The laminar position, morphology, and gene expression profiles of cortical astrocytes are influenced by time of birth from ventricular/subventricular progenitors.

Astrocytes that reside in superficial (SL) and deep cortical layers have distinct molecular profiles and morphologies, which may underlie specific functions. Here, we demonstrate that the production of SL and deep layer (DL) astrocyte populations from neural progenitor cells in the mouse is temporally regulated. Lineage tracking following in utero and postnatal electroporation with PiggyBac (PB) EGFP and birth dating with EdU and FlashTag, showed that apical progenitors produce astrocytes during late embryogenesis (E16.5) that are biased to the SL, while postnatally labeled (P0) astrocytes are biased to the DL. In contrast, astrocytes born during the predominantly neurogenic window (E14.5) showed a random distribution in the SL and DL. Of interest, E13.5 astrocytes birth dated at E13.5 with EdU showed a lower layer bias, while FT labeling of apical progenitors showed no bias. Finally, examination of the morphologies of "biased" E16.5- and P0-labeled astrocytes demonstrated that E16.5-labeled astrocytes exhibit different morphologies in different layers, while P0-labeled astrocytes do not. Differences based on time of birth are also observed in the molecular profiles of E16.5 versus P0-labeled astrocytes. Altogether, these results suggest that the morphological, molecular, and positional diversity of cortical astrocytes is related to their time of birth from ventricular/subventricular zone progenitors.

Multifaceted actions of Zeb2 in postnatal neurogenesis from the ventricular-subventricular zone to the olfactory bulb.

The transcription factor Zeb2 controls fate specification and subsequent differentiation and maturation of multiple cell types in various embryonic tissues. It binds many protein partners, including activated Smad proteins and the NuRD co-repressor complex. How Zeb2 subdomains support cell differentiation in various contexts has remained elusive. Here, we studied the role of Zeb2 and its domains in neurogenesis and neural differentiation in the young postnatal ventricular-subventricular zone (V-SVZ), in which neural stem cells generate olfactory bulb-destined interneurons. Conditional Zeb2 knockouts and separate acute loss- and gain-of-function approaches indicated that Zeb2 is essential for controlling apoptosis and neuronal differentiation of V-SVZ progenitors before and after birth, and we identified Sox6 as a potential downstream target gene of Zeb2. Zeb2 genetic inactivation impaired the differentiation potential of the V-SVZ niche in a cell-autonomous fashion. We also provide evidence that its normal function in the V-SVZ also involves non-autonomous mechanisms. Additionally, we demonstrate distinct roles for Zeb2 protein-binding domains, suggesting that Zeb2 partners co-determine neuronal output from the mouse V-SVZ in both quantitative and qualitative ways in early postnatal life.

Characterization of the ventricular-subventricular stem cell niche during human brain development.

Human brain development proceeds via a sequentially transforming stem cell population in the ventricular-subventricular zone (V-SVZ). An essential, but understudied, contributor to V-SVZ stem cell niche health is the multi-ciliated ependymal epithelium, which replaces stem cells at the ventricular surface during development. However, reorganization of the V-SVZ stem cell niche and its relationship to ependymogenesis has not been characterized in the human brain. Based on comprehensive comparative spatiotemporal analyses of cytoarchitectural changes along the mouse and human ventricle surface, we uncovered a distinctive stem cell retention pattern in humans as ependymal cells populate the surface of the ventricle in an occipital-to-frontal wave. During perinatal development, ventricle-contacting stem cells are reduced. By 7 months few stem cells are detected, paralleling the decline in neurogenesis. In adolescence and adulthood, stem cells and neurogenesis are not observed along the lateral wall. Volume, surface area and curvature of the lateral ventricles all significantly change during fetal development but stabilize after 1 year, corresponding with the wave of ependymogenesis and stem cell reduction. These findings reveal normal human V-SVZ development, highlighting the consequences of disease pathologies such as congenital hydrocephalus.

Dynamic Changes in the Neurogenic Potential in the Ventricular-Subventricular Zone of Common Marmoset during Postnatal Brain Development.

Even after birth, neuronal production continues in the ventricular-subventricular zone (V-SVZ) and hippocampus in many mammals. The immature new neurons ("neuroblasts") migrate and then mature at their final destination. In humans, neuroblast production and migration toward the neocortex and the olfactory bulb (OB) occur actively only for a few months after birth and then sharply decline with age. However, the precise spatiotemporal profiles and fates of postnatally born neurons remain unclear due to methodological limitations. We previously found that common marmosets, small nonhuman primates, share many features of V-SVZ organization with humans. Here, using marmosets injected with thymidine analogue(s) during various postnatal periods, we demonstrated spatiotemporal changes in neurogenesis during development. V-SVZ progenitor proliferation and neuroblast migration toward the OB and neocortex sharply decreased by 4 months, most strikingly in a V-SVZ subregion from which neuroblasts migrated toward the neocortex. Postnatally born neurons matured within a few months in the OB and hippocampus but remained immature until 6 months in the neocortex. While neurogenic activity was sustained for a month after birth, the distribution and/or differentiation diversity was more restricted in 1-month-born cells than in the neonatal-born population. These findings shed light on distinctive features of postnatal neurogenesis in primates.

Cyclin-Dependent Kinase 4 Regulates Adult Neural Stem Cell Proliferation and Differentiation in Response to Insulin.

Insulin is one of the standard components used to culture primary neurospheres. Although it stimulates growth of different types of cells, the effects of insulin on adult neural stem cells (NSCs) have not been well characterized. Here, we reveal that insulin stimulates proliferation, but not survival or self-renewal, of adult NSCs. This effect is mediated by insulin receptor substrate 2 (IRS2) and subsequent activation of the protein kinase B (or Akt), leading to increased activity of the G1-phase cyclin-dependent kinase 4 (Cdk4) and cell cycle progression. Neurospheres isolated from Irs2-deficient mice are reduced in size and fail to expand in culture and this impaired proliferation is rescued by introduction of a constitutively active Cdk4 (Cdk4R24C/R24C ). More interestingly, activation of the IRS2/Akt/Cdk4 signaling pathway by insulin is also necessary for the generation in vitro of neurons and oligodendrocytes from NSCs. Furthermore, the IRS2/Cdk4 pathway is also required for neuritogenesis, an aspect of neuronal maturation that has not been previously linked to regulation of the cell cycle. Differentiation of NSCs usually follows exit from the cell cycle due to increased levels of CDK-inhibitors which prevent activation of CDKs. In contrast, our data indicate that IRS2-mediated Cdk4 activity in response to a mitogen such as insulin promotes terminal differentiation of adult NSCs. Stem Cells 2017;35:2403-2416.

Mechanisms of neuronal migration in the adult brain.

Adult neurogenesis was first observed nearly 60 years ago, and it has since grown into an important neurochemistry research field. Much recent research has focused on the treatment of brain diseases through neuronal regeneration with endogenously generated neurons. In the adult brain, immature neurons called neuroblasts are continuously generated in the ventricular-subventricular zone (V-SVZ). These neuroblasts migrate rapidly through the rostral migratory stream to the olfactory bulb, where they mature and are integrated into the neuronal circuitry. After brain insult, some of the neuroblasts in the V-SVZ migrate toward the lesion to repopulate the injured tissue. This notable migratory capacity of V-SVZ-derived neuroblasts is important for efficiently regenerating neurons in remote areas of the brain. As these neurons migrate for long distances through adult brain tissue, they are supported by various guidance cues and structures that act as scaffolds. Some of these mechanisms are unique to neuroblast migration in the adult brain, and are not involved in migration in the developing brain. Here, we review the latest findings on the mechanisms of neuroblast migration in the adult brain under physiological and pathological conditions, and discuss various issues that still need to be resolved. This article is part of the mini review series "60th Anniversary of the Japanese Society for Neurochemistry".

The vasculature as a neural stem cell niche.

Neural stem cells (NSCs) are multipotent, self-renewing progenitors that generate progeny that differentiate into neurons and glia. NSCs in the adult mammalian brain are generally quiescent. Environmental stimuli such as learning or exercise can activate quiescent NSCs, inducing them to proliferate and produce new neurons and glia. How are these behaviours coordinated? The neurovasculature, the circulatory system of the brain, is a key component of the NSC microenvironment, or 'niche'. Instructive signals from the neurovasculature direct NSC quiescence, proliferation, self-renewal and differentiation. During ageing, a breakdown in the niche accompanies NSC dysfunction and cognitive decline. There is much interest in reversing these changes and enhancing NSC activity by targeting the neurovasculature therapeutically. Here we discuss principles of neurovasculature-NSC crosstalk, and the implications for the design of NSC-based therapies. We also consider the emerging contributions to this field of the model organism Drosophila melanogaster.

Decreased survival in glioblastomas is specific to contact with the ventricular-subventricular zone, not subgranular zone or corpus callosum.

The clinical effect of radiographic contact of glioblastoma (GBM) with neurogenic zones (NZ)-the ventricular-subventricular (VSVZ) and subgranular (SGZ) zones-and the corpus callosum (CC) remains unclear and, in the case of the SGZ, unexplored. We investigated (1) if GBM contact with a NZ correlates with decreased survival; (2) if so, whether this effect is associated with a specific NZ; and (3) if radiographic contact with or invasion of the CC by GBM is associated with decreased survival. We retrospectively identified 207 adult patients who underwent cytoreductive surgery for GBM followed by chemotherapy and/or radiation. Age, preoperative Karnofsky performance status score (KPS), and extent of resection were recorded. Preoperative MRIs were blindly analyzed to calculate tumor volume and assess its contact with VSVZ, SGZ, CC, and cortex. Overall (OS) and progression free (PFS) survivals were calculated and analyzed with multivariate Cox analyses. Among the 207 patients, 111 had GBM contacting VSVZ (VSVZ+GBMs), 23 had SGZ+GBMs, 52 had CC+GBMs, and 164 had cortex+GBMs. VSVZ+, SGZ+, and CC+ GBMs were significantly larger in size relative to their respective non-contacting controls. Multivariate Cox survival analyses revealed GBM contact with the VSVZ, but not SGZ, CC, or cortex, as an independent predictor of lower OS, PFS, and early recurrence. We hypothesize that the VSVZ niche has unique properties that contribute to GBM pathobiology in adults.

Influence of glioblastoma contact with the lateral ventricle on survival: a meta-analysis.

The ventricular-subventricular zone (V-SVZ), which lies in the walls of the lateral ventricles (LV), is the largest neurogenic niche within the adult brain. Whether radiographic contact with the LV influences survival in glioblastoma (GBM) patients remains unclear. We assimilated and analyzed published data comparing survival in GBM patients with (LV+GBM) and without (LV-GBM) radiographic LV contact. PubMed, EMBASE, and Cochrane electronic databases were searched. Fifteen studies with survival data on LV+GBM and LV-GBM patients were identified. Their Kaplan-Meier survival curves were digitized and pooled for generation of median overall (OS) and progression free (PFS) survivals and log-rank hazard ratios (HRs). The log-rank and reported multivariate HRs after accounting for the common predictors of GBM survival were analyzed separately by meta-analyses. The calculated median survivals (months) from pooled data were 12.95 and 16.58 (OS), and 4.54 and 6.25 (PFS) for LV+GBMs and LV-GBMs, respectively, with an overall log-rank HRs of 1.335 [1.204-1.513] (OS) and 1.387 [1.225-1.602] (PFS). Meta-analysis of log-rank HRs resulted in summary HRs of 1.58 [1.35-1.85] (OS, 10 studies) and 1.41 [1.22-1.64] (PFS, 5 studies). Meta-analysis of multivariate HRs resulted in summary HRs of 1.35 [1.14-1.58] (OS, 6 studies) and 1.64 [0.88-3.05] (PFS, 3 studies). Patients with GBM contacting the LV have lower survival. This effect may be independent of the common predictors of GBM survival, suggesting a clinical influence of V-SVZ contact on GBM biology.

Isolation, culture and analysis of adult subependymal neural stem cells.

Individual cells dissected from the subependymal neurogenic niche of the adult mouse brain proliferate in medium containing basic fibroblast growth factor (bFGF) and/or epidermal growth factor (EGF) as mitogens, to produce multipotent clonal aggregates called neurospheres. These cultures constitute a powerful tool for the study of neural stem cells (NSCs) provided that they allow the analysis of their features and potential capacity in a controlled environment that can be modulated and monitored more accurately than in vivo. Clonogenic and population analyses under mitogen addition or withdrawal allow the quantification of the self-renewing and multilineage potency of these cells and the identification of the mechanisms involved in these properties. Here, we describe a set of procedures developed and/or modified by our group including several experimental options that can be used either independently or in combination for the ex vivo assessment of cell properties of NSCs obtained from the adult subependymal niche.

Sonic hedgehog signaling in the postnatal brain.

Sonic hedgehog (Shh) is a pleiotropic factor in the developing central nervous system (CNS), driving proliferation, specification, and axonal targeting in multiple sites within the forebrain, hindbrain, and spinal cord. Studies in embryonic CNS have shown how gradients of this morphogen are translated by neuroepithelial precursors to determine the types of neurons and glial cells they produce [1,2]. Shh also has a well-characterized role as a mitogen for specific progenitor cell types in neural development [3,4]. As we begin to appreciate that Shh continues to act in the adult brain, a central question is what functional role this ligand plays when major morphogenetic and proliferative processes are no longer in operation. A second fundamental question is whether similar signaling mechanisms operate in embryonic and adult CNS. In the two major germinal zones of the adult brain, Shh signaling modulates the self-renewal and specification of astrocyte-like primary progenitors, frequently referred to as neural stem cells (NSCs). It also may regulate the response of the mature brain to injury, as Shh signaling has been variously proposed to enhance or inhibit the development of a reactive astrocyte phenotype. The identity of cells producing the Shh ligand, and the conditions that trigger its release, are also areas of growing interest; both germinal zones in the adult brain contain Shh-responsive cells but do not autonomously produce this ligand. Here, we review recent findings revealing the function of this fascinating pathway in the postnatal and adult brain, and highlight ongoing areas of investigation into its actions long past the time when it shapes the developing brain.

Roles of Wnt Signaling in the Neurogenic Niche of the Adult Mouse Ventricular-Subventricular Zone.

In many animal species, the production of new neurons (neurogenesis) occurs throughout life, in a specialized germinal region called the ventricular-subventricular zone (V-SVZ). In this region, neural stem cells undergo self-renewal and generate neural progenitor cells and new neurons. In the olfactory system, the new neurons migrate rostrally toward the olfactory bulb, where they differentiate into mature interneurons. V-SVZ-derived new neurons can also migrate toward sites of brain injury, where they contribute to neural regeneration. Recent studies indicate that two major branches of the Wnt signaling pathway, the Wnt/ß-catenin and Wnt/planar cell polarity pathways, play essential roles in various facets of adult neurogenesis. Here, we review the Wnt signaling-mediated regulation of adult neurogenesis in the V-SVZ under physiological and pathological conditions.

Ventricular-subventricular zone stem cell niche adaptations in a mouse model of post-infectious hydrocephalus.

Congenital post-infectious hydrocephalus (PIH) is a condition characterized by enlargement of the ventricular system, consequently imposing a burden on the associated stem cell niche, the ventricular-subventricular zone (V-SVZ). To investigate how the V-SVZ adapts in PIH, we developed a mouse model of influenza virus-induced PIH based on direct intracerebroventricular injection of mouse-adapted influenza virus at two distinct time points: embryonic day 16 (E16), when stem cells line the ventricle, and postnatal day 4 (P4), when an ependymal monolayer covers the ventricle surface and stem cells retain only a thin ventricle-contacting process. Global hydrocephalus with associated regions of astrogliosis along the lateral ventricle was found in 82% of the mice infected at P4. Increased ependymogenesis was observed at gliotic borders and throughout areas exhibiting intact ependyma based on tracking of newly divided cells. Additionally, in areas of intact ependyma, stem cell numbers were reduced; however, we found no significant reduction in new neurons reaching the olfactory bulb following onset of ventriculomegaly. At P4, injection of only the non-infectious viral component neuraminidase resulted in limited, region-specific ventriculomegaly due to absence of cell-to-cell transmission. In contrast, at E16 intracerebroventricular injection of influenza virus resulted in death at birth due to hypoxia and multiorgan hemorrhage, suggesting an age-dependent advantage in neonates, while the viral component neuraminidase resulted in minimal, or no, ventriculomegaly. In summary, we tracked acute adaptations of the V-SVZ stem cell niche following onset of ventriculomegaly and describe developmental changes that help mitigate the severity of congenital PIH.

Neonatal brain injury unravels transcriptional and signaling changes underlying the reactivation of cortical progenitors.

Germinal activity persists throughout life within the ventricular-subventricular zone (V-SVZ) of the postnatal forebrain due to the presence of neural stem cells (NSCs). Accumulating evidence points to a recruitment for these cells following early brain injuries and suggests their amenability to manipulations. We used chronic hypoxia as a rodent model of early brain injury to investigate the reactivation of cortical progenitors at postnatal times. Our results reveal an increased proliferation and production of glutamatergic progenitors within the dorsal V-SVZ. Fate mapping of V-SVZ NSCs demonstrates their contribution to de novo cortical neurogenesis. Transcriptional analysis of glutamatergic progenitors shows parallel changes in methyltransferase 14 (Mettl14) and Wnt/ß-catenin signaling. In agreement, manipulations through genetic and pharmacological activation of Mettl14 and the Wnt/ß-catenin pathway, respectively, induce neurogenesis and promote newly-formed cell maturation. Finally, labeling of young adult NSCs demonstrates that pharmacological NSC activation has no adverse effects on the reservoir of V-SVZ NSCs and on their germinal activity.

Which neurodevelopmental processes continue in humans after birth?

Once we are born, the number and location of nerve cells in most parts of the brain remain unchanged. These types of structural changes are therefore a significant form of flexibility for the neural circuits where they occur. In humans, the postnatal birth of neurons is limited; however, neurons do continue to migrate into some brain regions throughout infancy and even into adolescence. In human infants, multiple migratory pathways deliver interneurons to destinations across the frontal and temporal lobe cortex. Shorter-range migration of excitatory neurons also appears to continue during adolescence, particularly near the amygdala paralaminar nucleus, a region that follows a delayed trajectory of growth from infancy to adulthood. The significance of the timing for when different brain regions recruit new neurons through these methods is unknown; however, both processes of protracted migration and maturation are prominent in humans. Mechanisms like these that reconfigure neuronal circuits are a substrate for critical periods of plasticity and could contribute to distinctive circuit functionality in human brains.

Proteomic analysis of Girdin-interacting proteins in migrating new neurons in the postnatal mouse brain.

Neural stem cells continuously generate new neurons in the ventricular-subventricular zone (V-SVZ) of the postnatal and adult mammalian brain. New neurons born in the rodent V-SVZ migrate toward the olfactory bulb (OB), where they differentiate into interneurons. To reveal novel intracellular molecular mechanisms that control postnatal neuronal migration, we performed a global proteomic search for proteins interacting with Girdin, an essential protein for postnatal neuronal migration. Using GST pull-down and LC-MS/MS shotgun analysis, we identified cytoskeletal proteins, cytoskeleton-binding proteins, and signal-transduction proteins as possible participants in neuronal migration. Our results suggest that Girdin and Girdin-interacting proteins control neuronal migration by regulating actin and/or microtubule dynamics.

Corrigendum: PlexinD1 signaling controls domain-specific dendritic development in newborn neurons in the postnatal olfactory bulb.

[This corrects the article DOI: 10.3389/fnins.2023.1143130.].

Amphiphilic peptide-tagged N-cadherin forms radial glial-like fibers that enhance neuronal migration in injured brain and promote sensorimotor recovery.

The mammalian brain has very limited ability to regenerate lost neurons and recover function after injury. Promoting the migration of young neurons (neuroblasts) derived from endogenous neural stem cells using biomaterials is a new and promising approach to aid recovery of the brain after injury. However, the delivery of sufficient neuroblasts to distant injured sites is a major challenge because of the limited number of scaffold cells that are available to guide neuroblast migration. To address this issue, we have developed an amphiphilic peptide [(RADA)3-(RADG)] (mRADA)-tagged N-cadherin extracellular domain (Ncad-mRADA), which can remain in mRADA hydrogels and be injected into deep brain tissue to facilitate neuroblast migration. Migrating neuroblasts directly contacted the fiber-like Ncad-mRADA hydrogel and efficiently migrated toward an injured site in the striatum, a deep brain area. Furthermore, application of Ncad-mRADA to neonatal cortical brain injury efficiently promoted neuronal regeneration and functional recovery. These results demonstrate that self-assembling Ncad-mRADA peptides mimic both the function and structure of endogenous scaffold cells and provide a novel strategy for regenerative therapy.

PlexinD1 signaling controls domain-specific dendritic development in newborn neurons in the postnatal olfactory bulb.

Newborn neurons show immature bipolar morphology and continue to migrate toward their destinations. After the termination of migration, newborn neurons undergo spatially controlled dendrite formation and change into a complex morphology. The mechanisms of dendritic development of newborn neurons have not been fully understood. Here, we show that in the postnatal olfactory bulb (OB), the Sema3E-PlexinD1 signaling, which maintains bipolar morphology of newborn neurons, also regulates their dendritic development after the termination of migration in a dendritic domain-specific manner. Genetic ablation of Sema3E or PlexinD1 enhanced dendritic branching in the proximal domain of the apical dendrites of OB newborn granule cells, whereas PlexinD1 overexpression suppressed it in a Rho binding domain (RBD)-dependent manner. Furthermore, RhoJ, a small GTPase that directly binds to PlexinD1RBD in vascular endothelial cells, is expressed in migrating and differentiating newborn granule cells in the OB and is also involved in the suppression of proximal branching of their apical dendrites. These results suggest that the Sema3E-PlexinD1-RhoJ axis regulates domain-specific dendrite formation of newborn neurons in the postnatal OB.

High-efficiency pharmacogenetic ablation of oligodendrocyte progenitor cells in the adult mouse CNS.

Approaches to investigate adult oligodendrocyte progenitor cells (OPCs) by targeted cell ablation in the rodent CNS have limitations in the extent and duration of OPC depletion. We have developed a pharmacogenetic approach for conditional OPC ablation, eliminating >98% of OPCs throughout the brain. By combining recombinase-based transgenic and viral strategies for targeting OPCs and ventricular-subventricular zone (V-SVZ)-derived neural precursor cells (NPCs), we found that new PDGFRA-expressing cells born in the V-SVZ repopulated the OPC-deficient brain starting 12 days after OPC ablation. Our data reveal that OPC depletion induces V-SVZ-derived NPCs to generate vast numbers of PDGFRA+NG2+ cells with the capacity to proliferate and migrate extensively throughout the dorsal anterior forebrain. Further application of this approach to ablate OPCs will advance knowledge of the function of both OPCs and oligodendrogenic NPCs in health and disease.