Stem Cells In Subventricular Zone Research Articles

Novel analogs of allopregnanolone show improved efficiency and specificity in neuroprotection and stimulation of proliferation.

The natural neurosteroid allopregnanolone exerts beneficial effects in animal models of neurodegenerative diseases, nervous system injury and peripheral neuropathies. It not only has anti-apoptotic activity, but also promotes proliferation of progenitor cells. With respect to using it as a therapeutic tool, such pleiotropic actions might create unwanted side effects. Therefore, we have synthesized allopregnanolone analogs and analyzed their neuroprotective and proliferative effects to identify compounds with higher efficiency and less ambiguous biological actions. Proliferation-promoting effects of 3α and 3β isomers of 3-O-allyl-allopregnanolone and 12 oxo-allopregnanolone were studied in adult subventricular zone stem cell cultures and in primary hippocampal cultures by measuring 5-ethynyl-2'-deoxyuridine incorporation. Neuroprotective activity against amyloid beta 42-induced cell death was determined by quantifying caspase 3/7 activity. The 3α isomers significantly stimulated proliferation in all culture systems, whereas the 3β isomers were ineffective. The stimulatory effect of 3α-O-allyl-allopregnanolone was significantly higher than that of allopregnanolone. In neural stem cell cultures, 3α-O-allyl-allopregnanolone specifically enhanced proliferation of Nestin-positive progenitors. In addition, it promoted the differentiation of doublecortin-positive neurons. In neural stem cell cultures treated with amyloid beta 42, both the α and β isomers of O-allyl- allopregnanolone showed increased neuroprotective activity as compared to allopregnanolone, completely preventing amyloid-induced caspase 3/7 activation. The 12 oxo-allopregnanolone analogs were ineffective. These results identify structural allopregnanolone analogs with higher anti-apoptotic and proliferation-promoting activity than the natural neurosteroid. Interestingly, stereoisomers of the analogs were found to have distinct profiles of activity raising the possibility of exploiting the neuroprotective properties of neurosteroids with or without simultaneously stimulating neurogenesis. Cover Image for this issue: doi: 10.1111/jnc.13344.

Commentary: Impact of a deletion of the full-length and short isoform of p75NTR on cholinergic innervation and the population of postmitotic doublecortin positive cells in the dentate gyrus.

We read with great interest the article by Poser et al. (2015). Full-length human/murine P75NTR (FL-p75NTR; ENS0172229, ENSMU122, ENSRNO0007268) is encoded by six exons. Four cysteine-rich repeats span P75NTR extracellular domain and exhibit high-pro-neurotrophins-affinity and low-affinity to mature neurotrophins. Brain P75NTR plays pivotal roles in neurogenesis. Embryonic neurogenesis derives from ventricular zone radial glia (Mochida et al., 2007). Adult neurogenesis is localized to stem cell niches in hippocampal dentate gyrus and the sub-ventricular zone (SVZ). P75NTR is detected in deep layers of developing neocortex (Bishop et al., 2002) and defines adult neurogenesis stem cells in SVZ (Young et al., 2007) and hippocampus (Catts et al., 2008). P75NTR regulates synaptic plasticity through its interactions with pro-BNDF to produce long-term depression (Woo and Lu, 2009). P75NTR Interacts with Nogo/Lingo-1 leading to growth cone collapse, neurite retraction, and spine density decrease (Meeker and Williams, 2015). An alternative 62 kD P75NTR variant, named short isoform (s-p75NTR), claimed to arise by alternative splicing of P75NTR exon III, has been reported in the mouse and was claimed to also exist in rat and human species (von-Schack et al., 2001; Naumann et al., 2002). Exon III encodes cysteine-motifs 2–4 (Casaccia-Bonnefil et al., 1999). Its deletion would compromise P75NTR interaction with several molecules including Nogo (see above), sortilin and Trk. P75NTR-sortilin binding increases pro-neurotrophin affinity and promotes apoptosis while P75NTR-Trk interaction mediates pro-survival functions (Meeker and Williams, 2015). To verify the existence of the presumed mouse s-p75NTR, we conducted Rapid Amplification of cDNA Ends (RACE) cloning on mouse P75NTR mRNA and ascertained many exon III-containing clones without any evidence of continuous exon II/exon IV sequence with exon III-skipping (Figure ​(Figure1I).1I). Also we conducted RT-PCR using oligos flanking exon III and validated our results by sequencing the identified PCR fragments (Figure 1II). Additionally, we used the same discontinuous, exon III-skipping primers which were used in the original reports as “specific” oligos to the presumed s-75NTR variant (von-Schack et al., 2001). These specific primers contain exons 2/4 boundary sequences. Also, this approach did not identify any short, exon III-excluding isoform in the mouse (Figure 1II). Figure 1 Verifying P75NTR isoforms. (I) RACE cloning to explore potential additional P75NTR variants: RACE cloning was conducted using a 225 bp template representing mouse brain P75NTR exon 4 nucleotides 781–1005. A large number of clones were screened, ... Western blotting (WB) on mouse, rat, and human brain protein extracts using polyclonal antibodies specific to the intracellular region of P75NTR (Figure 1III) identified the 75 kD FL-p75NTR in the three species while a 62 kD band (corresponding to the claimed s-p75NTR) was not detected in any of the three species investigated. Apart from 75 kD FL-p75NTR, no other bands were detected in mouse and rat brains (Figure 1III). Of note is the fact that the report by (von-Schack et al., 2001) failed to show the protein band corresponding to the presumed 62 kD s-p75NTR in their WB. They rather showed a WB “smear” around 75 kD and argued that the 62 kD s-p75NTR band has been masked by the high level expression of FL-p75NTR! This is hardly convincing since the issue could have been easily resolved by simple technical improvements. It is noteworthy that our results, verifying only one murine FL-p75NTR band, are compatible with the documentations in major resources like Ensembl, NCBI, and UniProt database. In addition to the 75 kD FL-p75NTR, our WB results on control and Alzheimer's disease human brains also detected an additional, previously unreported, human 36 kD isoform, (Figure 1III). The 36 kD P75NTR isoform does not seem to be a marker for Alzheimer's disease since we detected the two isoforms (75 and 36 kD), either simultaneously or alternatively, in individual human samples from control or Alzheimer's brains (Figure 1III). It is notable that three protein-coding P75NTR human transcripts (ENS0172229, ENS0504201, and ENS0509200) are listed in ENSEMBL and UniProt. We are embarking on further analysis to determine the nature of our 36 kD isoform. It is seriously alarming that the report by von-Schack et al. (2001) did not show any experimental work to support their claim for the presence of a 62 kD s-p75NTR. In addition to the lack of WB evidence, they did not even sequence their RT-PCR fragments to prove their claims. Such unsubstantiated reporting caused considerable confusion in the neuroscience community since other groups (Fujii and Kunugi, 2009; Poser et al., 2015) innocently reiterated such un-validated reports and, moreover, interpreted their important results based on such unfounded claims (Poser et al., 2015).

The balance of Id3 and E47 determines neural stem/precursor cell differentiation into astrocytes.

Adult neural stem/precursor cells (NSPCs) of the subventricular zone (SVZ) are an endogenous source for neuronal replacement in CNS disease. However, adult neurogenesis is compromised after brain injury in favor of a glial cell fate, which is mainly attributed to changes in the NSPC environment. Yet, it is unknown how this unfavorable extracellular environment translates into a transcriptional program altering NSPC differentiation. Here, we show that genetic depletion of the transcriptional regulator Id3 decreased the number of astrocytes generated from SVZ-derived adult NSPCs in the cortical lesion area after traumatic brain injury. Cortical brain injury resulted in rapid BMP-2 and Id3 up-regulation in the SVZ stem cell niche. Id3(-/-) adult NSPCs failed to differentiate into BMP-2-induced astrocytes, while NSPCs deficient for the Id3-controlled transcription factor E47 readily differentiated into astrocytes in the absence of BMP-2. Mechanistically, E47 repressed the expression of several astrocyte-specific genes in adult NSPCs. These results identify Id3 as the BMP-2-induced transcriptional regulator, promoting adult NSPC differentiation into astrocytes upon CNS injury and reveal a molecular link between environmental changes and NSPC differentiation in the CNS after injury.

The effects of functional electrical stimulation on motor function and on the proliferation, migration and differentiation of neural stem cells in the subventricular zone

Objective To investigate the effects of functional electrical stimulation (FES) on motor function and on the expression, proliferation, migration and differentiation of endogenous neural stem cells in the subventricular zone (SVZ) after cerebral ischemia. Methods Middle cerebral artery occlusion (MCAO) was used to induce a model of cerebral ischemia in 108 rats using the modified Zea-Longa method of intraluminal filament occlusion. They were then randomly divided into an FES group, a placebo stimulation group and a control group with 36 cases in each. Superficial FES electrodes were pasted on the paralyzed forelimbs of the rats in the first two groups, though FES treatment was administered only to the FES group beginning on the 3rd day after the MCAO operation. The stimulation was designed to produce extension of the wrist and digits of the paralyzed forelimb. Before, and after 1, 3, 7 and 14 days of the treatment, the neurological deficit was evaluated using modified neurological severity scoring (mNSS). BrdU+ /GFAP+ , BrdU+ /DCX+ and BrdU+ /NeuN+ cells in the SVZ were detected using immunofluorescence technique. Results After 7 and 14 days of treatment, the average motor function of the rats in the FES group had improved significantly when compared with the averages of the other two groups. Compared with the other two groups, the average number of BrdU+ /GFAP+ positive cells in the ischemic SVZ was also significantly greater in the FES group after 7 and 14 days of treatment. After 14 days, BrdU+ /Dcx+ positive cells in the FES group had also increased significantly more, but only a few BrdU+ /NeuN+ cells had appeared in any of the three groups. Conclusion FES can improve motor function after cerebral ischemia, and promote proliferation and differentiation of neural stem cells in the SVZ. Key words: Electrical stimulation; Brain ischemia; Functional electrical stimulation; Neural stem cells; Subventricular zone

Defects in subventricular zone pigmented epithelium-derived factor niche signaling in the senescence-accelerated mouse prone-8.

We studied potential changes in the subventricular zone (SVZ) stem cell niche of the senescence-accelerated mouse prone-8 (SAM-P8) aging model. Bromodeoxyuridine (BrdU) assays with longtime survival revealed a lower number of label-retaining stem cells in the SAM-P8 SVZ compared with the SAM-Resistant 1 (SAM-R1) control strain. We also found that in SAM-P8 niche signaling is attenuated and the stem cell pool is less responsive to the self-renewal niche factor pigmented epithelium-derived factor (PEDF). Protein analysis demonstrated stable amounts of the PEDF ligand in the SAM-P8 SVZ niche; however, SAM-P8 stem cells present a significant expression decrease of patatin-like phospholipase domain containing 2, a receptor for PEDF (PNPLA2-PEDF) receptor, but not of laminin receptor (LR), a receptor for PEDF (LR-PEDF) receptor. We observed changes in self-renewal related genes (hairy and enhancer of split 1 (Hes1), hairy and enhancer of split 1 (Hes5), Sox2] and report that although these genes are down-regulated in SAM-P8, differentiation genes (Pax6) are up-regulated and neurogenesis is increased. Finally, sheltering mammalian telomere complexes might be also involved given a down-regulation of telomeric repeat binding factor 1 (Terf1) expression was observed in SAM-P8 at young age periods. Differences between these 2 models, SAM-P8 and SAM-R1 controls, have been previously detected at more advanced ages. We now describe alterations in the PEDF signaling pathway and stem cell self-renewal at a very young age, which could be involved in the premature senescence observed in the SAM-P8 model.

Gypenosides protected the neural stem cells in the subventricular zone of neonatal rats that were prenatally exposed to ethanol.

Fetal alcohol spectrum disorder (FASD) can cause severe mental retardation in children who are prenatally exposed to ethanol. The effects of prenatal and early postnatal ethanol exposure on adult hippocampal neurogenesis have been investigated; however, the effects of prenatal ethanol exposure on the subventricular zone (SVZ) have not. Gypenosides (GPs) have been reported to have neuroprotective effects in addition to other bioactivities. The effects of GPs on neural stem cells (NSCs) in the FASD model are unknown. Here, we test the effect of prenatal ethanol exposure on the neonatal SVZ, and the protection potential of GPs on NSCs in FASD rats. Our results show that prenatal ethanol exposure can suppress the cell proliferation and differentiation of neural stem cells in the neonatal SVZ and that GPs (400 mg/kg/day) can significantly increase the cell proliferation and differentiation of neural stem cells inhibited by ethanol. Our data indicate that GPs have neuroprotective effects on the NSCs and can enhance the neurogenesis inhibited by ethanol within the SVZ of neonatal rats. These findings provide new evidence for a potential therapy involving GPs for the treatment of FASD.

The transcription factor Fezf2 directs the differentiation of neural stem cells in the subventricular zone toward a cortical phenotype.

Postnatal neurogenesis in mammals is confined to restricted brain regions, including the subventricular zone (SVZ). In rodents, the SVZ is a lifelong source of new neurons fated to migrate to the olfactory bulb (OB), where the majority become GABAergic interneurons. The plastic capacity of neonatal and adult SVZ stem/progenitor cells is still largely unknown. By overexpressing the transcription factor Fezf2, a powerful master gene specifying the phenotype of glutamatergic subcerebral projecting neurons, we investigated whether the fate of postnatally generated SVZ neurons can be altered. Following lentiviral delivery of Fezf2 in the neonatal and adult SVZ niche, we showed that ectopic Fezf2 expression is sufficient to redirect the fate of SVZ stem cells. Thus, based on in vivo and in vitro experiments, we provide evidence that numerous Fezf2-positive OB neurons expressed glutamatergic pyramidal cell molecular markers instead of developing a GABAergic identity. Overexpression of Fezf2 had no effect on transit-amplifying progenitors or neuroblasts but was restricted to neural stem cells. Fezf2-respecified neurons bore features of pyramidal cells, exhibiting a larger cell body and a more elaborate dendritic tree, compared with OB granule cells. Patch-clamp recordings further indicated that Fezf2-respecified neurons had synaptic properties and a firing pattern reminiscent of a pyramidal cell-like phenotype. Together, the results demonstrate that neonatal and adult SVZ stem cells retain neuronal fate plasticity.

The expression and roles of Nde1 and Ndel1 in the adult mammalian central nervous system

Mental and neurological illnesses affect one in four people. While genetic linkage analyses have shown an association of nuclear distribution factor E (NDE1, or NudE) and its ohnolog NDE-like 1 (NDEL1, or Nudel) with mental disorders, the cellular mechanisms remain unclear. In the present study, we have demonstrated that Nde1 and Ndel1 are differentially localised in the subventricular zone (SVZ) of the forebrain and the subgranular zone (SGZ) of the hippocampus, two regions where neurogenesis actively occurs in the adult brain. Nde1, but not Ndel1, is localized to putative SVZ stem cells, and to actively dividing progenitors of the SGZ. The influence of these proteins on neural stem cell differentiation was investigated by overexpression in a hippocampal neural stem cell line, HCN-A94. Increasing Nde1 expression in this neural stem cell line led to increased neuronal differentiation while decreasing levels of astroglial differentiation. In primary cultured neurons and astrocytes, Nde1 and Ndel1 were found to have different but comparable subcellular localizations. In addition, we have shown for the first time that Nde1 is heterogeneously distributed in cortical astrocytes of human brains. Our data indicate that Nde1 and Ndel1 have distinct but overlapping distribution patterns in mouse brain and cultured nerve cells. They may function differently and therefore their dosage changes may contribute to some aspects of mental disorders.

Tis21 is required for adult neurogenesis in the subventricular zone and for olfactory behavior regulating cyclins, BMP4, Hes1/5 and Ids.

Bone morphogenic proteins (BMPs) and the Notch pathway regulate quiescence and self-renewal of stem cells of the subventricular zone (SVZ), an adult neurogenic niche. Here we analyze the role at the intersection of these pathways of Tis21 (Btg2/PC3), a gene regulating proliferation and differentiation of adult SVZ stem and progenitor cells. In Tis21-null SVZ and cultured neurospheres, we observed a strong decrease in the expression of BMP4 and its effectors Smad1/8, while the Notch anti-neural mediators Hes1/5 and the basic helix-loop-helix (bHLH) inhibitors Id1-3 increased. Consistently, expression of the proneural bHLH gene NeuroD1 decreased. Moreover, cyclins D1/2, A2, and E were strongly up-regulated. Thus, in the SVZ Tis21 activates the BMP pathway and inhibits the Notch pathway and the cell cycle. Correspondingly, the Tis21-null SVZ stem cells greatly increased; nonetheless, the proliferating neuroblasts diminished, whereas the post-mitotic neuroblasts paradoxically accumulated in SVZ, failing to migrate along the rostral migratory stream to the olfactory bulb. The ability, however, of neuroblasts to migrate from SVZ explants was not affected, suggesting that Tis21-null neuroblasts do not migrate to the olfactory bulb because of a defect in terminal differentiation. Notably, BMP4 addition or Id3 silencing rescued the defective differentiation observed in Tis21-null neurospheres, indicating that they mediate the Tis21 pro-differentiative action. The reduced number of granule neurons in the Tis21-null olfactory bulb led to a defect in olfactory detection threshold, without effect on olfactory memory, also suggesting that within olfactory circuits new granule neurons play a primary role in odor sensitivity rather than in memory.

Epidermal Growth Factor Treatment of the Adult Brain Subventricular Zone Leads to Focal Microglia/Macrophage Accumulation and Angiogenesis

SummaryOne of the major components of the subventricular zone (SVZ) neurogenic niche is the specialized vasculature. The SVZ vasculature is thought to be important in regulating progenitor cell proliferation and migration. Epidermal growth factor (EGF) is a mitogen with a wide range of effects. When stem and progenitor cells in the rat SVZ are treated with EGF, using intracerebroventricular infusion, dysplastic polyps are formed. Upon extended infusion, blood vessels are recruited into the polyps. In the current study we demonstrate how polyps develop through distinct stages leading up to angiogenesis. As polyps progress, microglia/macrophages accumulate in the polyp core concurrent with increasing cell death. Both microglia/macrophage accumulation and cell death peak during angiogenesis and subsequently decline following polyp vascularization. This model of inducible angiogenesis in the SVZ neurogenic niche suggests involvement of microglia/macrophages in acquired angiogenesis and can be used in detail to study angiogenesis in the adult brain.

Pten Loss in Olig2 Expressing Neural Progenitor Cells and Oligodendrocytes Leads to Interneuron Dysplasia and Leukodystrophy

Therapeutic modulation of phosphatidylinositol 3-kinase (PI3K)/PTEN signaling is currently being explored for multiple neurological indications including brain tumors and seizure disorders associated with cortical malformations. The effects of PI3K/PTEN signaling are highly cell context dependent but the function of this pathway in specific subsets of neural stem/progenitor cells generating oligodendroglial lineage cells has not been fully studied. To address this, we created Olig2-cre:Pten(fl/fl) mice that showed a unique pattern of Pten loss and PI3K activation in Olig2-lineage cells. Olig2-cre:Pten(fl/fl) animals progressively developed central nervous system white matter hypermyelination by 3 weeks of age leading to later onset leukodystrophy, chronic neurodegeneration, and death by 9 months. In contrast, during immediate postnatal development, oligodendroglia were unaffected but abnormal and accelerated differentiation of lateral subventricular zone stem cells produced calretinin-positive interneuron dysplasia. Neural stem cells isolated from Olig2-cre:Pten(fl/fl) mice also exhibited accelerated differentiation and proliferation into calretinin-positive interneurons and oligodendrocytes indicating such effects are cell autonomous. Opposition of the pathway by treatment of human primary neural progenitor cells (NPCs) with the PI3K inhibitor, NVP-BKM120, blocked in vitro differentiation of neurons and oligodendroglia indicating PI3K/PTEN effects on NPCs can be bidirectional. In summary, our results suggest Pten is a developmental rheostat regulating interneuron and oligodendroglial differentiation and support testing of PI3K modulating drugs as treatment for developmental and myelination disorders. However, such agents may need to be administered at ages that minimize potential effects on early stem/progenitor cell development.

Non-epithelial stem cells and cortical interneuron production in the human ganglionic eminences

GABAergic cortical interneurons underlie the complexity of neural circuits and are particularly numerous and diverse in humans. In rodents, cortical interneurons originate in the subpallial ganglionic eminences, but their developmental origins in humans are controversial. We characterized the developing human ganglionic eminences and found that the subventricular zone (SVZ) expanded massively during the early second trimester, becoming densely populated with neural stem cells and intermediate progenitor cells. In contrast with the cortex, most stem cells in the ganglionic eminence SVZ did not maintain radial fibers or orientation. The medial ganglionic eminence exhibited unique patterns of progenitor cell organization and clustering, and markers revealed that the caudal ganglionic eminence generated a greater proportion of cortical interneurons in humans than in rodents. On the basis of labeling of newborn neurons in slice culture and mapping of proliferating interneuron progenitors, we conclude that the vast majority of human cortical interneurons are produced in the ganglionic eminences, including an enormous contribution from non-epithelial SVZ stem cells.

CHD7 and retinoic acid signaling cooperate to regulate neural stem cell and inner ear development in mouse models of CHARGE syndrome

CHARGE syndrome is a multiple congenital anomaly disorder that leads to life-threatening birth defects, such as choanal atresia and cardiac malformations as well as multiple sensory impairments, that affect hearing, vision, olfaction and balance. CHARGE is caused by heterozygous mutations in CHD7, which encodes an ATP-dependent chromatin remodeling enzyme. Identification of the mechanisms underlying neurological and sensory defects in CHARGE is a first step toward developing treatments for CHARGE individuals. Here, we used mouse models of Chd7 deficiency to explore the function of CHD7 in the development of the subventricular zone (SVZ) neural stem cell niche and inner ear, structures that are important for olfactory bulb neurogenesis and hearing and balance, respectively. We found that loss of Chd7 results in cell-autonomous proliferative, neurogenic and self-renewal defects in the perinatal and mature mouse SVZ stem cell niche. Modulation of retinoic acid (RA) signaling prevented in vivo inner ear and in vitro neural stem cell defects caused by Chd7 deficiency. Our findings demonstrate critical, cooperative roles for RA and CHD7 in SVZ neural stem cell function and inner ear development, suggesting that altered RA signaling may be an effective method for treating Chd7 deficiency.

Hypoxia-inducible factor 1a is a Tsc1-regulated survival factor in newborn neurons in tuberous sclerosis complex

Tuberous sclerosis complex (TSC) is a genetic disorder caused by mutations in TSC1 or TSC2 resulting in hyperactivity of the mammalian target of rapamycin and disabling brain lesions. These lesions contain misplaced neurons enriched in hypoxia-inducible factor 1a (HIF1a). However, the relationship between TSC1/2 and HIF1a and the function of HIF1a in TSC neurons remain unexplored. Here, we examine the degree of HIF1a activity and its function in newborn Tsc1(null) neurons in a mouse model of TSC. Using single cell electroporation in the neurogenic subventricular zone (SVZ) of neonatal mice, we deleted Tsc1 and generated olfactory lesions containing misplaced Tsc1(null) neurons as previously reported. These newborn neurons displayed elevated HIF1a-mediated transcriptional activity when compared with Tsc1 heterozygote neurons and a marked resistance to cell death induced by a HIF1a antagonist. Electroporation of Hif1a targeting short hairpin RNA (shRNA) or dominant negative HIF1a constructs resulted in 80-90% loss of Tsc1(null) newborn neurons although sparing SVZ stem cells. Consistent with this later finding, induction of Hif1a shRNA expression during synaptic integration thus bypassing neuron production also resulted in newborn neuron death. Collectively, these results suggest that HIF1a acts as a molecular determinant of newborn neuron survival and that its TSC1-dependent up-regulation gave Tsc1(null) neurons a survival advantage, despite their misplacement in a novel microenvironment.

Distinct Roles of Nogo-A and Nogo Receptor 1 in the Homeostatic Regulation of Adult Neural Stem Cell Function and Neuroblast Migration

In the adult mammalian subventricular zone (SVZ), GFAP-positive neural stem cells (NSCs) generate neuroblasts that migrate tangentially along the rostral migratory stream (RMS) toward the olfactory bulb (OB). In the mouse brain, we found that the plasticity inhibitors Nogo-A and Nogo receptor 1 (NgR1) are differentially expressed in the SVZ-OB system, in which Nogo-A identifies immature neuroblasts and NgR1 germinal astrocytes. We therefore examined the role of Nogo-A and NgR1 in the regulation of neurogenesis. Pharmacological experiments show that Nogo-66/NgR1 interaction reduces the proliferation of NSCs. This is consistent with a negative-feedback loop, in which newly generated neurons modulate cell division of SVZ stem cells. Moreover, the Nogo-A-Δ20 domain promotes neuroblast migration toward the OB through activation of the Rho/ROCK (Rho-associated, coiled-coil containing protein kinase) pathway, without the participation of NgR1. Our findings reveal a new unprecedented function for Nogo-A and NgR1 in the homeostatic regulation of the pace of neurogenesis in the adult mouse SVZ and in the migration of neuroblasts along the RMS.

3-Dimensional Examination of the Adult Mouse Subventricular Zone Reveals Lineage-Specific Microdomains

Recent studies suggest that the subventricular zone (SVZ) of the lateral ventricle is populated by heterogeneous populations of stem and progenitor cells that, depending on their exact location, are biased to acquire specific neuronal fates. This newly described heterogeneity of SVZ stem and progenitor cells underlines the necessity to develop methods for the accurate quantification of SVZ stem and progenitor subpopulations. In this study, we provide 3-dimensional topographical maps of slow cycling “stem” cells and progenitors based on their unique cell cycle properties. These maps revealed that both cell populations are present throughout the lateral ventricle wall as well as in discrete regions of the dorsal wall. Immunodetection of transcription factors expressed in defined progenitor populations further reveals that divergent lineages have clear regional enrichments in the rostro-caudal as well as in the dorso-ventral span of the lateral ventricle. Thus, progenitors expressing Tbr2 and Dlx2 were confined to dorsal and dorso-lateral regions of the lateral ventricle, respectively, while Mash1+ progenitors were more homogeneously distributed. All cell populations were enriched in the rostral-most region of the lateral ventricle. This diversity and uneven distribution greatly impede the accurate quantification of SVZ progenitor populations. This is illustrated by measuring the coefficient of error of estimates obtained by using increasing section sampling interval. Based on our empirical data, we provide such estimates for all progenitor populations investigated in this study. These can be used in future studies as guidelines to judge if the precision obtained with a sampling scheme is sufficient to detect statistically significant differences between experimental groups if a biological effect is present. Altogether, our study underlines the need to consider the SVZ of the lateral ventricle as a complex 3D structure and define methods to accurately assess neural stem cells or progenitor diversity and population sizes in physiological or experimental paradigms.

Effects of protein restriction during gestation and lactation on cell proliferation in the hippocampus and subventricular zone: Functional implications. Protein restriction alters hippocampal/SVZ cell proliferation

There is no consensus about the effects of protein restriction on neurogenesis and behavior. Here, for the first time, we evaluated the effects of protein restriction during gestation and lactation, on the two major neurogenic regions of the adult brain, the subgranular zone (SGZ) of the hippocampal dentate gyrus and the subventricular zone (SVZ), simultaneously. We also assessed different types of behavior relevant to each region. After mating, pregnant Wistar rats were divided into a control group (CG) that received a normal diet (20% protein); and a protein-restriction group (PRG) that received a low-protein diet (8% protein). After birth, the same diets were provided to the mother and pups until weaning, when some rats were analyzed and others received a normal-protein diet until adulthood. Different sets of rats were used for cellular and behavioral studies in juvenile or adult age. Brains were processed for immunohistochemistry anti-BrdU, anti-Ki67, or anti-pHisH3. Juvenile and adult rats from distinct litters also underwent several behavioral tests. Our data show that early protein restriction results in a reduction of hippocampal progenitors and deficits in object recognition during adult life. Moreover, longer periods of immobility in the tail suspension and in the forced swimming tests revealed that PRG rats show a depressive behavior at 21 days of age (P21) and in adulthood. Furthermore, we suggest that despite the reduced number/proliferation of neural stem cells (B and/or E cells) in SVZ there is a compensatory mechanism in which the progenitors (types C and A cells) proliferate in a higher rate, without affecting olfactory ability in adulthood.

MiR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching.

Effects of functional electrical stimulation on motor function and the expression of bromodeoxyuridine + and glial fibrillary acid protein+ cells in the subventricular zone after cerebral infarction

Objective To investigate the effects of functional electrical stimulation (FES) on motor function and the expression of bromodeoxyuridine (Brdu) + and glial fibrillary acid protein (GFAP) + in the subventricular zone (SVZ) of rats with acute cerebral infarction,and to explore it's mechanism. Methods A rat model of cerebral infarction was established using Longa's technique for middle cerebral artery occlusion (MCAO) with an intraluminal filament.The rats were randomly divided into a FES group,a placebo stimulation group and a control group.In each group,rats were randomly allocated into 1 d,3 d,7 d and 14 d subgroups (6 rats/subgroup).Superficial electrodes were pasted on the paralyzed forelimbs of rats in the FES group for connecting with the FES instrument,and FES treatment was carried out with a current of 4-5 mA for 15 min on the third day after the MCAO operation to produce extension of the wrist and the digits of the paralyzed forelimb.The rats in the placebo stimulation group were pasted with electrodes,but no FES was administered and they received no other treatment.Neurological deficits were evaluated using the modified neurological severity score (mNSS) before treatment and on the 1 st,3rd,7th,and 14th day after treatment. BrdU and GFAP positive cells in the SVZ were detected by immunofluorescence techniques.Results After 7 or 14 days the motor function of rats in the FES group had improved significantly compared with the placebo stimulation and control groups.Compared with the other two groups,the expression levels of BrdU+ and GFAP+ cells in the ischemic SVZ in the FES group were significantly higher at the 3rd,7th and 14th day.Conclusion FES can improve motor function after acute cerebral infarction and also promote the proliferation and differentiation of neural stem cells in the SVZ. Key words: Functional electrical stimulation ; Cerebral infarction ; Neural plasticity ; Subventricularzone

An Endogenous Vitamin K-Dependent Mechanism Regulates Cell Proliferation in the Brain Subventricular Stem Cell Niche

Neural stem cells (NSC) persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ stem and progenitor cell proliferation are not fully elucidated. Vitamin K-dependent proteins (VKDPs) are mainly secreted factors that were initially discovered as major regulators of blood coagulation. Warfarin ((S(−)-3-acetonylbenzyl)-4-hydroxycoumarin)), a widespread anticoagulant, is a vitamin K antagonist that inhibits the production of functional VKDP. We demonstrate that the suppression of functional VKDPs production, in vitro, by exposure of SVZ cell cultures to warfarin or, in vivo, by its intracerebroventricular injection to mice, leads to a substantial increase in SVZ cell proliferation. We identify the anticoagulant factors, protein S and its structural homolog Gas6, as the two only VKDPs produced by SVZ cells and describe the expression and activation pattern of their Tyro3, Axl, and Mer tyrosine kinase receptors. Both in vitro and in vivo loss of function studies consisting in either Gas6 gene invalidation or in endogenous protein S neutralization, provided evidence for an important novel regulatory role of these two VKDPs in the SVZ neurogenic niche. Specifically, we show that while a loss of Gas6 leads to a reduction in the numbers of stem-like cells and in olfactory bulb neurogenesis, endogenous protein S inhibits SVZ cell proliferation. Our study opens up new perspectives for investigating further the role of vitamin K, VKDPs, and anticoagulants in NSC biology in health and disease. Stem Cells 2012; 30:719–731