Adult neurogenesis: From canaries to the clinic

Neuronal precursor cells persist in the adult vertebrate forebrain, residing primarily in the ventricular/subventricular zone (SZ). In vivo, SZ precursors yield progeny which may die or give rise to glia. Yet they may also generate neurons, which are recruited to restricted regions such as the avian telencephalon and mammalian olfactory bulb. The survival of neurons arising from adult progenitors is dictated by both the availability of a permissive pathway for migration and the environment into which migration occurs. In the songbird higher vocal center (HVC), both humoral and contact-mediated signals modulate the migration and survival of new neurons, through an orchestrated set of hormonally regulated paracrine interactions. New neurons of the songbird brain depart the SZ to enter the brain parenchyma by migrating upon radial guide fibers, which emanate from cell bodies in the ventricular epithelium. The radial guide cells coderive with new neurons from a common progenitor, which is widespread throughout the songbird SZ. Neural precursors are also widely distributed in the adult mammalian SZ, although it is unclear whether avian and mammalian progenitor cells are homologous: Whereas neuronal recruitment persists throughout much of the songbird forebrain, in mammals it is limited to the olfactory bulb. In humans, the adult SZ appears to largely cease neurogenesis in vivo, although it, too, can produce neurons in vitro. In both rats and humans, the differentiation and survival of neurons arising from the postnatal SZ may be regulated by access to postmitotic trophic factors. Indeed, serial application of fibroblast growth factor-2 (FGF-2) and brain-derived neurotrophic factor (BDNF) has allowed the generation and maintenance of neurons from the adult human SZ. This suggests the feasibility of inducing neurogenesis in the human brain, both in situ and through implanted progenitors. In this regard, using cell-specific neural promoters coupled to fluorescent reporters, defined progenitor phenotypes may now be isolated by fluorescence-activated cell sorting. Together, these findings give hope that structural brain repair through induced neurogenesis and neurogenic implants will soon be a clinical reality. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 267–286, 1998     

Ependymal/subependymal zone cells of postnatal and adult songbird brain generate both neurons and nonneuronal siblings in vitro and in vivo

The songbird forebrain continues to generate neurons in adulthood, from precursor cells located in the ependymal/subependymal zone (SZ) over the mediocaudal neostriatum. Precursor mitosis is followed by migration of neuronal daughter cells into the underlying forebrain, along radial fibers derived from the SZ. To define the ontogeny of both the new neurons and their radial guide cells, we employed retroviral insertion of the lacZ gene into neostriatal SZ precursor cells derived from postnatal and adult songbirds. We found that single SZ cells generate both neurons and substrate glia in vitro, and in an analogous fashion, both neurons and radial cells in vivo. This suggests that newly generated neurons and radial cells of the adult avian brain derive from a common pluripotential progenitor. © 1996 John Wiley & Sons, Inc.     

Hu protein as an early marker of neuronal phenotypic differentiation by subependymal zone cells of the adult songbird forebrain

The avian forebrain exhibits neurogenesis in adulthood, with neuronal production from ependymal/subependymal zone (SZ) precursor cells. To follow the commitment of newborn cells to neuronal lineage, we used their expression of the Hu family of neuronal RNA-binding proteins to identify them before their migration from the SZ. Adult canaries were injected with [³H]thymidine as a marker of DNA replication, sacrificed after varying intervals, stained for Hu, and autoradiographed. We found that Hu was not expressed by premitotic precursor cells, but rather appeared within hours in their neuronal progeny, which did not embark on parenchymal migration until 4 to 7 days later. Hu was expressed by all neurons, but not glia, both in vivo and in vitro, as determined by ultrastructural analysis as well as co-localization of Hu and cell-type selective antigens. In addition, co-staining for Hu and N-cadherin, whose expression is down-regulated on neuronal emigration from the SZ, revealed their initial co-expression by neuronal daughter cells still within the SZ. These results suggest that Hu expression may be used as a very early indicator of neuronal differentiation by SZ cells. Furthermore, the data indicate that in the adult avian brain, neuronal phenotype is established within hours of precursor mitosis, even though the neuronal daughter cells do not initiate parenchymal migration for at least 4 days thereafter, following their down-regulation of N-cadherin. © 1995 John Wiley & Sons, Inc.     

Nucleoside diphosphate kinase Nm23-M1 involves in oligodendroglial versus neuronal cell fate decision in vitro

The adult glial progenitor cells were recently shown to be able to produce neurons in central nervous system (CNS) and to become multipotent in vitro. Although the fate decision of glial progenitors was studied extensively, the signals and factors which regulate the timing of neuronal differentiation still remain unknown. To elucidate the mechanisms underlying the neuronal differentiation from glial progenitors, we modified the gene expression profile in NG2⁺ glial progenitor cells using enhanced retroviral mutagen (ERM) technique followed by phenotype screening to identify possible gene(s) responsible for glial-neuronal cell fate determination. Among the identified molecules, we found the gene named non-metastatic cell 1 which encodes a nucleoside diphosphate kinase protein A (Nm23-M1 or NME1). So far, the Nm23 members have been shown to be involved in various molecular processes including tumor metastasis, cell proliferation, differentiation and cell fate determination. In the present study, we provide evidence suggesting the role of NME1 in glial-neuronal cell fate determination in vitro. We showed that NME1 is widely expressed in neuronal structures throughout adult mouse CNS. Our immunohistochemical results revealed that NME1 is strongly colocalized with NF200 through white matter of spinal cord and brain. Interestingly, NME1 overexpression in oligodendrocyte progenitor OLN-93 cells potently induced the acquisition of neuronal fate, while its silencing was shown to promote oligodendrocyte differentiation. Furthermore, we demonstrated that dual-functional role of NME1 is achieved through cAMP-dependent protein kinase (PKA). Our data therefore suggested that NME1 acts as a switcher or reprogramming factor which involves in oligodentrocyte versus neuron cell fate specification in vitro.     

Neuronal precursors of the adult rat subependymal zone persist into senescence,with no decline in spatial extent or response to BDNF

The adult mammalian brain continues to harbor ependymal/subependymal zone (SZ) precursor cells, which can give rise to neurons in vitro. In adult rats, explants of the rostral 6–7 mm of the SZ give rise to neurons in vitro, and over this entire expanse, neuronal survival is supported specifically by brain-derived neurotrophic factor (BDNF). We asked whether either the (a) spatial distribution, (b) abundance, or (c) BDNF responsiveness of the neuronal precursor population was affected by age. Explants of three rostrocaudally defined regions were taken from both young and old rats (3 and 20 months old, respectively), and cultured in 2% fetal bovine serum-containing media with or without added BDNF (20 ng/mL). The extent of neuronal production by these explants varied only minimally with their level of derivation, such that substantial outgrowth was observed at each level tested. Neuronal outgrowth was marginally higher and more rapid in achieving its maximal extent in the 3-month-old rats compared with their aged counterparts, but neuronal outgrowth was robust at each age tested. The duration of survival of SZ-derived neurons did not differ between the young and old rats. At both ages, BDNF supported the survival of these new adult neurons. The extent of BDNF's influence was independent of both the age of the donor rat and the rostrocaudal level at which the parent SZ explant was taken. Thus, the neuronal precursors of the rat brain persist into senescence; the size of the precursor pool attenuates minimally with age, and its spatial extent remains constant. The neurons generated from these precursors can respond to BDNF throughout life. © 1997 John Wiley & Sons, Inc. J Neurobiol 32: 554–566, 1997     

Sequential RARβ and α signalling in vivo can induce adult forebrain neural progenitor cells to differentiate into neurons through Shh and FGF signalling pathways

We show here the role of retinoic acid receptor (RAR) β and α signalling in proliferation and differentiation of endogenous adult forebrain neural progenitor cells (NPCs). RARβ activation stimulates Sonic hedgehog signalling (Shh), and induces the proliferation of the NPCs. They can be induced to become Doublecortin (DCX) expressing migrating neuroblasts by RARα signalling, some of which differentiate into cholinergic neurons. The same signalling pathways cause the proliferation of embryonic forebrain NPCs. These cells express glial fibrillary acidic protein (GFAP) and are predominantly uni/bipolar, two characteristics of neuronal progenitor cells. We further show that fibroblast growth factor (FGF) signalling, induces the expression of the retinoic acid degrading enzyme cytochrome P450 (cyp) 26a1, and that one of its products, 4-oxo-RA, mimics the action of the RARα agonist in the differentiation of the NPCs into cholinergic neurons.     

Purinergic signaling regulates neural progenitor cell expansion and neurogenesis

Neural stem and progenitor cells typically exhibit a density-dependent survival and expansion, such that critical densities are required below which clonogenic progenitors are lost. This suggests that short-range autocrine factors may be critical for progenitor cell maintenance. We report here that purines drive the expansion of ventricular zone neural stem and progenitor cells, and that purine receptor activation is required for progenitor cells to be maintained as such. Neural progenitors expressed P2Y purinergic receptors and mobilized intracellular calcium in response to agonist. Receptor antagonists suppressed proliferation and permitted differentiation into neurons and glia in vitro, while subsequent removal of purinergic inhibition restored progenitor cell expansion. Real-time bioluminescence imaging of extracellular ATP revealed that the source of extracellular nucleotides are the progenitor cells themselves, which appear to release ATP in episodic burst events. Enzyme histochemistry of the adult rat brain for ectonucleotidase activity revealed that NTDPase, which acts to degrade active ATP and thereby clears it from areas of active purinergic transmission, was selectively localized to the subventricular zone and the dentate gyrus, regions in which neuronal differentiation proceeds from the progenitor cell pool. These data suggest that purine nucleotides act as proliferation signals for neural progenitor cells, and thereby serve as negative regulators of terminal neuronal differentiation. As a result, progenitor cell-derived neurogenesis is thus associated with regions of both active purinergic signaling and modulation thereof.     

Generation of a radial-like glial cell line

Rat C6 glioma is a cell line that has been used extensively as a model of astroglia. Although this cell line retains many of the properties of developing glia, it does not resemble morphologically the specialized form of glia found embryonically, the radial glia. In experiments designed to study a mutant form of receptor protein tyrosine phosphatase β, we isolated a subclone of C6 called C6-R which, like radial glia, assumes a highly polarized radial-like morphology in culture. C6-R cells and, to a somewhat lesser extent, C6 cells, express cytoskeletal proteins found in developing astroglia including glial fibrillary acidic protein and RC1. As seen with radial glia, cerebellar granule cell bodies and neurites migrated along radial processes of C6-R cells in culture. Morphological analysis of dye-labeled cells injected into the developing forebrain revealed that a large fraction (∼60%) of the C6-R cells in the cortex assumed a radial orientation and about half of these (∼30%) made contact with the pial surface. In contrast, the parental C6 cells generally formed aggregates and only displayed a radial alignment when associated with blood vessels. These results suggest that we have generated a stable cell line from C6 glioma which has adopted certain key features of radial glia, including the ability to promote neuronal migration in culture and integrate radially in vivo in response to local cues. This cell line may be particularly useful for studying receptors on radial glia that mediate neuronal migration. © 1998 John Wiley & Sons, Inc. J Neurobiol 37: 291–304, 1998     

In vitro survival and differentiation of neurons derived from epidermal growth factor-responsive postnatal hippocampal stem cells: Inducing effects of brain-derived neurotrophic factor

Neural stem cells proliferate in vitro and form neurospheres in the presence of epidermal growth factor (EGF), and are capable of differentiating into both neurons and glia when exposed to a substrate. We hypothesize that specific neurotrophic factors induce differentiation of stem cells from different central nervous system (CNS) regions into particular fates. We investigated differentiation of stem cells from the postnatal mouse hippocampus in culture using the following trophic factors (20 ng/mL): brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT-3) and glial-derived neurotrophic factor (GDNF). Without trophic factors, 32% of stem cells differentiated into neurons by 4 days in vitro (DIV), decreasing to 10% by 14 DIV. Addition of BDNF (starting at either day 0 or day 3) significantly increased neuron survival (31–43% by 14 DIV) and differentiation. Morphologically, many well-differentiated neurons resembled hippocampal pyramidal neurons. 5′-Bromodeoxyuridine labeling demonstrated that the pyramidal-like neurons originated from stem cells which had proliferated in EGF-containing cultures. However, similar application of NT-3 and GDNF did not exert such a differentiating effect. Addition of BDNF to stem cells from the postnatal cerebellum, midbrain, and striatum did not induce these neuronal phenotypes, though similar application to cortical stem cells yielded pyramidal-like neurons. Thus, BDNF supports survival of hippocampal stem cell-derived neurons and also can induce differentiation of these cells into pyramidal-like neurons. The presence of pyramidal neurons in BDNF-treated hippocampal and cortical stem cell cultures, but not in striatal, cerebellar, and midbrain stem cell cultures, suggests that stem cells from different CNS regions differentiate into region-specific phenotypic neurons when stimulated with an appropriate neurotrophic factor. © 1998 John Wiley & Sons, Inc. J Neurobiol 35: 395–425, 1998     

The neuronal stem cell of the olfactory epithelium

The vertebrate olfactory epithelium (OE) is a system in which behavior of neuronal progenitor cells can be observed and manipulated easily. It is morphologically and functionally similar to embryonic germinal neuroepithelia, but is simpler in that it produces large numbers of a single type of neuron, the olfactory receptor neuron (ORN). The OE is amenable to tissue culture, gene transfer, and in vivo surgical approaches, and these have been exploited in experiments aimed at understanding the characteristics of OE neuronal progenitor cells. This has led to the realization that the ORN lineage contains at least three distinct stages of proliferating neuronal progenitor cells (including a stem cell), each of which represents a point at which growth control can be exerted. Neurogenesis proceeds continually in the OE, and studies in vivo have shown that this is a regulated process that serves to maintain the number of ORNs at a particular level. These studies suggest that OE neuronal progenitors—which are in close physical proximity to ORNs—can “read” the number of differentiated neurons in their environment and regulate production of new neurons accordingly. Putative neuronal stem cells of the OE have been identified in vitro, and studies of these cells indicate that ORNs produce a signal that feeds back to inhibit neurogenesis. This inhibitory signal may be exerted at the level of the stem cell itself. Recent studies to identify this signal, as well as endogenous stimulatory signals that may be important in regulating OE neurogenesis, are also discussed. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 190–205, 1998     

Coordination of proliferation and neuronal differentiation by the retinoblastoma protein family

Once neurons enter the post‐mitotic G0 phase during central nervous system (CNS) development, they lose their proliferative potential. When neurons re‐enter the cell cycle during pathological situations such as neurodegeneration, they undergo cell death after S phase progression. Thus, the regulatory networks that drive cell proliferation and maintain neuronal differentiation are highly coordinated. In this review, the coordination of cell cycle control and neuronal differentiation during development are discussed, focusing on regulation by the Rb family of tumor suppressors (including p107 and p130), and the Cip/Kip family of cyclin dependent kinase (Cdk) inhibitors. Based on recent findings suggesting roles for these families in regulating neurogenesis and neuronal differentiation, I propose that the Rb family is essential for daughter cells of neuronal progenitors to enter the post‐mitotic G0 phase without affecting the initiation of neuronal differentiation in most cases, while the Cip/Kip family regulates the timing of neuronal progenitor cell cycle exit and the initiation of neuronal differentiation at least in the progenitor cells of the cerebral cortex and the retina. Rb's lack of involvement in regulating the initiation of neuronal differentiation may explain why Rb family‐deficient retinoblastomas characteristically exhibit neuronal features.     

Embryonic stem cell-derived neural progenitors as non-tumorigenic source for dopaminergic neurons

AIM:To find a safe source for dopaminergic neurons,we generated neural progenitor cell lines from human embryonic stem cells.METHODS:The human embryonic stem(hES)cell line H9 was used to generate human neural progenitor(HNP)cell lines.The resulting HNP cell lines were differentiated into dopaminergic neurons and analyzed by quantitative real-time polymerase chain reaction and immunofluorescence for the expression of neuronal differentiation markers,including beta-III tubulin(TUJ1)and tyrosine hydroxylase(TH).To assess the risk of teratoma or other tumor formation,HNP cell lines and mouse neuronal progenitor(MNP)cell lines were injected subcutaneously into immunodeficient SCID/beige mice.RESULTS:We developed a fairly simple and fast protocol to obtain HNP cell lines from hES cells.These cell lines,which can be stored in liquid nitrogen for several years,have the potential to differentiate in vitro into dopaminergic neurons.Following day 30 of differentiation culture,the majority of the cells analyzed expressed the neuronal marker TUJ1 and a high proportion of these cells were positive for TH,indicating differentiation into dopaminergic neurons.In contrast to H9 ES cells,the HNP cell lines did not form tumors in immunodeficient SCID/beige mice within 6 mo after subcutaneous injection.Similarly,no tumors developed after injection of MNP cells.Notably,mouse ES cells or neuronal cells directly differentiated from mouse ES cells formed teratomas in more than 90%of the recipients.CONCLUSION:Our findings indicate that neural progenitor cell lines can differentiate into dopaminergic neurons and bear no risk of generating teratomas or other tumors in immunodeficient mice.     

Notch3 is necessary for neuronal differentiation and maturation in the adult spinal cord

Notch receptors are key regulators of nervous system development and promoters of neural stem cells renewal and proliferation. Defects in the expression of Notch genes result in severe, often lethal developmental abnormalities. Notch3 is generally thought to have a similar proliferative, anti‐differentiation and gliogenic role to Notch1. However, in some cases, Notch3 has an opposite, pro‐differentiation effect. Here, we show that Notch3 segregates from Notch1 and is transiently expressed in adult rat and mouse spinal cord neuron precursors and immature neurons. This suggests that during the differentiation of adult neural progenitor cells, Notch signalling may follow a modified version of the classical lateral inhibition model, involving the segregation of individual Notch receptors. Notch3 knockout mice, otherwise neurologically normal, are characterized by a reduced number of mature inhibitory interneurons and an increased number of highly excitable immature neurons in spinal cord laminae I–II. As a result, these mice have permanently lower nociceptive thresholds, similar to chronic pain. These results suggest that defective neuronal differentiation, for example as a result of reduced Notch3 expression or activation, may underlie human cases of intractable chronic pain, such as fibromyalgia and neuropathic pain.     

TGF‐beta signalling in the adult neurogenic niche promotes stem cell quiescence as well as generation of new neurons

Members of the transforming growth factor (TGF)‐β family govern a wide range of mechanisms in brain development and in the adult, in particular neuronal/glial differentiation and survival, but also cell cycle regulation and neural stem cell maintenance. This clearly created some discrepancies in the field with some studies favouring neuronal differentiation/survival of progenitors and others favouring cell cycle exit and neural stem cell quiescence/maintenance. Here, we provide a unifying hypothesis claiming that through its regulation of neural progenitor cell (NPC) proliferation, TGF‐β signalling might be responsible for (i) maintaining stem cells in a quiescent stage, and (ii) promoting survival of newly generated neurons and their functional differentiation. Therefore, we performed a detailed histological analysis of TGF‐β1 signalling in the hippocampal neural stem cell niche of a transgenic mouse that was previously generated to express TGF‐β1 under a tetracycline regulatable Ca‐Calmodulin kinase promoter. We also analysed NPC proliferation, quiescence, neuronal survival and differentiation in relation to elevated levels of TGF‐β1 in vitro and in vivo conditions. Finally, we performed a gene expression profiling to identify the targets of TGF‐β1 signalling in adult NPCs. The results demonstrate that TGF‐β1 promotes stem cell quiescence on one side, but also neuronal survival on the other side. Thus, considering the elevated levels of TGF‐β1 in ageing and neurodegenerative diseases, TGF‐β1 signalling presents a molecular target for future interventions in such conditions.     

Large-scale generation of human iPSC-derived neural stem cells/early neural progenitor cells and their neuronal differentiation

Induced pluripotent stem cell (iPSC)-based technologies offer an unprecedented opportunity to perform high-throughput screening of novel drugs for neurological and neurodegenerative diseases. Such screenings require a robust and scalable method for generating large numbers of mature, differentiated neuronal cells. Currently available methods based on differentiation of embryoid bodies (EBs) or directed differentiation of adherent culture systems are either expensive or are not scalable. We developed a protocol for large-scale generation of neuronal stem cells (NSCs)/early neural progenitor cells (eNPCs) and their differentiation into neurons. Our scalable protocol allows robust and cost-effective generation of NSCs/eNPCs from iPSCs. Following culture in neurobasal medium supplemented with B27 and BDNF, NSCs/eNPCs differentiate predominantly into vesicular glutamate transporter 1 (VGLUT1) positive neurons. Targeted mass spectrometry analysis demonstrates that iPSC-derived neurons express ligand-gated channels and other synaptic proteins and whole-cell patch-clamp experiments indicate that these channels are functional. The robust and cost-effective differentiation protocol described here for large-scale generation of NSCs/eNPCs and their differentiation into neurons paves the way for automated high-throughput screening of drugs for neurological and neurodegenerative diseases.     

GABAergic excitation promotes neuronal differentiation in adult hippocampal progenitor cells

Hippocampal activity influences neurogenesis in the adult dentate gyrus; however, little is known about the involvement of the hippocampal circuitry in this process. In the subgranular zone of the adult dentate gyrus, neurogenesis involves a series of differentiation steps from radial glia-like stem/progenitor (type-1) cells, to transiently amplifying neuronal progenitor (type-2) cells, to postmitotic neurons. In this study, we conducted GFP-targeted recordings of progenitor cells in fresh hippocampal slices from nestin-GFP mice and found that neuronal progenitor (type-2) cells receive active direct neural inputs from the hippocampal circuitry. This input was GABAergic but not glutamatergic. The GABAergic inputs depolarized type-2 cells because of their elevated [Cl(-)](i). This excitation initiated an increase of [Ca(2+)](i) and the expression of NeuroD. A BrdU-pulse labeling study with GABA(A)-R agonists demonstrated the promotion of neuronal differentiation via this GABAergic excitation. Thus, it appears that GABAergic inputs to hippocampal progenitor cells promote activity-dependent neuronal differentiation.