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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.

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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.

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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.

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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.

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

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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.

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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.

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Primary cilia provide a specialized subcellular environment favoring ordered and timely interaction and modification of signaling molecules, necessary for the sensing and transduction of extracellular signals and environmental conditions. Crucial to the understanding of ciliary function is the knowledge of the signaling molecules composing the ciliary compartment. While proteomes of primary cilia have been published recently, the selective isolation of primary cilia from specific cell types and whole tissue still proves difficult, and many laboratories instead resort to the analysis of cultured cells, which may introduce experimental artifacts. Here we present a flow cytometry-based method to isolate and characterize primary cilia from the murine ventricular-subventricular zone. After deciliation, primary cilia are immunolabeled with antibodies against ciliary markers. As an example, we here use a double-staining with acetylated tubulin, which stains the ciliary axoneme, and ciliary membrane protein ADP-ribosylation-like factor 13b (Arl13b); additionally, we triple-labeled primary cilia using the ciliary marker adenylate cyclase 3 (AC3). Besides analysis at the single particle level, fluorescence activated cell sorting (FACS) allows collection of pure preparations of primary cilia suited for subsequent proteomic analyses like mass spectrometry or western blot. As an example of analytical application, we performed triple immunostaining and FACS analysis to reveal cilia heterogeneity. Thus, our cilia isolation method, which can readily be applied to other tissues or cell culture, will facilitate the study of this key cellular organelle and shed light on its role in normal conditions and disease.

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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.

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BACKGROUND: We previously identified Leucine-rich repeats and immunoglobulin-like domains 1 (Lrig1) as a marker of long-term neurogenic stem cells in the lateral wall of the adult mouse brain. The morphology of the stem cells thus identified differed from the canonical B1 type stem cells, raising a question about their cellular origin. Thus, we investigated the development of these stem cells in the postnatal and juvenile brain. Furthermore, because Lrig1 is a known regulator of quiescence, we also investigated the effect(s) of its deletion on the cellular proliferation in the lateral wall. METHODS: To observe the development of the Lrig1-lineage stem cells, genetic inducible fate mapping studies in combination with thymidine analog administration were conducted using a previously published Lrig1T2A-iCreERT2 mouse line. To identify the long-term consequence(s) of Lrig1 germline deletion, old Lrig1 knock-out mice were generated using two different Lrig1 null alleles in the C57BL/6J background. The lateral walls from these mice were analyzed using an optimized whole mount immunofluorescence protocol and confocal microscopy. RESULTS: We observed the Lrig1-lineage labeled cells with morphologies consistent with neurogenic stem cell identity in postnatal, juvenile, and adult mouse brains. Interestingly, when induced at postnatal or juvenile ages, morphologically distinct cells were revealed, including cells with the canonical B1 type stem cell morphology. Almost all of the presumptive stem cells labeled were non-proliferative at these ages. In the old Lrig1 germline knock-out mice, increased proliferation was observed compared to wildtype littermates without concomitant increase in apoptosis. CONCLUSIONS: Once set aside during embryogenesis, the Lrig1-lineage stem cells remain largely quiescent during postnatal and juvenile development until activation in adult age. The absence of premature proliferative exhaustion in the Lrig1 knock-out stem cell niche during aging is likely due to a complex cascade of effects on the adult stem cell pool. Thus, we suggest that the adult stem cell pool size may be genetically constrained via Lrig1.

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The concept of a perivascular niche has been proposed for neural stem cells (NSCs). This study examined endothelial colony-forming cell (ECFC)-secreted proteins as potential niche factors for NSCs. Intraventricle infusion with ECFC-secreted proteins increased the number of NSCs. ECFC-secreted proteins were more effective in promoting NSC self-renewal than marrow stromal cell (MSC)-secreted proteins. Differential proteomics analysis of MSC-secreted and ECFC-secreted proteins was performed, which revealed chitinase-like protein 3 (CHIL3; also called ECF-L or Ym1) as a candidate niche factor for NSCs. Experiments with recombinant CHIL3, small interfering RNA, and neutralizing antibodies demonstrated that CHIL3 stimulated NSC self-renewal with neurogenic propensity. CHIL3 was endogenously expressed in the neurogenic niche of the brain and retina as well as in the injured brain and retina. Transcriptome and phosphoproteome analyses revealed that CHIL3 activated various genes and proteins associated with NSC maintenance or neurogenesis. Thus, CHIL3 is a novel niche factor for NSCs.

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The ventricular-subventricular zone (V-SVZ) is a postnatal germinal niche. It holds a large population of neural stem cells (NSCs) that generate neurons and oligodendrocytes for the olfactory bulb and (primarily) the corpus callosum, respectively. These NSCs are heterogeneous and generate different types of neurons depending on their location. Positional identity among NSCs is thought to be controlled in part by intrinsic pathways. However, extrinsic cell signaling through the secreted ligand Sonic hedgehog (Shh) is essential for neurogenesis in both the dorsal and ventral V-SVZ. Here we used a genetic approach to investigate the role of the transcription factors GLI2 and GLI3 in the proliferation and cell fate of dorsal and ventral V-SVZ NSCs. We find that while GLI3 is expressed in stem cell cultures from both dorsal and ventral V-SVZ, the repressor form of GLI3 is more abundant in dorsal V-SVZ. Despite this high dorsal expression and the requirement for other Shh pathway members, GLI3 loss affects the generation of ventrally-, but not dorsally-derived olfactory interneurons in vivo and does not affect trilineage differentiation in vitro. However, loss of GLI3 in the adult dorsal V-SVZ in vivo results in decreased numbers of OLIG2-expressing progeny, indicating a role in gliogenesis.

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The adult mammalian brain entails a reservoir of neural stem cells (NSCs) generating glial cells and neurons. However, NSCs become increasingly quiescent with age, which hampers their regenerative capacity. New means are therefore required to genetically modify adult NSCs for re-enabling endogenous brain repair. Recombinant adeno-associated viruses (AAVs) are ideal gene-therapy vectors due to an excellent safety profile and high transduction efficiency. We thus conducted a high-throughput screening of 177 intraventricularly injected barcoded AAV variants profiled by RNA sequencing. Quantification of barcoded AAV mRNAs identified two synthetic capsids, peptide-modified derivative of wild-type AAV9 (AAV9_A2) and peptide-modified derivative of wild-type AAV1 (AAV1_P5), both of which transduce active and quiescent NSCs. Further optimization of AAV1_P5 by judicious selection of the promoter and dose of injected viral genomes enabled labeling of 30%-60% of the NSC compartment, which was validated by fluorescence-activated cell sorting (FACS) analyses and single-cell RNA sequencing. Importantly, transduced NSCs readily produced neurons. The present study identifies AAV variants with a high regional tropism toward the ventricular-subventricular zone (v-SVZ) with high efficiency in targeting adult NSCs, thereby paving the way for preclinical testing of regenerative gene therapy.

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Postnatal brain neural stem and progenitor cells (NSPCs) cluster in anatomically inaccessible stem cell niches, such as the subependymal zone (SEZ). Here, we describe a method for the isolation of NSPCs from live animals, which we term "milking." The intracerebroventricular injection of a release cocktail, containing neuraminidase, integrin-ß1-blocking antibody, and fibroblast growth factor 2, induces the controlled flow of NSPCs in the cerebrospinal fluid, where they are collected via liquid biopsies. Isolated cells retain key in vivo self-renewal properties and their cell-type profile reflects the cell composition of their source area, while the function of the niche is sustained even 8 months post-milking. By changing the target area more caudally, we also isolate oligodendrocyte progenitor cells (OPCs) from the corpus callosum. This novel approach for sampling NSPCs and OPCs paves the way for performing longitudinal studies in experimental animals, for more in vivo relevant cell culture assays, and for future clinical neuro-regenerative applications.

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BACKGROUND: An increasing number of rodent model systems use injection of DNA or viral constructs in the neonatal brain. However, approaches for reliable positioning and stereotaxic injection at this developmental stage are limited, typically relying on handheld positioning or molds that must be re-aligned for use in a given laboratory. NEW METHOD: A complete protocol and open-source software pipeline for generating 3D-printed head molds derived from a CT scan of a neonatal mouse head cast, together with a universal adapter that can be placed on a standard stereotaxic stage. RESULTS: A series of test injections with adenovirus encoding red fluorescent protein, or Fluorogold, were conducted using original clay molds and newly generated 3D printed molds. Several metrics were used to compare spread and localization of targeted injections. COMPARISON WITH EXISTING METHODS: The new method of head mold generation gave comparable results to the field standard, but also allowed the rapid generation of additional copies of each head mold with standardized positioning of the head each time. CONCLUSIONS: This 3D printing pipeline can be used to efficiently develop a series of head molds with standardized injection coordinates across multiple laboratories. More broadly, this pipeline can easily be adapted to other perinatal ages or species.

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The ventricular-subventricular zone (V-SVZ) is the principal neurogenic niche in the adult mammalian forebrain. Neural stem/progenitor cell (NSPC) activity within the V-SVZ is controlled by numerous of extrinsic factors, whose downstream effects on NSPC proliferation, survival and differentiation are transduced via a limited number of intracellular signaling pathways. Here, we investigated the relationship between age-related changes in NSPC output and activity of signaling pathways downstream of the epidermal growth factor receptor (EGFR), a major regulator of NSPC activity. Biochemical experiments indicated that age-related decline of NSPC activity in vivo is accompanied by selective deficits amongst various EGFR-induced signal pathways within the V-SVZ niche. Pharmacological loss-of-function signaling experiments with cultured NSPCs revealed both overlap and selectivity in the biological functions modulated by the EGFR-induced PI3K/AKT, MEK/ERK and mTOR signaling modules. Specifically, while all three modules promoted EGFR-mediated NSPC proliferation, only mTOR contributed to NSPC survival and only MEK/ERK repressed NSPC differentiation. Using a gain-of-function in vivo genetic approach, we electroporated a constitutively active EGFR construct into a subpopulation of quiescent, EGFR-negative neural stem cells (qNSCs); this ectopic activation of EGFR signaling enabled qNSCs to divide in 3-month-old early adult mice, but not in mice at middle-age or carrying familial Alzheimer disease mutations. Thus, (i) individual EGFR-induced signaling pathways have dissociable effects on NSPC proliferation, survival, and differentiation, (ii) activation of EGFR signaling is sufficient to stimulate qNSC cell cycle entry during early adulthood, and (iii) the proliferative effects of EGFR-induced signaling are dominantly overridden by anti-proliferative signals associated with aging and Alzheimer's disease.

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The ventricular-subventricular zone (V-SVZ) is located in the walls of the lateral ventricles and produces new neurons in the postnatal brain of mammals, including humans. Immature new neurons called "neuroblasts" generated by neural stem cells in the V-SVZ migrate toward their final destinations and contribute to brain development and plasticity. In this review, we describe recent progress in understanding the similarities and dissimilarities in postnatal neurogenesis and neuronal migration between rodents and primates. In rodents, most new V-SVZ-derived neurons migrate along the rostral migratory stream towards the olfactory bulb, where they differentiate into interneurons. In contrast, in humans, the extensive migration of new neurons towards the neocortex continues for several months after birth and might be involved in the development of the expanded neocortex. The mode of migration and the fate of neuroblasts seem to change depending on their environment, destination, and roles in the brain. A better understanding of these similarities and differences between rodents and primates will help translate important findings from animal models and may contribute to the development of clinical strategies for brain repair.

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Adult stem cells (SCs) transit between the cell cycle and a poorly defined quiescent state. Single neural SCs (NSCs) with quiescent, primed-for-activation, and activated cell transcriptomes have been obtained from the subependymal zone (SEZ), but the functional regulation of these states under homeostasis is not understood. Here, we develop a multilevel strategy to analyze these NSC states with the aim to uncover signals that regulate their level of quiescence/activation. We show that transitions between states occur in vivo and that activated and primed, but not quiescent, states can be captured and studied in culture. We also show that peripherally induced inflammation promotes a transient activation of primed NSCs (pNSCs) mediated by tumor necrosis factor α (TNF-α) acting through its receptor, TNF receptor 2 (TNFR2), and a return to quiescence in a TNF receptor 1 (TNFR1)-dependent manner. Our data identify a signaling pathway promoting NSC alertness and add to the emerging concept that SCs can respond to the systemic milieu.

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In the adult ventricular-subventricular zone (V-SVZ), neural stem cells (NSCs) generate new olfactory bulb (OB) neurons and glia throughout life. To map adult neuronal lineage progression, we profiled >56,000 V-SVZ and OB cells by single-cell RNA sequencing (scRNA-seq). Our analyses reveal the molecular diversity of OB neurons, including fate-mapped neurons, lineage progression dynamics, and an NSC intermediate enriched for Notum, which encodes a secreted WNT antagonist. SCOPE-seq technology, which links live-cell imaging with scRNA-seq, uncovers cell-size transitions during NSC differentiation and preferential NOTUM binding to proliferating neuronal precursors. Consistently, application of NOTUM protein in slice cultures and pharmacological inhibition of NOTUM in slice cultures and in vivo demonstrated that NOTUM negatively regulates V-SVZ proliferation. Timely, context-dependent neurogenesis demands adaptive signaling among neighboring progenitors. Our findings highlight a critical regulatory state during NSC activation marked by NOTUM, which attenuates WNT-stimulated proliferation in NSC progeny.

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