Quiescent Neural Stem Cells Research Articles

Dynamic interplay between GABAergic networks and developing neurons in the adult hippocampus

Neurogenesis is a powerful mechanism for structural and functional remodeling that occurs in restricted areas of the adult brain. Although different neurotransmitters regulate various aspects of the progression from neural stem cell quiescence to neuronal maturation, GABA is the main player. The developmental switch from excitation to inhibition combined with a heterogeneous population of GABAergic interneurons that target different subcellular compartments provides multiple points for the regulation of development and function of new neurons. This complexity is enhanced by feedback and feedforward networks that act as sensors and controllers of circuit activity, impinging directly or indirectly ontodeveloping granule cells and, subsequently, on mature neurons. Newly generated granule cells ultimately connect with input and output partners in a manner that is largely sculpted by the activity of local circuits.

Early stem cell aging in the mature brain.

Stem cell dysfunction drives many age-related disorders. Identifying mechanisms that initially compromise stem cell behavior represent early targets to promote tissue function later in life. Here, we pinpoint multiple factors that disrupt neural stem cell (NSC) behavior in the adult hippocampus. Clonal tracing showed that NSCs exhibit asynchronous depletion by identifying short-term NSCs (ST-NSCs) and long-term NSCs (LT-NSCs). ST-NSCs divide rapidly to generate neurons and deplete in the young brain. Meanwhile, multipotent LT-NSCs are maintained for months but are pushed out of homeostasis by lengthening quiescence. Single-cell transcriptome analysis of deep NSC quiescence revealed several hallmarks of molecular aging in the mature brain and identified tyrosine-protein kinase Abl1 as an NSC aging factor. Treatment with the Abl inhibitor imatinib increased NSC activation without impairing NSC maintenance in the middle-aged brain. Our study indicates that hippocampal NSCs are particularly vulnerable and adaptable to cellular aging.

Between Fate Choice and Self-Renewal-Heterogeneity of Adult Neural Crest-Derived Stem Cells.

Stem cells of the neural crest (NC) vitally participate to embryonic development, but also remain in distinct niches as quiescent neural crest-derived stem cell (NCSC) pools into adulthood. Although NCSC-populations share a high capacity for self-renewal and differentiation resulting in promising preclinical applications within the last two decades, inter- and intrapopulational differences exist in terms of their expression signatures and regenerative capability. Differentiation and self-renewal of stem cells in developmental and regenerative contexts are partially regulated by the niche or culture condition and further influenced by single cell decision processes, making cell-to-cell variation and heterogeneity critical for understanding adult stem cell populations. The present review summarizes current knowledge of the cellular heterogeneity within NCSC-populations located in distinct craniofacial and trunk niches including the nasal cavity, olfactory bulb, oral tissues or skin. We shed light on the impact of intrapopulational heterogeneity on fate specifications and plasticity of NCSCs in their niches in vivo as well as during in vitro culture. We further discuss underlying molecular regulators determining fate specifications of NCSCs, suggesting a regulatory network including NF-κB and NC-related transcription factors like SLUG and SOX9 accompanied by Wnt- and MAPK-signaling to orchestrate NCSC stemness and differentiation. In summary, adult NCSCs show a broad heterogeneity on the level of the donor and the donors’ sex, the cell population and the single stem cell directly impacting their differentiation capability and fate choices in vivo and in vitro. The findings discussed here emphasize heterogeneity of NCSCs as a crucial parameter for understanding their role in tissue homeostasis and regeneration and for improving their applicability in regenerative medicine.

Dynamic spatiotemporal coordination of neural stem cell fate decisions occurs through local feedback in the adult vertebrate brain.

SummaryNeural stem cell (NSC) populations persist in the adult vertebrate brain over a lifetime, and their homeostasis is controlled at the population level through unknown mechanisms. Here, we combine dynamic imaging of entire NSC populations in their in vivo niche over several weeks with pharmacological manipulations, mathematical modeling, and spatial statistics and demonstrate that NSCs use spatiotemporally resolved local feedback signals to coordinate their decision to divide in adult zebrafish brains. These involve Notch-mediated short-range inhibition from transient neural progenitors and a dispersion effect from the dividing NSCs themselves exerted with a delay of 9–12 days. Simulations from a stochastic NSC lattice model capturing these interactions demonstrate that these signals are linked by lineage progression and control the spatiotemporal distribution of output neurons. These results highlight how local and temporally delayed interactions occurring between brain germinal cells generate self-propagating dynamics that maintain NSC population homeostasis and coordinate specific spatiotemporal correlations.

Manipulation of EGFR-Induced Signaling for the Recruitment of Quiescent Neural Stem Cells in the Adult Mouse Forebrain.

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.

Maintenance of neural stem-progenitor cells by the lysosomal biosynthesis regulators TFEB and TFE3 in the embryonic mouse telencephalon.

Lysosomes have recently been implicated in regulation of quiescence in adult neural stem cells (NSCs). Whether lysosomes regulate the differentiation of neural stem-progenitor cells (NPCs) in the embryonic brain has remained unknown, however. We here show that lysosomes are more abundant in rapidly dividing NPCs than in differentiating neurons in the embryonic mouse neocortex and ganglionic eminence. The genes for TFEB and TFE3, master regulators of lysosomal biosynthesis, as well as other lysosome-related genes were also expressed at higher levels in NPCs than in differentiating neurons. Anatomic analysis revealed accumulation of lysosomes at the apical and basal endfeet of NPCs. Knockdown of TFEB and TFE3, or that of the lysosomal transporter Slc15a4, resulted in premature differentiation of neocortical NPCs. Conversely, forced expression of an active form of TFEB (TFEB-AA) suppressed neuronal differentiation of NPCs in association with upregulation of NPC-related genes. These results together point to a previously unappreciated role for TFEB and TFE3, and possibly for lysosomes, in maintenance of the undifferentiated state of embryonic NPCs. We further found that lysosomes are even more abundant in an NPC subpopulation that rarely divides and includes the embryonic origin of adult NSCs than in the majority of NPCs that divide frequently for construction of the embryonic brain, and that overexpression of TFEB-AA also suppressed the cell cycle of neocortical NPCs. Our results thus also implicate lysosomes in establishment of the slowly dividing, embryonic origin of adult NSCs.

Hes1 oscillation frequency correlates with activation of neural stem cells

Quiescent neural stem cells (NSCs) are occasionally activated to undergo proliferation and subsequent neuronal differentiation. It was previously shown that the transcriptional repressor Hes1 is involved in both active and quiescent states of NSCs: when Hes1 expression oscillates, it periodically represses the proneural gene Ascl1, thereby driving Ascl1 oscillations, which regulate the active state, while sustained Hes1 expression continuously suppresses Ascl1, promoting quiescence. However, it remains to be analyzed how the transition from quiescent to active states of NSCs is controlled. Here, we found that overexpression of the active form of Notch1 significantly activates NSCs in both in-vitro and in-vivo conditions and that its levels are proportional to NSC activation. The active form of Notch1 induces a burst of Hes1 oscillations in quiescent NSCs, and the frequency of Hes1 oscillations, rather than the Hes1 peak levels, correlates with the efficiency of NSC activation. These results raised the possibility that bursting Hes1 oscillations could increase the chance of Ascl1 oscillations in quiescent NSCs, suggesting that Notch1-induced Hes1 oscillation is a cue for a transition from quiescent to active states of NSCs.

Adult neural stem cell activation in mice is regulated by the day/night cycle and intracellular calcium dynamics

Neural stem cells (NSCs) in the adult brain transit from the quiescent state to proliferation to produce new neurons. The mechanisms regulating this transition in freely behaving animals are, however, poorly understood. We customized invivo imaging protocols to follow NSCs for several days up to months, observing their activation kinetics in freely behaving mice. Strikingly, NSC division is more frequent during daylight and is inhibited by darkness-induced melatonin signaling. The inhibition of melatonin receptors affected intracellular Ca2+ dynamics and promoted NSC activation. We further discovered a Ca2+ signature of quiescent versus activated NSCs and showed that several microenvironmental signals converge on intracellular Ca2+ pathways to regulate NSC quiescence and activation. Invivo NSC-specific optogenetic modulation of Ca2+ fluxes to mimic quiescent-state-like Ca2+ dynamics in freely behaving mice blocked NSC activation and maintained their quiescence, pointing to the regulatory mechanisms mediating NSC activation in freely behaving animals.

Absence of Both Thyroid Hormone Transporters MCT8 and OATP1C1 Impairs Neural Stem Cell Fate in the Adult Mouse Subventricular Zone.

SummaryAdult neural stem cell (NSC) generation in vertebrate brains requires thyroid hormones (THs). How THs enter the NSC population is unknown, although TH availability determines proliferation and neuronal versus glial progenitor determination in murine subventricular zone (SVZ) NSCs. Mice display neurological signs of the severely disabling human disease, Allan-Herndon-Dudley syndrome, if they lack both MCT8 and OATP1C1 transporters, or MCT8 and deiodinase type 2. We analyzed the distribution of MCT8 and OATP1C1 in adult mouse SVZ. Both are strongly expressed in NSCs and at a lower level in neuronal cell precursors but not in oligodendrocyte progenitors. Next, we analyzed Mct8/Oatp1c1 double-knockout mice, where brain uptake of THs is strongly reduced. NSC proliferation and determination to neuronal fates were severely affected, but not SVZ-oligodendroglial progenitor generation. This work highlights how tight control of TH availability determines NSC function and glial-neuron cell-fate choice in adult brains.

Intracrine Vascular Endothelial Growth Factor Maintains Hippocampal Neural Stem Cell Quiescence

In the dentate gyrus (DG) of the adult mouse hippocampus, neural stem cells (NSCs) balance self-renewal and differentiation to produce neurons that support hippocampal function. Vascular endothelial growth factor (VEGF) is a well-known supporting factor for adult neurogenesis, but conflicting studies have left it uncertain how VEGF signals to NSCs. Here, we identified a VEGF-VEGFR2 intracrine signaling mechanism within adult DG NSCs that prevents their exhaustion. We show both in vitro and in vivo that NSC-VEGF loss caused cell-autonomous exhaustion of adult DG NSCs. In contrast, extracellular VEGF was neither necessary nor sufficient to maintain NSC quiescence or to stimulate VEGFR2 signaling, most likely due to sheddase-mediated cleavage of extracellular VEGFR2 ligand binding domains. Our findings support an exclusively intracellular mechanism for VEGF signaling in adult DG NSCs, thereby providing resolution to previously conflicting studies and suggesting cellular source can dictate the functional impact of soluble ligands in DG NSCs.

The neurosphere assay: an effective in vitro technique to study neural stem cells.

The neurosphere assay: an effective in vitro technique to study neural stem cells.

High-resolution mouse subventricular zone stem-cell niche transcriptome reveals features of lineage, anatomy, and aging

Adult neural stem cells (NSC) serve as a reservoir for brain plasticity and origin for certain gliomas. Lineage tracing and genomic approaches have portrayed complex underlying heterogeneity within the major anatomical location for NSC, the subventricular zone (SVZ). To gain a comprehensive profile of NSC heterogeneity, we utilized a well-validated stem/progenitor-specific reporter transgene in concert with single-cell RNA sequencing to achieve unbiased analysis of SVZ cells from infancy to advanced age. The magnitude and high specificity of the resulting transcriptional datasets allow precise identification of the varied cell types embedded in the SVZ including specialized parenchymal cells (neurons, glia, microglia) and noncentral nervous system cells (endothelial, immune). Initial mining of the data delineates four quiescent NSC and three progenitor-cell subpopulations formed in a linear progression. Further evidence indicates that distinct stem and progenitor populations reside in different regions of the SVZ. As stem/progenitor populations progress from neonatal to advanced age, they acquire a deficiency in transition from quiescence to proliferation. Further data mining identifies stage-specific biological processes, transcription factor networks, and cell-surface markers for investigation of cellular identities, lineage relationships, and key regulatory pathways in adult NSC maintenance and neurogenesis.

Extracellular Vesicles, Influential Players of Intercellular Communication within Adult Neurogenic Niches.

Adult neurogenesis, involving the generation of functional neurons from adult neural stem cells (NSCs), occurs constitutively in discrete brain regions such as hippocampus, sub-ventricular zone (SVZ) and hypothalamus. The intrinsic structural plasticity of the neurogenic process allows the adult brain to face the continuously changing external and internal environment and requires coordinated interplay between all cell types within the specialized microenvironment of the neurogenic niche. NSC-, neuronal- and glia-derived factors, originating locally, regulate the balance between quiescence and self-renewal of NSC, their differentiation programs and the survival and integration of newborn cells. Extracellular Vesicles (EVs) are emerging as important mediators of cell-to-cell communication, representing an efficient way to transfer the biologically active cargos (nucleic acids, proteins, lipids) by which they modulate the function of the recipient cells. Current knowledge of the physiological role of EVs within adult neurogenic niches is rather limited. In this review, we will summarize and discuss EV-based cross-talk within adult neurogenic niches and postulate how EVs might play a critical role in the regulation of the neurogenic process.

Syndecan-1 Stimulates Adult Neurogenesis in the Mouse Ventricular-Subventricular Zone after Injury.

SummaryThe production of neurons from neural stem cells (NSCs) persists throughout life in the mouse ventricular-subventricular zone (V-SVZ). We have previously reported that NSCs from adult V-SVZ are contained in cell populations expressing the carbohydrate SSEA-1/LeX, which exhibit either characteristics of quiescent NSCs (qNSCs) or of actively dividing NSCs (aNSCs) based on the absence or the presence of EGF-receptor, respectively. Using the fluorescence ubiquitination cell cycle indicator-Cdt1 transgenic mice to mark cells in G0/G1 phase of the cell cycle, we uncovered a subpopulation of qNSCs which were primed to enter the cell cycle in vitro. Besides, we found that treatment with Syndecan-1, a heparan sulfate proteoglycan involved in NSC proliferation, hastened the division of qNSCs and increased proliferation of aNSCs shortening their G1 phase in vitro. Furthermore, administration of Syndecan-1 ameliorated the recovery of neurogenic populations in the V-SVZ after radiation-induced injury providing potential cure for neurogenesis decline during brain aging or after injury.

The significance of gene expression dynamics in neural stem cell regulation.

Neural stem cells (NSCs) actively proliferate and generate neurons and glial cells (active state) in the embryonic brain, whereas they are mostly dormant (quiescent state) in the adult brain. The expression dynamics of Hes1 are different between active and quiescent NSCs. In active NSCs, Hes1 expression oscillates and periodically represses the expression of proneural genes such as Ascl1, thereby driving their oscillations. By contrast, in quiescent NSCs, Hes1 oscillations maintain expression at higher levels even at trough phases (thus continuous), thereby continuously suppressing proneural gene expression. High levels of Hes1 expression and the resultant suppression of Ascl1 promote the quiescent state of NSCs, whereas oscillatory Hes1 expression and the resultant oscillatory Ascl1 expression regulate their active state. Furthermore, in other developmental contexts, high, continuous Hes1 expression induces astrocyte differentiation or the formation of boundaries, which function as signaling centers. Thus, the expression dynamics of Hes1 are a key regulatory mechanism generating and maintaining various cell types in the nervous system.

HCN Channel Activity Balances Quiescence and Proliferation in Neural Stem Cells and Is a Selective Target for Neuroprotection During Cancer Treatment.

Children suffering from neurologic cancers undergoing chemotherapy and radiotherapy are at high risk of reduced neurocognitive abilities likely via damage to proliferating neural stem cells (NSC). Therefore, strategies to protect NSCs are needed. We argue that induced cell-cycle arrest/quiescence in NSCs during cancer treatment can represent such a strategy. Here, we show that hyperpolarization-activated cyclic nucleotide-gated (HCN) ion channels are dynamically expressed over the cell cycle in NSCs, depolarize the membrane potential, underlie spontaneous calcium oscillations and are required to maintain NSCs in the actively proliferating pool. Hyperpolarizing pharmacologic inhibition of HCN channels during exposure to ionizing radiation protects NSCs cells in neurogenic brain regions of young mice. In contrast, brain tumor-initiating cells, which also express HCN channels, remain proliferative during HCN inhibition. IMPLICATIONS: Our finding that NSCs can be selectively rescued while cancer cells remain sensitive to the treatment, provide a foundation for reduction of cognitive impairment in children with neurologic cancers.

Lysosomes and signaling pathways for maintenance of quiescence in adult neural stem cells.

Quiescence is a cellular strategy for maintaining somatic stem cells in a specific niche in a low metabolic state without senescence for a long period of time. During development, neural stem cells (NSCs) actively proliferate and self-renew, and their progeny differentiate into both neurons and glial cells to form mature brain tissues. On the other hand, most NSCs in the adult brain are quiescent and arrested in G0/G1 phase of the cell cycle. Quiescence is essential in order to avoid the precocious exhaustion of NSCs, ensuring a sustainable source of available stem cells in the brain throughout the lifespan. After receiving activation signals, quiescent NSCs reenter the cell cycle and generate new neurons. This switching between quiescence and proliferation is tightly regulated by diverse signaling pathways. Recent studies suggest significant involvement of cellular proteostasis (homeostasis of the proteome) in the quiescent state of NSCs. Proteostasis is the result of integrated regulation of protein synthesis, folding, and degradation. In this review, we discuss regulation of quiescence by multiple signaling pathways, especially bone morphogenetic protein and Notch signaling, and focus on the functional involvement of the lysosome, an organelle governing cellular degradation, in quiescence of adult NSCs.

Age-related loss of neural stem cell O-GlcNAc promotes a glial fate switch through STAT3 activation

Increased neural stem cell (NSC) quiescence is a major determinant of age-related regenerative decline in the adult hippocampus. However, a coextensive model has been proposed in which division-coupled conversion of NSCs into differentiated astrocytes restrict the stem cell pool with age. Here we report that age-related loss of the posttranslational modification, O-linked β-N-acetylglucosamine (O-GlcNAc), in NSCs promotes a glial fate switch. We detect an age-dependent decrease in NSC O-GlcNAc levels coincident with decreased neurogenesis and increased gliogenesis in the mature hippocampus. Mimicking an age-related loss of NSC O-GlcNAcylation in young mice reduces neurogenesis, increases astrocyte differentiation, and impairs associated cognitive function. Using RNA-sequencing of primary NSCs following decreased O-GlcNAcylation, we detected changes in the STAT3 signaling pathway indicative of glial differentiation. Moreover, using O-GlcNAc-specific mass spectrometry analysis of the aging hippocampus, together with an in vitro site-directed mutagenesis approach, we identify loss of STAT3 O-GlcNAc at Threonine 717 as a driver of astrocyte differentiation. Our data identify the posttranslational modification, O-GlcNAc, as a key molecular regulator of regenerative decline underlying an age-related NSC fate switch.

Adult and endemic neurogenesis in the vestibular nuclei after unilateral vestibular neurectomy.

We previously revealed adult reactive neurogenesis in deafferented vestibular nuclei following unilateral vestibular neurectomy (UVN) in the feline model. We recently replicated the same surgery in a rodent model and aimed to elucidate the origin and fate of newly generated cells following UVN. We used specific markers of cell proliferation, glial reaction, and cell differentiation in the medial vestibular nucleus (MVN) of adult rats. UVN induced an intense cell proliferation and glial reaction with an increase of GFAP-Immunoreactive (Ir), IBA1-Ir and Olig2-Ir cells 3 days after the lesion in the deafferented MVN. Most of the newly generated cells survived after UVN and differentiated into oligodendrocytes, astrocytes, microglial cells and GABAergic neurons. Interestingly, UVN induced a significant increase in a population of cells colocalizing SOX2 and GFAP 3 days after lesion in the deafferented MVN indicating the probable presence of multipotent cells in the vestibular nuclei. The concomitant increase in BrdU- and SOX2-Ir cells with the presence of SOX2 and GFAP colocalization 3 days after UVN in the deafferented MVN may support local mitotic activity of endemic quiescent neural stem cells in the parenchyma of vestibular nuclei.

Life-Long Neural Stem Cells Are Fate-Specified at an Early Developmental Stage.

The origin and life-long fate of quiescent neural stem cells (NSCs) in the adult mammalian brain remain largely unknown. A few neural precursor cells in the embryonic brain elongate their cell cycle time and subsequently become quiescent postnatally, suggesting the possibility that life-long NSCs are selected at an early embryonic stage. Here, we utilized a GFP-expressing lentivirus to investigate the fate of progeny from individual lentivirus-infected NSCs by identifying the lentiviral integration site. Our data suggest that NSCs become specified to two or more lineages prior to embryonic day 13.5 in mice: one NSC lineage produces cells only for the cortex and another provides neurons to the olfactory bulb. The majority of neurosphere-forming NSCs in the adult brain are relatively dormant and generate very few cells, if any, in the olfactory bulb or cortex, and this NSC population could serve as a reservoir that is occasionally reactivated later in life.