Notch1 is required for maintenance of the reservoir of adult hippocampal stem cells.
ABSTRACT: Notch1 regulates neural stem cell (NSC) number during development, but its role in adult neurogenesis is unclear. We generated nestin-CreER(T2)/R26R-YFP/Notch1(loxP/loxP) [Notch1inducible knock-out (iKO)] mice to allow tamoxifen (TAM)-inducible elimination of Notch1 and concomitant expression of yellow fluorescent protein (YFP) in nestin-expressing Type-1 NSCs and their progeny in the adult hippocampal subgranular zone (SGZ). Consistent with previous research, YFP+ cells in all stages of neurogenesis were evident in the subgranular zone (SGZ) of wild-type (WT) mice (nestin-CreER(T2)/R26R-YFP/Notch1(w/w)) after tamoxifen (post-TAM), producing adult-generated YFP+ dentate gyrus neurons. Compared with WT littermates, Notch1 iKO mice had similar numbers of total SGZ YFP+ cells 13 and 30 d post-TAM but had significantly fewer SGZ YFP+ cells 60 and 90 d post-TAM. Significantly fewer YFP+ Type-1 NSCs and transiently amplifying progenitors (TAPs) resulted in generation of fewer YFP+ granule neurons in Notch1 iKO mice. Strikingly, 30 d of running rescued this deficit, as the total YFP+ cell number in Notch iKO mice was equivalent to WT levels. This was even more notable given the persistent deficits in the Type-1 NSC and TAP reservoirs. Our data show that Notch1 signaling is required to maintain a reservoir of undifferentiated cells and ensure continuity of adult hippocampal neurogenesis, but that alternative Notch- and Type-1 NSC-independent pathways compensate in response to physical activity. These data shed light on the complex relationship between Type-1 NSCs, adult neurogenesis, the neurogenic niche, and environmental stimuli.
Project description:Understanding the fate of adult-generated neurons and the mechanisms that influence them requires consistent labeling and tracking of large numbers of stem cells. We generated a nestin-CreER(T2)/R26R-yellow fluorescent protein (YFP) mouse to inducibly label nestin-expressing stem cells and their progeny in the adult subventricular zone (SVZ) and subgranular zone (SGZ). Several findings show that the estrogen ligand tamoxifen (TAM) specifically induced recombination in stem cells and their progeny in nestin-CreER(T2)/R26R-YFP mice: 97% of SGZ stem-like cells (GFAP/Sox2 with radial glial morphology) expressed YFP; YFP+ neurospheres could be generated in vitro after recombination in vivo, and maturing YFP+ progeny were increasingly evident in the olfactory bulb (OB) and dentate gyrus (DG) granule cell layer. Revealing an unexpected regional dissimilarity in adult neurogenesis, YFP+ cells accumulated up to 100 d after TAM in the OB, but in the SGZ, YFP+ cells reached a plateau 30 d after TAM. In addition, most SVZ and SGZ YFP+ cells became neurons, underscoring a link between nestin and neuronal fate. Finally, quantification of YFP+ cells in nestin-CreER(T2)/R26R-YFP mice allowed us to estimate, for example, that stem cells and their progeny contribute to no more than 1% of the adult DG granule cell layer. In addition to revealing the dynamic contribution of nestin-expressing stem cells to adult neurogenesis, this work highlights the utility of the nestin-CreER(T2)/R26R-YFP mouse for inducible gene ablation in stem cells and their progeny in vivo in the two major regions of adult neurogenesis.
Project description:HIF-1? is a hypoxia-inducible protein that regulates many cellular processes, including neural stem cell maintenance. Previous work demonstrated constitutive stabilization of HIF-1? in neural stem cells (NSCs) of the adult mouse subventricular zone (SVZ) and hippocampal subgranular zone (SGZ). Genetic inactivation of NSC-encoded HIF-1? in the adult SVZ results in gradual loss of NSCs, but whether HIF-1? is required for the maintenance of SGZ hippocampal progenitors and adult hippocampal neurogenesis has not been determined. Here we tested the hypothesis that HIF-1? plays an essential role in the maintenance of adult hippocampal neurogenesis using Nestin-CreERT2/R26R-YFP/Hif1afl/fl triple transgenic mice, in which HIF-1? was genetically inactivated in nestin+ hippocampal progenitors and their downstream progeny following tamoxifen exposure. We found that disruption of HIF-1? gene expression resulted in a marked 50% reduction of adult-generated dentate granule cells (DGCs) that was highly correlated with impaired hippocampal function, as assessed using two behavioral assays of pattern discrimination. These behavioral tests included the A-B contextual fear-conditioning task and the trial-unique, delayed nonmatching-to-location (TUNL) touch-screen operant chamber task. Our findings identify HIF-1? as a novel regulator of adult hippocampal neurogenesis under non-pathological conditions, and underscore the importance of neurogenesis for pattern discrimination learning.
Project description:Macroautophagy/autophagy is generally regarded as a cytoprotective mechanism, and it remains a matter of controversy whether autophagy can cause cell death in mammals. Here, we show that chronic restraint stress suppresses adult hippocampal neurogenesis in mice by inducing autophagic cell death (ACD) of hippocampal neural stem cells (NSCs). We generated NSC-specific, inducible <i>Atg7</i> conditional knockout mice and found that they had an intact number of NSCs and neurogenesis level under chronic restraint stress and were resilient to stress- or corticosterone-induced cognitive and mood deficits. Corticosterone treatment of adult hippocampal NSC cultures induced ACD via SGK3 (serum/glucocorticoid regulated kinase 3) without signs of apoptosis. Our results demonstrate that ACD is biologically important in a mammalian system <i>in vivo</i> and would be an attractive target for therapeutic intervention for psychological stress-induced disorders.<b>Abbreviations</b>: AAV: adeno-associated virus; ACD: autophagic cell death; ACTB: actin, beta; Atg: autophagy-related; ASCL1/MASH1: achaete-scute family bHLH transcription factor 1; BafA<sub>1</sub>: bafilomycin A<sub>1</sub>; BrdU: Bromodeoxyuridine/5-bromo-2'-deoxyuridine; CASP3: caspase 3; cKO: conditional knockout; CLEM: correlative light and electron microscopy; CORT: corticosterone; CRS: chronic restraint stress; DAB: 3,3'-diaminobenzidine; DCX: doublecortin; DG: dentate gyrus; GC: glucocorticoid; GFAP: glial fibrillary acidic protein; HCN: hippocampal neural stem; i.p.: intraperitoneal; MAP1LC3B: microtubule-associated protein 1 light chain 3 beta; MKI67/Ki67: antigen identified by monoclonal antibody Ki 67; MWM: Morris water maze; Nec-1: necrostatin-1; NES: nestin; NR3C1/GR: nuclear receptor subfamily 3, group C, member 1; NSC: neural stem cell; PCD: programmed cell death; PFA: paraformaldehyde; PX: Phox homology; PtdIns3P: phosphatidylinositol-3-phosphate; RBFOX3/NeuN: RNA binding protein, fox-1 homolog (C. elegans) 3; SGK: serum/glucocorticoid-regulated kinases; SGZ: subgranular zone; SOX2: SRY (sex determining region Y)-box 2; SQSTM1: sequestosome 1; STS: staurosporine; TAM: tamoxifen; Ulk1: unc-51 like kinase 1; TUNEL: terminal deoxynucleotidyl transferase dUTP nick end labeling; VIM: vimentin; WT: wild type; ZFYVE1: zinc finger, FYVE domain containing 1; Z-VAD/Z-VAD-FMK: pan-caspase inhibitor.
Project description:In the adult rodent brain, neural stem cells (NSCs) persist in the ventricular-subventricular zone (V-SVZ) and the subgranular zone (SGZ), which are specialized niches in which young neurons for the olfactory bulb (OB) and hippocampus, respectively, are generated. Recent studies have significantly modified earlier views on the mechanisms of NSC self-renewal and neurogenesis in the adult brain. Here, we discuss the molecular control, heterogeneity, regional specification and cell division modes of V-SVZ NSCs, and draw comparisons with NSCs in the SGZ. We highlight how V-SVZ NSCs are regulated by local signals from their immediate neighbors, as well as by neurotransmitters and factors that are secreted by distant neurons, the choroid plexus and vasculature. We also review recent advances in single cell RNA analyses that reveal the complexity of adult neurogenesis. These findings set the stage for a better understanding of adult neurogenesis, a process that one day may inspire new approaches to brain repair.
Project description:<h4>Background</h4>The porcine brain is gyrencephalic with similar gray and white matter composition and size more comparable to the human rather than the rodent brain; however, there is lack of information about neural progenitor cells derived from this model.<h4>Results</h4>Here, we isolated GFAP-positive porcine neural stem cells (NSCs) from the brain explant of a transgenic piglet, with expression of CreER<sup>T2</sup> under the control of the GFAP promoter (pGFAP-CreER<sup>T2</sup>). The isolated pGFAP-CreER<sup>T2</sup> NSCs showed self-renewal and expression of representative NSC markers such as Nestin and Sox2. Pharmacological inhibition studies revealed that Notch1 signaling is necessary to maintain NSC identity, whereas serum treatment induced cell differentiation into reactive astrocytes and neurons.<h4>Conclusions</h4>Collectively, these results indicate that GFAP promoter-driven porcine CreER<sup>T2</sup> NSCs would be a useful tool to study neurogenesis of the porcine adult central nervous system and furthers our understanding of its potential clinical application in the future. ᅟ.
Project description:The early postnatal period is a unique time of brain development, as diminishing amounts of neurogenesis coexist with waves of gliogenesis. Understanding the molecular regulation of early postnatal gliogenesis may provide clues to normal and pathological embryonic brain ontogeny, particularly in regards to the development of astrocytes and oligodendrocytes. Cyclin dependent kinase 5 (Cdk5) contributes to neuronal migration and cell cycle control during embryogenesis, and to the differentiation of neurons and oligodendrocytes during adulthood. However, Cdk5's function in the postnatal period and within discrete progenitor lineages is unknown. Therefore, we selectively removed Cdk5 from nestin-expressing cells and their progeny by giving transgenic mice (nestin-CreERT2/R26R-YFP/CDK5(flox/flox) [iCdk5] and nestin-CreERT2/R26R-YFP/CDK5(wt/wt) [WT]) tamoxifen during postnatal (P) days P2-P 4 or P7-P 9, and quantified and phenotyped recombined (YFP+) cells at P14 and P21. When Cdk5 gene deletion was induced in nestin-expressing cells and their progeny during the wave of cortical and hippocampal gliogenesis (P2-P4), significantly fewer YFP+ cells were evident in the cortex, corpus callosum, and hippocampus. Phenotypic analysis revealed the cortical decrease was due to fewer YFP+ astrocytes and oligodendrocytes, with a slightly earlier influence seen in oligodendrocytes vs. astrocytes. This effect on cortical gliogenesis was accompanied by a decrease in YFP+ proliferative cells, but not increased cell death. The role of Cdk5 in gliogenesis appeared specific to the early postnatal period, as induction of recombination at a later postnatal period (P7-P9) resulted in no change YFP+ cell number in the cortex or hippocampus. Thus, glial cells that originate from nestin-expressing cells and their progeny require Cdk5 for proper development during the early postnatal period.
Project description:To characterize the properties of adult neural stem cells (NSCs), we generated and analyzed Sox2-GFP transgenic mice. Sox2-GFP cells in the subgranular zone (SGZ) express markers specific for progenitors, but they represent two morphologically distinct populations that differ in proliferation levels. Lentivirus- and retrovirus-mediated fate-tracing studies showed that Sox2+ cells in the SGZ have potential to give rise to neurons and astrocytes, revealing their multipotency at the population as well as at a single-cell level. A subpopulation of Sox2+ cells gives rise to cells that retain Sox2, highlighting Sox2+ cells as a primary source for adult NSCs. In response to mitotic signals, increased proliferation of Sox2+ cells is coupled with the generation of Sox2+ NSCs as well as neuronal precursors. An asymmetric contribution of Sox2+ NSCs may play an important role in maintaining the constant size of the NSC pool and producing newly born neurons during adult neurogenesis.
Project description:<h4>Aim</h4>cAMP signal transduction cascade activation is important in regulating neurogenesis in adult rodents by increasing the proliferation of newborn cells. Although the ventricular-subventricular zone (V-SVZ) and subgranular zone (SGZ) both contain large populations of neural stem/precursor cells; it remains unclear whether an alternative target of cAMP, the exchange protein directly activated by cAMP (Epac2), is involved in adult neurogenesis in the V-SVZ and SGZ. Here, we investigated the cell-specific expression of Epac2 protein in the V-SVZ and SGZ of the adult mouse brain.<h4>Methods</h4>Immunohistochemical analyses were performed using antibodies against Epac2, glial fibrillary acidic protein (GFAP), doublecortin (DCX), and beta-catenin, to examine the co-localization of Epac2 protein and neural stem/precursor cells in the V-SVZ and SGZ in three 8-week-old male mice.<h4>Results</h4>In the V-SVZ of the lateral ventricle, most GFAP-positive adult neural stem cells (NSC, defined as type B cells) and 75% of DCX-positive migrating neuroblasts (type A cells) expressed Epac2 proteins. Ninety-three percent of beta-catenin-positive ependymal cells (type E cells), which are in direct contact with NSCs and the ventricles, also expressed Epac2 protein. Similarly, in the SGZ of the hippocampus, Epac2-immunopositive signals were shown by 83% of GFAP-positive radial-glia-like NSCs (type 1 cells), 86% of DCX-positive transiently amplifying cells (type 2 and type 3 cells), and 71% of DCX-positive immature neurons. The present data suggest that a PKA-independent cAMP signaling pathway via Epac2 may be party to adult neurogenesis in the V-SVZ and the SGZ.
Project description:New neurons are added to the adult hippocampus throughout life and contribute to cognitive functions, including learning and memory. It remains unclear whether ongoing neurogenesis arises from self-renewing neural stem cells (NSCs) or from multipotential progenitor cells that cannot self-renew in the hippocampus. This is primarily based on observations that neural precursors derived from the subventricular zone (SVZ) can be passaged long term, whereas hippocampal subgranular zone (SGZ) precursors are rapidly depleted by passaging. We demonstrate here that high levels of bone morphogenetic protein (BMP) signaling occur in hippocampal but not SVZ precursors in vitro, and blocking BMP signaling with Noggin is sufficient to foster hippocampal cell self-renewal, proliferation, and multipotentiality using single-cell clonal analysis. Moreover, NSC maintenance requires continual Noggin exposure, which implicates BMPs as crucial regulators of NSC aging. In vivo, Noggin is expressed in the adult dentate gyrus and limits BMP signaling in proliferative cells of the SGZ. Transgenic Noggin overexpression in the SGZ increases multiple precursor cell populations but proportionally increases the glial fibrillary acidic protein-positive cell population at the expense of other precursors, suggesting that Noggin acts on NSCs in vivo. To confirm this, we used a dual thymidine analog paradigm to repeatedly label slowly dividing cells over a long duration. We find that small populations of label-retaining cells exist in the SGZ and that Noggin overexpression increases their numbers. Thus, we propose that the adult hippocampus contains a population of NSCs, which can be expanded both in vitro and in vivo by blocking BMP signaling.
Project description:Genomic imprinting is implicated in the control of gene dosage in neurogenic niches. Here we address the importance of Igf2 imprinting for murine adult neurogenesis in the subventricular zone (SVZ) and in the subgranular zone (SGZ) of the hippocampus in vivo. In the SVZ, paracrine IGF2 is a cerebrospinal fluid and endothelial-derived neurogenic factor requiring biallelic expression, with mutants having reduced activation of the stem cell pool and impaired olfactory bulb neurogenesis. In contrast, Igf2 is imprinted in the hippocampus acting as an autocrine factor expressed in neural stem cells (NSCs) solely from the paternal allele. Conditional mutagenesis of Igf2 in blood vessels confirms that endothelial-derived IGF2 contributes to NSC maintenance in SVZ but not in the SGZ, and that this is regulated by the biallelic expression of IGF2 in the vascular compartment. Our findings indicate that a regulatory decision to imprint or not is a functionally important mechanism of transcriptional dosage control in adult neurogenesis.