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Neural stem cells (NSCs) persist in specific brain germinative niches and sustain neurogenesis throughout life in adult mammals. In addition to the two major stem cell niches in the subventricular zone and the hippocampal dentate gyrus, the area postrema located in the brainstem has been identified as a neurogenic zone as well. NSCs are regulated by signals from the microenvironment that adjust stem cell response to the needs of the organism. Evidence accumulated over the past decade indicates that Ca 2+ channels play pivotal functions in NSC maintenance. In this study, we explored in area postrema NSCs the presence and roles of a subset of Ca 2+ channels, the store-operated Ca 2+ channels (SOCs) that have the capacity to transduce extracellular signals into Ca 2+ signals. Our data show that NSCs derived from the area postrema express TRPC1 and Orai1, known to form SOCs, as well as their activator STIM1. Ca 2+ imaging indicated that NSCs exhibit store-operated Ca 2+ entries (SOCEs). Pharmacological blockade of SOCEs with SKF-96365, YM-58483 (also known as BTP2) or GSK-7975A resulted in decreased NSC proliferation and self-renewal, indicating a major role for SOCs in maintaining NSC activity within the area postrema. Furthermore, our results show that leptin, an adipose tissue-derived hormone whose ability to control energy homeostasis is dependent on the area postrema, decreased SOCEs and reduced self-renewal of NSCs in the area postrema. As aberrant SOC function has been linked to an increasing number of diseases, including brain disorders, our study opens new perspectives for NSCs in brain pathophysiology., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Ben Dhaou, Terrié, Déliot, Harnois, Cousin, Arnault, Constantin, Moyse and Coronas.)

Glioblastoma is the most frequent and deadly form of primary brain tumors. Despite multimodal treatment, more than 90% of patients experience tumor recurrence. Glioblastoma contains a small population of cells, called glioblastoma stem cells (GSC) that are highly resistant to treatment and endowed with the ability to regenerate the tumor, which accounts for tumor recurrence. Transcriptomic studies disclosed an enrichment of calcium (Ca 2+ ) signaling transcripts in GSC. In non-excitable cells, store-operated channels (SOC) represent a major route of Ca 2+ influx. As SOC regulate the self-renewal of adult neural stem cells that are possible cells of origin of GSC, we analyzed the roles of SOC in cultures of GSC previously derived from five different glioblastoma surgical specimens. Immunoblotting and immunocytochemistry experiments showed that GSC express Orai1 and TRPC1, two core SOC proteins, along with their activator STIM1. Ca 2+ imaging demonstrated that SOC support Ca 2+ entries in GSC. Pharmacological inhibition of SOC-dependent Ca 2+ entries decreased proliferation, impaired self-renewal, and reduced expression of the stem cell marker SOX2 in GSC. Our data showing the ability of SOC inhibitors to impede GSC self-renewal paves the way for a strategy to target the cells considered responsible for conveying resistance to treatment and tumor relapse.

The brain of adult mammals, including humans, contains neural stem cells (NSCs) located within specific niches of which the ventricular-subventricular zone (V-SVZ) is the largest one. Under physiological conditions, NSCs proliferate, self-renew and produce new neurons and glial cells. Several recent studies established that oncogenic mutations in adult NSCs of the V-SVZ are responsible for the emergence of malignant primary brain tumors called glioblastoma. These aggressive tumors contain a small subpopulation of cells, the glioblastoma stem cells (GSCs), that are endowed with proliferative and self-renewal abilities like NSCs from which they may arise. GSCs are thus considered as the cells that initiate and sustain tumor growth and, because of their resistance to current treatments, provoke tumor relapse. A growing body of studies supports that Ca 2+ signaling controls a variety of processes in NSCs and GSCs. Ca 2+ is a ubiquitous second messenger whose fluctuations of its intracellular concentrations are handled by channels, pumps, exchangers, and Ca 2+ binding proteins. The concerted action of the Ca 2+ toolkit components encodes specific Ca 2+ signals with defined spatio-temporal characteristics that determine the cellular responses. In this review, after a general overview of the adult brain NSCs and GSCs, we focus on the multiple roles of the Ca 2+ toolkit in NSCs and discuss how GSCs hijack these mechanisms to promote tumor growth. Extensive knowledge of the role of the Ca 2+ toolkit in the management of essential functions in healthy and pathological stem cells of the adult brain should help to identify promising targets for clinical applications., (Copyright © 2020 Coronas, Terrié, Déliot, Arnault and Constantin.)

The subventricular zone (SVZ) is the major stem cell niche in the brain of adult mammals. Within this region, neural stem cells (NSC) proliferate, self-renew and give birth to neurons and glial cells. Previous studies underlined enrichment in calcium signaling-related transcripts in adult NSC. Because of their ability to mobilize sustained calcium influxes in response to a wide range of extracellular factors, store-operated channels (SOC) appear to be, among calcium channels, relevant candidates to induce calcium signaling in NSC whose cellular activities are continuously adapted to physiological signals from the microenvironment. By Reverse Transcription Polymerase Chain Reaction (RT-PCR), Western blotting and immunocytochemistry experiments, we demonstrate that SVZ cells express molecular actors known to build up SOC, namely transient receptor potential canonical 1 (TRPC1) and Orai1, as well as their activator stromal interaction molecule 1 (STIM1). Calcium imaging reveals that SVZ cells display store-operated calcium entries. Pharmacological blockade of SOC with SKF-96365 or YM-58483 (also called BTP2) decreases proliferation, impairs self-renewal by shifting the type of SVZ stem cell division from symmetric proliferative to asymmetric, thereby reducing the stem cell population. Brain section immunostainings show that TRPC1, Orai1, and STIM1 are expressed in vivo, in SOX2-positive SVZ NSC. Injection of SKF-96365 in brain lateral ventricle diminishes SVZ cell proliferation and reduces the ability of SVZ cells to form neurospheres in vitro. The present study combining in vitro and in vivo approaches uncovers a major role for SOC in the control of SVZ NSC population and opens new fields of investigation for stem cell biology in health and disease. Stem Cells 2018;36:761-774., (© AlphaMed Press 2018.)

Adipocyte-derived hormone leptin has been recently implicated in the control of neuronal plasticity. To explore whether modulation of adult neurogenesis may contribute to leptin control of neuronal plasticity, we used the neurosphere assay of neural stem cells derived from the adult rat subventricular zone (SVZ). Endogenous expression of specific leptin receptor (ObRb) transcripts, as revealed by RT-PCR, is associated with activation of both ERK and STAT-3 pathways via phosphorylation of the critical ERK/STAT-3 amino acid residues upon addition of leptin to neurospheres. Furthermore, leptin triggered withdrawal of neural stem cells from the cell cycle as monitored by Ki67 labeling. This effect was blocked by pharmacological inhibition of ERK activation thus demonstrating that ERK mediates leptin effects on neural stem cell expansion. Leptin-dependent withdrawal of neural stem cells from the cell cycle was associated with increased apoptosis, as detected by TUNEL, which was preceded by cyclin D1 induction. Cyclin D1 was indeed extensively colocalized with TUNEL-positive, apoptotic nuclei. Cyclin-D1 silencing by specific shRNA prevented leptin-induced decrease of the cell number per neurosphere thus pointing to the causal relationship between leptin actions on apoptosis and cyclin D1 induction. Leptin target cells in SVZ neurospheres were identified by double TUNEL/phenotypic marker immunocytofluorescence as differentiating neurons mostly. The inhibition of neural stem cell expansion via ERK/cyclin D1-triggered apoptosis defines novel biological action of leptin which may be involved in adiposity-dependent neurotoxicity.

Neural stem cells, whose major reservoir in the adult mammalian brain is the subventricular zone (SVZ), ensure neuropoiesis, a process during which many generated cells die. Removal of dead cells and debris by phagocytes is necessary for tissue homeostasis. Using confocal and electron microscopy, we demonstrate that cultured SVZ cells phagocytose both 1 and 2 µm latex beads and apoptotic cell-derived fragments. We determine by flow cytometry that phagocytic cells represent more than 10% of SVZ cultured cells. Phenotyping of SVZ cells using nestin, GFAP, Sox2, or LeX/SSEA and quantification of aldehyde dehydrogenase (ALDH) activity, reveals that cells with neural stem-cell features phagocytose and represent more than 30% of SVZ phagocytic cells. In vivo, nestin-, Sox2-, and ALDH-expressing neural stem-like cells engulfed latex beads or apoptotic cell-derived fragments that were injected into mice lateral brain ventricles. We show also that SVZ cell phagocytic activity is an active process, which depends both on cytoskeleton dynamic and on recognition of phosphatidylserine eat-me signal, and is stimulated by the vitamin K-dependent factor protein S (ProS). ProS neutralizing antibodies inhibit SVZ cell phagocytic activity and exposure of SVZ cells to apoptotic cell-derived fragments induces a transient Mer tyrosine kinase receptor (MerTK) phosphorylation. Conversely, MerTK blocking antibodies impair both basal and ProS-stimulated SVZ cell phagocytic activity. By revealing that neural stem-like cells act within the SVZ neurogenic niche as phagocytes and that the ProS/MerTK path represents an endogenous regulatory mechanism for SVZ cell phagocytic activity, the present report opens-up new perspectives for both stem cell biology and brain physiopathology., (© 2014 AlphaMed Press.)

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., (Copyright © 2012 AlphaMed Press.)

Neural stem cells persist in the adult mammalian brain, within the subventricular zone (SVZ). The endogenous mechanisms underpinning SVZ neural stem cell proliferation, self-renewal, and differentiation are not fully elucidated. In the present report, we describe a growth-stimulatory activity of liver explant-conditioned media on SVZ cell cultures and identify hepatocyte growth factor (HGF) as a major player in this effect. HGF exhibited a mitogenic activity on SVZ cell cultures in a mitogen-activated protein kinase (MAPK) (ERK1/2)-dependent manner as U0126, a specific MAPK inhibitor, blocked it. Combining a functional neurosphere forming assay with immunostaining for c-Met, along with markers of SVZ cells subtypes, demonstrated that HGF promotes the expansion of neural stem-like cells that form neurospheres and self-renew. Immunostaining, HGF enzyme-linked immunosorbent assay and Madin-Darby canine kidney cell scattering assay indicated that SVZ cell cultures produce and release HGF. SVZ cell-conditioned media induced proliferation on SVZ cell cultures, which was blocked by HGF-neutralizing antibodies, hence implying that endogenously produced HGF accounts for a major part in SVZ mitogenic activity. Brain sections immunostaining revealed that HGF is produced by nestin-expressing cells and c-Met is expressed within the SVZ by immature cells. HGF intracerebroventricular injection promoted SVZ cell proliferation and increased the ability of these cells exposed in vivo to HGF to form neurospheres in vitro, whereas intracerebroventricular injection of HGF-neutralizing antibodies decreased SVZ cell proliferation. The present study unravels a major role, both in vitro and in vivo, for endogenous HGF in SVZ neural stem cell growth and self-renewal.

The gamma-carboxyglutamate-containing proteins are a family of secreted vitamin K-dependent proteins in which some glutamyl residues are post-translationally modified to gamma-carboxyglutamic acid residues. A vitamin K-dependent gamma-glutamyl carboxylase enzyme catalyses this post-translational modification. The gamma-carboxylase reaction requires vitamin K in its reduced form, vitamin K hydroquinone, and generates gamma-carboxyglutamate and vitamin K 2,3,-epoxide which is then recycled back to the hydroquinone form by a vitamin K reductase system. Warfarin blocks the vitamin K cycle and hence inhibits the gamma-carboxylase reaction, and this property of Warfarin has led to its wide use in anticoagulant therapy. Until recently, interest in vitamin K-dependent proteins was mostly restricted to the field of hematology. However, the discovery that the anti-coagulant factor protein S and its structural homologue Gas6 (growth arrest-specific gene 6), two vitamin K-dependent proteins, are ligands for the Tyro3/Axl/Mer family of related tyrosine kinase receptors has opened up a new area of research. Moreover, the phenotypes associated with the invalidation of genes encoding vitamin K-dependent proteins or their receptors revealed their implication in regulating phagocytosis during many cell differentiation phenomena such as retinogenesis, neurogenesis, osteogenesis, and spermatogenesis. Additionally, protein S was identified as the major factor responsible for serum-stimulated phagocytosis of apoptotic cells. Therefore, the elucidation of the molecular mechanisms underlying the role of vitamin K-dependent proteins in regulating apoptotic cell phagocytosis may lead to a better understanding of the physiopathology of cell differentiation and could form the framework of new therapeutic strategies aiming at a selective targeting of cell phagocytosis associated pathologies.

The small stress protein Hsp27 is expressed during mammalian neural development. We have analyzed the role of this protein in immortalized rat olfactory neuroblasts. In the presence of dopamine a fraction of these cells differentiate into neurons while the remaining cells undergo apoptosis. We report here that the dopamine induced differentiation and apoptosis are associated with a transient and specific accumulation of Hsp27. Moreover, transfection experiments have shown that Hsp27 overexpression drastically decreases the fraction of cells undergoing apoptosis. In contrast, reduction of the endogenous level of Hsp27 led to abortion of differentiation and, therefore, drastically increased the number of apoptotic cells. Furthermore, in the normal cell population we show that Hsp27 accumulation takes place only in differentiating cells that were not undergoing apoptosis. We therefore conclude that Hsp27 may represent a key protein that controls the decision of olfactory precursor cells to undergo either differentiation or cell death.