It has been widely established that neural stem cells (NSCs) exist in the adult mammalian brain. The area postrema (AP) and the ependymal cell layer of the central canal (CC) in the medulla were recently identified as NSC niches. There are two types of NSCs: astrocyte-like cells in the AP and tanycyte-like cells in the CC. However, limited information is currently available on the characteristics and functional significance of these NSCs and their progeny in the AP and CC. The AP is a part of the dorsal vagal complex (DVC), together with the nucleus of the solitary tract (Sol) and the dorsal motor nucleus of the vagus (10 N). DVC is the primary site for the integration of visceral neuronal and hormonal cues that act to inhibit food intake. Therefore, we examined the effects of high-fat diet (HFD) on NSCs and progenitor cells in the AP and CC. Eight-week-old male mice were fed HFD for short (1 week) and long periods (4 weeks). To detect proliferating cells, mice consecutively received intraperitoneal injections of BrdU for 7 days. Brain sections were processed with immunohistochemistry using various cell markers and BrdU antibodies. Our data demonstrated that adult NSCs and neural progenitor cells (NPCs) in the medulla responded more strongly to short-term HFD than to long-term HFD. HFD increased astrocyte density in the Sol and 10 N, and increased microglial/macrophage density in the AP and Sol. Furthermore, long-term HFD induced mild inflammation in the medulla, suggesting that it affected the proliferation of NSCs and NPCs.

Multiple sclerosis is a chronic inflammatory disease of the central nervous system characterized by autoimmune destruction of the myelin sheath, leading to irreversible and progressive functional deficits in patients. Pre-clinical studies involving the use of neural stem cells (NSCs) have already demonstrated their potential in neuronal regeneration and remyelination. However, the exclusive application of cell therapy has not proved sufficient to achieve satisfactory therapeutic levels. Recognizing these limitations, there is a need to combine cell therapy with other adjuvant protocols. In this context, extracellular vesicles (EVs) can contribute to intercellular communication, stimulating the production of proteins and lipids associated with remyelination and providing trophic support to axons. This study aimed to evaluate the therapeutic efficacy of the combination of NSCs and EVs derived from oligodendrocyte precursor cells (OPCs) in an animal model of multiple sclerosis. OPCs were differentiated from NSCs and had their identity confirmed by gene expression analysis and immunocytochemistry. Exosomes were isolated by differential ultracentrifugation and characterized by Western, transmission electron microscopy and nanoparticle tracking analysis. Experimental therapy of C57BL/6 mice induced with experimental autoimmune encephalomyelitis (EAE) were grouped in control, treated with NSCs, treated with OPC-derived EVs and treated with a combination of both. The treatments were evaluated clinically using scores and body weight, microscopically using immunohistochemistry and immunological profile by flow cytometry. The animals showed significant clinical improvement and weight gain with the treatments. However, only the treatments involving EVs led to immune modulation, changing the profile from Th1 to Th2 lymphocytes. Fifteen days after treatment revealed a reduction in reactive microgliosis and astrogliosis in the groups treated with EVs. However, there was no reduction in demyelination. The results indicate the potential therapeutic use of OPC-derived EVs to attenuate inflammation and promote recovery in EAE, especially when combined with cell therapy.

Adult neural stem cells (NSCs) in the hippocampal dentate gyrus continuously proliferate and generate new neurons throughout life. Although various functions of organelles are closely related to the regulation of adult neurogenesis, the role of endoplasmic reticulum (ER)-related molecules in this process remains largely unexplored. Here we show that Derlin-1, an ER-associated degradation component, spatiotemporally maintains adult hippocampal neurogenesis through a mechanism distinct from its established role as an ER quality controller. Derlin-1 deficiency in the mouse central nervous system leads to the ectopic localization of newborn neurons and impairs NSC transition from active to quiescent states, resulting in early depletion of hippocampal NSCs. As a result, Derlin-1-deficient mice exhibit phenotypes of increased seizure susceptibility and cognitive dysfunction. Reduced Stat5b expression is responsible for adult neurogenesis defects in Derlin-1-deficient NSCs. Inhibition of histone deacetylase activity effectively induces Stat5b expression and restores abnormal adult neurogenesis, resulting in improved seizure susceptibility and cognitive dysfunction in Derlin-1-deficient mice. Our findings indicate that the Derlin-1-Stat5b axis is indispensable for the homeostasis of adult hippocampal neurogenesis.

Autophagy plays a pivotal role in diverse biological processes, including the maintenance and differentiation of neural stem cells (NSCs). Interestingly, while complete deletion of Fip200 severely impairs NSC maintenance and differentiation, inhibiting canonical autophagy via deletion of core genes, such as Atg5, Atg16l1, and Atg7, or blockade of canonical interactions between FIP200 and ATG13 (designated as FIP200-4A mutant or FIP200 KI) does not produce comparable detrimental effects. This highlights the likely critical involvement of the non-canonical functions of FIP200, the mechanisms of which have remained elusive. Here, utilizing genetic mouse models, we demonstrated that FIP200 mediates non-canonical autophagic degradation of p62/sequestome1, primarily via TAX1BP1 in NSCs. Conditional deletion of Tax1bp1 in fip200 hGFAP conditional knock-in (cKI) mice led to NSC deficiency, resembling the fip200 hGFAP conditional knockout (cKO) mouse phenotype. Notably, reintroducing wild-type TAX1BP1 not only restored the maintenance of NSCs derived from tax1bp1-knockout fip200 hGFAP cKI mice but also led to a marked reduction in p62 aggregate accumulation. Conversely, a TAX1BP1 mutant incapable of binding to FIP200 or NBR1/p62 failed to achieve this restoration. Furthermore, conditional deletion of Tax1bp1 in fip200 hGFAP cKO mice exacerbated NSC deficiency and p62 aggregate accumulation compared to fip200 hGFAP cKO mice. Collectively, these findings illustrate the essential role of the FIP200-TAX1BP1 axis in mediating the non-canonical autophagic degradation of p62 aggregates towards NSC maintenance and function, presenting novel therapeutic targets for neurodegenerative diseases.
自噬在包括神经干细胞的维持和分化在内的各种生物过程中发挥着重要作用。虽然条件性敲除 Fip200 会严重损害神经干细胞的维持和分化，但通过缺失 Atg5、Atg16l1 和 Atg7 等自噬关键基因或阻断 FIP200 与 ATG13 相互作用来抑制经典自噬并不会产生类似的有害影响。这提示 FIP200 的非经典自噬功能可能具有关键的调控作用，而其作用机制仍知之甚少。该研究利用遗传修饰小鼠模型证明了 FIP200 主要通过 TAX1BP1 在神经干细胞中介导 p62 聚集体的非经典自噬降解。在 fip200 cKI 小鼠中条件性敲除 Tax1bp1 会导致小鼠出现与fip200 cKO 小鼠相似的神经干细胞缺陷。在fip200 cKI; tax1bp1 cKO 神经干细胞中重新引入野生型 TAX1BP1 不仅能恢复神经干细胞的自我更新能力，还能显著减少 p62 聚集体的积累；而无法与 FIP200 或 NBR1/p62 结合的 TAX1BP1 突变体则无法恢复神经干细胞的自我更新。此外，在 fip200 cKO 小鼠中进一步条件性敲除 Tax1bp1 会加剧神经干细胞缺陷和 p62 聚集体积累。总之，这些研究结果表明 FIP200-TAX1BP1 信号轴在介导 p62 聚集体的非经典自噬降解以维持神经干细胞功能方面的重要作用，为神经退行性疾病治疗提供了新的干预靶点。.

Many genes are known to regulate retinal regeneration after widespread tissue damage. Conversely, genes controlling regeneration after limited cell loss, as per degenerative diseases, are undefined. As stem/progenitor cell responses scale to injury levels, understanding how the extent and specificity of cell loss impact regenerative processes is important. Here, transgenic zebrafish enabling selective retinal ganglion cell (RGC) ablation were used to identify genes that regulate RGC regeneration. A single cell multiomics-informed screen of 100 genes identified seven knockouts that inhibited and 11 that promoted RGC regeneration. Surprisingly, 35 out of 36 genes known and/or implicated as being required for regeneration after widespread retinal damage were not required for RGC regeneration. The loss of seven even enhanced regeneration kinetics, including the proneural factors neurog1, olig2 and ascl1a. Mechanistic analyses revealed that ascl1a disruption increased the propensity of progenitor cells to produce RGCs, i.e. increased \'fate bias\'. These data demonstrate plasticity in the mechanism through which Müller glia convert to a stem-like state and context specificity in how genes function during regeneration. Increased understanding of how the regeneration of disease-relevant cell types is specifically controlled will support the development of disease-tailored regenerative therapeutics.

The maintenance and expansion of human neural stem cells (hNSCs) in 3D tissue scaffolds is a promising strategy in producing cost-effective hNSCs with quality and quantity applicable for clinical applications. A few biopolymers have been extensively used to fabricate 3D scaffolds, including hyaluronic acid, collagen, alginate, and chitosan, due to their bioactive nature and availability. However, these polymers are usually applied in combination with other biomolecules, leading to their responses difficult to ascribe to. Here, scaffolds made of chitosan, alginate, hyaluronic acid, or collagen, are explored for hNSC expansion under xeno-free and chemically defined conditions and compared for hNSC multipotency maintenance. This study shows that the scaffolds made of pure chitosan support the highest adhesion and growth of hNSCs, yielding the most viable cells with NSC marker protein expression. In contrast, the presence of alginate, hyaluronic acid, or collagen induces differentiation toward immature neurons and astrocytes even in the maintenance medium and absence of differentiation factors. The cells in pure chitosan scaffolds preserve the level of transmembrane protein profile similar to that of standard culture. These findings point to the potential of using pure chitosan scaffolds as a base scaffolding material for hNSC expansion in 3D.

Gangliosides are sialylated glycosphingolipids with essential but enigmatic functions in healthy and disease brains. GD3 is the predominant species in neural stem cells (NSCs) and GD3-synthase (sialyltransferase II; St8Sia1) knockout (GD3S-KO) revealed reduction of postnatal NSC pools with severe behavioral deficits including cognitive impairment, depression-like phenotypes, and olfactory dysfunction. Exogenous administration of GD3 significantly restored the NSC pools and enhanced the stemness of NSCs with multipotency and self-renewal, followed by restored neuronal functions. Our group discovered that GD3 is involved in the maintenance of NSC fate determination by interacting with epidermal growth factor receptors (EGFRs), by modulating expression of cyclin-dependent kinase (CDK) inhibitors p27 and p21, and by regulating mitochondrial dynamics via associating a mitochondrial fission protein, the dynamin-related protein-1 (Drp1). Furthermore, we discovered that nuclear GM1 promotes neuronal differentiation by an epigenetic regulatory mechanism. GM1 binds with acetylated histones on the promoter of N-acetylgalactosaminyltransferase (GalNAcT; GM2 synthase (GM2S); B4galnt1) as well as on the NeuroD1 in differentiated neurons. In addition, epigenetic activation of the GM2S gene was detected as accompanied by an apparent induction of neuronal differentiation in NSCs responding to an exogenous supplement of GM1. Interestingly, GM1 induced epigenetic activation of the tyrosine hydroxylase (TH) gene, with recruitment of Nurr1 and PITX3, dopaminergic neuron-associated transcription factors, to the TH promoter region. In this way, GM1 epigenetically regulates dopaminergic neuron specific gene expression, and it would modify Parkinson\'s disease. Multifunctional gangliosides significantly modulate lipid microdomains to regulate functions of important molecules on multiple sites: the plasma membrane, mitochondrial membrane, and nuclear membrane. Versatile gangliosides regulate functional neurons as well as sustain NSC functions via modulating protein and gene activities on ganglioside microdomains. Maintaining proper ganglioside microdomains benefits healthy neuronal development and millions of senior citizens with neurodegenerative diseases. Here, we introduce how to isolate GD3 and GM1 and how to administer them into the mouse brain to investigate their functions on NSC fate determination and nerve cell specification.

Increasing evidence shows that the administration of mesenchymal stem cells (MSCs) is a promising option for various brain diseases, including ischemic stroke. Studies have demonstrated that MSC transplantation after ischemic stroke provides beneficial effects, such as neural regeneration, partially by activating endogenous neural stem/progenitor cells (NSPCs) in conventional neurogenic zones, such as the subventricular and subgranular zones. However, whether MSC transplantation regulates the fate of injury-induced NSPCs (iNSPCs) regionally activated at injured regions after ischemic stroke remains unclear. Therefore, mice were subjected to ischemic stroke, and mCherry-labeled human MSCs (h-MSCs) were transplanted around the injured sites of nestin-GFP transgenic mice. Immunohistochemistry of brain sections revealed that many GFP+ cells were observed around the grafted sites rather than in the regions in the subventricular zone, suggesting that transplanted mCherry+ h-MSCs stimulated GFP+ locally activated endogenous iNSPCs. In support of these findings, coculture studies have shown that h-MSCs promoted the proliferation and neural differentiation of iNSPCs extracted from ischemic areas. Furthermore, pathway analysis and gene ontology analysis using microarray data showed that the expression patterns of various genes related to self-renewal, neural differentiation, and synapse formation were changed in iNSPCs cocultured with h-MSCs. We also transplanted h-MSCs (5.0 × 104 cells/µL) transcranially into post-stroke mouse brains 6 weeks after middle cerebral artery occlusion. Compared with phosphate-buffered saline-injected controls, h-MSC transplantation displayed significantly improved neurological functions. These results suggest that h-MSC transplantation improves neurological function after ischemic stroke in part by regulating the fate of iNSPCs.

Stem cells in vivo can transit between quiescence and activation, two metabolically distinct states. It is increasingly appreciated that cell metabolism assumes profound roles in stem cell maintenance and tissue homeostasis. However, the lack of suitable models greatly hinders our understanding of the metabolic control of stem cell quiescence and activation. In the present study, we have utilized classical signaling pathways and developed a cell culture system to model reversible NSC quiescence and activation. Unlike activated ones, quiescent NSCs manifested distinct morphology characteristics, cell proliferation, and cell cycle properties but retained the same cell proliferation and differentiation potentials once reactivated. Further transcriptomic analysis revealed that extensive metabolic differences existed between quiescent and activated NSCs. Subsequent experimentations confirmed that NSC quiescence and activation transition was accompanied by a dramatic yet coordinated and dynamic shift in RNA metabolism, protein synthesis, and mitochondrial and autophagy activity. The present work not only showcases the broad utilities of this powerful in vitro NSC quiescence and activation culture system but also provides timely insights for the field and warrants further investigations.

Poly-l-lysine (PLL) and Matrigel, both classical coating materials for culture substrates in neural stem cell (NSC) research, present distinct interfaces whose effect on NSC behavior at cellular and molecular levels remains ambiguous. Our investigation reveals intriguing disparities: although both PLL and Matrigel interfaces are hydrophilic and feature amine functional groups, Matrigel stands out with lower stiffness and higher roughness. Based on this diversity, Matrigel surpasses PLL, driving NSC adhesion, migration, and proliferation. Intriguingly, PLL promotes NSC differentiation into astrocytes, whereas Matrigel favors neural differentiation and the physiological maturation of neurons. At the molecular level, Matrigel showcases a wider upregulation of genes linked to NSC behavior. Specifically, it enhances ECM-receptor interaction, activates the YAP transcription factor, and heightens glycerophospholipid metabolism, steering NSC proliferation and neural differentiation. Conversely, PLL upregulates genes associated with glial cell differentiation and amino acid metabolism and elevates various amino acid levels, potentially linked to its support for astrocyte differentiation. These distinct transcriptional and metabolic activities jointly shape the divergent NSC behavior on these substrates. This study significantly advances our understanding of substrate regulation on NSC behavior, offering novel insights into optimizing and targeting the application of these surface coating materials in NSC research.