of prophylactic (or preconditioning) exposure. One caveat is that acute cerebral toxicity and other deleterious effects, partly due to the increased oxygen levels generating more reactive oxygen species, can occur following exposure to HBOT and so optimizing exposure duration to maximize the reward and decrease the detrimental effects of HBOT will be necessary. In this presentation, we will be providing an overview of our current knowledge and recommendations for future directions including prophylactic use and chronic treatment.
This study is supported by USF #140 Health Veteran PTSD and Traumatic Brain Injury Study.
B. Feinerman
Stem Cell Genetic Med, Wellington, FL, USA
Alzheimer's disease (AD) is a neurodegenerative disease that is associated with dementia, memory loss, impairment of judgment and lack of understanding on how to function. Patients may get lost even in familiar areas, often not able to do routine things around the house such as food preparation and doing laundry. Individuals cannot recognize familiar objects and people. Personal hygiene, eating, and dressing require assistance. Interacting in a social setting is lost. Withdrawing, restless, and even language abilities become lost. The disease usually occurs after 65 years of age but there are some individuals that have an early onset. The present proposed protocol is a stem cell and gene therapy for AD. Gene mutations that may be associated with the condition include amyloid precursor protein (APP), presenilin 1 (PSEN1), PSEN2, and apolipoprotein E (APOE). If an abnormal gene is present, the first step is to use a specific short hairpin RNA (shRNA) agent to knock out the mutation. The shRNA agent is transfected by an adeno-associated virus 2 (AAV-2) vector plus the gene mutation, which is given intrathecally. This is followed 48 h later by infusion of the normal gene plus neuron brain stem cells, brain-derived neurotrophic factor, nerve growth factor 100 μg, neurotrophins 10 μg, glial-derived neurotrophic factor 100 μg, and vascular endothelial growth factor 100 μg. In addition, NGR 0.1 mg/kg is also introduced into the cerebrospinal fluid to reduce amyloid plaque deposition and neuritic dystrophy. The intrathecal route allows the agents to by-pass the blood–brain barrier.
W. P. Flavin*†, L. Bousset‡, D. Wakeman§, J. Kordower§, R. Melki‡, E. M. Campbell†¶
*Stritch School of Medicine, Loyola University Chicago, Maywood, IL, USA
†Integrative Cell Biology Program, Loyola University Chicago, Maywood, IL, USA
‡Paris-Saclay Institute of Neuroscience, CNRS, Gif-sur-Yvette, France
§Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
¶Department of Microbiology and Immunology, Loyola University Chicago, Maywood, IL, USA
Numerous recent studies have established the ability of aggregated α-synuclein (a-syn) to spread from cell-to-cell in a prion-like fashion, whereby the propagation of both its misfolded conformation and higher order aggregation state from affected neurons to neighboring cells constitutes a central mechanism of pathological progression in Parkinson's disease. Understanding the cellular and molecular forces responsible for this transfer between cells is critical to developing treatment approaches designed to arrest or prevent disease progression. In the circular pathway of α-syn cell-to-cell transmission, our lab has addressed the previously unexplained phenomenon of endocytic vesicle exit by demonstrating that α-syn aggregates are able to induce lysosomal rupture, a type of cellular damage that causes cathepsinmediated oxidative stress and inflammasome activation. Further characterization of this disruptive cellular entry mechanism has resulted in our subsequent demonstration that aggregates of both wild-type and familial mutant α-syn are capable of significantly inducing vesicle rupture as measured by mCherry-galectin-3 (chGal3) relocalization in a human neuroblastoma cell line. Moreover, we show that differences in α-syn aggregate conformation as present in structurally well-defined α-syn strain assemblies result in different levels of vesicle rupture potency, implicating vesicle rupture as a mechanism for strain-specific differences in target cell pathology. Importantly, we have also validated α-syn-induced vesicle rupture with our chGal3 model using mature dopaminergic neurons derived from induced pluripotent stem cells. The consequences of vesicle rupture like lysosomal/autophagic dysfunction and oxidative stress are known driving forces behind the release of α-syn aggregates from an affected cell; therefore, we additionally investigated cell-to-cell transfer of α-syn in the context of aggregate-induced vesicle rupture by utilizing live cell fluorescent imaging. Ruptured vesicles containing α-syn can be observed in the extracellular environment and can be seen trafficking from an affected cell to a neighboring healthy cell. These results implicate α-syn-induced vesicle rupture as a detrimental consequence of endocytic entry, elucidate mechanistic differences dictating α-syn strain-specific pathology, and suggest that this form of cellular damage can serve as both a driving force and a vector for α-syn release and subsequent transmission to neighboring neurons.
A. D. Flowers*, H. Bell-Temin†, A. Cochran†, C. Hudson‡, S. Stevens†, P. Bickford*‡
*Department of Neurosurgery and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
†Department of Cell Biology, Microbiology and Molecular Biology, University of South Florida, College of Arts & Sciences, Tampa, FL, USA
‡James A. Haley Veterans Hospital, Tampa, FL, USA
In the last decade, the field of brain repair has made major leaps towards meaningful therapies and treatments for diseases ranging from the chronically degenerative to the acutely traumatic. These breakthroughs have come in part as we have gained a more detailed understanding of the progression and environment in which this damage occurs. While the ability to transplant or regenerate cells to facilitate repair is progressing rapidly, we must also focus on our ability to modulate the environment in which these cells are replaced. Replacing healthy cells into an active and ongoing degenerative process dramatically reduces the likelihood of success. Most idiopathic degenerative diseases of the central nervous system (CNS) occur concomitantly with age. Maintenance of homeostasis in the CNS is one function of the microglial cell, the resident immune cell of the brain. This long-lived cell is involved in several processes, including neurogenesis, synaptic pruning, ionic homeostasis, and response to injury or infection. These cells undergo dramatic changes with age, including changes in morphology and deregulated response to extracellular signals. The ability to integrate and adequately respond to their environment is a vital function of microglia, without which they are at best inefficient and at worst harmful. Their role in modulating synaptic connections and supporting cellular survival through the production of neurotrophins makes these cells central to the success of any cellular replacement or regenerative therapy. Our lab has performed the first hybrid stable isotope labeling by amino acids in cell culture (SILAC)/label free mass spectrometry analysis of microglia isolated from aged animals. This unique method overcame obstacles unique to proteomic analysis of primary microglia, which in culture do not proliferate, a requirement for SILAC labeling a highly accurate method of quantification of proteins. Analysis of mass spectrometry data requires a powerful bioinformatics platform. We utilized Ingenuity Pathway Analysis (IPA; Qiagen, Redwood City, CA, USA), a cutting-edge platform that combines common tools like pathway analysis with innovative features like upstream and downstream regulator analysis. The pathway analysis of aged microglia revealed significant alterations in pathways governing mitochondrial function, oxidative stress response, protein translation, and protein ubiquitination. Using IPA's upstream regulator analysis and molecular assays we identified and validated mechanistic target of rapamycin complex 2 (mTORC2) as an upstream regulator of several age-related cellular processes. The importance of this finding is twofold. First, nutrient-sensing pathways like mTOR are upstream of metabolic gene regulatory networks. In order for microglia to acquire their vast number of activation states, subtle switches in these networks must occur to allow for production of a variety of byproducts necessary for the intended cellular function. Second, the majority of research in nutrient-sensing deregulation has focused on mTORC1 not mTORC2. Mounting evidence suggests that it is in fact mTORC2 not mTORC1 that is responsible for several cell-intrinsic changes including insulin insensitivity, increased oxidative stress, and endoplasmic reticulum (ER) dysfunction. Our results suggest that mTORC2 activity is decreased in aged microglia and that this disruption directly impacts the cells' ability to appropriately respond to cytokines. Our results also suggest that it may be possible to target these pathways with presently available compounds to attenuate this age-dependent change in activity. We employed both pharmacological and molecular modulators of mTORC2 to demonstrate their ability to alter cellular function in a potentially advantageous way. Our future studies will aim to elucidate the exact mechanisms by which mTORC2 is affecting cellular activity and determine whether modulation of these pathways in vivo is sufficient to attenuate age-related microglial dysfunction. This work has the potential to greatly improve the success rates of future brain repair therapies.
T. Freeman
Department of Neurosurgery and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
Neural transplantation was first explored in the clinic for the treatment of Parkinson's disease (PD) and Huntington's disease (HD) based on extensive preclinical research. Translational issues included: donor staging, host volumetric scaling, volume of distribution, transplant immunology, tissue dissection and preparation methods, good manufacturing practice (GMP) issues, and human donation ethical, regulatory, and legal issues, among others. Clinical trials of neural transplants in both PD and HD have been only partially positive, due to both unexpected adverse events (AEs) as well as clinical trial issues. AEs in PD trials included off-time dyskinesias and variability in response rates; HD trials were complicated by subdural hematomas and poor clinical signal in only a few patients. Clinical trials were uniformly underpowered or overly reliant on descriptive rather than quantitative data. This has led to a rethinking of both basic science pillars as well as clinical trial design issues. Finally, transmission of the disease-specific pathologic proteins to grafts in patients with both PD and HD has led to questions about longevity of benefit and influence of the host transplant milieu, as well as shed light on the underlying mechanisms of the diseases. This circular interplay between the laboratory, clinic, clinical trials, and then going back to the laboratory will likely be similar with the use of stem cells in the treatment of PD and HD, as well as with additional cell therapies in the treatment of other neurological disorders.
A. Gharaibeh*†‡, R. Culver*†‡, A. Crane*†‡, R. Wyse*†§, A. Antcliff*†§, G. Shall*†‡, S. Moore*†‡, B. Srinageshwar*†‡, N. Kolli*†‡, D. Story*†‡, O. Lossia*†‡, L. Frollo*†‡, A. Eickholt*†‡, G. Dunbar*†‡¶, J. Rossignol*†§
*Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
†Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
‡Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
§College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
¶Field Neurosciences Institute, Saginaw, MI, USA
Huntington's disease (HD) is an inherited neurodegenerative disorder with no known effective treatment to delay its onset or progression. However, the use of induced pluripotent stem cells (iPSCs) holds significant promise as a potential treatment. The ultimate goal of our research is to replace the HD-induced loss of striatal neurons with iPSC-derived neurons and to preserve or restore normal functional outcomes. To this end, we developed mouse adenovirus-generated iPSCs [using octamer-binding transcription factor 4 (Oct4), sex determining region Y box 2 (Sox2), Krüppel-like factor 4 (Klf4) and c-Myc], which express all the genes and proteins of pluripotency, and transplanted them into the striata of wild-type (WT) and HD YAC128 mice. We then tested the efficacy of these transplanted iPSCs for (1) their ability to survive and differentiate into neuronal phenotypes (without forming tumors) and (2) their ability to reduce the significant motor deficits observed in this mouse model of HD. To assess the survivability, differentiation potential, and safety (potential tumor formation), we first transplanted iPSCs prelabeled with Hoechst 33258 bilaterally into the striata of female WT and HD YAC128 mice at 12 months age. Four- or 8-weeks following transplantation, the mice were perfused, and their brains were frozen, sectioned, and analyzed by immunohistochemistry. Our results revealed that iPSCs in both WT and HD mice had survived and showed evidence of differentiation into neuronal phenotypes, with colabeling of Hoechst with neuron-specific class III β-tubulin (clone Tuj1) and neuronal nuclei (NeuN) at both the 4- and 8-week posttransplant time points. In addition, we observed a reduction in reactive astrocytes [glial fibrillary acidic protein positive (GFAP+)] at 8 weeks compared to the 4 weeks at the site of transplantation and infiltrating areas where surviving cells were located. We also didn't see formation of tumors in the brains. To further analyze the functional efficacy of iPSC transplantation, 50 male and female WT and HD YAC128 mice were bilaterally transplanted at 10 months old with iPSCs prelabeled with Hoechst 33258 or vehicle control. Accelerod behavioral testing was performed 1 day before the transplantation, and then weekly for 10 weeks. The repeated-measures ANOVA and Tukey post hoc tests revealed a significant difference between WT and HD mice for the vehicle control groups, while both the WT and HD mice receiving the iPSC transplant performed at the levels of the control WT mice, indicating a complete preservation of normal motor functioning. Presently, we are evaluating the histological outcomes of this second study and will correlate these findings with the behavioral outcomes we have obtained. Collectively, our histological and behavioral data suggest that adenovirus-generated iPSCs may provide a safe and effective option for neuronal replacement therapy. Further research looking at the electrophysiological profiles of these iPSCs is under way and may further elucidate the mechanisms underlying the observed behavioral sparing produced by these iPSC transplants.
Support for this study was provided by the Office of Research and Sponsored Programs at CMU, the College of Medicine, the Field Neurosciences Institute, and the John G. Kulhavi Professorship in Neuroscience at CMU.
H. Ghuman*†, J. Donnelly‡, A. R. Massensini*§¶, S. F. Badylak*†#, M. Modo*†§
*McGowan Institute for Regenerative Medicine, University of Pittsburgh, Pittsburgh, PA, USA
†Department of Bioengineering, University of Pittsburgh, Pittsburgh, PA, USA
‡Department of Biological Sciences, University of Pittsburgh, Pittsburgh, PA, USA
§Department of Radiology, University of Pittsburgh, Pittsburgh, PA, USA
¶Department of Physiology and Biophysics, Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
#Department of Surgery, University of Pittsburgh, Pittsburgh, Pennsylvania, USA
Stroke is the leading cause of adult disability and a significant effort is under way to develop therapies to repair the damaged tissue. Biomaterials composed of mammalian extracellular matrix (ECM) promote constructive tissue remodeling with minimal scar formation. At ECM concentrations that have similar rheological properties as brain tissue, the biomaterial exists in fluid phase at room temperature, while forming hydrogels at body temperature. ECM with different concentrations (0, 1, 2, 3, 4, 8 mg/ml) was injected into the lesion cavity after stroke to support endogenous repair mechanisms. Retention and gelation of the ECM, as well as host cell invasion and phenotype, were analyzed at 1, 14, and 90 days postinjection using immunohistochemistry. Retention of ECM hydrogel within the cavity occurred at concentrations >3 mg/ml, with extensive diffusion into the host tissue at lower concentrations. A significant host cell invasion into the ECM hydrogel was seen at 1 day postinjection, with an average of over 35,000 cells invading in the 8 mg/ml concentration. As the acute inflammatory response was replaced with an ECM remodeling phase at later time points, there was a significant decrease in the total number of cells invading the biomaterial. Initial invading cells were of a microglia and macrophage phenotype and followed specific trails into the ECM biomaterial along topological features conducive to cell migration. The follow-on cells were neural and oligodendrocyte progenitor cells, which are essential for repopulation of the neural tissue. This characterization demonstrates that an ECM hydrogel can be readily injected and retained within the lesion cavity, while promoting an acute endogenous repair response. A behavioral study is necessary to evaluate the therapeutic potential of this approach.
T. Glaser*, R. Kageyama†‡, H. Ulrich*
*Department of Biochemistry, University of São Paulo, São Paulo, Brazil
†Institute of Chemistry, University of São Paulo, São Paulo, Brazil
‡Institute for Virus Research, Kyoto University, Kyoto, Japan
Oscillations of intracellular calcium concentrations participate in many cellular processes. Spatial and temporal patterns of calcium spike and wave activity are important for fate determination in the development of the nervous system and participate in neurotransmitter specification of differentiating neurons. As a suggested underlying mechanism for stem cell differentiation into defined phenotypes, a distinct transient calcium pattern in undifferentiated cells codes for the expression and activation of neural transcription factors, which then induce the expression of ion channels and other calcium-mobilizing mechanisms characteristic for an advanced stage of differentiation. Any interference with this transient calcium activity affects the fate of differentiation. Voltage-gated calcium channels and purinergic adenosine triphosphate (ATP)-activated receptors are already expressed at early development, and in vitro and in vivo data provided evidence for their participation in transient intracellular calcium signaling and control of neural differentiation. Using mouse embryonic stem cells (ESCs) as in vitro model for neuroectodermal differentiation into neurons and glial phenotype, we tracked transient calcium activity together with rhythmic neural transcription factor expression. Time-lapse imaging with calcium-sensitive fluorescent probes was combined with luminescence imaging of stable cells transfected with mammalian achaete scute homolog-1 (Mash-1) or neurogenin-2 promoter protein fused to a luciferase reporter construct. Spontaneous calcium transients observed as spikes or waves based on their frequency and duration exclusively depended on calcium mobilization from intracellular pools. Further, addition of ATP induced increases in cytosolic calcium concentration by activation of ionotropic and metabotropic purinergic receptors, and augmented frequencies and amplitudes of intracellular calcium oscillations together with rhythmic changes in Mash-1 and neurogenin-2 expression levels. Pharmacological tools led us to conclude that ESCs predifferentiated to neural stem cells or neural stem cells with augmented Mash-1 expression as a result of P2Y2 and/or P2Y4 purinergic receptor activity, while expression rates of this neural transcription factor were reduced as a consequence of P2X7 receptor activity. Mash-1 and neurogenin-2 oscillatory expression patterns depended on the voltage-gated calcium channel, as probed by the time-lapse fluorescence and luminescence imaging technique. Overall, our studies show that temporal oscillations of Mash-1 and neurogenin-2 expression patterns code for the neural phenotype (differentiation into neurons or astrocytes) as well as for neurotransmitter specification, providing novel insights into mechanisms of neurogenesis. Selected neural transcription factor activation allows enriching desired neuronal population in differentiating stem cells for transplantation purposes as well as mobilizations of endogenous stem cells for the treatment of neurodegenerative diseases.
Acknowledgment: Grants from Brazilian funding agencies FAPESP and CNPq.
R. Gonzalez*, I. Garitaonandia*, M. Poustovoitov*, T. Abramihina*, C. R. McEntire†, B. Culp†, J. Attwood†, A. Noskov*, A. Semechkin*, L. C. Laurent‡, J. D. Elsworth§¶, J. Sladek#, E. Y. Snyder**, D. E. Redmond Jr.†§¶, R. Kern*
*International Stem Cell Corporation, Carlsbad, CA, USA
†Axion Research Foundation, Hamden, CT, USA
‡Department of Reproductive Medicine, UC San Diego, La Jolla, CA, USA
§Department of Psychiatry, Yale University School of Medicine, New Haven, CT, USA
¶Department of Neurosurgery, Yale University School of Medicine, New Haven, CT, USA
#Department of Neurology, Pediatrics and Neuroscience, University of Colorado School of Medicine, Aurora, CO, USA
**Sanford-Burnham-Prebys Medical Discovery Institute, La Jolla, CA, USA
Clinical studies have shown that grafted fetal neural tissue can achieve considerable biochemical and clinical improvements in Parkinson's disease (PD). However, the source of fetal tissue grafts is limited and ethically controversial. Human parthenogenetic stem cells offer a good alternative because they are derived from unfertilized oocytes without destroying viable human embryos and can be used to generate an unlimited supply of neural stem cells for transplantation. We have previously reported that human parthenogenetic stem cell-derived neural stem cells (hpNSCs) successfully engraft, survive long-term, and increase midstriatum brain dopamine levels in nonhuman primate models of PD. Here we present additional biochemical, cell fate, and genomic analysis results from a long-term 12-month transplantation study of hpNSCs in 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-lesioned African green monkeys with moderate to severe clinical parkinsonian symptoms and a 9-month tumorigenicity and biodistribution study in athymic nude rats. hpNSCs manufactured under current good manufacturing practice (cGMP) conditions were injected bilaterally into the striatum and substantia nigra of immunosuppressed monkeys and rats. Our most recent biochemical, genomic, and cell fate analysis indicates that the transplanted hpNSCs promoted widespread recovery of dopamine levels, innervation, and number of dopaminergic neurons. These results provide further evidence for the clinical translation of hpNSCs for the treatment of PD.
K. M. Gresham, M. H. Theus
Department of Biomedical Sciences and Pathobiology, VirginiaMaryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
Adult neurogenesis occurs in the dentate gyrus (DG) of the hippocampus and is a highly coordinated process that regulates learning and memory through the survival, proliferation, and differentiation of neural stem/progenitor cells (NSPCs). Neurogenesis occurs when NSPCs in the subgranular layer are instructed by cues in their local microenvironment to divide and differentiate, resulting in the generation of newly born neurons. Traumatic brain injury (TBI) produces an initial loss of the NSPC population followed by cognitive recovery due to injury-induced neurogenesis. However, the mechanism(s) regulating this response is unclear. Recently, using endothelial cell (EC)-specific ephrin receptor (EphR) knockout mice, we observed a trend toward reduced terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the DG at 4 days post-TBI compared to wild type. This correlated with increased levels of the serine (s)368 phosphorylated form of gap junction-associated hemichannel protein connexin 43 (Cx43) in the subgranular layer of the DG. High levels of (p)s368 are associated with changes in gap junction permeability and enhanced proliferation in multiple cell types. We show that stimulation of cultured NSPCs with soluble ectopic EphR molecules suppresses (p)s368 levels, suggesting modulation of EphR in the DG following TBI may directly regulate NSPC growth by controlling Cx43 posttranslational phosphorylation. Moreover, using the C-terminus Cx43 memetic peptide aCT1, we show a significant reduction in cultured NSPC proliferation and survival. Protein kinase C (PKC has been shown to directly phosphorylate Cx43 at s368, and may play a potential role in this pathway. Based on our preliminary findings, we hypothesize that EC-specific EphR negatively regulates neurogenesis in the DG microenvironment by suppressing (p) s368 of Cx43 and modulating gap junction communication in NSPCs after trauma. Future studies will investigate this possibility as well as elucidate the pathways involved using coculture systems and in vivo gain- and reverse-of-function experiments. Knowledge gained from these studies will improve our basic understanding of the mechanism(s) controlling the neurogenic response in the DG following TBI.
B. Grimmig*, L. Daly†, C. Hudson*†‡, P. C. Bickford*†‡
*Department of Molecular Pharmacology and Physiology, 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
‡Research Service, James A. Haley Veterans Affairs Hospital, Tampa, FL, USA
Astaxanthin (AXT) is a natural carotenoid with diverse biological activities. Its unique chemical structure with a hydrophobic core capped by two polar ionone moieties allows it to span the lipid bilayer and integrate into the membrane, thus increasing its potency as an antioxidant. Although it is best known as a potent antioxidant, it also has a putative role as an anti-inflammatory agent and other properties, such as a capacity to increase neural progenitor cell proliferation, which implicate it as a potential neuroprotective agent. Given the role of oxidative stress and inflammation in the progression of many neurodegenerative diseases, we examined the potential of AXT to prevent 1-methyl-4-phenyl-1, 2,3,6-tetrahydropyridine (MPTP)-induced dopamine cell death in mice. Here we show that the administration of 3 mg/kg of algae-derived AXT reduced neurotoxicity in a mouse model of Parkinson's disease. After a 4-week dietary intervention, AXT-treated mice were protected against the loss of tyrosine hydroxylase (TH)-positive cells in the substantia nigra (SN) after MPTP exposure (10 mg/kg administered hourly for a total of 40 mg/kg) compared to the control diet. This effect of preserved TH immunoreactivity was also observed in the striatum. Furthermore, AXT administration was able to interrupt the neuroinflammatory process known to contribute to neurodegeneration in this model. We demonstrate that AXT neuroprotection was associated with attenuated microglial activation indicated by reduced immunohistochemical detection of ionized calcium binding adaptor molecule-1 (IBA-1) in the SN and striatum of AXT-treated mice. We are exploring a dose–response relationship of AXT's ability to rescue dopaminergic neurons from an MPTP insult. Taken together, these studies suggest that AXT has neuroprotective actions in the central nervous system (CNS) against MPTP neurotoxicity.
V. A. Guedes*, J.-Y. Lee*, M. Provenzano†, I. Antonucci†, L. Stuppia†, K. Richards*, N. Tajiri*, C. Cao‡, 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
†Laboratory of Molecular Genetics, DISPUTer, School of Medicine and Health Sciences, “G. d'Annunzio” University, Chieti-Pescara, Italy
‡Department of Pharmaceutical Sciences, College of Pharmacy, University of South Florida, Tampa, FL, USA
Stroke is a leading cause of death and disability in the US. The available treatment options are limited, making the development of new therapies an urgent need. It has been suggested that coffee and its active ingredient caffeine possess neuroprotective properties. However, large doses of coffee may need to be consumed in order to achieve such neuroprotection. Here we studied the possible protective effect of nanosized coffee (nanocoffee) in human amniotic fluid-derived stem cells (AFSCs). AFSCs were treated with different concentrations of nanocoffee under ambient cell culture condition or subsequently exposed to oxygen-glucose deprivation (OGD) (90-min duration) or tumor necrosis factor-α (TNF-α) treatment (10 ng/ml, 24 h). Cell viability was measured by calcein AM (acetoxymethyl) and MTT [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide] assays. An established cell culture protocol for OGD induction (Kaneko et al., 2014) was used. AFSC proliferation under ambient condition significantly increased with the nanocoffee treatment (p < 0.05). Additionally, nanocoffee significantly protected the AFSCs against OGD- and TNF-α-induced cell death (p < 0.05). Our results suggest that nanocoffee stands as a potent stem cell proliferative agent, and acts as a neuroprotectant against an in vitro experimental stroke. Studies about the mechanisms of action and therapeutic benefits of nanocoffee in an in vivo stroke model are in progress.
C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC, and NeuralStem Inc.
B. K. Harvey*, E. S. Wires*, K. A. Trychta*, X. Yan*, C. Richie*, M. Henderson*†
*Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, USA
†National Center for Advancing Translational Sciences, Rockville, MD, USA
The high calcium concentrations found in the endoplasmic reticulum (ER) of every cell are critical for cellular functions such as protein folding, lipid metabolism, and calcium signaling. Disruption of ER calcium homeostasis has been implicated in diverse pathologies, including substance abuse, neurodegeneration, cardiovascular diseases, and diabetes. Monitoring ER calcium has been limited to imaging techniques that are not amenable to longitudinal monitoring of ER calcium as a disease slowly progresses (e.g., neurodegenerative diseases). We recently developed a method to monitor ER calcium that was initiated by our structure/function studies of mesencephalic astrocyte-derived neurotrophic factor (MANF). MANF is an ER-localized, secreted ER stress response protein with neurotrophic activity. The C-terminus of MANF is sufficient to confer secretion of a protein specifically in response to ER calcium depletion. Using the C-terminus of MANF and Gaussia luciferase (GLuc) we created a novel reporter as a secreted ER calcium modulated protein (SERCaMP), or GLuc-SERCaMP. Using this reporter, it is now possible to study ER calcium fluctuations over extended periods of time, thereby enabling the investigation of the temporal relationship between ER calcium changes and disease progression. Monitoring the secretion of GLuc-SERCaMP provides a simple method to assess ER calcium homeostasis in vitro or in vivo by collecting and measuring extracellular fluids such as culture medium, cerebrospinal fluid, or blood. GLuc-SERCaMP is very sensitive, with detection of ER calcium depletion possible in as few as 20 cells. We demonstrated the utility of GLuc-SERCaMPs in characterizing inducers of ER stress, including hyperthermia and glutamate toxicity, in rat primary neurons. Our results suggest that SERCaMP-based reporters will have broad applications for the long-term monitoring of ER calcium homeostasis and the development of therapeutic approaches to counteract ER calcium dysregulation.
A. Házy, T. Brickler, I. C. Allen, M. Theus
Department of Biomedical Sciences and Pathobiology, VirginiaMaryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
Traumatic brain injury (TBI) is one of the most common types of acquired central nervous system (CNS) injuries in the US. Inflammation is a hallmark feature of TBI and plays a significant role in the progression of TBI pathology. However, the role of the innate immune response is poorly understood. Previous work from our lab has identified a unique subgroup of non-inflammasome-forming cellular proteins from the nucleotide-binding oligomerization domain-like (Nod-like) receptor (NLR) family of pattern recognition receptors that function as negative regulators of inflammation and other biological processes, including nuclear factor of kappa light polypeptide gene enhancer in B cells (NF-κB) signaling. We have found that NLRX1, a prototypical member of this subgroup, serves to protect the brain following TBI. Our data shows that Nlrx1 conventional knockout mice develop significantly larger lesions, increased neuronal cell death, and more pronounced functional defects compared to wild-type animals. The increased pathogenesis in these mice is accompanied by a massive increase in the microglia/macrophage population and increased proinflammatory signaling. Based on these findings, we hypothesize that NLRX1 suppresses microglia/macrophage activation (i.e., recruitment, proliferation, and/or survival) through the modulation of NF-κB signaling to quench over-zealous inflammation to prevent neuronal loss. Our future work will test this hypothesis utilizing unique conditional knockout mice to better define the mechanism(s) associated with NLRX1 function and protection in the CNS during TBI progression, with special emphasis on NF-κB modulation. Our current findings implicate the NLRX1 protein as a novel regulator of innate immune activation following TBI.
D. C. Hess
Department of Neurology, Medical College of Georgia, Georgia Regents University, Augusta, GA, USA
Preclinical work with stem cells and cell therapy is encouraging. While intravenous, intra-arterial, and stereotactic brain injection, and even intranasal routes, are potential routes of administration, the intravenous delivery of allogeneic stem cells would arguably be the most scalable and likely to get utilized in community hospitals. Mesenchymal stem cells (MSCs) and the related but distinct cell subset, Multistem® cells, are both promising for acute stroke within the first week. The Multistem® clinical trial funded by Athersys (Cleveland, OH, USA) is the largest randomized, blinded clinical trial of intravenous allogeneic cell therapy to date. Multistem® cells were well tolerated and safe and tended to reduce infections. While in the overall efficacy analysis there was no benefit, a post hoc analysis of patients treated “early” (24–36 h) had improved 90-day clinical outcomes. This supports testing Multistem® cells in a 24–36-h window in future trials.
B. M. Hiller, D. J. Marmion, J. P. Cioni, M. L. Russo, J. H. Kordower, D. R. Wakeman
Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
Huntington's disease (HD) is a fatal neurodegenerative disorder characterized by progressive cognitive and motor decline caused by expansion of CAG trinucleotide repeats, which induces the synthesis and aggregation of huntingtin. There are currently no treatments available for HD. As such, the development of a viable therapy is imperative. Preclinical studies have indicated that transplantation of fetal neurons may provide transient functional benefit in animal models, although this has not been convincingly demonstrated in HD patients. Induced pluripotent stem cells (iPSCs) hold great promise for treating a variety of neurological disorders such as Parkinson's disease and hold many advantages over fetal cells. Further, the cryopreservation of postmitotic neurons derived from iPSCs represents a significant vertical advancement for clinical translation of pluripotent stem cell technologies as they are reliably and reproducibly thawed, allowing for rapid access to large numbers of highly pure, patient-specific cells. In the present study, we examined the engraftment potential of iPSC-derived forebrain lineage neurons (iCell Neurons) after transplantation into the rodent and nonhuman primate brain. iCell Neurons were derived from human blood samples via episomal reprogramming and forebrain patterning then cryopreserved in large master cell banks. After thawing, iCell Neurons retained high viability (75%) and maintained gene and protein expression profiles consistent with the γ-aminobutyric acid (GABA)ergic and glutamatergic phenotypes in vitro. To determine in vivo survival, cryopreserved iCell Neurons were thawed and prepared for transplantation without manipulation or additional subculturing. iCell Neurons were injected bilaterally into the striatum of athymic-Rowett nude (RNU) rats (4.5 × 105 cells/hemisphere; one injection/hemisphere), transgenic R6/2 (CAG120) HD and wild-type littermate mice (3 × 105 cells/hemisphere; one injection/hemisphere), and cynomolgus macaques (3.75 × 106 cells/hemisphere; three injections/hemisphere). Rats were sacrificed at 3 or 9 months posttransplantation, and mice and monkeys were sacrificed at 5 weeks posttransplantation. Immunohistochemistry indicated robust graft survival and maintenance of neuronal phenotypes with extensive fiber outgrowth into the host parenchyma. Importantly, there was little to no cell proliferation, indicating safety in our initial studies. Future studies will be designed to ascertain whether cryopreserved iCell Neurons will provide functional benefit in transgenic mouse models of HD.
B. J. Hoffer*, Y. Luo*, A. Hoffer*, R. Baratz†, N. H. Greig‡, C. G. Pick†
*Department of Neurosurgery, Case Western Reserve University School of Medicine, Cleveland, OH, USA
†Department of Anatomy and Anthropology, Sackler School of Medicine, Tel Aviv University, Tel Aviv, Israel
‡Drug Design & Development Section, Translational Gerontology Branch, Intramural Research Program, National Institute on Aging, National Institutes of Health, Baltimore, MD, USA
Treatment of traumatic brain injury (TBI) represents an unmet medical need, as no effective pharmacological treatment currently exists. Development of such a treatment requires a fundamental understanding of the pathophysiological mechanisms that underpin the sequelae resulting from TBI, particularly the ensuing neuronal cell death and cognitive impairments. Tumor necrosis factor-α (TNF-α) is a cytokine that is a master regulator of systemic and neuroinflammatory processes. TNF-α levels are reported to be rapidly elevated post-TBI and, potentially, can lead to secondary neuronal damage. To evaluate the role of TNF-α in TBI, particularly as a drug target, the present study evaluated time-dependent TNF-α levels in the brain of a mouse closed head 50-g weight-drop mild TBI (mTBI) model in the presence and the absence of posttreatment with an experimental TNF-α synthesis inhibitor, 3,6′-dithiothalidomide. Brain TNF-α levels peaked at 12 h postinjury and returned to baseline by 18 h. This was accompanied by neuronal loss and an increase in astrocyte number [evaluated by neuronal nuclei (NeuN) and glial fibrillary acidic protein (GFAP) immunostaining, respectively], as well as an elevation in the apoptotic death marker BH3 interacting domain death agonist (BID) at 72 h. Impairments in measures of cognition, evaluated by novel object recognition and passive avoidance paradigms, were evident at 7 days after injury. Treatment with the TNF-α synthesis inhibitor 3,6′-dithiothalidomide 1 h postinjury prevented the mTBI-induced TNF-α elevation and ameliorated the neuronal loss (NeuN), elevations in astrocyte number (GFAP) and BID, and cognitive impairments. Cognitive impairments were prevented by treatment as late as 12 h post-mTBI but were not reversed when treatment was delayed until 18 h. These results suggest that pharmacologically limiting the generation of TNF-α post-mTBI may mitigate secondary damage and define a time window of up to 12 h to achieve this reversal.
K. Hosaka, K. Nowicki, S. Hourani, B. Hoh
Department of Neurosurgery, University of Florida, Gainesville, FL, USA
Cerebral aneurysms (CAs) are associated with chronic remodeling of the cerebral arterial wall. According to autopsy studies, cerebral aneurysms have a prevalence of up to 5% of the population in the US. In population-based studies, subarachnoid hemorrhage (SAH) caused by CA rupture comprises up to 7% of all strokes. Approximately half of all patients with SAH die, and of the surviving patients half have complications interfering with activities of daily living. Microsurgical clipping or endovascular coiling have been used in the treatment of ruptured and unruptured cerebral aneurysms. However, the mortality/morbidity rates are still high even after those treatments (up to 23.5%). It is apparent that better treatment options for aneurysms are needed. However, little is known about the pathophysiology of cerebral aneurysm formation and rupture. Inflammatory cells, such as macrophages, have been found in the walls of human cerebral aneurysms, but it is not known whether inflammation is an epiphenomenon to aneurysm formation. To develop novel treatments for aneurysm, better understanding of the mechanism for aneurysm formation is necessary. We have established two murine aneurysm models and an in vitro model to investigate our objectives: 1) define the mechanism of aneurysm formation, and 2) investigate the novel methods for aneurysm treatment to prevent its rupture. If a key factor in the CA inflammatory process can be identified, potential new therapies could be developed to prevent the development of CAs. The elastase-induced murine cerebral aneurysm model or murine carotid aneurysm model were used. Cytokine-releasing poly(lactic-co-glycolic acid) (PLGA)-coated platinum coils were used to investigate the aneurysm treatment. Green fluorescent protein-recombinant adeno-associated viruses (GFP-rAAVs) were used as a potential tool to investigate both aneurysm formation and treatment. Multiple cytokines were robustly expressed in elastase-induced murine aneurysms but not in control arteries. Expression of the same cytokines was also observed in human aneurysm samples. The blockade of those cytokines in murine model prior to CA induction inhibited CA formation. We have invented a cytokine-releasing PLGA-coated coil. We have found monocyte chemoattractant protein-1 (MCP-1)-releasing coil promoted significantly increased aneurysm healing after the implant. We have also revealed that interleukin-6 (IL-6) and osteopontin have some key roles in MCP-1-mediated aneurysm healing. While these results are preliminary, we have successfully transduced vascular cells (smooth muscle cells) in the selected area. We have found a few cytokines that have a role in the formation of CAs and the rupture of already developed CAs. We have also found MCP-1 and its downstream mediators as a key factor for aneurysm healing. These could be a potential therapeutic target for preventing the rupture of incidentally found unruptured CAs in patients. rAAV transduction could be a novel tool to research both aneurysm formation and treatment.
K.-W. Hsueh*†, Y.-C. Lin*, H.-J. Harn‡, S.-Z. Lin*
*Center for Neuropsychiatry, China Medical University Hospital, Taichung, Taiwan
†Ph.D. Program for Aging, China Medical University, Taichung, Taiwan
‡Department of Pathology, China Medical University Hospital, Taichung, Taiwan
Amyotrophic lateral sclerosis (ALS) displays loss of motor neurons in brain stem and spinal cord. Intraspinal neural progenitor cell transplantation in ALS patients stabilized patients' limb function (Riley et al., 2012), but patients' brain stem functions progressively deteriorated. In order to minimize the surgical trauma during the implantation procedures in the long segments of the spinal cord and improve both the survival of motor neurons in brain stem and spinal cord, we proposed that the combined intracerebral (IC) and intravenous (IV) deliveries of adipose tissue-derived mesenchymal stem cells (AD-MSCs) may slow down the deterioration of motor neuron loss in brain stem and spinal cord in ALS mice and prolong their life span. An ALS patient was treated in this way and followed up for 1 year after getting official approvals from Taiwan Ministry of Health and Welfare (TFDA) and Institutional Review Board (IRB). Male and female G93A transgenic mice overexpressing human mutant superoxide dismutase 1 (SOD1) were obtained from Jackson Laboratories. Animals were randomly distributed into three groups at 60 days of age: (1) untreated (n = 8); (2) riluzole-treated group (n = 4), in which the mice were treated with riluzole at a dosage of 16 mg/kg body weight once daily via intraperitoneal injection; (3) human AD-MSC-treated group, transplanted with AD-MSCs (2 × 106 cells/30 μl PBS; n = 8) via IC injection once at 60 days postnatal, and then transplanted with AD-MSCs (1 × 106 cells/150 μl PBS) IV at 90 days and 104 days postnatal. Immunohistochemical (IHC) staining for brain-derived neurotrophic factor (BDNF), chemokine, C-X-C motif, receptor 4 (CXCR4), and the number of motor neurons was performed in the brain stem and spinal cord of ALS mice. One male ALS patient received six IC infusions of a total of 1.7 × 108 AD-MSCs in 1.5 ml saline into the bilateral pericortical spinal tract (subcortical, basal ganglia, and thalamus) as well as four IV infusions of 2 × 108 AD-MSCs over the 2 weeks before and two after the IC injection. The life span of the mice in the AD-MSC-treated group (149.9 ± 4.8 days; p < 0.05) was much prolonged compared to the untreated group (126.4 ± 7.2 days) and the riluzole group (133.7 ± 6.4 days). IHC staining also showed that the AD-MSC-treated group had higher levels of BDNF and CXCR4, as well as volume of motor neurons in comparison to the other two groups. One ALS patient was treated with combined IC and IV deliveries of autologous AD-MSCs. Taiwan Ministry of Health and Welfare (TFDA) and Institutional Review Board (IRB) of China Medical University Hospital, Taiwan, approved this study. One year before transplantation the patient's ALS functional rating scale (ALSFRS) was 17, while 1 week before transplantation the score was 7. The postoperative ALSFRS score remained around 7–9 points for up to 6 months of follow-up, and, the ALSFRS score decreased to around 5 points at 9 and 12 months of follow-up. In conclusion, the results demonstrate that the combined IC and IV therapy of AD-MSCs extends the life span of ALS mice and slows down the deterioration rate of motor function in an ALS patient for around 12 months.
P. Jendelová*†, J. Růžička*†, L. M. Urdzíková*, J. Gillick‡, T. Amemori*, N. Romanyuk*, K. Kárová*, Š. Kubinová*, M. Jhanwar-Uniyal‡, E. Syková*
*Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
†Department of Neuroscience, Charles University, Second Faculty of Medicine, Prague, Czech Republic
‡New York Medical College, New York, NY, USA
Spinal cord injury (SCI) is a debilitating disease that results in paraplegia and has currently no effective treatment. A comparison of stem cell therapies in preclinical models is needed to understand the underlying mechanisms behind behavioral recovery, including stem cell effects on the growth factor expression, immune response, or glial scar modulation. In this investigation, we compared good manufacturing practice (GMP) manufactured human bone marrow mesenchymal stem cells (hMSCs) and two types of neural precursors (NPs) including a human conditional fetal spinal line (SPC-01), prepared in the same way as ReNeuron's CTX0E03 cell line currently used in a clinical trial of stroke patients, and human induced pluripotent derived neural progenitors (iPS-NPs) in the treatment of a balloon-induced spinal cord compression in rats. The effect and fate of three implanted human stem cell types was studied in rats that had undergone SCI. Rats received either intrathecal application of hMSCs (n = 37, 5 × 105/50 μl), saline (n = 28, 50 μl), or were implanted intraspinally at the level of the SCI with SPC-01 (n = 40, 5 × 105/5 μl), iPS-NPs (n = 39, 5 × 105/5 μl), or saline (n = 31, 5 μl). Behavioral evaluations were conducted, assessing the rats' locomotor skills (BBB, flat beam test, rotarod) and sensory response (plantar test). Tissue analyses of white/gray spared matter, axonal sprouting, and glial scar modulation were performed. qPCR [brain derived neurotrophic factor (Bdnf), nerve growth factor (Ngf), neurotrophin 3 (Nt3), fibroblast growth factor 2 (Fgf2), interferon regulatory factor 5 (Irf5), mannose receptor C type 1 (Mrc1), oligodendrocyte lineage transcription factor 2 (Olig2), caspase 3 (Casp3), growth-associated protein 43 (Gap43), glial fibrillary acidic protein (Gfap), vascular endothelial growth factor A (Vegfa), and ciliary neurotrophic factor (Cntf)] and Luminex multiplex cytokines [macrophage inflammatory protein-1α (MIP-1α), interleukin-4 (IL-4), IL-1β, IL-2, IL-6, IL-12p70, tumor necrosis factor-α (TNF-α), and regulated upon activation, normally T-expressed, and presumably secreted (RANTES)] were performed at 10, 14, 28, and 60 days after SCI, respectively, to detect host tissue responses to stem cell therapy. All stem cell-grafted rats showed increased locomotor recovery compared to controls. However, the iPS-NP-treated group also scored significantly better in the advanced locomotor tests. In all stem cell-treated groups white matter sparing was observed, while gray matter was preserved only in the iPS-NP-treated group. Both NPs significantly increased the number of GAP43+ axons and reduced astrogliosis. hMSCs, unlike both NPs, had transiently increased expression of growth factors (GFs) and decreased levels of inflammatory cytokines (IL-2, TNF-α) within 10 days after stem cell application. Conversely, both NPs downregulated expression of Casp3 and increased levels of cytokines (IL-6, IL-12). These findings correlate with the short survival of hMSCs (2 weeks); NPs survived longer (2 months) and matured slowly. From our results, we deduce that iPS-NPs are the most suitable candidate for treatment of SCI, having the highest locomotor performance compared to all other groups, due to their robust survival, tissue sparing, reduction of glial scarring, and increased axonal sprouting. The major advantage of hMSC use is its less invasive route of administration coupled with a strong anti-inflammatory effect, with repeated applications potentially overcoming its transiency.
Supported by 13-00939S, P304/12/G069, 7F14057.
X. Jiang*†, H. Pu*†, L. Mao*, J. Xia*, G. Wang*†, H. Zhang*, Y. Wu*, Y. Shi*, X. Hu*†‡, Y. Gao†, J. Chen*†‡
*Center of Cerebrovascular Disease Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
†State Key Laboratory of Medical Neurobiology and Institute of Brain Sciences, Fudan University, Shanghai, China
‡Geriatric Research, Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, PA, USA
Previous studies by others and us have shown that prophylactic long-term diet supplementation of omega-3 polyunsaturated fatty acids (n-3 PUFAs) remarkably ameliorates ischemic brain injury and promotes poststroke brain repair and neurological recovery. However, the therapeutic efficacy of poststroke administration of n-3 PUFAs, especially their effect on long-term neurological recovery, has not been determined thus far. In this study, we investigated whether poststroke treatment with n-3 PUFA diet supplementation alone or combined with docosahexaenoic acid (DHA) and eicosapentaenoic acid (EPA) injections could improve long-term stroke outcomes and enhance the brain repair processes, including neurogenesis, angiogenesis, and white matter restoration. Adult male C57BL/6J mice of 2–3 months of age were subjected to transient middle cerebral artery occlusion (tMCAO) for 60 min and then randomly and blindly assigned to four groups: 1) vehicle control; 2) EPA/DHA treatment alone (7 mgkg−1/3 mgkg−1, IP, 30 min after tMCAO, and then daily for 14 days); 3) n-3 PUFA diet supplementation alone (50 mg/g added to regular chew, beginning 5 days after tMCAO for up to 28 days), and 4) combination treatment of 2) and 3). Neurological deficits and poststroke brain tissue loss, neurogenesis, angiogenesis, and white matter integrity were assessed up to 28 days after tMCAO. The results revealed that both groups of 2) and 3) showed a marginal reduction in tissue loss (22.6 ± 6.7% and 20.8 ± 11.2% reduction in the EPA/DHA treatment group and the n-3 PUFA diet supplementation group, respectively, compared to vehicle treatment) and marginal improvement in the cylinder and rotarod tests (sensorimotor function) and the Morris water maze test (cognitive function) up to 28 days after tMCAO. Interestingly, animals that received a combined treatment of EPA/DHA and n-3 PUFA supplementation exhibited further reduced tissue loss (47.2 ± 6.5% reduction compared to the vehicle group) and improved neurobehavioral outcomes compared to either the EPA/DHA or n-3 PUFA treatment alone. Moreover, animals that received combined treatment of EPA/DHA and n-3 PUFA supplementation showed significantly greater beneficial effects on poststroke neurogenesis, angiogenesis, and preservation of white matter integrity, compared to either treatment alone. These results demonstrate an effective therapeutic regimen in promoting brain repair and improving long-term neurological recovery in the mouse model of tMCAO. Thus, combined poststroke treatment of EPA/DHA and dietary supplementation of n-3 PUFAs maybe a potential clinically translationable treatment for stroke or related brain disorders.
U. K. Jinwal*, M. Narayan*, K. W. Seeley†
*Department of Pharmaceutical Sciences, College of Pharmacy, Byrd Alzheimer's Institute, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
†Florida Center of Excellence for Drug Discovery & Innovation, University of South Florida, Tampa, FL, USA
Amyotrophic lateral sclerosis (ALS) is a rapidly progressing, debilitating, and fatal neurodegenerative disorder, which affects neurons in the motor cortex, brain stem, and spinal cord. While 10% of ALS cases are familial, the majority of ALS cases (90%) are sporadic with no clear risk factors associated with the disease. Given the sporadic nature of the disease, its severity, and rapidity of onset and progression of symptoms, the identification of biomarkers associated with ALS may lead to early detection and make it possible to intervene therapeutically. In the current study, we utilized fibroblasts from ALS patients and normal population controls and a mass spectrometry-based proteomic approach to identify differentially regulated proteins in ALS, which may serve as useful biomarkers for the disease. Stable isotope labeling by amino acids in cell culture (SILAC) coupled with mass spectrometry was used to quantitatively analyze the ALS and control fibroblast proteomes. A total of 861 proteins were identified in the analysis with 33 found to be differentially regulated including apolipoprotein B48 (ApoB48), heat shock protein 20 (Hsp20), and fibulin-1. ApoB48 and Hsp20 were found to be downregulated. Fibulin-1 was upregulated. The differential regulation of these proteins in ALS fibroblasts indicates their potential as novel biomarkers and possible drug targets for ALS.
V. Joers*†, G. Jeyaraj†, A. Weiss†‡, J. Bachevalier†‡, Y. Smith†§, M. Tansey*
*Department of Physiology, Emory University, Atlanta, GA, USA
†Yerkes Primate Center, Emory University, Atlanta, GA, USA
‡Department of Psychology, Emory University, Atlanta, GA, USA
§Department of Neurology, Emory University, Atlanta, GA, USA
Tumor necrosis factor (TNF) is a cytokine that belongs to a super-family of ligands that regulate neuroinflammatory responses. Elevated levels of TNF have been described in the cerebrospinal fluid (CSF) and brain tissue of Parkinson's disease (PD) patients, and are likely to be involved in the degeneration of nigral dopamine neurons in animal models of PD. Current FDA-approved anti-TNF therapies target both transmembrane (tm)TNF and soluble (sol)TNF, meaning that their use in treating neuroinflammatory diseases is undesirable in view of adverse findings including neurological deficits and demyelinating diseases in some patients. The novel TNF inhibitor XPro®1595 acts by a “dominant-negative” (DN) mechanism to eliminate only solTNF homotrimers through exchange of monomeric subunits between the compound and the native TNF. We recently demonstrated that systemically administered XPro®1595 crossed the blood–brain barrier (BBB), attenuated neuroinflammation, and protected against 6-hydroxydopamine-induced nigral neuron loss in rats (Barnum et al., 2014, J. Parkinson's Res., 4:349). Here we report the preliminary findings of XPro®1595 on motor and nonmotor symptoms of a low-dose chronic 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) monkey model of PD. We hypothesize that XPro®1595 would delay the development of motor and nonmotor symptoms in a chronic MPTP-treated nonhuman primate (NHP) model of PD. Five rhesus monkeys were given weekly doses of intramuscular MPTP (0.2–0.5 mg/kg) and evaluated weekly for changes in motor behavior and clinical rating scores. Peripheral treatment of XPro®1595 (10 mg/kg, n = 3) or vehicle (n = 2) began 11 weeks after the start of MPTP and continued every 3 days until the end of the study. Positron emission tomography (PET) scans were performed using fluorine-18 labeled N-acetyl-N-(2-fluoroethoxybenzyl)-2-phenoxy-5-pyridinamine ([18F]FEPPA) to evaluate microglial activation (peripheral benzodiazepine receptor/translocator protein 18 kDa ligand) and with fluorine-18 labeled 2β-carbomethoxy-3β-(4-chlorophenyl)-8-(2-fluoroethyl)nortropane ([18F]FECNT) to assess changes in dopamine transporters at baseline and at 8, 16, and approximately 28 weeks after the start of MPTP. Animals were tested before and after MPTP for cognitive flexibility and behavior inhibition using the intradimensional/extradimensional set-shifting task. Finally, blood and cerebrospinal fluid (CSF) were collected monthly to evaluate cytokine and XPro®1595 levels. The study ended when the animals developed moderate parkinsonism as determined by their clinical rating. Preliminary analysis of in vivo measures prior to treatment with XPro®1595 reveals that chronic MPTP dosing produced a progressive model in which deficits in cognitive performance occurred after 6 weeks of MPTP, prior to the development of PD motor signs. Increased systemic interleukin-6 (IL-6) levels correlated with the decrease in FECNT binding in the substantia nigra after 16 weeks of MPTP (R = 0.9954, p = 0.0004). We are currently evaluating the disease-modifying properties of systemically administered XPro®1595 on the remaining outcome measures.
Y. Kaneko*, C. Pappas†, N. Tajiri*, 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
†School of Aging Studies, University of South Florida, Tampa, FL, USA
Even with the advancements in stroke management, ischemicreperfusion (IR) injury is still a major cause of morbidity and mortality in patients. Oxytocin (OXT), a neuromodulator of intimacy, is secreted by the hypothalamus and has demonstrated neuroprotective effects against inflammation and oxidative stress in association with GABA (γ-aminobutyric acid) signaling transduction in neurons. However, the molecular mechanism by which oxytocin protects neuronal cells in stroke, especially the interaction between the oxytocin receptor and GABAA receptor (GABAAR), remains to be elucidated. Primary rat neuronal cells were exposed to OXT for 3 days before induction of experimental stroke model via oxygen-glucose deprivation (OGD) IR. Although oxytocin did not affect the glutathione-related cellular metabolism before OGD, oxytocin modulated the expression levels of GABAAR subunits, which function to remove excessive neuronal excitability via chloride ion influx. Oxytocin-pretreated cells significantly increased the chloride ion influx in response to GABA and GABA analog 4,5,6,7-tetrahydroisoxazolo(5,4-c)pyridin-3-ol (THIP, δ-GABAAR-specific agonist). This study provides evidence that oxytocin regulated GABAAR subunits in affording neuroprotection against IR injury. The study of signal transduction pathway between OXTR and GABAAR offers a new venue for developing treatment strategies against the early stage of stroke, and further reveals the pivotal role of OXT as a key regulatory hormone linking social interaction and stroke.
Funding: USF Department of Neurosurgery and Brain Repair Funds.
D. Kato, M. Mizuno, T. Sato, T. Toyoda, N. Matsukawa
Department of Neurology, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
Hippocampal cholinergic neurostimulating peptide (HCNP) induces the synthesis of acetylcholine in the medial septal nucleus. HCNP has a precursor protein, HCNP-pp, which can modulate signaling by binding to several proteins, including RAF-1, mitogen-activated protein kinase (MAPK)/extracellular signal regulated kinase, and G-protein-coupled receptor kinase 2. We recently demonstrated that HCNP-pp transgenic mouse had a psychobehavior character of a depressive-like state in the tail suspension test (TST). In this study, to further examine whether HCNP-pp may be involved in neuropsychiatric behavior, we investigated the tail suspension test (TST) in HCNP-pp conditional knockout (KO) mice by using Cre recombinase transgenic mice driven by the calmodulin kinase II promoter. For further insight into the period of influence of HCNP-pp reduction, we also examined the TST in mice with HCNP-pp knockdown in the hippocampus at 12–14 weeks old by use of an adeno-associated virus. Here we demonstrated that HCNP-pp reduction at 12–14 weeks old may be involved in the disease development of mania-like behavior. We confirmed the decrease of γ-aminobutyric acid (GABA) A receptor a3 subunit in this mouse model. These findings suggested that HCNP-pp in the hippocampus may be involved in bipolar disorder, depressive-like and/or mania-like behavior.
D.-K. Kim, S. Y. An, H. Nishida1, D. J. Prockop
*Institute for Regenerative Medicine, Texas A&M Health Science Center, College of Medicine, Temple, TX, USA
1Current address: Joint Department of Veterinary Medicine, Faculty of Applied Biological Sciences, Gifu University, Gifu, Japan
Thymic stromal lymphopoietin (TSLP) is an interleukin-7-like cytokine that activates myeloid dendritic cells to induce T cells to an unusual T helper 2 (Th2) inflammatory phenotype that plays a key role in allergy and asthma. Traumatic brain injury (TBI) or other injuries to the brain trigger innate and adaptive immune responses. In recent experiments we found that TSLP levels were increased in damaged cerebral cortex areas of the brain at 6 h after TBI was produced in mice by controlled cortical contusion. Mesenchymal stromal cells (MSCs) were shown by us and others to improve the effects of TBI in mice. Recent results suggest that the therapeutic effects of MSCs are at least in part explained by their secretion of extracellular vesicles (EVs) that contain cargos of cytokines, proteins, lipids, saccharides, and RNAs. In further experiments, we found that negatively charged EVs (n-EVs) chromatographically isolated from hMSCs decrease the level of TSLP in brain (p = 0.002) and blood (tendency, p = 0.065) at 6 h after TBI. Our results suggested that TSLP is a component of the inflammatory cascade produced in the brain by TBI, and that early administration of n-EVs isolated from hMSCs can interrupt the inflammatory cascade.
Supported in part by NIH grant P40OD011050.
S.-H. Kim, R. D. Shytle
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
Sulfate conjugation of hormones and neurotransmitters is necessary for normal growth and development as well as detoxification of xenobiotics. Sulfate may be an important element promoting autism phenotype, especially during early development. Recently, it has been reported that autistic children have lower sulfate levels and increased oxidative stress markers, suggesting that lower plasma sulfate levels may lead to accumulation of toxins and decreased antioxidant capacity in this population. Increased oxidative stress in autistic individuals has been reported. Glutathione (GSH) plays essential roles in detoxification and balancing redox homeostasis in brain. Cysteine (Cys) is the rate-limiting factor in GSH de novo synthesis, thereby performing an essential role in cellular protection against oxidative stress. In neurons, the majority of Cys is transported from extracellular space via excitatory amino acid transporter 3 (EAAT3), one of the glutamate transporters. Studies have suggested that disrupted EAAT3 expression resulted in neuronal death due to the excessive oxidative stress; furthermore, EAAT3 has been implicated in neurodegenerative diseases. Accordingly, our group hypothesize that autistic children may have lower detoxification capacity due to limited sulfation and GSH production, and therefore are more prone to drug-induced toxicity and oxidative stress. In our study, we determined if a mouse model of autism, the Black and Tan BRachyury (BTBR) line, exhibits differences when compared to C57BL/6J (C57) in plasma sulfate levels, EAAT3 expression, as well as GSH levels. Plasma sulfate levels, GSH levels in plasma and brain, and EAAT3 expression in frontal cortex were measured in BTBR and C57 mice (6 weeks old, male, n = 8). Our data showed that plasma sulfate levels were significantly lower in BTBR than C57 mice, which agrees with similar findings from another group. Interestingly, BTBR mice showed significantly lower EAAT3 expression levels in the prefrontal cortex. On the other hand, GSH and oxidized glutathione disulfide (GSSG) levels were similar between BTBR and C57 mice. Etiology of autism is unknown due to heterogeneity of the disorder. It has been suggested that genetic variations and various environmental factors may play a synergistic role in autism manifestation. Our findings suggest that lower plasma sulfate levels and neuronal EAAT3 expression may increase vulnerability to oxidative stress due to the diminished detoxification capacity and Cys availability, leading to GSH depletion in response to environmental toxins. The role of EAAT3 during development needs to be investigated in depth, and BTBR mice may be a favorable animal model to study the mechanism of EAAT3 modulation. Furthermore, how altered sulfate physiology affects normal brain development needs to be elucidated.
M. Kodali*†‡, B. Shuai*†‡, V. Mishra*†‡, S. Attaluri†‡, B. Hattiangady*†‡, A. K. Shetty*†‡
*Research Service, Olin E. Teague Veterans' Medical Center, Central Texas Veterans Health Care System, Temple, TX, USA
†Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine, Temple, TX, USA
‡Department of Molecular and Cellular Medicine, Texas A&M Health Science Center College of Medicine, College Station, TX, USA
Voluntary physical exercise has numerous health benefits, which comprise enhanced ability for cognition and forming new memories and better mood. Veterans afflicted with Gulf War illness (GWI) may also benefit from physical exercise, as persistent cognitive, memory, and mood impairments are the conspicuous brain alterations in GWI. Yet rigorous exercise may be detrimental in GWI because of comorbidities such as chronic fatigue and pain. Hence, using a rat model of GWI, we investigated the efficacy of moderate voluntary physical exercise (3 days/week) for reversing cognitive, memory, and mood dysfunction. We exposed young adult male Sprague–Dawley (SD) rats to GWI-related (GWIR) chemicals and mild stress (10 min of restraint) for 28 days. GWI-related chemicals comprised nerve gas prophylaxis drug pyridostigmine bromide (2.0 mg/kg, oral), and pesticides such as N,N-diethyl-meta-toluamide, (DEET; 60 mg/kg, dermal) and permethrin (0.2 mg/kg, dermal). Next, animals were randomly assigned to running exercise or sedentary groups (GWI-RE group, n = 9; GWI-SED group, n = 7). Rats in GWI-RE group were housed individually in larger cages fitted with running wheels for 3 days every week for 13 weeks (until euthanasia), whereas rats in GWI-SED group were housed in standard cages. Another cohort of age-matched rats housed in standard cages were included as a naive control group for comparison (NC group, n = 12). Rats in GWI-RE group ran an average of 3.4 km per day (mean ± SEM = 3.4 ± 0.5) during the entire 39 days of running period (over 13 weeks). Animals were examined with a battery of behavioral tests, commencing ~6 weeks after the exposure to GWIR chemicals and stress to elucidate RE-related modifications in cognitive, memory, and mood function. Animals in GWI-SED group exhibited inability for novel object recognition (in novel object recognition test, NORT), pattern separation (in pattern separation test, PST) and place recognition (in object location test, OLT) and depressive-like behavior (in novelty suppressed feeding test, NSFT), implying cognitive, memory, and mood dysfunction in these rats. In contrast, animals in GWI-RE group displayed capability for novel object recognition and pattern separation and reduced depressive-like behavior, demonstrating improved cognitive, memory, and mood function. However, these rats displayed inability for place recognition in OLT, akin to that seen in GWI-SED rats. Analyses through doublecortin (DCX) immunostaining and stereological counting of newly born DCX+ neurons in the dentate subgranular zone-granule cell layer revealed that hippocampal neurogenesis in the GWI-RE group is increased in comparison to GWI-SED group (1.6-fold increase, p < 0.05) and reached closer to values seen in the NC group. Thus, moderate physical exercise is beneficial for alleviating cognitive, memory, mood, and hippocampal neurogenesis impairments observed in GWI.
Supported by Merit Review and Research Career Scientist Awards from the Department of Veteran Affairs to A.K.S.
C. Kurien, * N. Tajiri*, A. Thomson*, J. Alookaran*, G. Steiner*, S. Abraham*, G. James*, A. Mahendrasah*, S. H. Appel†, C. V. Borlongan*, 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
†Department of Neurology, Houston Methodist Neurological Institute, Houston, TX, 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
Amyotrophic lateral sclerosis (ALS) is a disease characterized by degeneration of motor neurons in the brain and spinal cord. Vascular pathology, including damage of the blood–brain barrier (BBB) and blood–spinal cord barrier (BSCB) via endothelial cell degeneration, has recently been recognized as one hallmark of ALS pathogenesis. Repairing vascular damage by replacement of endothelial cells via systemic cell administration may be a new therapeutic approach for ALS. The aim of this study was to characterize the effect of intravenous transplantation of human bone marrow cluster of differentiation 34-positive (CD34+; hBM34+) cells, a source of endothelial progenitor cells, into symptomatic mutated superoxide dismutase 1 (G93A SOD1) mice on disease progression and motor neuron survival. Three different doses of hBM34+ cells (5 × 104, 5 × 105, or 1 × 106) were transplanted intravenously via jugular vein into G93A mice at 13 weeks of age. Mice underwent weekly pretransplant and posttransplant behavioral testing (extension reflex, rotarod, and grip strength tests) and monitoring of body weight. Cell-treated, media-treated, and control mice were euthanized at 17 weeks of age, corresponding to 4 weeks after initial treatment at symptomatic disease stage. The cervical and lumbar spinal cords were removed, postfixed, cut in a cryostat, and then stained with cresyl violet. Histological evaluation and stereological count of motor neurons in the spinal cord were performed in the ventral horn. The results demonstrated that systemic administration of hBM34+ cells into symptomatic ALS mice at different doses delayed disease progression as determined by behavioral outcomes and motor neuron survival at 4 weeks posttransplant. The beneficial effects were supported by maintained body weight, as a valuable marker for detecting progression of muscle atrophy, and delayed deterioration of hindlimb extension in all cell-treated ALS mice versus media for the entire posttransplant period. Also, a delayed loss in muscle strength (grip test) was determined in cell-treated G93A mice. ALS mice with cell treatment at different doses also demonstrated longer latency on rotarod test for the entire posttransplant period versus media mice. Although more significant functional improvements in the late symptomatic stage (17 weeks of age) were determined mainly in mice receiving 1 × 106 cells, there were no significant differences detected between cell-treated mice during the 3 weeks after cell transplantation. Delayed disease progression in G93A mice via hBM34+ cell transplantation at symptomatic stage was confirmed by superior motor neuron survival in the ventral horns of lumbar spinal cords in all cell-treated animals. Studies are in progress to determine in vivo-administered cell differentiation into endothelial cells and cell engraftment, particularly within the vascular lumen, to determine possible repair of vascular damage in ALS mice. Also, while the present study is limited to transplantation of hBM34+ cells, an additional mouse treatment group receiving bone marrow-derived endothelial progenitor cells/endothelial cells (BM-EPC/ECs) is being processed to determine the most beneficial cell type in restoration of vascular integrity in ALS.
Supported by the NIH, NINDS (1R01NS090962) grant.
J.-Y. Lee, H. Nguyen, N. Tajiri, S. A. Acosta, 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
The occurrence of retinal ischemia in animals subjected to middle cerebral artery occlusion (MCAO) has been recently suggested owing in part to the circulatory juxtaposition of the ophthalmic artery to the MCA. In this study, we used laser Doppler to evaluate the brain and retinal perfusion at baseline (prior to MCAO), during MCAO, and after MCAO. Retina-relevant behavioral and histological outcomes were performed at 3 days, 14 days, and 28 days post-MCAO. We also studied the retina-sensitive task performance and the density of retinal ganglion cells and the optic nerve depth after mesenchymal stromal cell (MSC) treatment. Laser Doppler revealed typical reduction in brain perfusion in the ipsilateral front parietal cortical area of at least 80% reduction during MCAO compared to baseline, which returned to near baseline levels after MCAO. A significant defect in the retinal perfusion in the ipsilateral eye was detected with at least 30% reduction in perfusion during MCAO compared to baseline, which was restored to near baseline levels during reperfusion. Behavioral performance in light stimulus-mediated place preference was significantly impaired in MCAO rats compared to control animals. Retinal ganglion cell density and optic nerve depth were significantly decreased in the ipsilateral eye. Retinal perfusion defects closely parallel the timing of cerebral blood flow alterations in the acute stages of MCAO in adult rats. Behavioral and histological impairments further reveal the contribution of retinal dysfunction in stroke outcomes. Interestingly, these functional and neurostructural deficits were significantly attenuated when MSCs were intravenously transplanted immediately after MCAO induction. In summary, stroke may present with visual deficits, which can be detected by retinal perfusion using laser Doppler measurements, complementing the diagnosis of stroke onset and progression. Stem cell transplantation may provide relief from such stroke-induced retinal dysfunctions.
C.V.B. is supported by NIH NINDS 1R01NS071956-01, NIH NINDS 1 R21 NS089851-01, DOD TATRC W81XWH-11-1-0634, James and Esther King Foundation for Biomedical Research Program 1KG01-33966, SanBio Inc., Celgene Cellular Therapeutics, KMPHC. and NeuralStem Inc.
R. Lim*†, E. Wallace*†
*Department of Obstetrics and Gynaecology, Monash University, Clayton, Australia
†The Ritchie Centre, Hudson Institute of Medical Research, Clayton, Australia
A strong link exists between intrauterine inflammation, preterm birth, and cerebral palsy. In surviving preterm babies, the most common neuropathologies are cerebral white matter abnormalities such as lesions to the periventricular white matter, so-called periventricular leukomalacia (PVL). A significant proportion of preterm babies with this form of white matter injury subsequently develop cerebral palsy. Human amnion epithelial cells (hAECs) are a stem-like cell obtained from the membrane of placenta discarded at birth, and have a number of important properties that make them of interest for a neuroprotective treatment. They have low immunogenicity and can prevent the activation of both innate and adaptive immune systems. hAECs suppress proinflammatory cytokines, regulate macrophage recruitment, and secrete factors that inhibit the chemotactic activity of neutrophils and macrophages. In vitro, they have neuroprotective properties in a model of ischemic and oxidative stress injury, and they have the ability to differentiate into a variety of cell types, including neurons. This talk will describe some of the work that we have done to assess the efficacy of hAECs in small- and large-animal models of cerebral palsy as well as their safety profile in humans.
A. Lindenmair, A. Banerjee, A. Weidinger, A. Lemke, S. Wolbank, C. Gabriel, H. Redl
Ludwig Boltzmann Institute for Experimental and Clinical Traumatology, Austrian Cluster for Tissue Regeneration, Vienna, Austria
Human amnion represents an abundantly available and uncontroversial source of stem cells and can be used as human amniotic membrane (hAM) with or without cells. Cell therapy, applying stem cells with characteristics such as differentiation and immunomodulatory capacity, has become an important strategy in regenerative medicine. For this reason, we have isolated as well as expanded and thoroughly characterized amniotic cells cultured in different media. We found that the expression of cell surface antigens clearly depends on the culture medium and has a relatively high donor dependency. Regardless of the medium used for expansion, no adipogenic differentiation could be induced. We also found that there are differences in the cells and their mitochondrial activity depending on the anatomic localization on the membrane (placental and reflected area). hAM is consistently gaining access to new areas in regenerative medicine. We have studied optimal storage conditions and developed a good manufacturing practice (GMP) cryopreservation procedure. We found the antifibrotic properties in vivo (coop. Ornella Parolini) and started using it as a gliding tissue. Since hAM represents a preformed sheet of stem cells, there is no need for additional carrier material. Hence, we studied a new approach for bone and cartilage tissue engineering via differentiation of hAM as a whole. Furthermore, we evaluated the potential for the ectodermal lineage by differentiating hAM towards Schwann cell-like cells. Under osteogenic conditions mineralization was observed and most of the cells expressed osteocalcin and had increased calcium content. Increased glycosaminoglycan (GAG) production of hAM was observed under chondrogenic conditions. These findings were attended by quantification of the GAG content. Differentiation towards an ectodermal lineage, such as Schwann cell-like cells, was demonstrated by positive expression of glial fibrillary acidic protein (GFAP) and low-affinity nerve growth factor receptor (p75). Thus, these different results are further promising steps for a versatile use of hAM for tissue regeneration.
D. Ma*, J. L. Harris†‡, B. Snyder*, W. M. Brooks*‡§, S. M. Shapiro§¶#, J. A. Stanford*
*Department of Molecular & Integrative Physiology, University of Kansas Medical Center, Kansas City, KS, USA
†Department of Anatomy & Cell Biology, University of Kansas Medical Center, Kansas City, KS, USA
‡Hoglund Brain Imaging Center, University of Kansas Medical Center, Kansas City, KS, USA
§Department of Neurology, University of Kansas Medical Center, Kansas City, KS, USA
¶Department of Pediatrics, University of Kansas Medical Center, Kansas City, KS, USA
#Department of Pediatrics, Children's Mercy Hospital, Kansas City, MO, USA
Bilirubin-induced neurological dysfunction (BIND) is implicated in a wide range of neurodevelopmental disorders. In jaundiced Gunn rats (jjs), hyperbilirubinemia (HB) peaks between 16 and 21 days of age, and jjs exhibit hyperactivity and gait abnormalities implicating cerebellar dysfunction. Determining cerebellar effects of HB at the time of high exposure, and subsequently, may provide biomarkers to diagnose BIND and new targets for intervention. In order to do this, we used proton magnetic resonance spectroscopy (1H-MRS) in a 9.4T magnetic resonance imager (MRI) to quantify neurochemicals related to oxidative stress, astroglial activation, inflammation, excitatory neurotransmission, bioenergetics, and neuronal integrity in the cerebellum of jjs and their non-jaundiced (Nj) littermates at 21 days and 4 months of age. We also measured body weight, total plasma bilirubin, locomotor activity, and MRI cerebellar volume. Bilirubin levels in jjs were 13.5 ± 1.1 mg/dl and 13.3 ± 0.4 mg/dl in the 21 days and 4 months imaging groups, respectively. At 21 days jjs exhibited significant increases in neurochemicals related to glutamate turnover (glutamine and glutamine/glutamate ratio), excitotoxicity (serine), astroglial activation/inflammation (myo-inositol), oxidative stress (ascorbate, glutathione), cell membrane turnover and breakdown (glycerophosphocholine, phosphocholine), and significant decreases in bioenergetic function (aspartate), and neuronal integrity (N-acetylaspartate, taurine). At 4 months, markers of decreased neuronal integrity were still present, and glutamate and macromolecules were decreased in jjs, but the other markers were no longer different between jjs and Njs. Cerebellar volume was significantly decreased in jjs at both ages, and in 21-day jjs, cerebellar glucose was significantly correlated positively with myo-inositol (r = 0.86) and negatively with body weight (r = −0.76) and cerebellar volume (r = −0.77). In summary, we found abnormal neurochemical events in the cerebellum of jjs. The transitory effects at the time of peak HB, including the potential relationships between glucose and measures of astroglial activation/inflammation, oxidative stress, cerebellar volume, and body weight, provide therapeutic targets for intervention in BIND, and long-term abnormalities suggest possible 1H-MRS biomarkers in older children and/or adults to implicate BIND.
Supported by Children's Mercy Hospital and by the Kansas Intellectual & Developmental Disabilities Research Center grant HD02528.
F. P. Manfredsson*†‡, M. J. Benskey*‡, B. D. Gulbransen‡§, X. Bian¶, N. C. Kuhn*, J. J. Galligan‡¶
*Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
†Mercy Health Saint Mary's, Hauenstein Neuroscience Center, Grand Rapids, MI, USA
‡Neuroscience Program, Michigan State University, East Lansing, MI, USA
§Department of Physiology, Michigan State University, East Lansing, MI, USA
¶Department of Pharmacology and Toxicology, Michigan State University, East Lansing, MI, USA
Our current understanding of the molecular etiology of Parkinson's disease (PD) is incomplete and is likely the product of multiple interacting factors. The best-validated participant in the molecular pathology of PD is the protein α-synuclein (a-syn). Mutations and multiplication of the gene encoding α-syn result in inherited forms of PD. In addition, the presence of aggregated α-syn in Lewy bodies and Lewy neurites provides evidence for its association with idiopathic PD. Although PD is most often thought of as a central nervous system (CNS) disorder, PD is also characterized by gastrointestinal (GI) dysfunction and peripheral neuropathy. For example, aggregated α-syn has been observed throughout the enteric nervous system (ENS) of PD patients; thus, it has been proposed that α-syn pathology in the ENS is the cause of GI dysfunction in PD. This hypothesis has been supported in transgenic (a-syn-overexpressing mice) that do indeed display GI dysmotility. However, a key confound with these studies is the fact that α-syn overexpression is produced in neuronal populations throughout the animals; thus, it is not clear whether the GI phenotype observed in these animals is due to α-syn pathology in the CNS, ENS, or both. To that end, we developed a novel gene therapy approach using direct injections of adeno-associated virus (AAV) to the ENS. This approach results in robust transduction of enteric neurons and glia, with no viral genomes detected in the CNS. To determine if α-syn pathology specifically within neurons of the ENS is sufficient to produce GI dysfunction, we utilized this approach to overexpress human wild-type α-syn in enteric neurons. Adult male rats received direct injections of AAV expressing either human wild-type α-syn or a green fluorescent protein (GFP) control transgene into the descending colon. Four and eight weeks postsurgery we assessed colonic motility in ambulatory subjects. The α-syn-overexpressing animals had significantly impaired colonic motility compared to animals that received AAV-GFP. In order to better understand the role of pathological α-syn in enteric neuron dysfunction we performed a battery of ex vivo measures on transduced colonic tissue: α-syn overexpression was associated with 1) impaired neurogenic contraction and relaxation of colonic circular muscle; 2) increased neuromuscular inhibition junction potential; 3) decreased basal levels of calcium in both neurons and glia. Importantly, a quantitative histological examination of transduced tissue revealed that α-syn overexpression did not produce any neurodegeneration. Together, these findings suggest that ENS α-syn overexpression results in alterations in neurotransmission, which is in agreement with a presumptive role of α-syn in neurotransmitter production and handling. To further test this we are currently performing single cell electrophysiological recordings of transduced ENS neurons. In summary, α-syn overexpression in neurons of the ENS has a profound effect on GI physiology. This work provides a unique platform upon which to model and study GI dysfunction associated with PD and other synucleinopathies.
E. McKay*†, J. S. Beck*, M. E. Winn‡, K. Dykema‡, A. P. Lieberman§, H. L. Paulson¶, S. E. Counts*†#**
*Department of Translational Science & Molecular Medicine, Van Andel Research Institute, Grand Rapids, MI, USA
†Neuroscience Program, Van Andel Research Institute, Grand Rapids, MI, USA
‡Bioinformatics and Biostatistics Core, Van Andel Research Institute, Grand Rapids, MI, USA
§Department of Pathology, University of Michigan, Ann Arbor, MI, USA
¶Deptartment of Neurology, University of Michigan, Ann Arbor, MI, USA
#Department of Family Medicine, Michigan State University, Grand Rapids, MI, USA
**Hauenstein Neuroscience Institute, Mercy Health Saint Mary's, Grand Rapids, MI, USA
As the percentage of senior adults in the worldwide population has continued to grow, so has the incidence of dementias in this aged population. Alzheimer's disease (AD) and vascular dementia (VaD) account for the first and second leading causes of dementia, respectively, yet the pathogenic mechanisms that differentiate these two diseases are unclear. To begin to understand these differences, we used Agilent human 8×60K v2 microarrays (Santa Clara, CA, USA) to examine gene expression profiles of frozen frontal cortex (BA 10) tissue samples harvested postmortem from individuals who died with probable AD, VaD (multi-infarct subtype), or no dementia (n = 11–12/group). Samples were matched for age, postmortem interval, and gender. Of the 24,127 genes featured on the microarray platform, 3,495 genes were uniquely dysregulated in AD compared to VaD and controls (1,344 up; 2,151 down). By contrast, 413 genes were dysregulated in VaD (221 up; 192 down) compared to AD and controls. Preliminary network analysis revealed that genes specifically downregulated in AD but not in VaD were heavily enriched for synaptic function (e.g., synapsin I, RAB3A), axonal transport (e.g., dynein, bassoon), and glutamatergic signaling [e.g., glutamate receptor, ionotropic, N-methyl D-aspartate (NR2), metabotropic glutamate receptor 5 (mGluR5)], whereas VaD cases exhibited a unique and significant upregulation of the histone acetyltransferase cAMP-response element binding protein (CREB)-binding protein (CREBBP) [log fold change = 0.4, false discovery rate (FDR) adjusted p < 0.02], which served as a modulatory hub for several upregulated genes encoding core histones (e.g., H2, H4) and regulators of chromatin remodeling [e.g., special AT-rich sequence binding protein 1 (SATB1)]. These findings suggest that distinct epigenetic programs are activated in VaD compared to AD. Intriguingly, network analysis also revealed a VaD-specific upregulation of the oxytocin receptor (OXTR, log fold change = 0.86, FDR adjusted p = 0.034), a Gq-protein-coupled receptor linked to phospholipase C activation that has been shown to reduce infarct size in models of ischemic heart disease. Real-time quantitative PCR validation assays showed a 2.6-fold upregulation in OXTR transcript levels in VaD compared to controls, while levels in AD did not differ significantly from controls [F(2, 27) = 3.89, p = 0.03, via one-way ANOVA], thus confirming the microarray results. These initial findings provide valuable clues to potential mechanisms differentiating AD and VaD. Moreover, the results suggest that OXTR upregulation is involved in VaD pathophysiology and may offer a potential target for therapy.
Funding: NIH AG 014449, AG044712, Saint Mary's Foundation.
S. Misumi, Y. Ueda, A. Ishida, C-G. Jung, H. Hida
Department of Neurophysiology & Brain Science, Nagoya City University Graduate School of Medical Sciences, Nagoya, Japan
We reported a white matter injury (WMI) model of mild hindlimb dysfunction generated by right common carotid artery occlusion and 6% oxygen for 60 min at P3, in which actively proliferating oligodendrocyte (OL) progenitors are mainly damaged (Misumi et al., Cell Transplant., 2015). The pathological response to mild hypoxic-ischemia (H-I) in neurons and OL lineage cells and myelination failure were first investigated in the WMI model. In this model, coordinated motor function, as assessed by the accelerating rotarod test, was impaired. The dysfunction was accompanied by myelination failure in layer I–IV of the sensorimotor cortex. Some oligo2-positive OLs in both the cortex and white matter were immunostained by active caspase 3 at 24 h after H-I, but few neuronal nuclei (NeuN)-positive neurons were apoptotic. Argyrophil-III staining for damaged neurons revealed no increase in the number of degenerating cells in the WMI model. Moreover, the total number of NeuN-positive neurons in the cortex was comparable to that of controls. Retrograde labeling of the corticospinal tract with Fluoro-Gold revealed no significant loss of layer V neurons. In addition, no decrease in the numbers of cortical projecting neurons and layer V–VI neurons in both motor and sensory areas was observed. Notably, the numbers of inhibitory γ-aminobutyric acid (GABA)ergic cells immunoreactive for parvalbumin, calretinin, or somatostatin were preserved in the P26 cortex. Thus, we found that impaired motor coordination was not induced by neuron loss, but by myelination failure in layer I–IV. We then examined gene expression at P5 in the ipsilateral H-I side of the neonatal WMI model. We found that 98 genes were upregulated and 65 genes were downregulated in the sensorimotor cortex. Among the increasing factors, we focused on insulin-like growth factor (Igf)-2 and potassium channel, inwardly rectifying subfamily J, member 13 (Kcnj13) that is a gene for the inward rectifier K+ channel, Kir7.1. The expression of both factors was strongly detected in glial fibrillary acidic protein (GFAP)-positive cells and oligodendrocyte lineage transcription factor 2 (olig2)-positive cells in the corpus callosum (CC) of the WMI model. Data suggest that myelination failure in layer I–IV rather than neuron loss in the cortex is a main cause for hindlimb dysfunction in neonatal WMI model rat, which might be related to increased expression of IGF-2 and Kir7.1 at P5 of the model. If possible, data for the transplantation of OL progenitors into this WMI model might also be presented.
H. Nguyen*, S. Reyes*, H. J. Yeon*, S. Mashkouri*, D. Aum*, Q. Colburn*, X. Kaya*, J. Y. Lee*, S. A. Acosta*, W. Quillen†, N. Tajiri*†, C. V. Borlongan*
*Department of Neurosurgery and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
†School of Physical Therapy, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
Traumatic brain injury (TBI) is the signature of war for our soldiers returning from Middle East battle zones. Rehabilitation therapy, which involves physical training or exercise, has been the single most utilized treatment for TBI patients. The use of exercise as a prophylactic regimen to reduce TBI functional deficits has been shown to be effective. However, the mechanism of action remains poorly understood. Here, we examined the role pre-TBI of exercise in enhancing brain angiogenesis/vasculogenesis in an effort to test our hypothesis that functional recovery post-TBI may be augmented by physical training-mediated preservation of the cerebrovasculature. TBI is associated with devastating damage to the cerebrovasculature, so treatments designed to strengthen blood vessels may prove beneficial against TBI. Adult Sprague-Dawley rats were initially exposed to 1 h of forced running wheel exercise daily for 3 days, then subsequently subjected to a moderate controlled cortical impact model of TBI. Laser Doppler readings were recorded at baseline (prior to TBI) and immediately after TBI. Behavioral tasks, using neurological exams, elevated body swing test, and rotorod test, were also conducted at baseline and at day 1 after TBI induction. Results revealed significant increments of 35% and 105% cerebral blood flow at both baseline and immediately after TBI in exercised rats compared to those that did not receive physical training (p < 0.05). In addition, neurological scores and rotorod performance, but not biased swing activity, were significantly improved in exercised rats compared to non-exercised animals (p < 0.05). These findings showed that exercise may attenuate TBI-induced neurological and motor coordination deficits by enhancing the cerebrovasculature integrity. Cellular assessment of the cerebrovasculature is ongoing.
B. Okyere, A. Hazy, T. Brickler, X. Wang, M. Theus
Department of Biomedical Sciences & Pathobiology, VirginiaMaryland Regional College of Veterinary Medicine, Blacksburg, VA, USA
Leptomeningeal anastomoses or collaterals play a critical role in regulating vascular reperfusion following ischemic disease. However, the mechanisms regulating early collateral development remains under investigation. Our current findings indicate that ephrin type A receptor 4 (EphA4) is a novel negative regulator of collaterogenesis. We demonstrate that EphA4 is highly expressed on pial arteriole collaterals at postnatal day (P) 1 and 7, but significantly reduced by P21. To address the role of EphA4 in collateral formation, we generated endothelial cell (EC)-specific EphA4 knockout mice, EphA4f/f/Tie2::Cre (KO), then analyzed collateral development using vessel painting. EphA4 ablation on ECs increased the density but not diameter of pial collaterals at P1 to adulthood. Interestingly, cultured brain-derived ECs isolated from KO mice displayed a threefold increase in proliferation, enhanced migration, and tube formation compared to EphA4f/f (WT) ECs. These findings correspond with an increase in the level of phospho-Akt. Inhibiting phospho-Akt in KO ECs using LY294002 attenuated the proliferative and migration effects. RNAseq analysis also revealed altered expression patterns for genes that regulate cell proliferation, vascular development, extracellular matrix, and immune-mediate responses. Further analysis using real-time polymerase chain reaction showed increased expression in KO EC monocyte chemoattractant protein 1 (Mcp-1), matrix metallopeptidase 2 (Mmp2), and angiopoietin-1 (Ang-1). Lastly, we show that arterial collateral remodeling and reperfusion significantly increase after induction of stroke, using the permanent middle cerebral occlusion murine model, in KO mice compared to WT mice. This resulted in improved memory and sensory and motor activity of KO mice. These findings demonstrate a novel role for EphA4 in the early development of the pial collateral network and suggests a role in regulating vascular remodeling after arterial obstruction.
E. Ouellet*, G. Tharmarajah*, O. Seira†‡, J. Liu†‡, A. Thomas*, T. Leaver*, A. Wild*, Y. Li§, Y. T. Wang§, W. Tetzlaff†‡, C. Hansen¶, P. Cullis#, J. R. Taylor*, E. Ramsay*
*Precision NanoSystems Inc., Vancouver, BC, Canada
†International Collaboration on Repair Discoveries (ICORD), University of British Columbia, BC, Canada
‡Department of Zoology, University of British Columbia, BC, Canada
§Brain Research Center, University of British Columbia, BC, Canada
¶Department of Physics and Astronomy, University of British Columbia, BC, Canada
#Department of Biochemistry and Molecular Biology, University of British Columbia, BC, Canada
Lipid nanoparticles (LNPs) have demonstrated efficient nucleic acid delivery in vitro and in vivo, as well as in clinical development. They exploit endogenous delivery pathways, by co-opting apolipoprotein E (apoE), to mediate effective delivery of the encapsulated nucleic acids into cells via the low-density lipoprotein receptor (LDLR). However, use of LNPs from the bench to the clinic has been considerably limited by challenges in manufacturing at both small and large scales. Here, we bridge that gap by describing the robust manufacture and use of clinical-grade lipid-based nanoparticles for highly efficient delivery of nucleic acids at scales suitable for both in vitro screening and in vivo applications. RNA-LNPs manufactured using an optimized microfluidic platform enables efficient encapsulation of nucleic acids [e.g. short interfering RNA (siRNA), messenger RNA (mRNA), plasmid DNA (pDNA)] into biocompatible “solid-core” nanoparticles (~50 nm). The resultant nanoparticles can then be applied to cell cultures in vitro or administered in vivo. The following reports a comprehensive set of studies conducted to evaluate the merits of the technology and further provide insights for delivering siRNA and mRNA in difficult-to-transfect cells both in vitro and in vivo. RNA-LNPs were formulated to encapsulate a potent siRNA directed against phosphatase and tensin homolog (PTEN)—a clinically relevant gene associated with neural regeneration. Exceptional cellular uptake (>98%) with minimal toxicity was observed in both primary rat hippocampal and mixed cortical cell cultures. High transfection efficency (>95%) of the encapsulated material resulted in concomitant high-level (>85%) PTEN knockdown within the first 4 h of a low dose (100 ng/ml) treatment; that level of knockdown was further sustained for 21 days. Similarly, RNA-LNPs encapsulating mRNA were also found to mediate early (<4 h) and sustained gene expression (>75% for 7 days) following a single (500 ng/ml) treatment in primary rat mixed cortical cultures. Strategies for locally administering RNA-LNPs into the brain and spinal cord of adult Sprague–Dawley rats were also investigated. Controlled localized injections of PTEN-encaspulated siRNA into the motor cortex resulted in significant and sustained (7 days) knockdown. Similarly, local administration at the site of a cervical spinal cord injury significantly reduced target PTEN expression 10 days later. Visible uptake of RNA-LNPs characterized by their presence in the soma of neurons found in the red nucleus provides further insights into a retrograde transport mechanism involving the axons. Collectively, these studies reflect the simplicity and efficacy of this commercially available technology in presenting a cost-effective and advantageous avenue for screening and validating new targeted nucleic acid therapies.
F. E. Padovan-Neto*, S. Chakroborty*, A. M. Dec*, C. J. Schmidt†, A. R. West*
*Department of Neuroscience, Rosalind Franklin University of Medicine and Science, North Chicago, IL, USA
†Pfizer Global Research and Development, Eastern Point Road, Groton, CT, USA
Cyclic nucleotide phosphodiesterase (PDE) enzymes catalyze the hydrolysis and inactivation of cyclic nucleotides [cyclic adenosine monophosphate (cAMP)/cyclic guanosine monophosphate (cGMP)] in the brain. Several classes of PDE enzymes with distinct tissue distributions, cyclic nucleotide selectivity, and regulatory factors are highly expressed in brain regions subserving cognitive and motor processes affected by diverse brain diseases, including Parkinson's disease (PD) and Huntington's disease (HD). Further, small-molecule inhibitors of several different PDE family members alter cyclic nucleotide levels and favorably enhance motor function and cognition in animal disease models. This presentation will explore the roles and therapeutic potential of selective PDE9A and PDE10A inhibitors on neural processing in fronto-subcortical circuits in wild-type animals and models of HD and L-3,4-dihydroxyphenylalanine (L-DOPA)-induced dyskinesias associated with PD. In studies of HD models, the impact of selective PDE9A and PDE10A inhibitors on spontaneous and cortically evoked firing recorded in the striatum of wild-type and full-length bacterial artificial chromosome (BAC) transgenic HD rats (BACHD) and Q175 mice will be reviewed. New data on the efficacy of PDE10A inhibitors for reversing behavioral and electrophysiological correlates of L-DOPA-induced dyskinesias in a rat model of PD will also be presented. Together, these data will highlight the potential of novel PDE inhibitors for treatment of movement disorders associated with abnormal corticostriatal transmission.
O. Parolini
Centro Ricerca E. Menni-Fondazione Poliambulanza, Brescia, Italy
The past literature has substantiated the therapeutic effects of the amniotic membrane and, more recently, also its derivatives such as cells and conditioned medium have proven to possess the same beneficial effects. The preclinical studies and even clinical trials that support treatment with the amniotic membrane and its derivatives have done so in a plethora of different disease models, and their applications have been described in burns, diabetic ulcers, wounds, lung, and liver fibrosis, autoimmune diseases, and ischemia. In vitro and in vivo immunological studies have shown that amniotic derivatives can reduce the Th1 and Th17 inflammatory immune response and enhance the T regulatory cell population. Moreover, these studies have shown that amnion can also act by inhibiting dendritic cell differentiation and by controlling macrophage activation, switching them from the classical inflammatory (M1) versus the alternative (M2) macrophage phenotype. In the aim to understand which diseases could benefit most from treatment with amniotic membrane and its derivatives, surely the common denominator of those treated successfully thus far is inflammation, or simply an altered immune response. The relevant issue is now to understand the optimal treatment time and regimen, since thus far the results available suggest peak benefit when administered during the acute phase of disease. This underlines the importance of a future stratification of diseases that could benefit most from amnion and its derivatives, and the regimen that should be used to obtain therapeutic effects.
N. K. Polinski*†, L. Congdon†, S. M. Nakashima†, D. L. Fischer*†, F. P. Manfredsson*, M. Benskey*, C. J. Kemp*, N. C. Kuhn*, K. Steece-Collier*, K. L. Paumier*, C. E. Sortwell*
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Neuroscience Program, Michigan State University, Grand Rapids, MI, USA
Clinical trials currently examine the efficacy of viral vector-mediated gene delivery for treating age-related neurodegenerative diseases such as Parkinson's disease (PD). While viral vector strategies have been successful in preclinical studies, to date human clinical trials have disappointed. This may be partially due to the fact that preclinical studies fail to account for aging as an important covariate despite the fact that aging is the primary risk factor for PD. Previously, we found that gene transfer utilizing recombinant adeno-associated virus serotype 2/5 (rAAV2/5) results in decreased transduction efficiency in the aged (20 month) rat nigrostriatal system compared to the young adult (3 month) rat (PMID: 25457448). In the present series of experiments, we investigated whether this phenomenon of deficient transduction is generalizable to additional vector constructs and other PD-relevant circuitry. To accomplish this, we compared the transduction efficiency of rAAV2/5, 2/2, 2/9, or lentivirus (LV) expressing green fluorescent protein (GFP) in young adult or aged male Fischer344 rats (n = 10/group) following injections into the substantia nigra (SN) or the striatum, the structure most often targeted in PD gene therapy clinical trials. In order to assess transduction efficiency, we quantified GFP protein in the anterograde structure through Western blots, GFP-positive (GFP+) cells at the injection site, GFP+/tyrosine hydroxylase+ neurons in the SN (nigral injections) or GFP immunoreactivity in the striatum (striatal injections), and GFP mRNA at the injection site using in situ hybridization. Our primary outcome measure was the level of GFP protein in the anterograde structure. Four weeks after injection into the SN, three of the four vector constructs (rAAV2/5, 2/2, and LV) were less efficient in transducing the aged nigrostriatal system, whereas rAAV2/9 GFP was equally as efficient between ages. Furthermore, 4 weeks following injection into the striatum, all three rAAV serotypes were deficient in transducing the striatonigral system. Results investigating the efficiency of LV in the striatonigral system are pending. Ongoing experiments will examine the effect of age on rAAV2/2-mediated expression of glial cell line-derived neurotrophic factor (GDNF) as well as offer mechanistic insight into the age-related transduction deficiencies. These results demonstrate that the aged nigrostriatal and striatonigral systems are less amenable to viral vector-mediated gene transfer. In addition, we propose a comprehensive and systematic approach for assessing transduction efficiency across viral vector constructs that focuses on the level of protein delivery. Understanding how age impacts transduction efficiency and identifying vector constructs that efficiently transduce the aged brain will be required for successful outcomes in gene therapy clinical trials for age-related diseases.
This research was supported by the Michigan State University Graduate School (N.K.P.) and Pearl J Aldrich Endowment in Aging Related Research (C.E.S.), Mercy Health Saint Mary's (F.P.M.), and the Morris K. Udall Center of Excellence for Parkinson's Disease Research at Michigan State University NS058830 (T.J.C.).
K. Pollock*, H. Stewart*, H. Nelson*, K. D. Fink*, W. Cary*, K. Hendrix*, P. Deng*, A. Torrest*, J. Gutierrez*, C. Nacey*, K. Pepper*, W. Gruenloh*, G. Bauer*, G. Annett*, T. Tempkin†, V. Wheelock†, J. A. Nolta*
*Stem Cell Program and Institute for Regenerative Cures, University of California Davis Health System, Sacramento, CA, USA
†Department of Neurology, University of California Davis Health System, Sacramento, CA, USA
Huntington's disease (HD) is a terminal neurodegenerative autosomal dominant disease that causes neuronal death and is characterized by striatal atrophy. Brain-derived neurotrophic factor (BDNF) has been shown to prevent cell death and to stimulate the growth and migration of new neurons in the brain, making it a lead candidate for the treatment of HD. Human bone marrow mesenchymal stem cells (MSCs) were transduced with a lentivirus designed to overproduce BDNF. The MSC/BDNF product was manufactured following good manufacturing practice standard operating procedures to facilitate translation to future clinical trials. No alterations in cell growth, differentiation capacity, cell size, or phenotype were observed after transduction by the BDNF vector, and the karyotype remained stable. Double-blinded studies were performed to examine the effects of intrastriatal transplantation of MSC/BDNF on disease progression in two strains of HD transgenic mice: YAC 128 and R6/2. MSC/BDNF treatment decreased striatal atrophy in YAC128 mice. MSC/BDNF treatment also significantly reduced anxiety as measured in the open field assay. A significant correlation between striatal size and anxiety-like behaviors was observed, suggesting the validity of these endpoints for our planned future cellular trial. Both MSC and MSC/BDNF treatments induced a significant increase in neurogenesis-like activity in R6/2 mice. Our genetically modified MSC/BDNF set a precedent for stem cell-based neurotherapeutics and could potentially be modified for other neurodegenerative disorders such as amyotrophic lateral sclerosis, Alzheimer's disease, and some forms of Parkinson's disease. These cells provide a platform delivery system for future studies involving corrective gene editing strategies.
Support for this project was provided by the California Institute for Regenerative Medicine (CIRM) DR2-05415 (V. Wheelock/J. A. Nolta), NIH Director's transformative award 1R01GM099688 (J. A. Nolta), a NIH NRSA Postdoctoral Fellowship No. F32NS090722 (K. D. Fink), and philanthropic donors from the HD and JHD communities, including the Dake Foundation, Roberson family and Team KJ.
H. Pu*†, W. Zhu*, L. Huang†, G. Wang†, X. Jiang*†, M. Xu*†, L. Zhang*, Y. Wang†, W. Zhang†, Y. Shi*, Y. Gao*†, Jun Chen*†
*Pittsburgh Institute of Brain Disorders & Recovery, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
†State Key Laboratory of Medical Neurobiology and Institute of Brain Science, Fudan University, Shanghai, China
Traumatic brain injury (TBI) can lead to long-term motor and cognitive dysfunctions, which are attributed, at least in part, to blood–brain barrier (BBB) disruption and white matter injury (WMI). The mechanisms underlying post-TBI WMI and BBB disruption, however, are poorly understood thus far. Na+-K+-2Cl- cotransporter isoform 1 (NKCC1) is a universally expressed ion transporter that maintains intracellular ion homeostasis by increasing intracellular K+ and Cl-. Having been characterized in stroke models, NKCC1 is activated in various types of cells in the ischemic brain, and thought to mediate BBB disruption, brain edema, and neuronal cell death. In this study, we tested the hypothesis that pharmacological inhibition of NKCC1 may protect against BBB damage and WMI and improve long-term neurological outcomes in a mouse model of TBI. Adult male C57BL/6J mice of 2–3 months of age were subjected to controlled cortical impact (CCI), and then randomly and blindly assigned to vehicle or NKCC1 inhibitor treatment group. Bumetanide, a selective NKCC1 inhibitor, was administrated (25 mg/kg, IP) immediately after CCI and then every 6 h (six doses in total). Sensorimotor deficits were evaluated by hang wire, cylinder, and foot fault tests at 1–35 days after CCI. BBB integrity was examined at 48–72 h post-CCI by measuring Evans blue extravasation, brain water content, and expression levels of tight junction proteins. WMI was assessed at 35 days post-CCI by immunohistochemical examination of the myelin basic protein and amyloid precursor protein and electrophysiological measurement of the combined action potentials of WM myelinated nerve fibers. The results revealed that post-CCI treatment with bumetanide significantly improved sensorimotor functions after CCI, although it did not reduce the overall cortical lesion size in CCI mice. Bumetanide treatment also markedly decreased brain water content and BBB leakage, likely by reducing matrix metallopeptidase 9 (MMP-9) expression and preventing the degradation of tight junction proteins. Moreover, bumetanide treatment attenuated WMI after CCI, exhibiting significantly improved integrity of myelin sheaths, axons, and WM electric conductivity. These results suggest that NKCC1 mediates BBB disruption and WMI after TBI. Thus, NKCC1 inhibition may offer the potential for improving long-term neurological outcomes in clinical TBI.
S. Reyes*, H. Nguyen*, H. J. Yeon*, S. Mashkouri*, D. Aum*, Q. Colburn*, X. Kaya*, J. Y. Lee*, S. A. Acosta*, W. Quillen†, N. Tajiri*†, C. V. Borlongan*
*Department of Neurosurgery and Brain Repair, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
†School of Physical Therapy, University of South Florida, Morsani College of Medicine, Tampa, FL, USA
Stroke remains a significant unmet clinical need with limited therapeutic options. Exercise has been shown to afford therapeutic benefits in stroke patients, but the exact mechanism of action is yet to be fully determined. With the interruption in the brain blood circulation being a major trigger of ischemic stroke, finding treatments catered towards maintaining the potency of the cerebrovasculature may prove therapeutic against stroke. The present study assessed the effects of short bouts of exercise prior to stroke induction and characterized in vivo the cerebral blood flow and concomitant motor functions. Adult Sprague–Dawley rats were initially exposed to 30-min or 60-min forced running wheel exercise daily for 3 days, then subsequently subjected to transient intraluminal occlusion of the middle cerebral artery (MCAo). Non-exercised stroke rats served as controls. Laser Doppler cerebral blood flow (CBF) readings were recorded at baseline (prior to MCAo), during MCAo, and during reperfusion. The elevated body swing test was conducted at baseline, day 0 (day of stroke), and at days 1 and 3 after stroke. Results revealed significant 50% increments in CBF at baseline in exercised rats compared to controls (p < 0.05). In addition, while CBF did not significantly differ during MCAo across groups (p > 0.05), significant 10%–30% elevated CBF during reperfusion accompanied exercised rats compared to controls (p < 0.05). Moreover, the animals that received 60-min exercise displayed significantly improved motor performance compared to the 30-min exercise and non-exercised rats (p < 0.05). The present data highlight the importance of exercise as a prophylactic treatment against stroke, which possibly acts on improving cerebrovascular integrity and function. Immunohistochemical assays targeting the status of the cerebrovasculature to reveal the cellular effects of exercise are currently being analyzed.
C. T. Richie, P. Koivula, J. Necarsulmer, H. A. Baldwin, L. V. Fortuno, Y. Zhang, B. K. Harvey
Optogenetic and Transgenic Technology Core, National Institute on Drug Abuse, Baltimore, MD, USA
The development of clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized genome engineering and facilitated the genetic customization of model organisms. Although this technology's greatest impact comes from its use in germline editing and the construction of transgenic animals, it is continuously being adapted for gene therapy with viral vectors, despite the relatively large size of the CRISPR-associated protein 9 (Cas9) protein. Although both Streptococcus pyogenes Cas9 (SpCas9) and the smaller Staphylococcus aureus Cas9 (SaCas9) have been successfully delivered to rodents using adeno-associated virus (AAV), the ability to restrict CRISPR activity to specific tissues has been limited to the handful of promoters that are themselves small enough to accommodate the Cas9 payload. As an initial proof-of-principle, we disrupted the expression of tyrosine hydroxylase (TH) in rat brains by simultaneously administering two viruses [one expressing Cas9, and the other expressing green fluorescent protein (GFP) as well as a chimeric guide RNA (gRNA) targeting TH] by unilateral injection. These animals exhibit a directional bias in methamphetamine-induced rotation assays when compared to control animals that received a single virus, indicating a CRISPR-dependent dopamine imbalance has occurred. Immunohistochemistry with anti-TH antibodies revealed that expression of TH was reduced in the striatum of the injected hemisphere. These results demonstrate that virally delivered CRISPR can be used to effect changes in behavior. We are currently developing a set of AAV vectors that will confine the transcription of guide RNAs to cells that express Cre recombinase in a tissue-specific manner. When combined with the existing repertoire of Cre-driver mice and rats, this mechanism will allow for the permanent disruption of target genes in specific cell types of adult animals. This tool will bypass the costly requirement of creating a conditional knockout allele of the gene of interest. When implemented using the smaller SaCas9 homolog, this strategy has the potential to fit into a single AAV viral particle.
I. M. Sandoval, B. Daley, N. Kuhn, N. Marckini, A. C. Strauss, J. W. Lipton, F. P. Manfredsson, T. J. Collier
Department of Translational Science and Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
Customizable nucleases can be used to introduce frame-shifting mutations to knock out a desired gene with high precision. The newest class of nucleases, CRISPR (clustered regularly interspaced short palindromic repeats)/CRISPR-associated protein 9 (Cas9) system, has been widely used in a variety of cells and microorganisms, but its application in mature neuronal tissue has been limited. We sought to investigate whether we could achieve efficient CRISPR/Cas9-mediated gene inactivation in vivo, specifically in dopaminergic neurons of the substantia nigra, the cells affected in Parkinson's disease (PD). We selected to target four proteins: tyrosine hydroxylase (TH)—the rate-limiting enzyme in the production of the neurotransmitter dopamine—as a proof of concept, as well as α-synuclein, vesicular transport protein 35 (VPS35), and eukaryotic translation initiation factor 4 gamma (EIF4G1), all of which are linked to familial forms of PD. We designed guide RNAs (gRNAs) against exon 1 and exon 2 of the TH gene and confirmed genomic DNA cleavage efficiency and reduction in protein levels in PC12 cells in vitro. Using a dual recombinant adeno-associated virus (rAAV) vector system we delivered the protein Cas9, together with the gRNAs as well as green fluorescent protein (GFP) as a transduction marker into the substantia nigra of 2-month-old Sprague–Dawley rats. Immunostaining of brain sections collected at 6 weeks after surgery showed a robust decrease of TH protein expression in CRISPR/gRNA(TH)-treated brains compared to “unguided” control injected rats. Stereological quantification of TH-immunoreactive neurons revealed a 48% reduction of TH expressing nigral neurons in the treated animals. These results validate the use of CRISPR technology in vivo in the nigrostriatal dopaminergic system in mature brains. Ongoing experiments are using the same strategy to target the aforementioned PD-related genes: α-synuclein, VPS35, and EIF4G1. Upon completion we will have built a powerful toolbox to study gene function specifically in nigral dopamine neurons while still in their native context.
This work was supported by the office of the MSU Vice President for Research and Graduate Studies (F.P.M., I.M.S.), NS058830 The Udall Center of Excellence at Michigan State University, and the Edwin Brophy Endowment in Central Nervous System Disorders (T.J.C.).
A. J. Schwab, M. R. Meade, A. D. Ebert
Medical College of Wisconsin, Milwaukee, WI, USA
Parkinson's disease (PD) is a progressive degenerative disorder of the central nervous system typically characterized by the loss of dopamine neurons in the substantia nigra. Although the mechanisms underlying dopamine neuron loss in PD are not well understood, mitochondrial dysfunction has been repeatedly implicated as a potential contributor. We hypothesize that mitochondrial dysfunction may also be observed in other neurons, such as sensory and glutamatergic neurons, which may explain why PD patients suffer from a variety of motor and nonmotor symptoms that are independent of dopamine neuron loss. In order to study mitochondrial dysfunction in dopamine, sensory and glutamatergic neurons in a human system, we utilized human induced pluripotent stem cells (iPSCs) from two homozygous leucine-rich repeat kinase 2 (LRRK2) G2019S patients and one asymptomatic heterozygous LRRK2 G2019S patient. We followed previously published differentiation protocols to generate dopamine, sensory and glutamatergic neurons from all PD iPSCs as well as three unaffected control iPSCs. Analysis of neuron morphology revealed neurite abnormalities in homozygous and heterozygous LRRK2 G2019S dopamine, sensory, and glutamatergic neurons. Interestingly, LRRK2 G2019S sensory and glutamatergic neurons displayed increased neurite aggregations whereas dopamine neurons displayed shortened neurites and reduced neurite branching. We next found that LRRK2 G2019S dopamine, sensory, and glutamatergic neurons each displayed a significant decrease in mitochondrial respiration, although mitochondrial trafficking was only impaired within dopamine neurons. Interestingly, LRRK2 kinase inhibition did not improve mitochondrial respiration and trafficking in any of the neuronal contexts. The increased severity of respiration and mitochondrial trafficking deficits observed in LRRK2 G2019S dopamine neurons may explain the heightened vulnerability of dopamine neurons in PD, but our data support the idea that PD pathology exhibits nondopaminergic attributes as well. Determining the cause and consequences of neurite and mitochondrial deficits in multiple neuronal subtypes may provide insight into the underlying mechanisms of motor and nonmotor symptoms in PD, thereby aiding therapeutic development.
S. Seiler, S. Di Santo, H. R. Widmer
Department of Neurosurgery, Research Laboratory and Regenerative Neuroscience Cluster, University of Bern and University Hospital Bern, Bern, Switzerland
Transplantation of fetal human ventral mesencephalic dopaminergic neurons into the striatum is a promising strategy to compensate for the characteristic dopamine deficit observed in Parkinson's disease (PD). However, this therapeutic approach is currently limited by the high number of fetuses needed for transplantation and the poor survival and functional integration of grafted dopaminergic neurons into the host brain. Accumulating evidence indicates that contrasting inhibitory signals endowed in the central nervous system might support neuronal regeneration. Hence, in the present study we aimed at improving survival and integration of grafted cells in the host brain by neutralizing Nogo-A, one of the most potent neurite growth inhibitors in the central nervous system. For that purpose, ventral mesencephalic tissue cultures were transplanted into rats with a 6-hydroxydopamine lesion and concomitantly treated for 2 weeks with intraventricular infusion of neutralizing anti-Nogo-A antibodies. Motor behavior using the cylinder test was assessed prior to and after transplantation as the functional outcome. At the end of the experimental period, the number of dopaminergic fibers growing into the host brain and the number of surviving dopaminergic neurons in the grafts, as well as graft size, were examined. We found that anti-Nogo-A antibody infusion significantly improved the asymmetrical forelimb use observed after lesions as compared to controls. Importantly, a significantly threefold higher dopaminergic fiber outgrowth from the transplants was detected in the Nogo-A antibody-treated group compared to controls. Furthermore, Nogo-A neutralization showed a tendency for increased survival of dopaminergic neurons (by twofold) in the grafts. No significant differences were observed for graft volume and the number of dopaminergic neurons coexpressing G-protein-coupled inward rectifier potassium channel subunit 2 between groups. In sum, our findings support the view that neutralization of Nogo-A in the host brain may offer a novel and therapeutically meaningful intervention for cell transplantation approaches in PD.
This study was supported by the Swiss National Science Foundation (grant No. 31003A-135565) and the HANELA Foundation.
R. C. Sellnow*†, E. Flores-Barrera‡, A. R. West‡, K. Steece-Collier*, M. J. Benskey*, I. M. Sandoval*, N. Kuhn*, K. Y. Tseng‡, F. P. Manfredsson*
*Department of Translational Science and Molecular Medicine, Michigan State University, Grand Rapids, MI, USA
†Cell and Molecular Biology Program, Michigan State University, Grand Rapids, MI, USA
‡Department of Cellular and Molecular Pharmacology, Rosalind Franklin University, North Chicago, IL, USA
The current gold standard treatment for the motor symptoms in Parkinson's disease (PD) is levodopa (L- DOPA). L-DOPA acts as a replacement for the lost dopamine following degeneration of cells in the substantia nigra. Unfortunately, chronic treatment with L-DOPA inevitably leads to the development of levodopa-induced dyskinesias (LIDs) in a majority of PD patients. LIDs are involuntary motor behaviors that include chorea, dystonias, and limb hyperkinesia that are distinct from parkinsonian motor behaviors. It has been shown previously by our and other groups that an ectopic induction of the transcription factor nuclear receptor related 1 (Nurr1) in striatal medium spiny neurons (MSNs) is associated with LIDs. Preliminary data from our laboratory suggest that recombinant adeno-associated virus (rAAV)-mediated overexpression of Nurr1 exacerbates the severity of LIDs, while rAAV-mediated knockdown of Nurr1 attenuates LIDs. These data show that Nurr1 overexpression in the striatum may be an important mediator of LID development. In order to better understand the role of Nurr1 in LIDs, we are currently investigating the effects of Nurr1 upregulation on striatal physiology. The goal of these studies is to determine if Nurr1 expression in the striatum—a region where Nurr1 is not normally expressed—can change 1) electrophysiological activity of the striatum, and/or 2) morphology of striatal MSNs. In the first study, 6-hydroxydopamine (6-OHDA)-lesioned Sprague–Dawley rats received either rAAV-Nurr1 or rAAV-green fluorescent protein (GFP) injections in the striatum. Animals were not treated with L-DOPA and thus did not become dyskinetic. The local field potential (LFP) of the striatum following cortical stimulation was measured following a single dose of L-DOPA. Animals treated with GFP showed an inhibition of the corticostriatal response to L-DOPA while Nurr1-treated animals showed a potentiation of the response. Interestingly, the LFP profile of rAAV-Nurr1 rats was virtually identical to that of subjects with established dyskinesias. This suggests that ectopic Nurr1 expression induces physiologic changes in the striatum similar to those observed in dyskinetic animals. Ongoing studies are directed at expanding these electrophysiological findings, as well as examining whether Nurr1 upregulation in the absence of L-DOPA also induces the hallmark LID-associated morphological changes (e.g., dendritic spine morphology) we have previously observed.
Y. Shi*†, L. Zhang*†, H. Pu*, L. Mao*, X. Hu*†, X. Jiang*, N. Xu*, R. A. Stetler*†, R. K. Leak‡, R. F. Keep§, J. Chen*†
*Center of Cerebrovascular Disease Research, University of Pittsburgh School of Medicine, Pittsburgh, PA, USA
†Geriatric Research, Educational and Clinical Center, Veterans Affairs Pittsburgh Health Care System, Pittsburgh, PA, USA
‡Division of Pharmaceutical Sciences, Mylan School of Pharmacy, Duquesne University, Pittsburgh, PA, USA
§Department of Neurosurgery, University of Michigan, Ann Arbor, MI, USA
The mechanism and long-term consequences of early blood–brain barrier (BBB) disruption after cerebral ischemic/reperfusion (I/R) injury are poorly understood. In this study, we identified an early breach of the BBB within 30 min after cerebral I/R and investigated the accompanying changes in brain microvascular endothelial cells (BMECs). Mice were randomly assigned to groups and all measurements were made by investigators blinded to experimental groups. Transient focal cerebral ischemia (tFCI) was induced by 60-min middle cerebral artery occlusion and reperfusion. BBB breakdown was examined by the extravasation of injected tracers from peripheral circulation into the brain parenchyma. We discovered that I/R induced subtle leakage of small tracers (≤3 kDa) through the BBB within 30–60 min, likely independent of matrix metalloproteinase (MMP) activities. Using an in vitro BBB model comprised of BMECs, we found that the early loss of BBB integrity was caused by activation of rho-associated, coiled-coil containing protein kinase 1/myosin light chain (ROCK/MLC) signaling, persistent actin polymerization (shown by a 2.04-fold increase of the filamentous/globular (F/G)-actin ratio and formation of F-actin+ stress fibers within 1 h), and the disassembly of junctional proteins [e.g., occludin, claudin, vascular endothelial (VE)-cadherin] in BMECs. The weakened BBB permitted the infiltration of peripheral immune cells, including MMP-9-producing neutrophils/macrophages, which further damage the BBB. Inactivation of actin depolymerizing factor (ADF), a major endogenous inhibitor of actin polymerization, caused sustained actin polymerization in BMECs, whereas lentivirus-mediated overexpression of a constitutively active mutant ADF (ADFm) reduced actin polymerization within BMECs and prevented junctional protein disassembly. Furthermore, overexpression of ADFm in EC-targeted transgenic mice attenuated BBB disruption at both early (1 h) and late (24 h) reperfusion stages (p < 0.01). ADFm overexpression reduced neutrophil infiltration by 70.4% and downregulated 11 proinflammatory cytokines tested (p < 0.05) at 24 h, thereby alleviating secondary tissue injury. Brain infarct was reduced by 45% at 48 h, and sensorimotor (p < 0.01 in the cylinder and corner tests) and cognitive functions (p < 0.05 in the water maze test) were robustly improved in ADFm overexpressors up to 28 days after tFCI. Together, we identify a previously unexplored role for early BBB disruption in permanent neurovascular damage and long-term stroke outcomes, whereby BBB rupture may be a cause rather than a consequence of parenchymal cell injury. Prevention of early cytoskeletal changes in BMECs breaks the progression of permanent neurovascular damage and offers long-lasting neurovascular protection after stroke.
J. Shultz*†, H. Resnikoff*, V. Bondarenko*, J. Holden‡, T. Barnhardt‡, P. Lao‡, B. Christian‡, J. Nickles‡, M. Emborg*†‡
*Wisconsin National Primate Research Center, University of Wisconsin–Madison, Madison, WI, USA
†Cellular and Molecular Pathology Graduate Program, University of Wisconsin–Madison, Madison, WI, USA
‡Department of Medical Physics, University of Wisconsin–Madison, Madison, WI, USA
Cardiac autonomic dysfunction (dysautonomia) is a common Parkinson's disease (PD) nonmotor symptom that greatly impacts a patient's quality of life. Cardiac dysautonomia is associated with loss of postganglionic sympathetic innervation to the heart, decreased circulating catecholamines, and arterial baroreflex failure. Disease-modifying strategies for cardiac dysautonomia are not available. Biomarkers of cardiac sympathetic neurodegeneration are needed for early or differential diagnosis, understanding pathophysiological mechanisms of cardiac sympathetic loss, and evaluation of emerging neuroprotective therapies. Central and peripheral catecholaminergic neuronal loss in PD is associated with inflammation and oxidative stress, and can be mimicked in animals by dosing of the catecholaminergic neurotoxin 6-hyroxydopamine (6-OHDA). Interestingly, activation of the peroxisome proliferator-activated receptor g (PPARg) decreases the inflammatory response (microglial, macrophage), increases mitochondrial biogenesis, and decreases oxidative stress. Here we report our studies in a nonhuman primate (NHP) model of cardiac dysautonomia induced by systemic dosing of 6-OHDA. We aimed to assess the feasibility of using positron emission tomography (PET) with radioligands specific to catecholaminergic innervation {[11C]meta-hydroxyephedrine (MHED)}, inflammation {[11C] N-acetyl-N-(2-methoxybenzyl)-2-phenoxy-5-pyridinamine (PBR28)}, and oxidative stress {[61Cu]bis(thiosemicarbazone) (ATSM)} to visualize in vivo mechanisms of neurodegeneration and target validation. We hypothesized that PPARg activation by pioglitazone modulates the 6-OHDA-induced inflammatory response and production of reactive oxidative species, resulting in preservation of cardiac catecholaminergic innervation. Ten adult male rhesus monkeys received intravenous 6-OHDA (50 mg/kg) and 24 h later were randomly assigned to receive daily oral dosing of placebo (n = 5) or pioglitazone (5 mg/kg; n = 5). At baseline, 1, and 12 weeks post-6-OHDA all animals were PET scanned with MHED, PBR28, and ATSM (total: 90 scans) in a Siemens micro-PET Focus 220 under isofluorane anesthesia and constant veterinarian monitoring of vital signs. Analysis of MHED PET data showed a significant loss of uptake at 1 week post-neurotoxin in both placebo-treated (86%) and pioglitazone-treated (82%) animals with partial recovery by 12 weeks that was significantly greater in the pioglitazone [54% retention deficit (%RD)] animals compared to placebo (70 %RD; ANOVA p < 0.05). Preliminary analysis of PBR28 and ATSM data revealed some uptake of the radioligands at baseline. One week post-neurotoxin placebo-treated compared to pioglitazone-treated animals had increased uptake of both radioligands. PBR28 and ATSM values returned to baseline levels by 12 weeks post-6-OHDA. Our results demonstrate that PET imaging with MHED, PBR28, and ATSM is a sensitive method to visualize changes in catecholaminergic innervation of the left ventricle and for monitoring neurodegeneration and target validation.
Research was supported by NIH grants P51 P51OD011106, UL1TR000427, and R21NS084158 and the VCRGE and the Department of Medical Physics, University of Wisconsin–Madison.
V. M. Spruance, L. Zholudeva, T. Bezdudnaya, V. Marchenko, K. M. Negron, M. A. Lane
Drexel University College of Medicine, Department of Neurobiology and Anatomy, Philadelphia, PA, USA
Impaired breathing is a devastating consequence of cervical spinal cord injury (SCI) that increases morbidity and the risk of mortality. Injuries at high-to-mid cervical levels (C1–4) result in the most severe deficits as the phrenic motor circuitry—controlling the diaphragm—is directly compromised, typically resulting in dependence on assisted ventilation. While there is mounting evidence for spontaneous respiratory improvement, the extent of recovery—or functional plasticity—remains limited. Thus, there is a need to develop therapeutic strategies for enhancing repair and recovery of respiratory pathways. Our ongoing research aims to elucidate spinal and supraspinal changes that may influence respiration post-SCI, and assess whether treatments can harness ongoing neuroplasticity to improve function postinjury. These studies have identified that spinal interneurons represent a potential therapeutic target for enhancing plasticity and recovery of phrenic motor function. With a particular focus on the phrenic motor system, the goal of the present work is to assess whether transplantation of neural precursor and stem cells (NPCs/NSCs) can facilitate repair of the injured adult rat cervical spinal cord and promote lasting, functional recovery. We hypothesize that spinally derived NPCs, rich in interneuronal precursors, will provide a source of neurons that facilitate a novel neuronal relay capable of restoring input to phrenic motoneurons. Adult, female Sprague–Dawley rats (~250 g) received lateralized C3/4 contusions (200 kilodynes; Infinite Horizons Pneumatic Impactor). One week postinjury, NPCs derived from developing rat spinal cord (E13.5 Sprague–Dawley or E13.5 Fisher rat, expressing green fluorescent protein) were injected directly into the injury cavity (~1 million cells). Transplanted animals are compared against injured, untreated animals. Four weeks or 1 year later, a transynaptic, retrograde tracer (pseudorabies virus) was delivered to the ipsilateral hemidiaphragm or directly into the transplant. Tracing revealed synaptic integration between donor neurons and host phrenic circuitry at 1 month following transplantation. However, evidence for this connectivity is lost at 1 year following transplantation. Interestingly, phrenic function assessed by terminal electrophysiology revealed enhanced phrenic and diaphragm recovery at both time points in those animals that received NPC transplants following cervical contusion injury. These ongoing studies are providing insight into the therapeutic potential for NPC therapy in the injured spinal cord.
K. Steece-Collier*, J. T. Keeney*, A. Cole-Strauss*, J. A. Stancati*, T. J. Collier*, M. E. Winn†, C. E. Sortwell*, F. P. Manfredsson*, J. W. Lipton*, I. E. Vega*
*Department of Translational Science & Molecular Medicine, College of Human Medicine, Michigan State University, Grand Rapids, MI, USA
†Bioinformatics & Biostatistics Core, Van Andel Research Institute, Grand Rapids, MI, USA
Levodopa-induced dyskinesia (LID) is an undesirable consequence of pharmacotherapy for Parkinson's disease (PD). However, some patients are more resistant to developing LID than others. Depending on the course of levodopa (LD) administration, a range of LID severity and incidence is similarly observed in parkinsonian rats. We have attempted to utilize this variability in LID development/resistance to chronic LD as a strategy to profile differential expression, at the transcript and protein level, to gain insights about molecular targets that may be involved in LID induction, maintenance, or resistance. To find such targets, we conducted two separate studies that capitalized on this LID variability to chronic LD in 6-hydroxydopamine (6-OHDA)-induced parkinsonian rats. The first examined individual variation in response to LD in an outbred rat strain (Sprague–Dawley, SD), and a second examined differential resistance to LID development in two inbred rat strains: Fischer 344 (F344) and Lewis RT.1 (Lewis) rat strains, where unilaterally parkinsonian F344s exhibit robust LID while Lewis' display a marked LID resistance over a 3-week daily LD regimen. For both studies, rats were rendered parkinsonian with 6-OHDA and 4–5 weeks later began treatment with daily high-dose LD (12 mg/kg) for 3 weeks. In the first study we used a transcriptomic approach to identify expression differences in response to LD in the striatum and substantia nigra (SN) of parkinsonian, LD-treated subjects that developed dyskinesias (LID+) and those that did not (LID-). Differential expression of genes associated with LID was found in the striatum but not in the SN, where out of the 258 differentially expressed genes, those showing among the largest increase in expression were Nr4a2 [nuclear receptor subfamily 4, group A, member 2 or Nurr 1 (nuclear receptor related 1)], Trh (thyrotropin-releasing hormone), and Inhba (inhibin-beta A). In our proteomics analysis of the striatum of the two inbred lines we identified 20 candidate proteins showing differential levels of abundance. Two proteins, DJ-1 (Parkinson protein 7; PARK7) and ubiquitin C-terminal hydrolase L1 (UCHL1), have been selected as candidates for further characterization because of their relevance to PD and their differential profiles in Lewis versus F344 rats, which suggests a potential association with LID resistance. We contend that direct comparison of gene expression profiles, in conjunction with comparison of protein abundance profiles in rats that do, or do not, develop LID provides a useful platform for understanding the biology of LID and identification of targets for development of interventions that may diminish this unwanted side effect of the best medical therapy for PD.
Supported by NINDS Udall award P50NS058830; MSU DFI award.
A. N. Stewart*†, J. J. Matyas*†, R. M. Welchko*†, A. Goldsmith*†, E. D. Peterson*†, S. E. Zeiler*†, N. M. Brandi*†, M. Lu*†‡, Z. Nan*†‡, J. Rossignol*†§, G. L. Dunbar*†‡¶
*Field Neurosciences Institute Laboratory for Restorative Neurology, Central Michigan University, Mount Pleasant, MI, USA
†Program in Neuroscience, Central Michigan University, Mount Pleasant, MI, USA
‡Department of Psychology, Central Michigan University, Mount Pleasant, MI, USA
§College of Medicine, Central Michigan University, Mount Pleasant, MI, USA
¶Field Neurosciences Institute, Saginaw, MI, USA
Stromal-derived factor-1α (SDF-1α) is a known chemotactic molecule functioning to elicit prosurvival and migratory behaviors of various cell types of the nervous system, including astrocytes, microglia, neural stem cells, growth cones, and oligodendrocytes, leading to a role of SDF-1α in neurogenesis, gliosis, axonal guidance and myelination. Utilizing the genetic overexpression of various progrowth/prosurvival molecules in mesenchymal stem cells (MSCs) as a combinatorial treatment has shown promise in enhancing the regenerative potential of the nervous system and to promoting improved behavioral outcomes. In the present study, MSCs were engineered to secrete SDF-1a, and the paracrine effects were explored on neuronal stem cells (NSCs) in vitro, utilizing two coculture experiments, the under agarose migration assay and the transwell migration assay. Subacute transplantation into the lesion of a contusive rat model of spinal cord injury (SCI) was followed by 7 or 15 weeks of survival, during which behavioral analyses were conducted, including utilizing the Basso, Beattie, Bresnahan (BBB) locomotor rating scale and a novel assessment of rearing ability. Gliosis, inflammation, white matter sparing, and axonal growth were characterized with optical densitometry following histological/immunohistochemical development. Results from these experiments demonstrate that the overexpression of SDF-1α by MSCs can enhance both the migration of NSCs and process outgrowth from differentiating NSCs in vitro, compared to vehicle or MSCs alone. Our in vivo results indicated a significant increase in the density of growth-associated protein 43 (GAP-43) immunohistochemical labeling, which was observed inside the lesion/graft site, beyond that of unmodified MSCs. Although the transplantation of both SDF-1 MSCs and unmodified MSCs improved behavioral performance on the BBB at 7 weeks postinjury, the overexpression of SDF-1α offered no additional benefits compared to MSCs alone on preserving motor outcomes. No significant differences were found between groups on measures of gliosis or white matter sparing, while inflammation was increased in the SDF-1 MSC group compared to unmodified MSC-treated animals. The results from these experiments indicate that utilizing SDF-1α to enhance growth and regeneration of axons offers a promising therapeutic approach for traumatic SCI.
Support for this study was provided by the Office of Research and Sponsored Programs at CMU, the College of Medicine, the Field Neurosciences Institute, and the John G. Kulhavi Professorship in Neuroscience at CMU.
S. Strom*, K. Kannisto*, R. Srinivasan*, T. Miki†, R. Gramignoli*
*Department of Laboratory Medicine, Karolinska Institutet, Stockholm, Sweden
†Department of Biochemistry and Molecular Biology, University of Southern California, Los Angeles, CA, USA
Our group initiated allogeneic hepatocyte transplants for the cellular therapy of liver disease in patients. However, this therapy is still limited by the availability of useful cells for transplants. We began investigating stem cell sources of cells for these transplants. We reported that human amnion epithelial cells (hAECs) express surface markers and have a gene expression profile similar to pluripotent stem cells. However, hAECs do not express telomerase, are not immortal, and do not form tumors when transplanted. We investigated the ability of hAECs to correct two different mouse models of metabolic liver disease. Mice with maple syrup urine disease (MSUD) or phenylketonuria (PKU) were transplanted with hAECs beginning at birth and continuing through weaning. In both mouse models, the transplantation of hAECs provided a greater than 60% correction of the amino acid and neurotransmitter disturbances characteristic of the untreated animals, and increases survival of the MSUD affected mice from 0%, in untreated mice to 82% in hAEC-transplanted mice at day 100. In the mouse model of PKU, hAEC transplants reduced brain phenylalanine (Phe) levels to those not significantly different from wild-type, normal animals. Initial studies suggest that hAEC transplants will also provide similar levels of improvement in a mouse model of the urea cycle defect, ornithine transcarbamylase deficiency. The reported safety of the cellular therapy and the strong preclinical evidence of efficacy in faithful mouse models of human metabolic liver diseases supported an application to use this cellular therapy in patients. Permission was granted to begin an investigation of the safety and efficacy of hAEC therapy in up to 10 patients with liver diseases that would normally be considered candidates for hepatocyte transplants.
E. Sykova*†, S. Forostyak*†, A. Homola*†, S. Konradova*‡, K. Ruzickova*‡, M. Syka*‡, P. Jendelova*†
*Institute of Experimental Medicine, Academy of Sciences of the Czech Republic, Prague, Czech Republic
†Department of Neuroscience, 2nd Faculty of Medicine, Charles University, Prague, Czech Republic
‡Bioinova s.r.o, Vídenská 1083, Prague, Czech Republic
Amyotrophic lateral sclerosis (ALS) is a progressive neurodegenerative disorder leading to the death of upper and lower motoneurons (MNs). Its clinical diagnosis is primarily based on the symptoms presented, and until now there has been no known way to prevent, cure, or even block its development. Cell therapy may present a new possibility to cure ALS by providing neurotrophic support to host MNs and/or replace dysfunctional glial cells. In our preclinical study, 500,000 human bone marrow mesenchymal stromal cells (hMSCs), from Bioinova s.r.o., suspended in 50 μl of growth media were delivered intrathecally (via the cistern magna) into symptomatic mutant human superoxide dismutase 1 (SOD1 G93A) transgenic rats (n = 11). Sham-treated animals (n = 9) were injected with growth media. Overall survival in the hMSC-treated group was prolonged by 13.6 days compared with the sham-treated group. We found that the cell-treated rats showed significantly better motility and grip strength when compared to vehicle-injected animals. Electrophysiological evaluation of transgenic SOD1 rats showed a progressive loss of motor units in the gastrocnemius muscle, with no major changes in nerve conduction velocity. Quantitative analyses of Wisteria floribunda agglutinin (WFA) fluorescence intensity measured in the ventral horns of the cervical and lumbar levels of the spinal cord revealed significantly greater numbers of perineuronal nets (PNNs) in the hMSC-treated animals when compared with the sham-treated group. The clinical trial titled “Autologous multipotent mesenchymal stem cells from 3rd passage, A Prospective, Non-randomized, Open Label Study to Assess the Safety and the Efficacy of Autologous Multipotent Mesenchymal Stem Cells in the Treatment of Amyotrophic Lateral Sclerosis” (EudraCT Vo. 2011-000362-35) and sponsored by Bioinova s.r.o. is currently being evaluated. Twenty-six patients were enrolled in the study (phase I/IIa) and have undergone application of MSCs via lumbar puncture. No serious adverse reactions have been observed. So far, 20 ALS patients completed the study and have been evaluated for efficacy. The disease progression on the ALS functional rating scale slowed down or stopped in 60% of the patients (n = 12) for a time period of 3 months, and in 30% of patients (n = 6) for a time period of 6 months. Several patients (n = 8) had some temporary functional improvement in swallowing, speech, or movement. Stable values of muscular strength in upper and lower extremities for a time period of 3 months were observed in 75% of the patients. In 85% of the patients (n = 17) 6 months' stabilization and in 60% of the patients (n = 12) 1 year stabilization of the forced vital capacity (FVC) was found, and in all of these patients FVC values remained above 65%. Our results demonstrate that hMSCs can, at least temporarily, slow down the progression of ALS. We are currently innovating our hMSCs product and preparing phase IIb of the ALS clinical trial.
Supported by: GAČR 14-10504P, 15-06958S, P304/12/G069.
G. Toshkezi, M. Kyle, S. Longo, L. Chin, L.-R. Zhao
Department of Neurosurgery, SUNY Upstate Medical University, Syracuse, NY, USA
Traumatic brain injury (TBI) has a high incidence in young adults and creates a major cause of long-term disability and death in the US. The subacute phase of TBI is a unique time period when both the secondary damage and the repair process occur in the brain after injury. The lack of pharmaceutical therapy for subacute TBI remains a crucial medical challenge. Stem cell factor (SCF) and granulocyte-colony stimulating factor (G-CSF), two key hematopoietic growth factors, have shown neuroprotective and neurorestorative effects in experimental stroke. The objective of this study is to determine the therapeutic efficacy of SCF + G-CSF in subacute TBI. Young adult male C57BL mice were subject to TBI in the right hemisphere. After induction of TBI, mice were randomly divided into two groups: a vehicle control group and a SCF + G-CSF treatment group. In addition, mice without TBI served as sham operative controls. Treatment was given 2 weeks after TBI induction. This time point was chosen because it corresponds to the subacute phase of TBI. SCF (200 μg/kg) and G-CSF (50 μg/kg) or an equal volume of vehicle solution was subcutaneously injected daily for 7 days. Neurobehavioral tests were performed during the period of 2 to 9 weeks after treatment. Three months after treatment, neurodegeneration was determined through Fluoro-Jade C staining, and neural networks were identified with immunohistochemistry. In the water maze test, SCF + G-CSF-treated TBI mice showed a significantly reduced latency to find the hidden platform compared to the TBI vehicle controls. The mice in the TBI vehicle control group spent significantly longer time to find the hidden platform than those of sham controls, while the escape latency appeared to be no different between the sham controls and SCF + G-CSF-treated TBI mice. The findings from the elevated plus maze test displayed a significant reduction of the posttraumatic anxiety and risk-taking behavior in the SCF + G-CSF-treated TBI mice when compared to the TBI vehicle controls. No difference was observed between the SCF + G-CSF-treated TBI mice and sham control mice in the elevated plus maze test. Histological data showed that SCF + G-CSF significantly reduced degenerating neurons and increased dendritic density. SCF + G-CSF treatment in the subacute phase of TBI inhibits TBI-induced neurodegeneration, enhances neural network rewiring, improves recovery in spatial learning and memory, and prevents the posttraumatic anxiety. These findings suggest a therapeutic potential of hematopoietic growth factors in brain repair after TBI.
This study was supported by Neurosurgery Research and Education Foundation (2015-16NREF).
D. Upadhya*†‡, B. Hattiangady*†‡, B. Shuai*†‡, A. Bates*†‡, A. K. Shetty*†‡
*Institute for Regenerative Medicine, Texas A&M Health Science Center College of Medicine, Temple, TX, USA
†Research Service, Olin E. Teague Veterans' Medical Center, Central Texas Veterans Health Care System, Temple, TX, USA
‡Department of Molecular and Cellular Medicine, Texas A&M Health Science Center College of Medicine, College Station, TX, USA
Status epilepticus (SE) evolves into chronic temporal lobe epilepsy (TLE) via numerous epileptogenic changes in the hippocampus. The features of TLE include complex partial seizures, hippocampal neurodegeneration, and comorbidities such as cognitive, memory, and mood impairments. Decreases in the number of various subclasses of γ-amino butyric acid-positive (GABAergic) interneurons and their axon terminals, and/or altered functional inhibition in the hippocampus are palpable brain modifications thought to contribute to the permanence of spontaneous recurrent seizures (SRS) and other comorbidities in TLE. This has led to a view that replacement of the lost GABAergic interneurons would enhance inhibitory synaptic neurotransmission in the epileptic hippocampus and thereby prevent/reduce the incidence of SRS. In this study, using young male rats, we probed the efficiency of grafting human GABAergic progenitors [derived from human induced pluripotent stem cells (hiPSCs) through directed differentiation methods] into the hippocampus early after SE for alleviating SRS and the associated comorbidities. We induced SE through graded intraperitoneal injections of kainic acid, terminated acute seizures 2 h after SE by an injection of diazepam, and grafted hiPSC-derived GABAergic progenitor cells into the hippocampus (3 grafts/side, ~50,000 live cells/graft) a week after the SE episode. An additional cohort of age-matched rats that underwent similar SE was maintained as the “epilepsy-only” control group. Evaluation of behavioral SRS at 3 months after transplantation revealed that animals transplanted with hiPSC-derived GABAergic progenitors displayed reduced frequency and intensity of SRS, in comparison to “epilepsy-only” controls. The reductions were ~56% for the frequency of all SRS, ~71% for Stage-V SRS (the most severe form of SRS), and ~56% for the percentage of time spent in seizure activity. Furthermore, investigation through behavioral tests for novel object recognition (via novel object recognition test, NORT), place recognition (by object location test, OLT), and pattern separation (via pattern separation test, PST) confirmed preservation of both cognitive and memory function in rats that received human GABAergic progenitor cell grafts into the hippocampus after SE. Similarly, examining through behavioral tests for anhedonia/depressive-like behavior (via sucrose preference test, SPT; and novelty suppressed feeding test, NSFT) uncovered normal mood function in rats receiving human GABAergic progenitor cell grafts. In contrast, rats in the “epilepsy-only” group displayed considerable cognitive, memory, and mood impairments when examined through the above tests. Analyses of graft cell survival and differentiation and the effects of grafting on the host are currently in progress. Collectively, the results obtained so far underscore that early grafting intervention after SE with human GABAergic progenitors is efficacious not only for restraining SRS but also for preventing SE-induced cognitive, memory and mood impairments.
Supported by Emerging Technology Funds from the State of Texas to A.K.S and Department of Veterans Affairs Merit Award to A.K.S.
S. C. Vermilyea*†, S. Guthrie†, M. Meyer†, K. Smuga-Otto†, K. Braun†, S. Howden‡, J. A. Thomson‡, S-C. Zhang§, T. G. Golos†¶#, M. E. Emborg*†**
*Neuroscience Training Program, University of Wisconsin–Madison, Madison, WI, USA
†Wisconsin National Primate Research Center, University of Wisconsin–Madison, Madison, WI, USA
‡Morgridge Institute for Research, University of Wisconsin–Madison, Madison, WI, USA
§Department of Neuroscience, University of Wisconsin–Madison, Madison, WI, USA
¶Department of Comparative Biosciences, University of Wisconsin–Madison, Madison, WI, USA
#Department of Obstetrics and Gynecology, University of Wisconsin–Madison, Madison, WI, USA
**Department of Medical Physics, University of Wisconsin–Madison, Madison, WI, USA
The common marmoset monkey (Callithrix jacchus) is an ideal species for modeling age-related disorders, such as Parkinson's disease (PD), due to their shorter life span compared to macaques. Neuronal differentiation for regenerative medicine approaches has been achieved from marmoset embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) derived from fetal tissues, yet production of patterned midbrain floorplate dopaminergic (DAergic) neurons from adult marmoset fibroblast-derived iPSCs have not been reported. The aim of this study was to pattern floorplate-derived midbrain DAergic neurons, and characterize the expression of pluripotent, neural ectoderm, neuroprogenitor, and mature neuronal subtype genes throughout the differentiation process of both marmoset ESCs and iPSCs. iPSCs were reprogrammed from adult marmoset skin fibroblasts. Pluripotency of ESC and iPSC colonies selected by morphological criteria was verified by: 1) RT-PCR performed using primers for marmoset octamer binding transcription factor 4 (OCT4), sex determining region Y box 2 (SOX2), NANOG, Krüppel-like factor 4 (KLF4), LIN28, and C-MYC genes; 2) triple immunofluorescent staining for OCT4/SOX2/NANOG; and 3) teratoma formation by cell line injection into the kidney capsule or testis of immune-compromised mice. A9 dopaminergic differentiation ventral patterning was achieved after sonic hedgehog (SHH) was added at 500 ng/ml for the first 4 days, followed by purmorphamine at 5 μM until day 16, when it was reduced to 0.2 μM and fibroblast growth factor 8b (FGF8b) at 100 ng/ml was added until day 28. Floorplate derivation was confirmed by a shift of expression from paired box 6 (PAX6) to forkhead box A2 (FOXA2). Anterior–posterior patterning was accomplished using CHIR99021 (0.4 μM). Colocalization of ortho-denticle homeobox 2 (OTX2) and engrailed homeobox 1 (EN-1) confirmed midbrain identity. For DAergic differentiation and maturation, suspension neurospheres were transferred to adherent culture on day 28 and exposed to brain-derived neurotrophic factor (BDNF), glial cell line-derived neurotrophic factor (GDNF), transforming growth factor (TGF)-β3, cyclic adenosine monophosphate (cAMP), and ascorbic acid in neural basal media. Immunocytochemistry for nestin, βIII-tubulin, and tyrosine hydroxylase (TH) confirmed neuroprogenitor and mature neural fates. Our results demonstrate marmoset iPSCs can be generated from adult marmoset skin fibroblasts and that marmoset ESCs and iPSCs can be patterned to become floorplate-derived midbrain DAergic neurons. These cells are currently being used as platforms to assess the impact of genomic editing of PD-related genes.
Acknowledgments: NIH grants P51OD011106, UL1TR000427, R01NS076352, R24OD019803, T32GM007507 and UW-Madison Bridge Funds.
D. R. Wakeman*, B. M. Hiller*, D. J. Marmion*, C. McMahon†, G. T. Corbett*, J. Ma†, J. H. Kordower*
*Department of Neurological Sciences, Rush University Medical Center, Chicago, IL, USA
†Cellular Dynamics International Inc., Madison, WI, USA
Cryopreservation of postmitotic, induced pluripotent stem cell-derived midbrain lineage dopamine (iPSC-mDA) neurons is a significant advancement for cell therapy in Parkinson's disease (PD). Here we demonstrate that cryopreserved iPSC-mDA neurons are reliably thawed with excellent viability and maintain biochemical and physiological signatures indicative of human midbrain dopamine neurons. We also examined the engraftment potential of iPSC-mDA neurons after transplantation into both the parkinsonian rodent brain up to 6 months postgrafting and the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) nonhuman primate brain up to 3 months posttransplantation. Immunohistochemical analysis demonstrated robust graft survival and maintenance of the midbrain dopaminergic phenotype with extensive fiber innervation into the host parenchyma. A long-term functional study revealed significant reversal in motor deficits in the 6-hydroxydopamine (6-OHDA)-lesioned rat model of PD that persisted for up to 6 months posttransplantation. Moreover, we found no evidence of cell proliferation, indicating safety in our initial studies. Investigational new drug (IND)-enabling studies are currently under way to ascertain whether cryopreserved iPSC-mDA neurons are both safe and efficacious at longer time points in both rodent and nonhuman primate models of PD. These results indicate considerable promise for the development of pluripotent cell-based therapies in PD.
D. Wang, S. Longo, M. Kyle, L.-R. Zhao
Department of Neurosurgery, State University of New York Upstate Medical University, Syracuse, NY, USA
S100 calcium-binding protein A9 (S100A9) has been regarded as a signature marker for inflammation. The biological function of S100A9 has been extensively studied in the immune system. However, the role of S100A9 in the central nervous system remains poorly understood. The current study is aimed at determining whether S100A9 is involved in neuron injury in the setting of experimental stroke. Cerebral cortical ischemia was used as a stroke model in this study. Male adult S100A9-/- and wild-type (WT) mice (C57BL/6) were subjected to cortical ischemia. Additional S100A9-/- mice and C57BL/6 mice without cortical ischemia served as sham operative controls. Cortical infarct volume was determined 48 h after ischemia through tetrazolium chloride (TTC) staining. Neutrophil infiltration into the ischemic brain was detected 1 week postischemia though determination of the protein levels of myeloperoxidase (MPO) using Western blot. Neurodegeneration was examined by Fluoro-Jade C staining 1 week after brain ischemia. During the period of 1 month to 6 months after induction of brain ischemia, several neurobehavioral tests were performed to evaluate poststroke depression (forced swimming), cognitive impairment (new object recognition), impairments in motor activity and exploratory behavior (open field), and deficits in somatosensory and motor function (adhesive tape removal). Mice lacking S100A9 showed a significant increase in infarct volume. As expected, the levels of MPO were significantly reduced in S100A9-/- mice 1 week postischemia. However, at the same time point (1 week poststroke), Fluoro-Jade C-positive degenerative neurons in the peri-infarct cortex were significantly greater in the S100A9-/- mice than those of WT mice. In the forced swimming testing, freezing time was significantly increased in S100A9-/- mice. In the new object recognition testing, S100A9-/- mice spent significantly less time in the new object zone. During the open field testing, a significant increase in the immobile time at the edge and corner zone was observed in S100A9-/-mice. Moreover, S100A9-/- mice showed a significantly prolonged time to remove the adhesive tape from the affected forepaws compared to C57BL/6 mice. These findings suggest that S100A9 is required for neuroprotection in the condition of brain ischemia. Preventing poststroke inflammation appears to exacerbate neuron loss. The mechanisms underlying S100A9-mediated neuroprotection after stroke will be further explored in future studies.
This study was supported by NIHNINDS (R01 NS060911) and George W. Perkins endowment.
A. E. Willing*†‡, R. Deichert§, R. Wood§, S. A. Girling¶, J. Gonzalez¶, D. F. Hernandez¶, E. Foran*, K. Kip¶#
*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
‡College of Pharmacy, University of South Florida, Tampa, FL, USA
§Tampa Jiu Jitsu, Tampa, FL, USA
¶College of Nursing, University of South Florida, Tampa, FL, USA
#Department of Epidemiology and Biostatistics, College of Public Health, University of South Florida, Tampa, FL, USA
Posttraumatic stress disorder (PTSD) is a disabling anxiety disorder that may occur after witnessing a traumatic event. While existing therapies often require a long-term, time-intensive, and costly commitment from the patient, the overall success rate remains low. Therefore, there is a need for alternative therapies that can effectively help our veterans and service personnel overcome the effects of PTSD and lead fully productive lives as they reintegrate into society. Anecdotal evidence suggests that routine practice of Jiu Jitsu, a martial art that focuses on self-defense and control, can reduce symptoms of PTSD, both psychological and physiological. The purpose of this project is to examine whether Jiu Jitsu effectively reduces symptoms of PTSD among US service members and veterans. To accomplish this objective, 7 male US active duty service members and veterans participated in and completed a 5-month Jiu Jitsu training program (the program is ongoing). The participants attended two 70-min classes per week for 20 weeks. Self-report measures were used to examine changes in PTSD and psychopathology symptoms over the course of the study. Study participants, the majority of whom were veterans (82%), ranged in age from 22 to 60 (mean = 34.5 ± 13.1 years). While most branches of the military were represented in our sample, the majority of participants served in either the Navy (29%) or Marine Corps (35%). Fifty percent served at least one overseas tour with 56.3% of all participants experiencing combat. The majority were deployed to either Iraq (30%) or Afghanistan (35%). Fully 58% of study participants reported previously seeking treatment for PTSD with treatments ranging from prescription medication to intensive psychotherapy. These individuals demonstrated a substantial, clinically meaningful, and statistically significant decrease in scores on the PTSD checklist for diagnostic and statistical manual of mental disorders (DSM) 5 (PCL-5), our primary instrument for measuring PTSD symptoms. In addition, there were significant decreases in global psychopathology, as measured by the 125-item Psychiatric Diagnostic Screening Questionnaire. Improvements on both measures were continuous throughout the study. These preliminary data suggest that Jiu Jitsu training may be a beneficial, cost-effective, and easily accessible therapy to assist service members and veterans with reintegration into civilian life.
Supported by the State of Florida, Senate Education Appropriations.
K.-J. Wu*, S.-J. Yu*, M. Airavaara†, Y. Wang*
*Center for Neuropsychiatric Research, National Health Research Institutes, Zhunan, Taiwan
†Institute of Biotechnology, Viikki Biocenter, University of Helsinki, Helsinki, Finland
We previously reported that pretreatment with 9-cis retinoic acid (9cRA) reduced brain infarction in stroke brain (Shen et al., J. Neurosci. Res., 2009). The purpose of this study was to examine the neuroregenerative effect of 9cRA in an animal model of stroke. Adult male rats received a 60-min right middle cerebral artery occlusion (MCAo). Animals were separated into two groups with similar infarction sizes, based on magnetic resonance imaging on day 2 after MCAo. 9cRA or vehicle was given noninvasively via an intranasal route starting from day 3 to day 15 after MCAo. We found that intranasal administration of 9cRA increased brain 9cRA level at 1 h after delivery. Behavioral measurements were carried out in automated activity chambers for 24 h at 2 (before administration of 9cRA or vehicle), 7, and 14 days after MCAo. No difference was found before 9cRA or vehicle injection on day 2. Rats receiving poststroke 9cRA treatment show enhanced recovery in motor function demonstrated by significant increases in horizontal activity, total distance traveled, number of movements, and vertical activity, compared to the control group. To further characterize the mechanisms of this functional recovery, animals were treated with bromodeoxyuridine (BrdU) or vehicle. We found that posttreatment with 9cRA significantly enhanced BrdU immunoreactivity in the subventricular zone and lesioned cortex in stroke rats. 9cRA significantly increased the density of BrdU+ cells coexpressing nestin and neuronal nuclei (NeuN) in the lesioned cortex. Taken together, our data support that 9cRA has a neuroregenerative effect in stroke animals. The functional recovery may relate to the de novo genesis of neurocircuits in the ischemic brain.
X. Yan*, M. Henderson*†, K. Trychta*, A. Yasgar†, A. Jadhav†, D. Maloney†, H. Sun†, A. Simeonov†, B. Harvey*
*Intramural Research Program, National Institute on Drug Abuse, Baltimore, MD, USA
†National Center for Advancing Translational Sciences, Rockville, MD, USA
The endoplasmic reticulum (ER) is essential for protein production and lipid metabolism. The ER also serves as the primary intracellular store of calcium. The ER calcium concentration is estimated to be 5,000-fold higher than the cytosolic calcium concentration. Evidence for the disruption of this gradient can be observed in various diseases including neurodegeneration, cardiovascular diseases, and diabetes. Understanding the role of ER calcium dysregulation in disease pathogenesis has been limited by the available techniques to longitudinally monitor ER calcium. Our laboratory recently developed a novel reporter protein, secreted ER calcium monitoring protein (SERCaMP), to study the ER [Ca2+]. In collaboration with the National Center for Advancing Translational Sciences, we developed a high throughput screen (HTS) to identify compounds capable of stabilizing ER calcium. Additional characterization of compounds identified through the HTS demonstrated robust neuroprotective effects in an in vitro oxygen glucose deprivation (OGD) model and has implications for treating brain ischemia. Overall, we describe a novel approach to monitoring ER calcium dysregulation and therapeutically targeting ER calcium to treat neurodegenerative disorders.
T. Yasuhara, M. Kameda, T. Agari, H. Takeuchi, J. Morimoto, M. Okazaki, K. Kin, A. Shinko, T. Baba, T. Morimoto, N. Tajiri, I. Date
Department of Neurological Surgery, Okayama University Graduate School of Medicine, Okayama, Japan
Electrical stimulation, including deep brain stimulation (DBS) and spinal cord stimulation (SCS), is an established treatment for Parkinson's disease (PD) and intractable pain. We have explored neuroprotective, neurorescue, and neurorestorative effects of electrical stimulation for stroke and PD model of rats. Electrical stimulation using an epidural electrode exerted neuroprotective effects in an acute stroke model of rats induced by transient middle cerebral artery occlusion as determined both behaviorally and immunohistochemically. Parenchymal stimulation showed neurorestorative effects in a chronic stroke model of rats at least partly through neurogenic effects. Electrical stimulation increased the intraparenchymal concentration of several trophic factors and growth factors, such as glial cell line-derived neurotrophic factor, brain-derived neurotrophic factor, and vascular endothelial growth factor. These factors might affect the neurotrophic, neurorestorative, and neurogeneic effects of electrical stimulation. Recently, migratory effects of electrical stimulation are explored. Epidural electrical stimulation at the ischemic penumbra increased the migratory distances of mesenchymal stem cells transplanted into the nonischemic cortex. Additionally, hippocampal long-term potentiation (LTP) is explored using the hypoperfusion model of rats induced by bilateral carotid artery occlusion. LTP enhanced neurogenesis in the dentate gyrus. An hour-long daily SCS with an epidural electrode at C1/2 level for 2 weeks exerted neuroprotective effects in a PD model of rats as determined both behaviorally and immunohistochemically. The neuroprotective effects were mediated at least partly by upregulation of vascular endothelial growth factor. We are currently exploring the neurorescue effects of SCS. Neurorescue effects of SCS have been partly demonstrated immunohistochemically. The therapeutic effects of stimulation might depend on the stimulation conditions including parameter (frequency, strength, and duration), stimulation timing, and stimulation site. Electrical stimulation might be a strong therapeutic option, although further experiments are required to explore the mechanisms and optimum conditions and to clarify the safety and efficacy.
D. R. Yavagal*†‡§
*Clinical Neurology and Neurosurgery, University of Miami & Jackson Memorial Hospitals, Miami, FL, USA
†Interventional Neurology, University of Miami & Jackson Memorial Hospitals, Miami, FL, USA
‡Endovascular Neurosurgery, University of Miami & Jackson Memorial Hospitals, Miami, FL, USA
§Interdisciplinary Stem Cell Institute, University of Miami & Jackson Memorial Hospitals, Miami, FL, USA
Stem cell therapy for ischemic stroke shows robust promise in preclinical and early clinical studies. Mesenchymal stromal cells (MSCs) are considered as one of the most attractive cell types for clinical application in stroke due to the absence of controversy, easy production, and low risk of tumorgenicity. Cells can be delivered to the brain via direct stereotactic, intraventricular (ICV), intravenous (IV) and intra-arterial (IA) delivery. Our research has focused on IA MSC delivery for stroke, in order to circumvent the lungs and liver, and to achieve maximal delivery of cells to the ischemic brain. Our previous study demonstrated that there was significantly lower infarction volume and better neurological outcome in IA MSC-treated rats. Microvascular ischemia from the MSCs can be completely avoided by lowering their dose adequately. We have also evaluated IA MSCs in a canine stroke model showing safety up to a maximal tolerated dose of 20 million cells per dose. We have also shown safety of IA autologous ALD-401 cells (bone marrow-derived adult stem cells that express high levels of aldehyde dehydrogenase; Aldagen/Cytomedix, Inc., Durham, NC, USA) in a phase 2a doubleblind, multicenter trial RECOVER-Stroke (NCT01273337). There is also a post hoc signal of effect seen in the trial. Thus, we propose next phase clinical trials of IA MSC-based stroke therapy while continuing to answer translational questions in our interventional stroke laboratory.
X. Yuan*, J. T. Rosenberg*†, Y. Liu*, A.-C. Tsai*, S. C. Grant*†, T. Ma*
*Chemical and Biomedical Engineering, The Florida State University, Tallahassee, FL, USA
†The National High Magnetic Field Laboratory, The Florida State University, Tallahassee, FL, USA
In the US, stroke is the primary cause of severe disability and the third leading cause of death. To date, the only FDA-approved drug for stroke is tissue plasminogen activator (tPA), a thrombolytic agent that has limited benefits to patients. Human mesenchymal stem cells (hMSCs) have emerged as an important cell source in stroke treatment and clinical trials have yielded promising results. Originally isolated from bone marrow as the progenitor cells responsible for the repair and regeneration of mesenchymal tissues, hMSC-based cell therapy is promising for stroke due to their trophic effects that promote endogenous tissue repair by forming a proregenerative molecular milieu. However, hMSCs have low cell survival and reduced secretory function posttransplantation in the stroke lesion, which significantly reduced their therapeutic potency. Studies have demonstrated that in vitro hMSC preconditioning by exposure to sublethal hypoxia and 3D aggregation can significantly improve hMSC migration, secretory functions, and resistance to ischemic stress posttransplantation. 3D aggregation also reprograms hMSCs and increases multipotentiality and expression of hypoxic-induced factor-1α (HIF-1α). High-field magnetic resonance imaging (MRI) posttransplantation shows that in vitro preconditioning enhances hMSC permanence and stroke lesion recovery in stroke rats. This talk will discuss the mechanism of in vitro preconditioning on hMSC properties and its effects on hMSC in vivo migration, stroke lesion recovery, as well as improvement in behavioral recovery.
X. Zeng*†, A. E. Ropper*†, J. E. Anderson*†, Z. Aljuboori*†, H. J. Lee‡, E. Y. Snyder§, R. L. Sidman¶, S. U. Kim‡, Y. D. Teng*†#
*Department of Neurosurgery, Harvard Medical School/Brigham and Women's Hospital, Boston, MA, USA
†Division of SCI Research, Veterans Affairs Boston Healthcare System, Boston, MA, USA
‡Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Korea
§Center for Stem Cells & Regenerative Medicine, Sanford-Burnham Medical Research Institute, La Jolla, CA, USA
¶Department of Neurology, Harvard Medical School/BIDMC, Boston, MA, USA
#Department of PM&R, Harvard Medical School/Spaulding Rehabilitation Hospital, Boston, MA, USA
Current clinical regimen of surgery, chemotherapy, and radiation shows very limited efficacy for treating spinal cord gliomas. To tackle the challenge, we, by C6 implantation of G55 tumor cells, established the first model of spinal cord glioma that manifested both somatomotosensory and autonomic dysfunctions. Human neural stem cells (hNSCs) were engineered to express either cytosine deaminase (F3.CD) or CD-thymidine kinase (F3.CD-TK); tumor tropic F3.CD and F3.CD-TK kill cancer cells locally via converting nontoxic 5-fluorocytosine (5-FC) and 5-FC + ganciclovir (GCV) into 5-fluorouracil (5-FU) and 5-FU + GCV-triphosphate, respectively, to trigger DNA fragmentation and apoptosis. Using in vitro assays we determined that F3.CD-TK treatment had a stronger oncolytic effect relative to F3.CD. We next transplanted DiI-labeled F3.CD-TK, F3.CD, or cell debris 1 mm rostral and caudal to C6 tumor mass 1 week post-G55 injection (n = 6/group), followed by 5-FC (500 mg/kg/5 ml/day) and GCV (25 mg/kg/1 ml/day) administrations (×5, IP). Evaluation of autonomic parameters (i.e., respiratory function, blood pressure, and body temperature) and hindlimb locomotion was performed. All rats showed pathophysiological signs typical for cervical spinal glioma. Compared to F3.CD or cell debris-injected controls, rats treated with F3.CD-TK plus 5-FC and GCV demonstrated significantly increased survival that was defined by retention of hindlimb stepping ability (p < 0.05; ANOVA). The treatment also significantly mitigated respiratory deficit and arterial blood pressure abnormality. Analytical assays revealed that F3.CD-TK extensively infiltrated the tumor mass and, after prodrug dosing, markedly impeded glioma growth, resulting in a topology of tumor microstructure that favored sparing of neural functions. Our data suggest that CD-TK dual gene-engineered hNSCs may be developed into an effective therapy for spinal cord gliomas.
J. H. Zhang
Loma Linda University, Loma Linda, CA, USA
The most popular ischemic stroke animal model is the middle cerebral artery occlusion and reperfusion (MCAO-R) model. Most neuroprotective or other brain protective agents are tested using this model for reperfusion brain injury. In this MCAO-R model, 25–50% infarction occurs after 2 h of ischemia and 22 h of reperfusion. However, clinically about 5% of stroke patients even in industrialized countries are treated to recanalize the occluded arteries to generate reperfusion. Globally less than 1% ischemic stroke patients are treated in time to reopen the arteries. After recanalization, patients' outcomes could be divided into three subtypes. In subtype A, patients are treated within the therapeutic window and end up with small or no infarction and good outcome. In subtype B, either severe stroke occurs or patients are treated too late, large infarction and hemorrhage occurs, and patients have poor outcomes. In subtype C, recanalization failed to reduce but enlarged infarction and led to disability. Only subtype C matches with the MCAO-R model in rodents or other animals, regardless of whether the model is produced by thrombus or by a suture. The majority of ischemic stroke patients, up to 90% or more, are not recanalization patients, and if reperfusion brain injury is different from ischemic brain injury, the MCAO-R model does not represent the majority of stroke patients. Without recanalization, most drug treatment could not effectively reach the ischemic core and penumbra, different from the recanalized MCAO-R model. If we divide no recanalization stroke patients into three subtypes, up to 30% are subtype A, which is a small stroke with relatively good outcome; more than 50% are subtype B, which is a large infarct with disability; and about 20% are subtype C, a large infarct with massive brain swelling and death. New animal models are needed to mimic subtypes B and C. The delayed recanalization rodent model, brain tissue evacuation rodent model, and awake or remote awake rodent model will be discussed in the presentation. The philosophy and overall approach are from bedside back to bench.
K. Zhang*†, J. Yan†, L. Wang†, X. Tian†, T. Zhang†, L. Guo†, B. Li*, W. Wang‡, X. Liu†
*Department of Biochemistry and Molecular and Cellular Biology, Georgetown University, Washington, DC, USA
†Department of Neurology, the Second Hospital of Hebei Medical University, Shijiazhuang, China
‡Mitochondria and Metabolism Center, University of Washington, Seattle, WA, USA
Mitochondrial dysfunction caused by Ca2+ overload plays an important role in ischemic-induced brain damage. The mitochondrial calcium uniporter (MCU), located on the mitochondrial inner membrane, is the major channel responsible for mitochondrial Ca2+ uptake. Activated proline-rich tyrosine kinase 2 (Pyk2) can directly phosphorylate MCU, which enhances mitochondrial Ca2+ uptake. It has been suggested that the Pyk2/MCU pathway is a novel therapy target in stress-induced cell apoptosis. Herein, we explored the role of the Pyk2/MCU pathway in ischemic stroke. Results from our study showed that Pyk2 and MCU expression increased in rats subjected to cerebral ischemia and in neuronal cells of the female neuroblastoma SH-SY5Y cell line exposed to glutamate-mediated excitotoxicity. Moreover, MCU-dependent mitochondrial Ca2+ accumulation contributed to neuronal mitochondrial swelling and morphological changes. At the same time we observed the functional impairment of neuronal mitochondria, including decrease of mitochondrial membrane potential (Ψm) and increase of intracellular free [Ca2+] ([Ca2+]i). Increasing expression of activated apoptotic proteins suggested that the consequence of this series of reactions is neuron death. The increase in [Ca2+]i and reactive oxygen species (ROS) generation and decrease in Ψm observed in glutamate-pretreated cells was abolished in the presence of PF-431396 (an inhibitor of Pyk2). In addition, both neuronal injury and neuronal apoptosis were blocked. This suggests that the Pyk2/MCU pathway is activated in the rat cerebral ischemia model and neuron excitotoxicity model, and that it is responsible for mitochondrial dysfunction and neuron apoptosis. Finally, our results showed that human urinary kallidinogenase (HUK) alleviates ischemic neuron injury possibly by inhibiting the Pyk2/MCU pathway involved in stabilizing mitochondrial membrane potential, reducing calcium influx, protecting mitochondria, inhibiting the generation of ROS, and decreasing the apoptosis of neurons. Elucidation of this new regulatory mechanism underlying neuron damage could provide novel neuron targets for the treatment of cerebral infarction.
L. V. Zholudeva, V. M. Spruance, T. Bezdudnaya, K. M. Negron, I. Fischer, M. A. Lane
Department of Neurobiology and Anatomy, Drexel University College of Medicine, Philadelphia, PA, USA
More than half of all spinal cord injuries (SCIs) occur at the cervical level resulting in some degree of respiratory deficiency. Although there has been increasing evidence for spontaneous neuroplasticity and improvements in respiration, functional recovery remains limited. Thus, there is an urgent need for the development of therapies targeting restoration of respiratory function following SCI. One therapeutic strategy gaining translational recognition is transplantation of neural precursor cells (NPCs) to repair the injured spinal cord. NPC transplants have been shown to survive, differentiate, form synaptic connections between donor and host cells, and enhance functional recovery. However, little is known about the development of donor neurons transplanted into the injured spinal cord, and whether the injured adult spinal cord selects for specific neuronal phenotypes that could limit therapeutic potential. The present work begins to track the development of cultured and noncultured NPCs 3, 7, 14, and 30 days after transplantation into a lateral, C3/4 contusion injury in the adult rat. Results from these ongoing studies reveal that grafted cells survive and differentiate into mature neurons and glia within the injured spinal cord 14 days posttransplantation. However, cell survival and neurite outgrowth appears to be greater in noncultured grafts. The neuronal precursor phenotype also differs between cultured and noncultured donor NPCs. These ongoing experiments are beginning to identify donor cell phenotypes and offer insight into how the internal milieu of the injured spinal cord might select for specific neurons and their outgrowth into host tissue.
A. Chou*†, N. Day*‡, T. Jopson*‡, F. Cho*†, C. Sidrauski§¶, P. Walter¶#, S. Rosi*†‡§
*Brain and Spinal Injury Center, University of California, San Francisco, CA, USA
†Neuroscience Graduate Program, University of California, San Francisco, CA, USA
‡Department of Physical Therapy Rehabilitation Science, University of California, San Francisco, CA, USA
§Department of Neurological Surgery, University of California, San Francisco, CA, USA
¶Department of Biochemistry and Biophysics, University of California, San Francisco, CA, USA
#Howard Hughes Medical Institute, University of California, San Francisco, CA, USA
The integrated stress response (ISR) controls mRNA translation by phosphorylation of the eukaryotic translation initiation factor eIF2. ISRIB is a drug-like small-molecule ISR inhibitor (in-cell EC50 = 5 nM) that enhances memory consolidation in normal animals. Loss of cognitive functions and sustained ISR are associated with numerous neurological conditions, including traumatic brain injury (TBI). We investigated the efficacy of ISRIB on the cognitive deficits induced by TBI using two different animal models tested in two different cognitive tasks. First, focal contusion injury was induced by controlled cortical impact (CCI) in C57B6 mice. Spatial learning and memory retention were measured in the radial arm water maze starting 28 days after injury. Either ISRIB (2.5 mg/kg) or vehicle was administered intraperitoneally the day prior to and at the end of each training day for a total of three injections. In agreement with previous reports, TBI animals receiving vehicle only failed to learn the location of the escape platform. In striking contrast, ISRIB-treated TBI animals learned as well as the noninjured animals. Memory consolidation was measured 24 h and 7 days after training in the absence of any additional treatment. At both times, ISRIB-treated TBI animals remembered the location of the hidden platform indistinguishable from noninjured controls. Thus, ISRIB completely restored the ability of the injured animals to learn and remember a new task. Most importantly, this memory was fully consolidated, as it could be recalled without further treatment. Second, diffuse TBI was modeled by closed-head injury (CHI) in C57B6 mice and the delayed matching-to-place paradigm was used in a dry maze (modified Barnes maze) to assess working/episodic-like learning and memory. Fourteen days after injury either ISRIB (2.5 mg/kg) or vehicle was administered intraperitoneally prior to and then again at the end of each training day for a total of four injections. As for the CCI experiments, ISRIB-treated animals performed indistinguishable from uninjured controls, indicating that ISRIB treatment completely reversed the deficits induced by CHI. We conclude that in these models targeting the ISR at time points late after injury can completely reverse chronic loss of cognitive functions induced by head trauma.
O. Glushakova, A. Glushakov, R. Hayes
Banyan Biomarkers, Alachua, FL, USA
Traumatic brain injury (TBI) is a significant risk factor for chronic traumatic encephalopathy (CTE), Alzheimer's disease (AD), and Parkinson's disease (PD). Cerebral microbleeds, focal inflammation, and white matter damage are associated with many neurological and neurodegenerative disorders including CTE, AD, PD, vascular dementia, stroke, and TBI. This talk will summarize studies on microvascular abnormalities observed at acute and chronic stages following TBI in rats induced by controlled cortical impact (CCI) and examines pathological processes associated with these abnormalities including neuroinflammation, caspase 3-mediated apoptosis, tau pathologies, and abnormal angiogenesis. The experimental TBI resulted in focal microbleeds that were related to the magnitude of injury. At the lower magnitude of injury, microbleeds gradually increased over the 3-month duration of the study. Immunohistochemistry (IHC) revealed TBI-induced focal abnormalities including blood–brain barrier (BBB) damage (IgG), endothelial damage [intercellular adhesion molecule 1 (ICAM1)], activation of reactive microglia [ionized calcium binding adaptor molecule 1 (Iba1)], gliosis [glial fibrillary acidic protein (GFAP)], and macrophage-mediated inflammation [cluster of differentiation 68 (CD68)], all showing different temporal profiles. At chronic stages (up to 3 months), apparent myelin loss (Luxol fast blue) and scattered deposition of microbleeds were observed. Microbleeds were surrounded by glial scars and colocalized with CD68 and IgG puncta stainings, suggesting that localized BBB breakdown and inflammation were associated with vascular damage. In addition, TBI resulted in increased level of cleaved caspase 3 in both white and gray matter at the latest stages following injury. IHC staining of cleaved caspase 3 was gradually increased over the 3-month duration of the study in the corpus callosum and thalamus. In corpus callosum, IHC with CD34 revealed TBI-induced microvascular abnormalities that were characterized by proliferation, irregular capillary formation, and atypical structure of the new vessels. Furthermore, increased perivascular accumulation levels of caspase-cleaved tau was observed at 2 and 3 month after injury. In addition, fluorescent costaining experiments demonstrated colocalization of caspase 3 with SMI-71 (antibody for a BBB-specific endothelial protein) and cleaved caspase 3 with CD68, suggesting involvement of apoptosis and delayed neuroinflammation in the mechanisms of microvascular damage in the corpus callosum. In conclusion, these results indicate that evolving white matter degeneration following experimental TBI is associated with significantly delayed microvascular damage and focal microbleeds that are temporally and regionally associated with development of punctate BBB breakdown and progressive inflammatory responses, abnormal angiogenesis, and perivascular tau accumulation. Increased understanding of the mechanisms underlying delayed microvascular damage and delayed apoptosis following TBI could provide novel insights into chronic pathological responses to TBI and potential common mechanisms underlying TBI and neurodegenerative diseases.