Kasum Azim

GSK3β negatively regulates oligodendrocyte differentiation and myelination in vivo

Glycogen synthase kinase 3β (GSK3β) is an essential integrating molecule for multiple proliferation and differentiation signals that regulate cell fate. Here, we have examined the effects of inhibiting GSK3β on the development of oligodendrocytes (OLs) from their oligodendrocyte precursors (OP) in vivo by injection into the lateral ventricle of postnatal mice and ex vivo in organotypic cultures of isolated intact rodent optic nerve. Our results show that a range of GSK3β inhibitors (ARA‐014418, lithium, indirubin, and L803‐mt) increase OPs and OLs and promote myelination. Inhibition of GSK3β stimulates OP proliferation and is prosurvival and antiapoptotic. The effects of GSK3β inhibition in OPs is via the canonical Wnt signaling pathway by stimulating nuclear translocation of β‐catenin. However, direct comparison of the effects of Wnt3a and GSK3β inhibition in optic nerves shows that they have opposing actions on OLs, whereby GSK3β inhibition strikingly increases OL differentiation, whereas Wnt3a inhibits OL differentiation. Notably, GSK3β inhibition overrides the negative effects of Wnt3a on OLs, indicating novel GSK3β signaling mechanisms that negatively regulate OL differentiation. We identify that two mechanisms of GSK3β inhibition are to stimulate cAMP response element binding (CREB) and decrease Notch1 signaling, which positively and negatively regulate OL differentiation and myelination, respectively. A key finding is that GSK3β inhibition has equivalent effects in the adult and stimulates the regeneration of OLs and remyelination following chemically induced demyelination. This study identifies GSK3β as a profound negative regulator of OL differentiation that contributes to inefficient regeneration of OLs and myelin repair in demyelination.

Persistent Wnt/β-Catenin Signaling Determines Dorsalization of the Postnatal Subventricular Zone and Neural Stem Cell Specification into Oligodendrocytes and Glutamatergic Neurons

In the postnatal and adult central nervous system (CNS), the subventricular zone (SVZ) of the forebrain is the main source of neural stem cells (NSCs) that generate olfactory neurons and oligodendrocytes (OLs), the myelinating cells of the CNS. Here, we provide evidence of a primary role for canonical Wnt/β‐catenin signaling in regulating NSC fate along neuronal and oligodendroglial lineages in the postnatal SVZ. Our findings demonstrate that glutamatergic neuronal precursors (NPs) and oligodendrocyte precursors (OPs) are derived strictly from the dorsal SVZ (dSVZ) microdomain under the control of Wnt/β‐catenin, whereas GABAergic NPs are derived mainly from the lateral SVZ (lSVZ) microdomain independent of Wnt/β‐catenin. Transcript analysis of microdissected SVZ microdomains revealed that canonical Wnt/β‐catenin signaling was more pronounced in the dSVZ microdomain. This was confirmed using the β‐catenin‐activated Wnt‐reporter mouse and by pharmacological stimulation of Wnt/β‐catenin by infusion of the specific glycogen synthase kinase 3β inhibitor, AR‐A014418, which profoundly increased the generation of cycling cells. In vivo genetic/pharmacological stimulation or inhibition of Wnt/β‐catenin, respectively, increased and decreased the differentiation of dSVZ‐NSCs into glutamatergic NPs, and had a converse effect on GABAergic NPs. Activation of Wnt/β‐catenin dramatically stimulated the generation of OPs, but its inhibition had no effect, indicating other factors act in concert with Wnt/β‐catenin to fine tune oligodendrogliogenesis in the postnatal dSVZ. These results demonstrate a role for Wnt/β‐catenin signaling within the dorsal microdomain of the postnatal SVZ, in regulating the genesis of glutamatergic neurons and OLs. Stem Cells 2014;32:1301–1312

Adding a spatial dimension to postnatal ventricular-subventricular zone neurogenesis

Neurogenesis does not stop abruptly at birth, but persists in specific brain regions throughout life. The neural stem cells (NSCs) located in the largest germinal region of the forebrain, the ventricular-subventricular zone (V-SVZ), replenish olfactory neurons throughout life. However, V-SVZ NSCs are heterogeneous: they have different embryonic origins and give rise to distinct neuronal subtypes depending on their location. In this Review, we discuss how this spatial heterogeneity arises, how it affects NSC biology, and why its consideration in future studies is crucial for understanding general principles guiding NSC self-renewal, differentiation and specification. Summary: This review highlights the contribution of geometry to the regulation of the neurogenic niche in the mouse and human adult brain for continuous neuronal replacement throughout life.

Intraventricular injection of FGF-2 promotes generation of oligodendrocyte-lineage cells in the postnatal and adult forebrain.

FGF2 is considered a key factor in the generation of oligodendrocytes (OLs) derived from neural stem cells (NSCs) located within the subventricular zone (SVZ). Here, we have examined FGF2 signaling in the forebrain of postnatal and adult mice. Using qPCR of microdissected microdomains of the dorsal SVZ (dSVZ) and lateral SVZ (lSVZ), and prominin1‐sorted NSCs purified from these microdomains, we show that transcripts for FGF receptor 1 (FGFR1) and FGFR2 are enriched in the dSVZ, from which OLs are largely derived, whereas FGFR3 are significantly enriched within prominen1‐sorted NSC of the lSVZ, which mainly generate olfactory interneurons. We show that direct administration of FGF2 into the lateral ventricle increased the generation of oligodendrocyte progenitors (OPCs) throughout the SVZ, both within the dSVZ and ectopically in the lSVZ and ependymal wall of the SVZ. Furthermore, FGF2 stimulated proliferation of neural progenitors (NPs) and their differentiation into OPCs. The results indicate that FGF2 increased specification of OPCs, inducing NPs to follow an oligodendrocyte developmental pathway. Notably, FGF2 did not block OPC differentiation and increased the number of oligodendrocytes in the periventricular white matter (PVWM) and cortex. However, FGF2 markedly disrupted myelination in the PVWM. A key finding was that FGF2 had equivalent actions on the generation of OPCs and myelin disruption in postnatal and adult mice. This study demonstrates a central role for FGF2 in promoting oligodendrocyte generation in the developing and adult brain.

GSK3β regulates oligodendrogenesis in the dorsal microdomain of the subventricular zone via Wnt-β-catenin signaling

Oligodendrocytes, the myelinating cells of the CNS, are derived postnatally from oligodendrocyte precursors (OPs) of the subventricular zone (SVZ). However, the mechanisms that regulate their generation from SVZ neural stem cells (NSC) are poorly understood. Here, we have examined the role of glycogen synthase kinase 3β (GSK3β), an effector of multiple converging signaling pathways in postnatal mice. The expression of GSK3β by rt‐qPCR was most prominent in the SVZ and in the developing white matter, around the first 1–2 weeks of postnatal life, coinciding with the peak periods of OP differentiation. Intraventricular infusion of the GSK3β inhibitor ARA‐014418 in mice aged postnatal day (P) 8–11 significantly increased generation of OPs in the dorsal microdomain of the SVZ, as shown by expression of cell specific markers using rt‐qPCR and immunolabelling. Analysis of stage specific markers revealed that the augmentation of OPs occurred via increased specification from earlier SVZ cell types. These effects of GSK3β inhibition on the dorsal SVZ were largely attributable to stimulation of the canonical Wnt/β‐catenin signaling pathway over other pathways. The results indicate GSK3β is a key endogenous factor for specifically regulating oligodendrogenesis from the dorsal SVZ microdomain under the control of Wnt‐signaling. GLIA 2014;62:778–789

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.

Transcriptional Hallmarks of Heterogeneous Neural Stem Cell Niches of the Subventricular Zone

Throughout postnatal life in mammals, neural stem cells (NSCs) are located in the subventricular zone (SVZ) of the lateral ventricles. The greatest diversity of neuronal and glial lineages they generate occurs during early postnatal life in a region‐specific manner. In order to probe heterogeneity of the postnatal SVZ, we microdissected its dorsal and lateral walls at different postnatal ages and isolated NSCs and their immediate progeny based on their expression of Hes5‐EGFP/Prominin1 and Ascl1‐EGFP, respectively. Whole genome comparative transcriptome analysis revealed transcriptional regulators as major hallmarks that sustain postnatal SVZ regionalization. Manipulation of single genes encoding for locally enriched transcription factors (loss‐of‐function or ectopic gain‐of‐function in vivo) influenced NSC specification indicating that the fate of regionalized postnatal SVZ‐NSCs can be readily modified. These findings reveal the pronounced transcriptional heterogeneity of the postnatal SVZ and provide targets to recruit region‐specific lineages in regenerative contexts. Stem Cells 2015;33:2232–2242

Early Decline in Progenitor Diversity in the Marmoset Lateral Ventricle

The lateral ventricle (LV) of the adult rodent brain harbors neural stem cells (NSCs) that continue to generate new neurons throughout life. NSCs located in defined areas of the LV walls generate progenitors with distinct transcriptional profiles that are committed to specific neuronal fates. Here, we assessed if such diversity of NSCs also exist in the adult common marmoset, a widely used primate species in basic and clinical neuroscience research. We first investigated the 3D distributions of proliferative progenitors and committed neuroblasts in the marmoset forebrain. In addition to these maps, we assessed the spatial presence of divergent progenitor populations based on their expression of defined transcription factors, that is, Dlx2, Pax6, Tbr2, and Ngn2 which are differentially expressed by γ-aminobutyric acidergic versus glutamatergic progenitors in the adult rodent forebrain. In striking contrast to rodents, glutamatergic progenitors were only sparse in neonates and absent from the adult LV, whilst present in the hippocampus. Our analyses highlight major differences in the diversity of NSCs of the marmoset LV compared with rodents and emphasize the need to address NSCs diversity in evolutionary higher order mammals concomitantly to rodents.

Pharmacogenomic identification of small molecules for lineage specific manipulation of subventricular zone germinal activity

Strategies for promoting neural regeneration are hindered by the difficulty of manipulating desired neural fates in the brain without complex genetic methods. The subventricular zone (SVZ) is the largest germinal zone of the forebrain and is responsible for the lifelong generation of interneuron subtypes and oligodendrocytes. Here, we have performed a bioinformatics analysis of the transcriptome of dorsal and lateral SVZ in early postnatal mice, including neural stem cells (NSCs) and their immediate progenies, which generate distinct neural lineages. We identified multiple signaling pathways that trigger distinct downstream transcriptional networks to regulate the diversity of neural cells originating from the SVZ. Next, we used a novel in silico genomic analysis, searchable platform-independent expression database/connectivity map (SPIED/CMAP), to generate a catalogue of small molecules that can be used to manipulate SVZ microdomain-specific lineages. Finally, we demonstrate that compounds identified in this analysis promote the generation of specific cell lineages from NSCs in vivo, during postnatal life and adulthood, as well as in regenerative contexts. This study unravels new strategies for using small bioactive molecules to direct germinal activity in the SVZ, which has therapeutic potential in neurodegenerative diseases.

E-proteins orchestrate the progression of neural stem cell differentiation in the postnatal forebrain

BackgroundNeural stem cell (NSC) differentiation is a complex multistep process that persists in specific regions of the postnatal forebrain and requires tight regulation throughout life. The transcriptional control of NSC proliferation and specification involves Class II (proneural) and Class V (Id1-4) basic helix-loop-helix (bHLH) proteins. In this study, we analyzed the pattern of expression of their dimerization partners, Class I bHLH proteins (E-proteins), and explored their putative role in orchestrating postnatal subventricular zone (SVZ) neurogenesis.ResultsOverexpression of a dominant-negative form of the E-protein E47 (dnE47) confirmed a crucial role for bHLH transcriptional networks in postnatal neurogenesis by dramatically blocking SVZ NSC differentiation. In situ hybridization was used in combination with RT-qPCR to measure and compare the level of expression of E-protein transcripts (E2-2, E2A, and HEB) in the neonatal and adult SVZ as well as in magnetic affinity cell sorted progenitor cells and neuroblasts. Our results evidence that E-protein transcripts, in particular E2-2 and E2A, are enriched in the postnatal SVZ with expression levels increasing as cells engage towards neuronal differentiation. To investigate the role of E-proteins in orchestrating lineage progression, both in vitro and in vivo gain-of-function and loss-of-function experiments were performed for individual E-proteins. Overexpression of E2-2 and E2A promoted SVZ neurogenesis by enhancing not only radial glial cell differentiation but also cell cycle exit of their progeny. Conversely, knock-down by shRNA electroporation resulted in opposite effects. Manipulation of E-proteins and/or Ascl1 in SVZ NSC cultures indicated that those effects were Ascl1 dependent, although they could not solely be attributed to an Ascl1-induced switch from promoting cell proliferation to triggering cell cycle arrest and differentiation.ConclusionsIn contrast to former concepts, suggesting ubiquitous expression and subsidiary function for E-proteins to foster postnatal neurogenesis, this work unveils E-proteins as being active players in the orchestration of postnatal SVZ neurogenesis.