Endogenous factors derived from embryonic cortex regulate proliferation and neuronal differentiation of postnatal subventricular zone cell cultures

In rodents, the subventricular zone (SVZ) harbours neural stem cells that proliferate and produce neurons throughout life. Previous studies showed that factors released by the developing cortex promote neurogenesis in the embryonic ventricular zone. In the present report, we studied in the rat the possible involvement of endogenous factors derived from the embryonic cortex in the regulation of the development of postnatal SVZ cells. To this end, SVZ neurospheres were maintained with explants or conditioned media (CM) prepared from embryonic day (E) 13, E16 or early postnatal cortex. We demonstrate that early postnatal cortex-derived factors have no significant effect on SVZ cell proliferation or differentiation. In contrast, E13 and E16 cortex release diffusible, heat-labile factors that promote SVZ cell expansion through increased proliferation and reduced cell death. In addition, E16 cortex-derived factors stimulate neuronal differentiation in both early postnatal and adult SVZ cultures. Fibroblast growth factor (FGF)-2- but not epidermal growth factor (EGF)-immunodepletion drastically reduces the mitogenic effect of E16 cortex CM, hence suggesting a major role of endogenous FGF-2 released by E16 cortex in the stimulation of SVZ cell proliferation. The evidence we provide here for the regulation of SVZ cell proliferation and neuronal differentiation by endogenous factors released from embryonic cortex may be of major importance for brain repair research.     

HIV-1-infected and/or immune-activated macrophage-secreted TNF-alpha affects human fetal cortical neural progenitor cell proliferation and differentiation

Neurogenesis, tied to the proliferation, migration and differentiation of neural progenitor cells (NPC) is affected during neurodegenerative diseases, but how neurogenesis is affected during HIV-1 associated dementia (HAD) has not been fully addressed. Here we test the hypothesis that HIV-1-infected and/or immune-activated brain macrophages affect NPC proliferation and differentiation through the regulation of cytokines. We showed that human monocyte-derived macrophages (MDM) conditioned medium (MCM) induces a dose dependent increase in NPC proliferation. Conditioned media from lipopolysaccharide (LPS)-activated MDM (LPS-MCM) or HIV-infected MCM (HIV-MCM) induced a profound increase in NPC proliferation. HIV-infected and LPS-activated MCM (HIV+LPS-MCM) induced the most robust increase in NPC proliferation. Moreover, LPS-MCM and HIV+LPS-MCM decreased beta-III-tubulin and increased GFAP expression, demonstrating an induction of gliogenesis and inhibition of neurogenesis. The increase of NPC proliferation and gliogenesis correlated with increases in production of TNF-alpha by infected/activated MDM. Although both IL-1beta and TNF-alpha induced NPC proliferation and gliogenesis, these effects were only partially abrogated by soluble TNF-alpha receptors R1 and R2 (TNF-R1R2), but not by the IL-1 receptor antagonist (IL-1ra). This indicated that the HIV-1-infected/LPS-activated MCM-mediated effects were, in part, through TNF-alpha. These observations were confirmed in severe combined immunodeficient (SCID) mice with HIV-1 encephalitis (HIVE). In these HIVE mice, NPC injected with HIV-infected MDM showed more astrocyte differentiation and less neuronal differentiation compared to NPC injection alone. These observations demonstrated that HIV-1-infected and immune-activated MDM could affect neurogenesis through induction of NPC proliferation, inhibition of neurogenesis, and activation of gliogenesis.     

Glutamate enhances proliferation and neurogenesis in human neural progenitor cell cultures derived from the fetal cortex

Excitatory amino acids such as glutamate play important roles in the central nervous system. We previously demonstrated that a neurosteroid, dehydroepiandrosterone (DHEA), has powerful effects on the cell proliferation of human neural progenitor cells (hNPC) derived from the fetal cortex, and this effect is modulated through NMDA receptor signaling. Here, we show that glutamate can significantly increase the proliferation rates of hNPC. The increased proliferation could be blocked by specific NMDA receptor antagonists, but not other glutamate antagonists for kainate-AMPA or metabotropic receptors. The NR1 subunit of the NMDA receptor was detectable in elongated bipolar or unipolar cells with small cell bodies. These NR1-positive cells were colocalized with GFAP immunoreactivity. Detection of the phosphorylation of cAMP response element-binding protein (pCREB) revealed that a subset of NR1-positive hNPC could respond to glutamate. Furthermore, we hypothesized that glutamate treatment may affect mainly the hNPC with a radial morphology and found that glutamate as well as DHEA selectively affected elongated hNPC; these elongated cells may be a type of radial glial cell. Finally we asked whether the glutamate-responsive hNPC had an increased potential for neurogenesis and found that glutamate-treated hNPC produced significantly more neurons following differentiation. Together these data suggest that glutamate stimulates the division of human progenitor cells with neurogenic potential.     

Human embryonic neural stem cell transplantation increases subventricular zone cell proliferation and promotes peri‐infarct angiogenesis after focal cerebral ischemia

Neurogenesis and angiogenesis are two important processes that may contribute to the repair of brain injury after stroke. This study was designed to investigate whether transplantation of human embryonic neural stem cells (NSCs) into cortical peri‐infarction 24 h after ischemia effects cell proliferation in the subventricular zone (SVZ) and angiogenesis in the peri‐infarct zone. NSCs were prepared from embryonic human brains at 8 weeks gestation. Focal cerebral ischemia was induced by permanent occlusion of the middle cerebral artery of adult rats. Animals were randomly divided into two groups (n = 30, each) at 24 h after ischemia: NSC‐grafted and medium‐grafted groups. Toluidine blue staining and 5′‐bromo‐2′‐deoxyuridine (BrdU) or von Willebrand factor (vWF) immunohistochemistry were performed at 7, 14 and 28 days after transplantation. NSC transplantation increased the number of BrdU‐positive cells in the ischemic ipsilateral SVZ compared with the medium control at 7 days (P < 0.01). This difference in SVZ cell proliferation persisted at 14 days (P < 0.01), but was not significant at 28 days (P > 0.05). In addition, angiogenesis, as indicated by BrdU and vWF staining in cortical peri‐infarct regions, was augmented by 46% and 65% in NSC‐grafted rats versus medium‐grafted rats at 7 and 14 days, respectively (P < 0.05). However, this increase became non‐significant at 28 days (P > 0.05). Our results indicate that NSC transplantation enhances endogenous cell proliferation in the SVZ and promotes angiogenesis in the peri‐infarct zone, even if it is performed in the acute phase of ischemic injury.     

Alzheimer's disease modifies progenitor cell expression of monoamine oxidase B in the subventricular zone

In the adult brain, progenitor cells remaining in the subventricular zone (SVZ) are frequently identified as glial fibrillary acidic protein (GFAP)‐positive cells that retain attributes reminiscent of radial glia. Because the very high expression of monoamine oxidase B (MAO‐B) in the subventricular area has been related to epithelial and astroglial expression, we sought to ascertain whether it was also expressed by progenitor cells of human control and Alzheimer's disease (AD) patients. In the SVZ, epithelial cells and astrocyte‐like cells presented rich MAO‐B activity and immunolabeling. Nestin‐positive cells were found in the same area, showing a radial glia‐like morphology. When coimmunostaining and confocal microscopy were performed, most nestin‐positive cells showed MAO‐B activity and labeling. The increased progenitor activity in SVZ proposed for AD patients was confirmed by the positive correlation between the SVZ nestin/MAO‐B ratio and the progression of the disease. Nestin/GFAP‐positive cells, devoid of MAO‐B, can represent a distinct subpopulation of an earlier phase of maturation. This would indicate that MAO‐B expression takes place in a further step of nestin/GFAP‐positive cell differentiation. In the early AD stages, the discrete MAO‐B reduction, different from the severe GFAP decrease, would reflect the capacity of this population of MAO‐B‐positive progenitor cells to adapt to the neurodegenerative process. © 2010 Wiley‐Liss, Inc.     

Stromal cell-derived factor 1-mediated CXCR4 signaling in rat and human cortical neural progenitor cells

Stromal cell-derived factor 1 (SDF-1) and the chemokine receptor CXCR4 are highly expressed in the nervous system. Knockout studies have suggested that both SDF-1 and CXCR4 play essential roles in cerebellar, hippocampal, and neocortical neural cell migration during embryogenesis. To extend these observations, CXCR4 signaling events in rat and human neural progenitor cells (NPCs) were examined. Our results show that CXCR4 is expressed in abundance on rat and human NPCs. Moreover, SDF-1alpha induced increased NPCs levels of inositol 1,4,5-triphosphate, extracellular signal-regulated kinases 1/2, Akt, c-Jun N-terminal kinase, and intracellular calcium whereas it diminished cyclic adenosine monophosphate. Finally, SDF-1alpha can induce human NPC chemotaxis in vitro, suggesting that CXCR4 plays a functional role in NPC migration. Both T140, a CXCR4 antagonist, and pertussis toxin (PTX), an inactivator of G protein-coupled receptors, abrogated these events. Ultimately, this study suggested that SDF-1alpha can influence NPC function through CXCR4 and that CXCR4 is functional on NPC.     

Dopamine D3 receptor activation promotes neural stem/progenitor cell proliferation through AKT and ERK1/2 pathways and expands type‐B and ‐C cells in adult subventricular zone

The neurotransmitter dopamine acts on the subventricular zone (SVZ) to regulate both prenatal and postnatal neurogenesis, in particular through D₃ receptor (D₃R) subtype. In this study, we explored the cellular mechanism(s) underlying D₃R‐mediated cell proliferation and tested if systemic delivery of a D₃R agonist would induce SVZ multipotent neural stem/precursor cell (NSC/NPC) proliferation in vivo. We found that treatment with the D₃R agonist, 7‐OH‐DPAT, enhances cell proliferation in a dose‐dependent manner in cultured SVZ neurospheres from wild‐type, but not D₃R knock‐out mice. Furthermore, D₃R activation also stimulates S‐phase and enhances mRNA and protein levels of cyclin D1 in wild‐type neurospheres, a process which requires cellular Akt and ERK1/2 signaling. Moreover, chronic treatment with low dose 7‐OH‐DAPT in vivo increases BrdU⁺ cell numbers in the adult SVZ, but this effect was not seen in D₃R KO mice. Additionally, we probed the cell type specificity of D₃R agonist‐mediated cell proliferation. We found that in adult SVZ, GFAP⁺ astrocytes, type‐B GFAP⁺/nestin⁺ and type‐C EGF receptor (EGFR⁺)/nestin⁺ cells express D₃R mRNA, but type‐A Doublecortin (Dcx)⁺ neuroblasts do not. Using flow cytometry and immunofluorescence, we demonstrated that D₃R activation increases GFAP⁺ type‐B and EGFR⁺ type‐C cell numbers, and the newly divided Dcx⁺ type‐A cells. However, BrdU⁺/Dcx⁺ cell numbers were decreased in D₃R KO mice compared to wildtype, suggesting that D₃R maintains constitutive NSC/NPCs population in the adult SVZ. Overall, we demonstrate that D₃R activation induces NSC/NPC proliferation through Akt and ERK1/2 signaling and increases the numbers of type‐B and ‐C NSC/NPCs in the adult SVZ. © 2013 Wiley Periodicals, Inc.     

Secreted amyloid precursor proteins promote proliferation and glial differentiation of adult hippocampal neural progenitor cells

Amyloid precursor protein (APP) is an integral membrane glycoprotein present at high levels in nerve cells. Two soluble secreted forms, sAPPα and sAPPβ, are processed from APP by two mutually exclusive proteolytic pathways. sAPPα shows a range of neuroprotective and growth factor properties, including reduction of neuronal injury and improvement in memory performance, in contrast to the generally less potent sAPPβ. In addition, sAPPα has been shown to increase the proliferation of both embryonic neural stem cells and neural progenitor cells (NPCs) derived from the subventricular zone (SVZ) of the adult brain. However, an effect of sAPPα (or sAPPβ) on adult hippocampal progenitor cell proliferation and differentiation has not previously been observed. In this study, we examined the effect of both the α- and β-cleaved ectodomains of sAPP on adult NPCs isolated from the subgranular zone (SGZ) of the rat hippocampus in the presence or absence of depolarizing conditions. Assays were performed to examine the effect of sAPPα and sAPPβ on SGZ-derived adult NPC proliferation in parallel with SVZ-derived cells and on differentiation with SGZ-derived cells. We observed both sAPPα and sAPPβ increased the proliferation of SGZ-derived NPCs in vitro. Further, treatment of SGZ-derived NPCs with either sAPPα or sAPPβ increased the number of cells expressing the astrocytic marker GFAP and promoted cell survival. The effect on differential fate was observed in both the presence and absence of depolarizing conditions. Thus, both sAPPα and sAPPβ exert a complex range of effects on SGZ-derived adult NPCs, including increasing NPC proliferation, maintaining cell viability, yet promoting glial over neuronal differentiation. These findings provide the first direct support for the secreted forms of APP regulating SGZ-derived NPCs, and raise the possibility some or all of the effects may have therapeutic benefit in models of neurological disease.     

Age-related proteomic changes in the subventricular zone and their association with neural stem/progenitor cell proliferation

In the mammalian central nervous system, generation of new neurons persists in the subventricular zone (SVZ) throughout life. However, the capacity for neurogenesis in this region declines with aging. Recent studies have examined the degree of these age-related neurogenic declines and the changes of cytoarchitecture of the SVZ with aging. However, little is known about the molecular changes in the SVZ with aging. In this study, we dissected the SVZs from rats aged postnatal day 28, 3 months, and 24 months. The SVZ tissues were processed for 2-D gel electrophoresis to identify protein changes following aging. Protein spots were subsequently subjected to mass spectrometry analysis to compare age-related alterations in the SVZ proteome. We also examined the level of cell proliferation in the SVZ in animals of these three age groups by using bromodeoxyuridine labeling. We found significant age-related changes in the expression of several proteins that play critical roles in the proliferation and survival of neural stem/progenitor cells in the SVZ. Among these proteins, glial fibrillary acidic protein, ubiquitin carboxy terminal hydrolase 1, glutathione S-transferase omega, and preproalbumin were increased with aging, whereas collapsin response-mediated protein 4 (CRMP-4), CRMP-5, and microsomal protease ER60 exhibited declines with aging. We have also observed a significant decline of neural stem/progenitor cell proliferation in the SVZ with aging. These alterations in protein expression in the SVZ with aging likely underlie the diminishing proliferative capacity of stem/progenitor cells in the aging brain.     

NG2 cells are distinct from neurogenic cells in the postnatal mouse subventricular zone

NG2 cells express the chondroitin sulfate proteoglycan NG2 and are a fourth type of glia distinct from astrocytes, oligodendrocytes, and microglia. NG2 cells generate oligodendrocytes but have also been reported to represent neuronal progenitor cells in the postnatal mouse subventricular zone (SVZ). We performed a detailed immunohistochemical analysis of NG2 cells in the mouse SVZ, rostral migratory stream (RMS), and olfactory bulb granule cell layer (OB GCL), which constitute a neurogenic niche in the postnatal forebrain. NG2 cells in the SVZ and RMS expressed the oligodendrocyte precursor cell antigen platelet‐derived growth factor receptor‐α but did not express antigens known to be expressed by neuronogenic cells in the SVZ, such as doublecortin, PSA‐NCAM, beta‐tubulin, Dlx2, or GFAP. More than 99.5% of the proliferating cells in the SVZ were NG2 negative. In the olfactory bulb, NG2 cells were found to generate primarily oligodendrocytes and a small number of astrocytes but not neurons. In the SVZ and RMS, NG2 cells were sparse and made up a much smaller fraction of the cells compared with the surrounding nonneurogenic parenchyma. Parenchymal NG2 cells were often located along the border of the SVZ and RMS. The abundance of NG2 cells increased in the distal parts of the RMS and especially in the OB GCL, where NG2 cell processes were seen in close proximity to many maturing interneurons. Our findings indicate that NG2 cells do not represent neuronal progenitor cells in the postnatal SVZ but are likely to be oligodendrocyte precursor cells. J. Comp. Neurol. 512:702–716, 2009. © 2008 Wiley‐Liss, Inc.     

Enhanced neuronal differentiation in a three-dimensional collagen-hyaluronan matrix

Efficient 3D cell systems for neuronal induction are needed for future use in tissue regeneration. In this study, we have characterized the ability of neural stem/progenitor cells (NS/PC) to survive, proliferate, and differentiate in a collagen type I-hyaluronan scaffold. Embryonic, postnatal, and adult NS/PC were seeded in the present 3D scaffold and cultured in medium containing epidermal growth factor and fibroblast growth factor-2, a condition that stimulates NS/PC proliferation. Progenitor cells from the embryonic brain had the highest proliferation rate, and adult cells the lowest, indicating a difference in mitogenic responsiveness. NS/PC from postnatal stages down-regulated nestin expression more rapidly than both embryonic and adult NS/PC, indicating a faster differentiation process. After 6 days of differentiation in the 3D scaffold, NS/PC from the postnatal brain had generated up to 70% neurons, compared with 14% in 2D. NS/PC from other ages gave rise to approximately the same proportion of neurons in 3D as in 2D (9-26% depending on the source for NS/PC). In the postnatal NS/PC cultures, the majority of betaIII-tubulin-positive cells expressed glutamate, gamma-aminobutyric acid, and synapsin I after 11 days of differentiation, indicating differentiation to mature neurons. Here we report that postnatal NS/PC survive, proliferate, and efficiently form synapsin I-positive neurons in a biocompatible hydrogel.     

Distribution of ionotropic glutamate receptor subunit mRNAs in the rat hypothalamus

The excitatory amino acid neurotransmitter glutamate participates in the control of most (and possibly all) neuroendocrine systems in the hypothalamus. This control is exerted by binding to two classes of membrane receptors, the ionotropic and metabotropic receptor families, which differ in their structure and mechanisms of signal transduction. To gain a better understanding about the precise sites of action of glutamate and the subunit compositions of the receptors involved in the glutamatergic neurotransmission in the hypothalamus and septum, in situ hybridization was used with 35S-labeled cRNA probes for the different ionotropic receptor subunits, including glutamate receptor subunits 1-4 (GluR1-GluR4), kainate-2, GluR5-GluR7, N-methyl-D-aspartate (NMDA) receptor 1 (NMDAR1), and NMDAR2A-NMDAR2D. The results showed that subunits of alpha-amino-3-hydroxy-5-methyl-4-isoxazole-propionate-preferring, kainate-preferring, and NMDA-preferring receptor subunits are distributed widely but heterogeneously and that the GluR1, GluR2, kainate-2, NMDAR1, NMDAR2A, and NMDAR2B subunits are the most abundant in the hypothalamus. Thus, GluR1 subunit mRNA was prominent in the lateral septum, preoptic area, mediobasal hypothalamus, and tuberomammillary nucleus, whereas kainate-2 subunit mRNA was abundant in the medial septum-diagonal band, median and anteroventral preoptic nuclei, and supraoptic nuclei as well as the magnocellular portion of the posterior paraventricular nucleus. Regions that contained the highest levels of NMDAR1 subunit mRNA included the septum, the median preoptic nucleus, the anteroventral periventricular nucleus, and the supraoptic and suprachiasmatic nuclei as well as the arcuate nucleus. Together, the extensive distribution of the different GluR subunit mRNAs strengthen the view that glutamate is a major excitatory neurotransmitter in the hypothalamus. The overlap in the distribution of the various subunit mRNAs suggests that many neurons can express GluR channels that belong to different families, which would allow a differential regulation of the target neurons by glutamate.     

Propofol and remifentanil at moderate and high concentrations affect proliferation and differentiation of neural stem/progenitor cells

Propofol and remifentanil alter intracellular Ca2+ concentration([Ca2+]i) in neural stem/progenitor cells by activating γ-aminobutyric acid type A receptors and by reducing testosterone levels.However,whether this process affects neural stem/progenitor cell proliferation and differentiation remains unknown.In the present study,we applied propofol and remifentanil,alone or in combination,at low,moderate or high concentrations(1,2–2.5 and 4–5 times the clinically effective blood drug concentration),to neural stem/progenitor cells from the hippocampi of newborn rat pups.Low concentrations of propofol,remifentanil or both had no noticeable effect on cell proliferation or differentiation; however,moderate and high concentrations of propofol and/or remifentanil markedly suppressed neural stem/progenitor cell proliferation and differentiation,and induced a decrease in [Ca2+]i during the initial stage of neural stem/progenitor cell differentiation.We therefore propose that propofol and remifentanil interfere with the proliferation and differentiation of neural stem/progenitor cells by altering [Ca2+]i.Our findings suggest that propofol and/or remifentanil should be used with caution in pediatric anesthesia.     

Age-dependent effect of nitric oxide on subventricular zone and olfactory bulb neural precursor proliferation

Nitric oxide (NO) synthase (NOS) is developmentally regulated in the embryonic brain, where NO participates in cell proliferation, survival, and differentiation. In adults, NO inhibits neurogenesis under physiological conditions. This work investigates whether the NO action is preserved all along development up to adulthood or whether its effects in adults are a new feature acquired during brain maturation. The relationship between nitrergic neurons and precursors, as well as the functional consequences of pharmacological NOS inhibition, were comparatively analyzed in the subventricular zone (SVZ) and olfactory bulb (OB) of postnatal (P7) and adult (>P60) mouse brains. The SVZ was markedly reduced between P7 and adults, and, at both ages, neurons expressing neuronal NOS (nNOS) were found in its striatal limits. In postnatal mice, these nitrergic neurons contained PSA-NCAM, and their projections were scarce, whereas, in adults, mature nitrergic neurons, devoid of PSA-NCAM, presented abundant neuropil. In the OB, local proliferation almost disappeared in the transition to adulthood, and periglomerular nitrergic neurons, some of which were PSA-NCAM positive, were found in postnatal and adult mice. Administration of the NOS inhibitor L-NAME did not affect cell proliferation in the SVZ or in the OB of postnatal mice, whereas it significantly enhanced the number of mitotic cells in both regions in adults. Thus, the NO action on SVZ neurogenesis is a phenomenon that appears after the postnatal age, which is probably due to the germinal layer size reduction, allowing exposure of the NO-sensitive neural precursors to the NO produced in the SVZ-striatum limits.     

Reactive astrogliosis induces astrocytic differentiation of adult neural stem/progenitor cells in vitro

Neural stem cells reside in defined areas of the adult mammalian brain, including the dentate gyrus of the hippocampus. Rat neural stem/progenitor cells (NSPCs) isolated from this region retain their multipotency in vitro and in vivo after grafting into the adult brain. Recent studies have shown that endogenous or grafted NSPCs are activated after an injury and migrate toward lesioned areas. In these areas, reactive astrocytes are present and secrete numerous molecules and growth factors; however, it is not currently known whether reactive astrocytes can influence the lineage selection of NSPCs. We investigated whether reactive astrocytes could affect the differentiation, proliferation, and survival of adult NSPCs by modelling astrogliosis in vitro, using mechanical lesion of primary astrocytes. Initially, it was found that conditioned medium from lesioned astrocytes induced astrocytic differentiation of NSPCs without affecting neuronal or oligodendrocytic differentiation. In addition, NSPCs in coculture with lesioned astrocytes also displayed increased astrocytic differentiation and some of these NSPC-derived astrocytes participated in glial scar formation in vitro. When proliferation and survival of NSPCs were analyzed, no differential effects were observed between lesioned and nonlesioned astrocytes. To investigate the molecular mechanisms of the astrocyte-inducing activity, the expression of two potent inducers of astroglial differentiation, ciliary neurotrophic factor and leukemia inhibitory factor, was analyzed by Western blot and shown to be up-regulated in conditioned medium from lesioned astrocytes. These results demonstrate that lesioned astrocytes can induce astroglial differentiation of NSPCs and provide a mechanism for astroglial differentiation of these cells following brain injury.