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Although neural stem cells (NSCs) sustain continuous neurogenesis throughout the adult lifespan of mammals,they progressively exhibit proliferation defects that contribute to a sharp reduction in subventricular neurogenesis during aging. However,little is known regarding the early age-related events in neurogenic niches. Using a fluorescence-activated cell sorting technique that allows for the prospective purification of the main neurogenic populations from the subventricular zone (SVZ),we demonstrated an early decline in adult neurogenesis with a dramatic loss of progenitor cells in 4 month-old young adult mice. Whereas the activated and quiescent NSC pools remained stable up to 12 months,the proliferative status of activated NSCs was already altered by 6 months,with an overall extension of the cell cycle resulting from a specific lengthening of G1. Whole genome analysis of activated NSCs from 2- and 6-month-old mice further revealed distinct transcriptomic and molecular signatures,as well as a modulation of the TGFβ signalling pathway. Our microarray study constitutes a cogent identification of new molecular players and signalling pathways regulating adult neurogenesis and its early modifications. View Publication

We determined the embryonic origins of adult forebrain subventricular zone (SVZ) stem cells by Cre-lox fate mapping in transgenic mice. We found that all parts of the telencephalic neuroepithelium,including the medial ganglionic eminence and lateral ganglionic eminence (LGE) and the cerebral cortex,contribute multipotent,self-renewing stem cells to the adult SVZ. Descendants of the embryonic LGE and cortex settle in ventral and dorsal aspects of the dorsolateral SVZ,respectively. Both populations contribute new (5-bromo-2'-deoxyuridine-labeled) tyrosine hydroxylase- and calretinin-positive interneurons to the adult olfactory bulb. However,calbindin-positive interneurons in the olfactory glomeruli were generated exclusively by LGE-derived stem cells. Thus,different SVZ stem cells have different embryonic origins,colonize different parts of the SVZ,and generate different neuronal progeny,suggesting that some aspects of embryonic patterning are preserved in the adult SVZ. This could have important implications for the design of endogenous stem cell-based therapies in the future. View Publication

Memantine is classified as an NMDA receptor antagonist. We recently reported that memantine promoted the proliferation of neural progenitor cells and the production of mature granule neurons in the adult hippocampus. However,the molecular mechanism responsible for the memantine-induced promotion of cellular proliferation remains unknown. In this study we searched for a factor that mediates memantine-induced cellular proliferation,and found that pigment epithelium-derived factor (PEDF),a broad-acting neurotrophic factor,is up-regulated in the dentate gyrus of adult mice after the injection of memantine. PEDF mRNA expression increased significantly by 3.5-fold at 1 day after the injection of memantine. In addition,the expression level of PEDF protein also increased by 1.8-fold at 2 days after the injection of memantine. Immunohistochemical study using anti-PEDF antibody showed that the majority of the PEDF-expressing cells were protoplasmic and perivascular astrocytes. Using a neurosphere assay,we confirmed that PEDF enhanced cellular proliferation under the presence of fibroblast growth factor-2 (FGF-2) and epidermal growth factor (EGF) but was not involved in the multilineage potency of hippocampal progenitor cells. Over expression of PEDF by adeno-associated virus,however,did not stimulate cellular proliferation,suggesting PEDF per se does not promote cellular proliferation in vivo. These findings suggest that the memantine induced PEDF up-regulation is involved in increased proliferation of hippocampal progenitor cells. View Publication

Insight into the normal function of PrP(C),and how it can be subverted to produce neurotoxic effects,is provided by PrP molecules carrying deletions encompassing the conserved central region. The most neurotoxic of these mutants,Δ105-125 (called ΔCR),produces a spontaneous neurodegenerative illness when expressed in transgenic mice,and this phenotype can be dose-dependently suppressed by co-expression of wild-type PrP. Whether the toxic activity of ΔCR PrP and the protective activity or wild-type PrP are cell-autonomous,or can be exerted on neighboring cells,is unknown. To investigate this question,we have utilized co-cultures of differentiated neural stem cells derived from mice expressing ΔCR or wild-type PrP. Cells from the two kinds of mice,which are marked by the presence or absence of GFP,are differentiated together to yield neurons,astrocytes,and oligodendrocytes. As a surrogate read-out of ΔCR PrP toxicity,we assayed sensitivity of the cells to the cationic antibiotic,Zeocin. In a previous study,we reported that cells expressing ΔCR PrP are hypersensitive to the toxic effects of several cationic antibiotics,an effect that is suppressed by co-expression of wild type PrP,similar to the rescue of the neurodegenerative phenotype observed in transgenic mice. Using this system,we find that while ΔCR-dependent toxicity is cell-autonomous,the rescuing activity of wild-type PrP can be exerted in trans from nearby cells. These results provide important insights into how ΔCR PrP subverts a normal physiological function of PrP(C),and the cellular mechanisms underlying the rescuing process. View Publication

INTRODUCTION: Ischemic stroke is a leading cause of death and disability,but treatment options are severely limited. Cell therapy offers an attractive strategy for regenerating lost tissues and enhancing the endogenous healing process. In this study,we investigated the use of human embryonic stem cell-derived neural precursors as a cell therapy in a murine stroke model.backslashnbackslashnMETHODS: Neural precursors were derived from human embryonic stem cells by using a fully adherent SMAD inhibition protocol employing small molecules. The efficiency of neural induction and the ability of these cells to further differentiate into neurons were assessed by using immunocytochemistry. Whole-cell patch-clamp recording was used to demonstrate the electrophysiological activity of human embryonic stem cell-derived neurons. Neural precursors were transplanted into the core and penumbra regions of a focal ischemic stroke in the barrel cortex of mice. Animals received injections of bromodeoxyuridine to track regeneration. Neural differentiation of the transplanted cells and regenerative markers were measured by using immunohistochemistry. The adhesive removal test was used to determine functional improvement after stroke and intervention.backslashnbackslashnRESULTS: After 11 days of neural induction by using the small-molecule protocol,over 95% of human embryonic stem-derived cells expressed at least one neural marker. Further in vitro differentiation yielded cells that stained for mature neuronal markers and exhibited high-amplitude,repetitive action potentials in response to depolarization. Neuronal differentiation also occurred after transplantation into the ischemic cortex. A greater level of bromodeoxyuridine co-localization with neurons was observed in the penumbra region of animals receiving cell transplantation. Transplantation also improved sensory recovery in transplant animals over that in control animals.backslashnbackslashnCONCLUSIONS: Human embryonic stem cell-derived neural precursors derived by using a highly efficient small-molecule SMAD inhibition protocol can differentiate into electrophysiologically functional neurons in vitro. These cells also differentiate into neurons in vivo,enhance regenerative activities,and improve sensory recovery after ischemic stroke. View Publication

Gaucher disease (GD) is caused by insufficient activity of acid $\$-glucosidase (GCase) resulting from mutations in GBA1. To understand the pathogenesis of the neuronopathic GD,induced pluripotent stem cells (iPSCs) were generated from fibroblasts isolated from three GD type 2 (GD2) and 2 unaffected (normal and GD carrier) individuals. The iPSCs were converted to neural precursor cells (NPCs) which were further differentiated into neurons. Parental GD2 fibroblasts as well as iPSCs,NPCs,and neurons had similar degrees of GCase deficiency. Lipid analyses showed increases of glucosylsphingosine and glucosylceramide in the GD2 cells. In addition,GD2 neurons showed increased $\$-synuclein protein compared to control neurons. Whole cell patch-clamping of the GD2 and control iPSCs-derived neurons demonstrated excitation characteristics of neurons,but intriguingly,those from GD2 exhibited consistently less negative resting membrane potentials with various degree of reduction in action potential amplitudes,sodium and potassium currents. Culture of control neurons in the presence of the GCase inhibitor (conduritol B epoxide) recapitulated these findings,providing a functional link between decreased GCase activity in GD and abnormal neuronal electrophysiological properties. To our knowledge,this study is first to report abnormal electrophysiological properties in GD2 iPSC-derived neurons that may underlie the neuropathic phenotype in Gaucher disease. View Publication

Reactive oxygen species (ROS) damage brain lipids,carbohydrates,proteins,as well as DNA and may contribute to neurodegeneration. We previously reported that ER- and oxidative stress cause neuronal apoptosis in infantile neuronal ceroid lipofuscinosis (INCL),a lethal neurodegenerative storage disease,caused by palmitoyl-protein thioesterase-1 (PPT1) deficiency. Polyunsaturated fatty acids (PUFA) are essential components of cell membrane phospholipids in the brain and excessive ROS may cause oxidative damage of PUFA leading to neuronal death. Using cultured neurons and neuroprogenitor cells from mice lacking Ppt1,which mimic INCL,we demonstrate that Ppt1-deficient neurons and neuroprogenitor cells contain high levels of ROS,which may cause peroxidation of PUFA and render them incapable of providing protection against oxidative stress. We tested whether treatment of these cells with omega-3 or omega-6 PUFA protects the neurons and neuroprogenitor cells from oxidative stress and suppress apoptosis. We report here that both omega-3 and omega-6 fatty acids protect the Ppt1-deficient cells from ER- as well as oxidative stress and suppress apoptosis. Our results suggest that PUFA supplementation may have neuroprotective effects in INCL. View Publication

Types A and B Niemann-Pick disease (NPD) are lysosomal storage disorders resulting from loss of acid sphingomyelinase (ASM) activity. We have used a knockout mouse model of NPD (ASMKO mice) to evaluate the effects of direct intracerebral transplantation of bone marrow-derived mesenchymal stem cells (MSCs) on the progression of neurological disease in this disorder. MSCs were transduced with a retroviral vector to overexpress ASM and were injected into the hippocampus and cerebellum of 3-week-old ASMKO pups. Transplanted cells migrated away from the injection sites and survived at least 6 months after transplantation. Seven of 8 treated mice,but none of the untreated controls,survived for textgreater or = 7 months after transplant. Survival times were greater in sex-matched than in sex-mismatched transplants. Transplantation significantly delayed the Purkinje cell loss that is characteristic of NPD,although the protective effect declined with distance from the injection site. Overall ASM activity in brain homogenates was low,but surviving Purkinje cells contained the retrovirally expressed human enzyme,and transplanted animals showed a reduction in cerebral sphingomyelin. These results reveal the potential of treating neurodegenerative lysosomal storage disorders by intracerebral transplantation of bone marrow-derived MSCs. View Publication

The role of epigenetic regulators in the control of adult neurogenesis is largely undefined. We show that the histone demethylase enzyme Kdm5b (Jarid1b) negatively regulates neurogenesis from adult subventricular zone (SVZ) neural stem cells (NSCs) in culture. shRNA-mediated depletion of Kdm5b in proliferating adult NSCs decreased proliferation rates and reduced neurosphere formation in culture. When transferred to differentiation culture conditions,Kdm5b-depleted adult NSCs migrated from neurospheres with increased velocity. Whole-genome expression screening revealed widespread transcriptional changes with Kdm5b depletion,notably the up-regulation of reelin (Reln),the inhibition of steroid biosynthetic pathway component genes and the activation of genes with intracellular transport functions in cultured adult NSCs. Kdm5b depletion increased extracellular reelin concentration in the culture medium and increased phosphorylation of the downstream reelin signaling target Disabled-1 (Dab1). Sequestration of extracellular reelin with CR-50 reelin-blocking antibodies suppressed the increase in migratory velocity of Kdm5b-depleted adult NSCs. Chromatin immunoprecipitation revealed that Kdm5b is present at the proximal promoter of Reln,and H3K4me3 methylation was increased at this locus with Kdm5b depletion in differentiating adult NSCs. Combined the data suggest Kdm5b negatively regulates neurogenesis and represses Reln in neural stem cells from the adult SVZ. View Publication