Characterization of the Netrin/RGMa receptor neogenin in neurogenic regions of the mouse and human adult forebrain

In the adult rodent forebrain, astrocyte‐like neural stem cells reside within the subventricular zone (SVZ) and give rise to progenitors and neuroblasts, which then undergo chain migration along the rostral migratory stream (RMS) to the olfactory bulb, where they mature into fully functional interneurons. Neurogenesis also occurs in the adult human SVZ, where neural precursors similar to the rodent astrocyte‐like stem cell and neuroblast have been identified. A migratory pathway equivalent to the rodent RMS has also recently been described for the human forebrain. In the embryo, the guidance receptor neogenin and its ligands netrin‐1 and RGMa regulate important neurogenic processes, including differentiation and migration. We show in this study that neogenin is expressed on neural stem cells (B cells), progenitor cells (C cells), and neuroblasts (A cells) in the adult mouse SVZ and RMS. We also show that netrin‐1 and RGMa are ideally placed within the neurogenic niche to activate neogenin function. Moreover, we find that neogenin and RGMa are also present in the neurogenic regions of the human adult forebrain. We show that neogenin is localized to cells displaying stem cell (B cell)‐like characteristics within the adult human SVZ and RMS and that RGMa is expressed by the same or a closely apposed cell population. This study supports the hypothesis that, as in the embryo, neogenin regulates fundamental signalling pathways important for neurogenesis in the adult mouse and human forebrain. J. Comp. Neurol. 518:3237–3253, 2010. © 2010 Wiley‐Liss, Inc.     

IGF-I promotes neuronal migration and positioning in the olfactory bulb and the exit of neuroblasts from the subventricular zone

While insulin-like growth factor-I (IGF-I) supports neuronal and glial differentiation in the CNS, it is largely unknown whether IGF-I also influences neuronal migration and positioning. We show here that the pattern of olfactory bulb (OB) layering is altered in Igf-I ^−/− mice. In these animals, Tbr1⁺-glutamatergic neurons are misplaced in the mitral cell layer (ML) and the external plexiform layer (EPL). In addition, there are fewer interneurons in the glomerular layer and the EPL of the Igf-I ^−/− mice, and fewer newborn neurons are incorporated into the OB from the forebrain subventricular zone (SVZ). Indeed, neuroblasts accumulate in the postnatal/adult SVZ of Igf-I ^−/− mice. Significantly, the positioning of Tbr1⁺-cells in a primitive ML is stimulated by IGF-I in cultured embryonic OB slices, an effect that is partially repressed by the phosphoinositide 3-kinase (PI3K) inhibitor. In OB cell cultures, IGF-I increases the phosphorylation of disabled1 (P-Dab1), an adaptor protein that is a target of Src family kinases (SFK) in the reelin signalling pathway, whereas reduced P-Dab1 levels were found in Igf-I ^−/− mice. Neuroblast migration from the rostral migratory stream (RMS) explants of postnatal Igf-I ^−/− was similar to that from Igf-I ^+/+ explants. However, cell migration was significantly enhanced by IGF-I added to the explants, an effect that was repressed by PI3K and SFK inhibitors. These findings suggest that IGF-I promotes neuronal positioning in the OB and support a role for IGF-I in stimulating neuroblast exit from the SVZ into the RMS, thereby promoting the incorporation of newly formed neurons into the OB.     

Conditional ablation and recovery of forebrain neurogenesis in the mouse

Forebrain neurogenesis persists throughout life in the rodent subventricular zone (SVZ) and hippocampal dentate gyrus (DG). Several strategies have been employed to eliminate adult neurogenesis and thereby determine whether depleting adult‐born neurons disrupts specific brain functions, but some approaches do not specifically target neural progenitors. We have developed a transgenic mouse line to reversibly ablate adult neural stem cells and suppress neurogenesis. The nestin‐tk mouse expresses herpes simplex virus thymidine kinase (tk) under the control of the nestin 2nd intronic enhancer, which drives expression in neural progenitors. Administration of ganciclovir (GCV) kills actively dividing cells expressing this transgene. We found that peripheral GCV administration suppressed SVZ‐olfactory bulb and DG neurogenesis within 2 weeks but caused systemic toxicity. Intracerebroventricular GCV infusion for 28 days nearly completely depleted proliferating cells and immature neurons in both the SVZ and DG without systemic toxicity. Reversibility of the effects after prolonged GCV infusion was slow and partial. Neurogenesis did not recover 2 weeks after cessation of GCV administration, but showed limited recovery 6 weeks after GCV that differed between the SVZ and DG. Suppression of neurogenesis did not inhibit antidepressant responsiveness of mice in the tail suspension test. These findings indicate that SVZ and DG neural stem cells differ in their capacity for repopulation, and that adult‐born neurons are not required for antidepressant responses in a common behavioral test of antidepressant efficacy. The nestin‐tk mouse should be useful for studying how reversible depletion of adult neurogenesis influences neurophysiology, other behaviors, and neural progenitor dynamics. J. Comp. Neurol. 514:567–582, 2009. © 2009 Wiley‐Liss, Inc.     

Characterization of the canine rostral ventricular‐subventricular zone: Morphological,immunohistochemical, ultrastructural,and neurosphere assay studies

The mammalian ventricular‐subventricular zone (V‐SVZ) presents the highest neurogenic potential in the brain of the adult individual. In rodents, it is mainly composed of chains of neuroblasts. In humans, it is organized in layers where neuroblasts do not form chains. The aim of this study is to describe the cytoarchitecture of canine V‐SVZ (cV‐SVZ), to assess its neurogenic potential, and to compare our results with those previously described in other species. We have studied by histology, immunohistochemistry (IHC), electron microscopy and neurosphere assay the morphology, cytoarchitecture and neurogenic potential of cV‐SVZ. Age groups of animals were performed. Histological and ultrastructural studies indicated that the cV‐SVZ is organized in layers as in humans, but including migratory chains as in rodents. Neural progenitors were organized in niches in the subependymal area and a decline in their number was observed with age. Adult‐young dogs contained migratory cells capable to expand and differentiate in vitro according with previous results obtained in rodents, primates, humans, pigs, and dogs. Some adult animals presented perivascular niches outside the V‐SVZ. Our observations evidence a great similarity between canine and human V‐SVZ indicating that the dog may be better representative of neurogenic events in humans, compared with rodents. Accordingly with our results, we conclude that dogs are a valuable animal model of adult neurogenesis in comparative and preclinical studies.     

Differential vascular permeability along the forebrain ventricular neurogenic niche in the adult murine brain

Adult neurogenesis is influenced by blood‐borne factors. In this context, greater or lesser vascular permeability along neurogenic niches would expose differentially neural stem cells (NSCs), transit amplifying cells (TACs), and neuroblasts to such factors. Here we evaluate endothelial cell morphology and vascular permeability along the forebrain neurogenic niche in the adult brain. Our results confirm that the subventricular zone (SVZ) contains highly permeable, discontinuous blood vessels, some of which allow the extravasation of molecules larger than those previously reported. In contrast, the rostral migratory stream (RMS) and the olfactory bulb core (OBc) display mostly impermeable, continuous blood vessels. These results imply that NSCs, TACs, and neuroblasts located within the SVZ are exposed more readily to blood‐borne molecules, including those with very high molecular weights, than those positioned along the RMS and the OBc, subregions in which every stage of neurogenesis also takes place. These observations suggest that the existence of specialized vascular niches is not a precondition for neurogenesis to occur; specialized vascular beds might be essential for keeping high rates of proliferation and/or differential differentiation of neural precursors located at distinct domains. © 2015 Wiley Periodicals, Inc.     

Green fluorescent protein bone marrow cells express hematopoietic and neural antigens in culture and migrate within the neonatal rat brain

Finding a reliable source of alternative neural stem cells for treatment of various diseases and injuries affecting the central nervous system is a challenge. Numerous studies have shown that hematopoietic and nonhematopoietic progenitors derived from bone marrow (BM) under specific conditions are able to differentiate into cells of all three germ layers. Recently, it was reported that cultured, unfractionated (whole) adult BM cells form nestin-positive spheres that can later initiate neural differentiation (Kabos et al., 2002). The identity of the subpopulation of BM cells that contributes to neural differentiation remains unknown. We therefore analyzed the hematopoietic and neural features of cultured, unfractionated BM cells derived from a transgenic mouse that expresses green fluorescent protein (GFP) in all tissues. We also transplanted the BM cells into the subventricular zone (SVZ), a region known to support postnatal neurogenesis. After injection of BM cells into the neurogenic SVZ in neonatal rats, we found surviving GFP+ BM cells close to the injection site and in various brain regions, including corpus callosum and subcortical white matter. Many of the grafted cells were detected within the rostral migratory stream (RMS), moving toward the olfactory bulb (OB), and some cells reached the subependymal zone of the OB. Our in vitro experiments revealed that murine GFP+ BM cells retained their proliferation and differentiation potential and predominantly preserved their hematopoietic identity (CD45, CD90, CD133), although a few expressed neural antigens (nestin, glial fibrillary acdiic protein, TuJ1).     

Transiently impaired neurogenesis in MPTP mouse model of Parkinson's disease

It is still being debated whether neurogenesis in the subventricular zone (SVZ) is enhanced in response to 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) injury in the adult mouse brain. Our previous studies provided evidence that MPTP induces apoptosis of migrating neuroblasts (neural progenitor cells, A cells) in the SVZ and rostral migratory stream (RMS). We investigated cellular kinetics in the adult SVZ and olfactory bulb (OB) after MPTP damage. Cells were labeled with bromodeoxyuridine (BrdU), and the effects of MPTP on the survival and fate of migrating and residing neuroblasts were evaluated. Two days after BrdU labeling and MPTP treatment, the number of BrdU-positive cells in the SVZ and OB of MPTP-treated mice was significantly lower than in the SVZ and OB of saline controls. Additionally, fewer BrdU-positive cells migrated to the OB of treated mice than to that of saline controls, and the cells that did migrate diffused radially into the granule cell layer (GCL) when observed at 7, 14, and 28 days. In the OB GCL, the differentiation of BrdU-positive cells into mature neurons significantly attenuated 14 and 28 days after MPTP injury. Moreover, the impaired neurogenesis was followed by a recovery of A cells in the SVZ and OB, suggesting activation of the self-repair process as a result of MPTP-induced depletion of BrdU-positive cells. Our findings clarify the kinetics underlying neurogenesis in MPTP-treated mice and may contribute to the development of an animal model of Parkinson's disease, and the demonstration of cellular kinetics in SVZ may also provide a new insight into assessing neurogenesis in MPTP-treated mouse.     

Absence of striatal newborn neurons with mature phenotype following defined striatal and cortical excitotoxic brain injuries

Experimental stroke and excitotoxic brain lesion to the striatum increase the proliferation of cells residing within the ventricular wall and cause subsequent migration of newborn neuroblasts into the lesioned brain parenchyma. In this study, we clarify the different events of neurogenesis following striatal or cortical excitotoxic brain lesions in adult rats. Newborn cells were labeled by intraperitoneal injection of bromo–deoxy–uridine (BrdU), or by green fluorescent protein (GFP)-expressing lentiviral vectors injected into the subventricular zone (SVZ). We show that only neural progenitors born the first 5 days in the SVZ reside and expand within this neurogenic niche over time, and that these early labeled cells are more prone to migrate towards the striatum as neuroblasts. However, these neuroblasts could not mature into NeuN⁺ neurons in the striatum. Furthermore, we found that cortical lesions, close or distant from the SVZ, could not upregulate SVZ cell proliferation nor promote neurogenesis. Our study demonstrates that both the time window for labeling proliferating cells and the site of lesion are crucial when assessing neurogenesis following brain injury.     

Cytoarchitecture of the lateral ganglionic eminence and rostral extension of the lateral ventricle in the human fetal brain

The fetal development of the anterior subventricular zone (SVZ) involves the transformation of radial glia into neural stem cells, in addition to the migration of neuroblasts from the SVZ towards different regions in the brain. In adult rodents this migration from the anterior SVZ is restricted to the olfactory bulb following a rostral migratory stream (RMS) formed by chains of migratory neuroblasts. Similar to rodents, an RMS has been suggested in the adult human brain, where the SVZ remains as an active proliferative region. Nevertheless, a human fetal RMS has not been described and the presence of migratory neuroblasts in the adult remains controversial. Here we describe the cytoarchitecture of the human SVZ at the lateral ganglionic eminence late in the second trimester of development (23-24 weeks postconception). Cell organization in this region is heterogeneous along the ventricular wall, with GFAP-positive cells aligned to the ventricle. These cells coexpress markers for radial glia like GFAPδ, nestin, and vimentin. We also show the presence of abundant migratory neuroblasts in the anterior horn SVZ forming structures here denominated cell throngs. Interestingly, a ventral extension of the lateral ventricle suggests the presence of a putative RMS. Nevertheless, in the olfactory bulb neuroblast throngs or chain-like structures were not observed. The lack of these structures closer to the olfactory bulb could indicate a destination for the migratory neuroblasts outside the olfactory bulb in the human brain.     

Drebrin E regulates neuroblast proliferation and chain migration in the adult brain

F‐actin‐binding protein drebrin has two major isoforms: drebrin A and drebrin E. Drebrin A is the major isoform in the adult brain and is highly concentrated in dendritic spines, regulating spine morphology and synaptic plasticity. Conversely, drebrin E is the major isoform in the embryonic brain and regulates neuronal morphological differentiation, but it is also expressed in neurogenic regions of the adult brain. The subventricular zone (SVZ) is one of the brain regions where adult neurogenesis occurs. Neuroblasts migrate to the olfactory bulb (OB) and integrate into existing neuronal networks, after which drebrin expression changes from E to A, suggesting that drebrin E plays a specific role in neuroblasts in the adult brain. Therefore, to understand the role of drebrin E in the adult brain, we immunohistochemically analyzed adult neurogenesis using drebrin‐null‐mutant (DXKO) mice. In DXKO mice, the number of neuroblasts and cell proliferation decreased, although cell death remained unchanged. These results suggest that drebrin E regulates cell proliferation in the adult SVZ. Surprisingly, the decreased number of neuroblasts in the SVZ did not result in less neurons in the OB. This was because the survival rate of newly generated neurons in the OB increased in DXKO mice. Additionally, when neuroblasts reached the OB, the change in the migratory pathway from tangential to radial was partly disturbed in DXKO mice. These results suggest that drebrin E is involved in a chain migration of neuroblasts.     

Glial conversion of SVZ-derived committed neuronal precursors after ectopic grafting into the adult brain

In the adult mouse forebrain, large numbers of neuronal precursors, destined to become GABA- and dopamine-producing interneurons of the olfactory bulb (OB), are generated in the subventricular zone (SVZ). Although this neurogenic system represents a potential reservoir of stem and progenitor cells for brain repair approaches, information about the survival and differentiation of SVZ-derived cells in ectopic brain regions is still fragmentary. We show here that ectopic grafting of SVZ tissue gave rise to two morphologically distinguishable cell types displaying oligodendrocytic or astrocytic characteristics. Since SVZ tissue contains neuronal and glial progenitors, we used magnetic cell sorting to deplete A2B5+ glial progenitors from the dissociated SVZ and to positively select cells that express PSA-NCAM. This procedure allowed the purification of neuronal precursors expressing TUJ1, DCX and GAD65/67. Transplantation of these cells led again to the generation of the same two glial cell types, showing that committed interneuron precursors undergo glial differentiation outside their normal environment.     

Olfactory bulb core is a rich source of neural progenitor and stem cells in adult rodent and human

The olfactory bulb (OB) core is an extension of the rostral migratory stream and thus is a potential source of neural progenitor and neural stem cells. We characterized in vivo and in vitro neuronal progenitor and neural stem cells in the adult OB core. In mouse and rat, bromodeoxyuridine (BrdU) labeling showed that the OB core accumulates newly replicated cells. Nestin, a neuroepithelial stem cell marker, was enriched in the OB core. BrdU-positive cells were immunolabeled for nestin and TUC4, a marker for early postmitotic neurons. The distributions of cells labeled for BrdU, TUC4, and nestin were similarly concentrated in the OB core. Nestin- and TUC4-positive cells were also found in the OB of young and aged humans. Isolated and cultured OB core cells from adult rat and mouse had the capacity to generate numerous neurospheres. Adult OB core neurospheres were cryopreserved and subsequently cultured. Single cell clonal analysis of neurospheres revealed the capacity for self-renewal and multipotency. Cultured adult OB core cells differentiated into neurons, astrocytes, and oligodendrocytes. Some neurons expressed choline acetlytransferase, substance P, and glutamic acid decarboxylase. Basic fibroblast growth factor potentiated the self-renewal of cells and beta-nerve growth factor stimulated differentiation. OB-derived neural stem cells in coculture with skeletal muscle cells were induced to become neurons expressing choline acetyltransferase and substance P and formed neuromuscular synaptic junctions on myocytes displaying acetylcholinesterase-positive motor end plates. Cocultured OB-derived neural stem cells with myoblast cells also generated nonneural cell progeny. We conclude that the adult mammalian OB core is a reservoir of neural progenitor cells and pluripotent neural stem cells.     

A diacylglycerol lipase-CB2 cannabinoid pathway regulates adult subventricular zone neurogenesis in an age-dependent manner

The subventricular zone (SVZ) is a major site of neurogenesis in the adult. We now show that ependymal and proliferating cells in the adult mouse SVZ express diacylglycerol lipases (DAGLs), enzymes that synthesise a CB1/CB2 cannabinoid receptor ligand. DAGL and CB2 antagonists inhibit the proliferation of cultured neural stem cells, and the proliferation of progenitor cells in young animals. Furthermore, CB2 agonists stimulate progenitor cell proliferation in vivo, with this effect being more pronounced in older animals. A similar response was seen with a fatty acid amide hydrolase (FAAH) inhibitor that limits degradation of endocannabinoids. The effects on proliferation were mirrored in changes in the number of neuroblasts migrating from the SVZ to the olfactory bulb (OB). In this context, CB2 antagonists reduced the number of newborn neurons appearing in the OB in the young adult animals while CB2 agonists stimulated this in older animals. These data identify CB2 receptor agonists and FAAH inhibitors as agents that can counteract the naturally observed decline in adult neurogenesis that is associated with ageing.     

Expression of trophinin and bystin identifies distinct cell types in the germinal zones of adult rat brain

In the adult brain, the subventricular zone (SVZ) is one of the regions where active neurogenesis occurs. Relatively few specific markers are available to distinguish different types of cells in the SVZ and rostral migratory stream (RMS) of adult brain. Here, we showed that trophinin and bystin, both of which are required for early embryo implantation during development, were expressed in the SVZ and RMS of the adult rat brain, but not in the brain of embryos and early postnatal animals. Trophinin-expressing cells were immunopositive for both Ki-67 and nestin in the SVZ. Some of the trophinin-positive cells did not express either the type A cell marker polysialylated weakly adhesive form of the neural cell adhesion molecule (PSA-NCAM) or the type B cell marker glial fibrillary acidic protein (GFAP). Double-label immunohistochemistry revealed that bystin-positive cells co-expressed GFAP, Ki-67 and nestin, but not PSA-NCAM, suggesting that they are likely type B cells. Intracerebroventricular infusion of cytosine-beta-d-arabiofuranoside (Ara-C) eliminated trophinin-positive cells in the SVZ. Following its depletion, however, the remaining bystin-positive cells continued to divide and generate actively dividing trophinin-positive cells that were negative for PSA-NCAM, leading to reconstruction of SVZ network. These characteristics indicate that this subset of trophinin-positive cells in the SVZ is type C cells. Conversely in the RMS, trophinin co-localized with nestin and PSA-NCAM, suggesting that it is expressed in neuroblasts. Cultured neural precursor cells derived from the adult SVZ also expressed both trophinin and bystin. These findings provide insight into the molecular basis of adult neurogenesis in the SVZ and RMS.     

Telomerase activity in the subventricular zone of adult mice

The subventricular zone (SVZ) is the most active site for the production of new neurons in the adult mouse brain. Neural stem cells in the adult SVZ give rise to neuroblasts that travel via the rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into interneurons. The enzyme telomerase has been identified in other population of stem cells and is necessary for the synthesis of telomeric DNA to prevent chromosomal shortening, end-to-end fusions, and apoptosis during successive rounds of cell division. However, previous studies have failed to detect telomerase in the adult mammalian brain. Here we demonstrate that telomerase is expressed by all brain regions shortly after birth, but becomes restricted to the SVZ and olfactory bulb in the adult mouse brain. Cultures of neural precursor cells or of migratory neuroblasts purified from the SVZ were each found to possess telomerase activity. After elimination of migrating neuroblasts and immature precursor cells in vivo by treatment with cytosine-beta-D-arabinofuranoside (Ara-C), telomerase activity was still detectable in the remaining SVZ, which includes a population of neural stem cells. Following withdrawal of Ara-C, telomerase activity subsequently increased with a time course that parallels regeneration of the SVZ network and RMS. Finally, intracranial surgery alone, which has previously been shown to increase the number of cells in the SVZ, produced higher telomerase levels in the SVZ. We conclude that telomerase is active in neural precursor cells of the adult mouse and suggest that its regulation is an important parameter for cellular proliferation to occur in the mammalian brain.     

Focal cerebral ischemia induces a multilineage cytogenic response from adult subventricular zone that is predominantly gliogenic

The purpose of this study was to ascertain the relative contribution of neural stem/progenitor cells (NSPCs) of the subventricular zone (SVZ) to lineages that repopulate the injured striatum following focal ischemia. We utilized a tamoxifen‐inducible Cre/loxP system under control of the nestin promoter, which provides permanent YFP labeling of multipotent nestin⁺ SVZ‐NSPCs prior to ischemic injury and continued YFP expression in all subsequent progeny following stroke. YFP reporter expression was induced in adult male nestin‐CreER^T2:R26R‐YFP mice by tamoxifen administration (180 mg kg⁻¹, daily for 5 days). Fourteen days later, mice were subjected to 60‐min transient middle cerebral artery occlusion (MCAO) and sacrificed at 2 days, 2 weeks, or 6 weeks post‐MCAO for phenotypic fate mapping of YFP⁺ cells using lineage‐specific markers. Migration of YFP⁺ cells from SVZ into the injured striatal parenchyma was apparent at 2 and 6 weeks, but not 2 days, post‐MCAO. At 2 weeks post‐MCAO, the average percent distribution of YFP⁺ cells within the injured striatal parenchyma was as follows: 10% Dcx⁺ neuroblasts, 15–20% oligodendrocyte progenitors, 59% GFAP⁺ astrocytes, and only rare NeuN⁺ postmitotic neurons. A similar phenotypic distribution was observed at 6 weeks, except for an increased average percentage of YFP⁺ cells that expressed Dcx⁺ (20%) or NeuN (5%). YFP⁺ cells did not express endothelial markers, but displayed unique anatomical relationships with striatal vasculature. These results indicate that nestin⁺ NSPCs within the SVZ mount a multilineage response to stroke that includes a gliogenic component more predominant than previously appreciated. © 2010 Wiley‐Liss, Inc.     

How a radial glial cell decides to become a multiciliated ependymal cell

The V‐SVZ adult neurogenic niche is located in the wall of the lateral ventricles and contains neural stem cells, with self‐renewing and differentiating ability and postmitotic multiciliated ependymal cells, an important structural and trophic component of the niche. The niche is established at postnatal stages from a subpopulation of radial glial cells, determined during embryogenesis. Radial glial cells constitute a heterogeneous population, which give rise, in addition to niche cellular components, to neurons and glial cells. The mechanisms that direct their fate commitment towards V‐SVZ niche cells are largely unknown. In the present review, we discuss recent findings on the signaling networks governing fate commitment decisions of radial glial cells towards multiciliated ependymal cells. We highlight the role of two novel factors: McIdas and GemC1/Lynkeas and the molecular pathways which they activate in order to promote ependymal cell differentiation. Finally, we discuss a possible crosstalk of known signaling pathways, such as Notch, STAT3, and BMPs, for the specification of ependymal versus adult neural stem cells in the V‐SVZ niche. GLIA 2017;65:1032–1042