Quiescent Neural Stem Cells Research Articles

Modelling quiescence exit of neural stem cells reveals a FOXG1-FoxO6 axis.

The molecular mechanisms controlling the balance of quiescence and proliferation in adult neural stem cells (NSCs) are often deregulated in brain cancers such as glioblastoma (GBM). Previously, we reported that FOXG1, a forebrain-restricted neurodevelopmental transcription factor, is frequently upregulated in glioblastoma stem cells (GSCs) and limits the effects of cytostatic pathways, in part by repression of the tumour suppressor Foxo3. Here, we show that increased FOXG1 upregulates FoxO6, a more recently discovered FoxO family member with potential oncogenic functions. Although genetic ablation of FoxO6 in proliferating NSCs has no effect on the cell cycle or entry into quiescence, we find that FoxO6-null NSCs can no longer efficiently exit quiescence following FOXG1 elevation. Increased FoxO6 results in the formation of large acidic vacuoles, reminiscent of Pak1-regulated macropinocytosis. Consistently, Pak1 expression is upregulated by FOXG1 overexpression and downregulated upon FoxO6 loss in proliferative NSCs. These data suggest a pro-oncogenic role for FoxO6, downstream of GBM-associated elevated FOXG1, in controlling quiescence exit, and shed light on the potential functions of this underexplored FoxO family member.

SUMOylation of Warts kinase promotes neural stem cell reactivation

A delicate balance between neural stem cell (NSC) quiescence and proliferation is important for adult neurogenesis and homeostasis. Small ubiquitin-related modifier (SUMO)-dependent post-translational modifications cause rapid and reversible changes in protein functions. However, the role of the SUMO pathway during NSC reactivation and brain development is not established. Here, we show that the key components of the SUMO pathway play an important role in NSC reactivation and brain development in Drosophila. Depletion of SUMO/Smt3 or SUMO conjugating enzyme Ubc9 results in notable defects in NSC reactivation and brain development, while their overexpression leads to premature NSC reactivation. Smt3 protein levels increase with NSC reactivation, which is promoted by the Ser/Thr kinase Akt. Warts/Lats, the core protein kinase of the Hippo pathway, can undergo SUMO- and Ubc9-dependent SUMOylation at Lys766. This modification attenuates Wts phosphorylation by Hippo, leading to the inhibition of the Hippo pathway, and consequently, initiation of NSC reactivation. Moreover, inhibiting Hippo pathway effectively restores the NSC reactivation defects induced by SUMO pathway inhibition. Overall, our study uncovered an important role for the SUMO-Hippo pathway during Drosophila NSC reactivation and brain development.

A cholinergic signaling pathway underlying cortical circuit activation of quiescent neural stem cells in the lateral ventricle.

Neural stem cells (NSCs) in the subventricular zone (SVZ) located along the lateral ventricles (LVs) of the mammalian brain continue to self-renew to produce new neurons after birth and into adulthood. Quiescent LV cells, which are situated close to the ependymal cells lining the LVs, are activated by choline acetyltransferase-positive (ChAT+) neurons within the subependymal (subep) region of the SVZ when these neurons are stimulated by projections from the anterior cingulate cortex (ACC). Here, we uncovered a signaling pathway activated by the ACC-subep-ChAT+ circuit responsible for the activation and proliferation of quiescent LV NSCs specifically in the ventral area of the SVZ. This circuit activated muscarinic M3 receptors on quiescent LV NSCs, which subsequently induced signaling mediated by the inositol 1,4,5-trisphosphate receptor type 1 (IP3R1). Downstream of IP3R1 activation, which would be expected to increase intracellular Ca2+, Ca2+-/calmodulin-dependent protein kinase II δ and the MAPK10 signaling pathway were stimulated and required for the proliferation of quiescent LV NSCs in the SVZ. These findings reveal the mechanisms that regulate quiescent LV NSCs and underscore the critical role of projections from the ACC in promoting their proliferative activity within the ventral SVZ.

EphA4 Targeting Peptide-Conjugated Extracellular Vesicles Rejuvenates Adult Neural Stem Cells and Exerts Therapeutic Benefits in Aging Rats.

Aging and various neurodegenerative diseases cause significant reduction in adult neurogenesis and simultaneous increase in quiescent neural stem cells (NSCs), which impact the brain's regenerative capabilities. To deal with this challenging issue, current treatments involve stem cell transplants or prevention of neurodegeneration; however, the efficacy or success of this process remains limited. Therefore, extensive and focused investigation is highly demanding to overcome this challenging task. Here, we have designed an efficient peptide-based EphA4 receptor-targeted ligand through an in silico approach. Further, this strategy involves chemical conjugation of the peptide with adipose tissue stem cell-derived EV (Exo-pep-11). Interestingly, our newly designed engineered EV, Exo-pep-11, targets NSC through EphA4 receptors, which offers promising therapeutic advantages by stimulating NSC proliferation and subsequent differentiation. Our result demonstrates that NSC successfully internalized Exo-pep-11 in both in vitro culture conditions as well as in the in vivo aging rats. We found that the uptake of Exo-pep-11 decreased by ∼2.3-fold when NSC was treated with EphA4 antibody before Exo-pep-11 incubation, which confirms the receptor-specific uptake of Exo-pep-11. Exo-pep-11 treatment also increases NSC proliferation by ∼1.9-fold and also shows ∼1.6- and ∼2.4-fold increase in expressions of Nestin and ID1, respectively. Exo-pep-11 also has the potential to increase neurogenesis in aging rats, which is confirmed by ∼1.6- and ∼1.5-fold increases in expressions of TH and Tuj1, respectively, in rat olfactory bulb. Overall, our findings highlight the potential role of Exo-pep-11 for prospective applications in combating age-related declines in NSC activity and neurogenesis.

SOX10 mediates glioblastoma cell-state plasticity

Phenotypic plasticity is a cause of glioblastoma therapy failure. We previously showed that suppressing the oligodendrocyte-lineage regulator SOX10 promotes glioblastoma progression. Here, we analyze SOX10-mediated phenotypic plasticity and exploit it for glioblastoma therapy design. We show that low SOX10 expression is linked to neural stem-cell (NSC)-like glioblastoma cell states and is a consequence of temozolomide treatment in animal and cell line models. Single-cell transcriptome profiling of Sox10-KD tumors indicates that Sox10 suppression is sufficient to induce tumor progression to an aggressive NSC/developmental-like phenotype, including a quiescent NSC-like cell population. The quiescent NSC state is induced by temozolomide and Sox10-KD and reduced by Notch pathway inhibition in cell line models. Combination treatment using Notch and HDAC/PI3K inhibitors extends the survival of mice carrying Sox10-KD tumors, validating our experimental therapy approach. In summary, SOX10 suppression mediates glioblastoma progression through NSC/developmental cell-state transition, including the induction of a targetable quiescent NSC state. This work provides a rationale for the design of tumor therapies based on single-cell phenotypic plasticity analysis.

DNA methylation controls stemness of astrocytes in health and ischaemia

Astrocytes are the most abundant cell type in the mammalian brain and provide structural and metabolic support to neurons, regulate synapses and become reactive after injury and disease. However, a small subset of astrocytes settles in specialized areas of the adult brain where these astrocytes instead actively generate differentiated neuronal and glial progeny and are therefore referred to as neural stem cells1–3. Common parenchymal astrocytes and quiescent neural stem cells share similar transcriptomes despite their very distinct functions4–6. Thus, how stem cell activity is molecularly encoded remains unknown. Here we examine the transcriptome, chromatin accessibility and methylome of neural stem cells and their progeny, and of astrocytes from the striatum and cortex in the healthy and ischaemic adult mouse brain. We identify distinct methylation profiles associated with either astrocyte or stem cell function. Stem cell function is mediated by methylation of astrocyte genes and demethylation of stem cell genes that are expressed later. Ischaemic injury to the brain induces gain of stemness in striatal astrocytes7. We show that this response involves reprogramming the astrocyte methylome to a stem cell methylome and is absent if the de novo methyltransferase DNMT3A is missing. Overall, we unveil DNA methylation as a promising target for regenerative medicine.

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine filamentous actin (F-actin) structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of myocardin-related transcription factor, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G protein-coupled receptor Smog, G protein αq subunit, Rho1 guanosine triphosphatase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand folded gastrulation to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, an NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

Epigenetic maintenance of adult neural stem cell quiescence in the mouse hippocampus via Setd1a

Quiescence, a hallmark of adult neural stem cells (NSCs), is required for maintaining the NSC pool to support life-long continuous neurogenesis in the adult dentate gyrus (DG). Whether long-lasting epigenetic modifications maintain NSC quiescence over the long term in the adult DG is not well-understood. Here we show that mice with haploinsufficiency of Setd1a, a schizophrenia risk gene encoding a histone H3K4 methyltransferase, develop an enlarged DG with more dentate granule cells after young adulthood. Deletion of Setd1a specifically in quiescent NSCs in the adult DG promotes their activation and neurogenesis, which is countered by inhibition of the histone demethylase LSD1. Mechanistically, RNA-sequencing and CUT & RUN analyses of cultured quiescent adult NSCs reveal Setd1a deletion-induced transcriptional changes and many Setd1a targets, among which down-regulation of Bhlhe40 promotes quiescent NSC activation in the adult DG in vivo. Together, our study reveals a Setd1a-dependent epigenetic mechanism that sustains NSC quiescence in the adult DG.

Combined deletion of Mct8 and Dio2 impairs SVZ neurogliogenesis and olfactory function in adult mice

Within the adult mouse subventricular zone (SVZ), neural stem cells (NSCs) produce neuroblasts and oligodendrocyte precursor cells (OPCs). T3, the active thyroid hormone, influences renewal and commitment of SVZ progenitors. However, how regulators of T3 availability affect these processes is less understood. Using Mct8/Dio2 knockout mice, we investigated the role of MCT8, a TH transporter, and DIO2, the T3-generating enzyme, in regulating adult SVZ-neurogliogenesis. Single-cell RNA-Seq revealed Mct8 expression in various SVZ cell types in WT mice, while Dio2 was enriched in neurons, astrocytes, and quiescent NSCs. The absence of both regulators in the knockout model dysregulated gene expression, increased the neuroblast/OPC ratio and hindered OPC differentiation. Immunostainings demonstrated compromised neuroblast migration reducing their supply to the olfactory bulbs, impairing interneuron differentiation and odor discrimination. These findings underscore the pivotal roles of MCT8 and DIO2 in neuro- and oligodendrogenesis, offering targets for therapeutic avenues in neurodegenerative and demyelinating diseases.

Classification of Neural Stem Cell Activation State In Vitro using Autofluorescence.

Neural stem cells (NSCs) divide and produce newborn neurons in the adult brain through a process called adult neurogenesis. Adult NSCs are primarily quiescent, a reversible cell state where they have exited the cell cycle (G0) yet remain responsive to the environment. In the first step of adult neurogenesis, quiescent NSCs (qNSCs) receive a signal and activate, exiting quiescence and re-entering the cell cycle. Thus, understanding the regulators of NSC quiescence and quiescence exit is critical for future strategies targeting adult neurogenesis. However, our understanding of NSC quiescence is limited by technical constraints in identifying quiescent NSCs (qNSCs) and activated NSCs (aNSCs). This protocol describes a new approach to identify and enrich qNSCs and aNSCs generated in in vitro cultures by imaging NSC autofluorescence. First, this protocol describes how to use a confocal microscope to identify autofluorescent markers of qNSCs and aNSCs to classify NSC activation state using autofluorescence intensity. Second, this protocol describes how to use a fluorescent activated cell sorter (FACS) to classify NSC activation state and enrich samples for qNSCs or aNSCs using autofluorescence intensity. Third, this protocol describes how to use a multiphoton microscope to perform fluorescence lifetime imaging (FLIM) at single-cell resolution, classify NSC activation state, and track the dynamics of quiescent exit using both autofluorescence intensitiesand fluorescence lifetimes. Thus, this protocol provides a live-cell, label-free, single-cell resolution toolkit for studying NSC quiescence and quiescence exit.

Deciphering molecular heterogeneity and dynamics of human hippocampal neural stem cells at different ages and injury states

While accumulated publications support the existence of neurogenesis in the adult human hippocampus, the homeostasis and developmental potentials of neural stem cells (NSCs) under different contexts remain unclear. Based on our generated single-nucleus atlas of the human hippocampus across neonatal, adult, aging, and injury, we dissected the molecular heterogeneity and transcriptional dynamics of human hippocampal NSCs under different contexts. We further identified new specific neurogenic lineage markers that overcome the lack of specificity found in some well-known markers. Based on developmental trajectory and molecular signatures, we found that a subset of NSCs exhibit quiescent properties after birth, and most NSCs become deep quiescence during aging. Furthermore, certain deep quiescent NSCs are reactivated following stroke injury. Together, our findings provide valuable insights into the development, aging, and reactivation of the human hippocampal NSCs, and help to explain why adult hippocampal neurogenesis is infrequently observed in humans.

Deciphering molecular heterogeneity and dynamics of human hippocampal neural stem cells at different ages and injury states.

While accumulated publications support the existence of neurogenesis in the adult human hippocampus, the homeostasis and developmental potentials of neural stem cells (NSCs) under different contexts remain unclear. Based on our generated single-nucleus atlas of the human hippocampus across neonatal, adult, aging, and injury, we dissected the molecular heterogeneity and transcriptional dynamics of human hippocampal NSCs under different contexts. We further identified new specific neurogenic lineage markers that overcome the lack of specificity found in some well-known markers. Based on developmental trajectory and molecular signatures, we found that a subset of NSCs exhibit quiescent properties after birth, and most NSCs become deep quiescence during aging. Furthermore, certain deep quiescent NSCs are reactivated following stroke injury. Together, our findings provide valuable insights into the development, aging, and reactivation of the human hippocampal NSCs, and help to explain why adult hippocampal neurogenesis is infrequently observed in humans.

Autofluorescence is a biomarker of neural stem cell activation state

Neural stem cells (NSCs) must exit quiescence to produce neurons; however, our understanding of this process remains constrained by the technical limitations of current technologies. Fluorescence lifetime imaging (FLIM) of autofluorescent metabolic cofactors has been used in other cell types to study shifts in cell states driven by metabolic remodeling that change the optical properties of these endogenous fluorophores. Using this non-destructive, live-cell, and label-free strategy, we found that quiescent NSCs (qNSCs) and activated NSCs (aNSCs) have unique autofluorescence profiles. Specifically, qNSCs display an enrichment of autofluorescence localizing to a subset of lysosomes, which can be used as a graded marker of NSC quiescence to predict cell behavior at single-cell resolution. Coupling autofluorescence imaging with single-cell RNA sequencing, we provide resources revealing transcriptional features linked to deep quiescence and rapid NSC activation. Together, we describe an approach for tracking mouse NSC activation state and expand our understanding of adult neurogenesis.

Prosaposin maintains adult neural stem cells in a state associated with deep quiescence

In most vertebrates, adult neural stem cells (NSCs) continuously give rise to neurons in discrete brain regions. A critical process for maintaining NSC pools over long periods of time in the adult brain is NSC quiescence, a reversible and tightly regulated state of cell-cycle arrest. Recently, lysosomes were identified to regulate the NSC quiescence-proliferation balance. However, it remains controversial whether lysosomal activity promotes NSC proliferation or quiescence, and a finer influence of lysosomal activity on NSC quiescence duration or depth remains unexplored. Using RNA sequencing and pharmacological manipulations, we show that lysosomes are necessary for NSC quiescence maintenance. In addition, we reveal that expression of psap, encoding the lysosomal regulator Prosaposin, is enriched in quiescent NSCs (qNSCs) that reside upstream in the NSC lineage and display a deep/long quiescence phase in the adult zebrafish telencephalon. We show that shRNA-mediated psap knockdown increases the proportion of activated NSCs (aNSCs) as well as NSCs that reside in shallower quiescence states (signed by ascl1a and deltaA expression). Collectively, our results identify the lysosomal protein Psap as a (direct or indirect) quiescence regulator and unfold the interplay between lysosomal function and NSC quiescence heterogeneities.

Astrocytes control quiescent NSC reactivation via GPCR signaling-mediated F-actin remodeling.

The transitioning of neural stem cells (NSCs) between quiescent and proliferative states is fundamental for brain development and homeostasis. Defects in NSC reactivation are associated with neurodevelopmental disorders. Drosophila quiescent NSCs extend an actin-rich primary protrusion toward the neuropil. However, the function of the actin cytoskeleton during NSC reactivation is unknown. Here, we reveal the fine F-actin structures in the protrusions of quiescent NSCs by expansion and super-resolution microscopy. We show that F-actin polymerization promotes the nuclear translocation of Mrtf, a microcephaly-associated transcription factor, for NSC reactivation and brain development. F-actin polymerization is regulated by a signaling cascade composed of G-protein-coupled receptor (GPCR) Smog, G-protein αq subunit, Rho1 GTPase, and Diaphanous (Dia)/Formin during NSC reactivation. Further, astrocytes secrete a Smog ligand Fog to regulate Gαq-Rho1-Dia-mediated NSC reactivation. Together, we establish that the Smog-Gαq-Rho1 signaling axis derived from astrocytes, a NSC niche, regulates Dia-mediated F-actin dynamics in NSC reactivation.

A Reversible Neural Stem Cell Quiescence and Activation Culture System for Metabolic Study.

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.

Protein homeostasis and degradation in quiescent neural stem cells.

Tissue stem cells are maintained in the adult body throughout life and are crucial for tissue homeostasis as they supply newly functional cells. Quiescence is a reversible arrest in the G0/G1 phase of the cell cycle and a strategy to maintain the quality of tissue stem cells. Quiescence maintains stem cells in a self-renewable and differentiable state for a prolonged period by suppressing energy consumption and cell damage and depletion. Most adult neural stem cells in the brain maintain the quiescent state and produce neurons and glial cells through differentiation after activating from the quiescent state to the proliferating state. In this process, proteostasis, including proteolysis, is essential to transition between the quiescent and proliferating states associated with proteome remodeling. Recent reports have demonstrated that quiescent and proliferating neural stem cells have different expression patterns and roles as proteostatic molecules and are affected by age, indicating differing processes for protein homeostasis in these two states in the brain. This review discusses the multiple regulatory stages from protein synthesis (protein birth) to proteolysis (protein death) in quiescent neural stem cells.

Identification of genes with oscillatory expression in glioblastoma: the paradigm of SOX2

Quiescence, a reversible state of cell-cycle arrest, is an important state during both normal development and cancer progression. For example, in glioblastoma (GBM) quiescent glioblastoma stem cells (GSCs) play an important role in re-establishing the tumour, leading to relapse. While most studies have focused on identifying differentially expressed genes between proliferative and quiescent cells as potential drivers of this transition, recent studies have shown the importance of protein oscillations in controlling the exit from quiescence of neural stem cells. Here, we have undertaken a genome-wide bioinformatic inference approach to identify genes whose expression oscillates and which may be good candidates for controlling the transition to and from the quiescent cell state in GBM. Our analysis identified, among others, a list of important transcription regulators as potential oscillators, including the stemness gene SOX2, which we verified to oscillate in quiescent GSCs. These findings expand on the way we think about gene regulation and introduce new candidate genes as key regulators of quiescence.

Serine and Arginine-Rich Splicing Factor 3 Promotes the Activation of Quiescent Mouse Neural Stem Cells.

The quiescence and activation of adult stem cells are regulated by many kinds of molecular mechanisms, and RNA alternative splicing participates in regulating many cellular processes. However, the relationship between stem cell quiescence and activation regulation and gene alternative splicing has yet to be studied. In this study, we aimed to elucidate the regulation of stem cell quiescence and activation by RNA alternative splicing. The upregulated genes in activated mouse neural stem cells (NSCs), muscle stem cells, and hematopoietic stem cells were collected for Gene Ontology and Kyoto Encyclopedia of Genes and Genomes analysis. The genes from three tissue stem cells underwent Venn analysis. The mouse NSCs were used for quiescence and reactivation induction. The immunostaining of cell-specific markers was performed to identify cell properties. The reverse transcription-polymerase chain reaction and western blotting were used to detect the gene expression and protein expression, respectively. We found that the upregulated genes in activated stem cells from three tissues were all enriched in RNA splicing-related biological processes; the upregulated RNA splicing-related genes in activated stem cells displayed tissue differences; mouse NSCs were successfully induced into quiescence and reactivation in vitro without losing differentiation potential; serine and arginine-rich splicing factor 3 (Srsf3) was highly expressed in the activated mouse NSCs, and the overexpression of SRSF3 protein promoted the activation of quiescent mouse NSCs and increased the neural cell production. Our data indicate that the alternative splicing change may underline the transition of quiescence and activation of stem cells. The manipulation of the splicing factor may benefit tissue repair by promoting the activation of quiescent stem cells.

Characteristics of quiescent adult neural stem cells induced by the bFGF/BMP4 combination or BMP4 alone in vitro.

Bone morphogenetic protein-4 (BMP4) is involved in regulation of neural stem cells (NSCs) proliferation, differentiation, migration and survival. It was previously thought that the treatment of NSCs with BMP4 alone induces astrocytes, whereas the treatment of NSCs with the bFGF/BMP4 combination induces quiescent neural stem cells (qNSCs). In this study, we performed bulk RNA sequencing (RNA-Seq) to compare the transcriptome profiles of BMP4-treated NSCs and bFGF/BMP4-treated NSCs, and found that both NSCs treated by these two methods were Sox2 positive qNSCs which were able to generate neurospheres. However, NSCs treated by those two methods exhibited different characteristics in state and the potential for neuronal differentiation based on transcriptome analysis and experimental results. We found that BMP4-treated NSCs tended to be in a deeper quiescent state than bFGF/BMP4-treated NSCs as the percentage of ki67-positive cells were lower in BMP4-treated NSCs. And after exposure to differentiated environment, bFGF/BMP4-treated NSCs generated more DCX-positive immature neurons and MAP2-positive neurons than BMP4-treated NSCs. Our study characterized qNSCs treated with BMP4 alone and bFGF/BMP4 combination, providing a reference for the scientific use of BMP4 and bFGF/BMP4-induced qNSCs models.