In vivo clonal analysis reveals self-renewing and multipotent adult neural stem cell characteristics.

Neurogenesis and gliogenesis continue in discrete regions of the adult mammalian brain. A fundamental question remains whether cell genesis occurs from distinct lineage-restricted progenitors or from self-renewing and multipotent neural stem cells in the adult brain. Here, we developed a genetic marking strategy for lineage tracing of individual, quiescent, and nestin-expressing radial glia-like (RGL) precursors in the adult mouse dentate gyrus. Clonal analysis identified multiple modes of RGL activation, including asymmetric and symmetric self-renewal. Long-term lineage tracing in vivo revealed a significant percentage of clones that contained RGL(s), neurons, and astrocytes, indicating capacity of individual RGLs for both self-renewal and multilineage differentiation. Furthermore, conditional Pten deletion in RGLs initially promotes their activation and symmetric self-renewal but ultimately leads to terminal astrocytic differentiation and RGL depletion in the adult hippocampus. Our study identifies RGLs as self-renewing and multipotent neural stem cells and provides novel insights into in vivo properties of adult neural stem cells.

Mechanosensory neuron regeneration in adult Drosophila.

Auditory and vestibular mechanosensory hair cells do not regenerate following injury or aging in the adult mammalian inner ear, inducing irreversible hearing loss and balance disorders for millions of people. Research on model systems showing replacement of mechanosensory cells can provide mechanistic insights into developing new regenerative therapies. Here, we developed lineage tracing systems to reveal the generation of mechanosensory neurons in the Johnston's organ (JO) of intact adult Drosophila, which are the functional counterparts to hair cells in vertebrates. New JO neurons develop cilia and target central brain circuitry. Unexpectedly, mitotic recombination clones point to JO neuron self-replication as a likely source of neuronal plasticity. This mechanism is further enhanced upon treatment with experimental and ototoxic compounds. Our findings introduce a new platform to expedite research on mechanisms and compounds mediating mechanosensory cell regeneration, with nascent implications for hearing and balance restoration.

Genome-wide analysis of facial skeletal regionalization in zebrafish.

Patterning of the facial skeleton involves the precise deployment of thousands of genes in distinct regions of the pharyngeal arches. Despite the significance for craniofacial development, how genetic programs drive this regionalization remains incompletely understood. Here we use combinatorial labeling of zebrafish cranial neural crest-derived cells (CNCCs) to define global gene expression along the dorsoventral axis of the developing arches. Intersection of region-specific transcriptomes with expression changes in response to signaling perturbations demonstrates complex roles for Endothelin 1 (Edn1) signaling in the intermediate joint-forming region, yet a surprisingly minor role in ventralmost regions. Analysis of co-variance across multiple sequencing experiments further reveals clusters of co-regulated genes, with in situ hybridization confirming the domain-specific expression of novel genes. We then created loss-of-function alleles for 12 genes and uncovered antagonistic functions of two new Edn1 targets, follistatin a (fsta) and emx2, in regulating cartilaginous joints in the hyoid arch. Our unbiased discovery and functional analysis of genes with regional expression in zebrafish arch CNCCs reveals complex regulation by Edn1 and points to novel candidates for craniofacial disorders.

Function and Dysfunction of Adult Hippocampal Neurogenesis in Regeneration and Disease.

The hippocampus is the only known brain region where physiological neurogenesis continues into adulthood across mammalian species and in humans. However, disease and injury can change the level of adult hippocampal neurogenesis, which plays an important role in regulating cognitive and emotional abilities. Alterations in hippocampal neurogenesis can mediate treatment of mental illness or affect the brain's capacity for repair and regeneration. In the present review, we evaluate how adult neurogenesis contributes to the repair and regeneration of hippocampal circuitry in the face of diseases and injuries. We also discuss possible future directions for harnessing adult neurogenesis for therapeutic use.

Neuronal circuitry mechanism regulating adult quiescent neural stem-cell fate decision.

Adult neurogenesis arises from neural stem cells within specialized niches. Neuronal activity and experience, presumably acting on this local niche, regulate multiple stages of adult neurogenesis, from neural progenitor proliferation to new neuron maturation, synaptic integration and survival. It is unknown whether local neuronal circuitry has a direct impact on adult neural stem cells. Here we show that, in the adult mouse hippocampus, nestin-expressing radial glia-like quiescent neural stem cells (RGLs) respond tonically to the neurotransmitter γ-aminobutyric acid (GABA) by means of γ2-subunit-containing GABAA receptors. Clonal analysis of individual RGLs revealed a rapid exit from quiescence and enhanced symmetrical self-renewal after conditional deletion of γ2. RGLs are in close proximity to terminals expressing 67-kDa glutamic acid decarboxylase (GAD67) of parvalbumin-expressing (PV+) interneurons and respond tonically to GABA released from these neurons. Functionally, optogenetic control of the activity of dentate PV+ interneurons, but not that of somatostatin-expressing or vasoactive intestinal polypeptide (VIP)-expressing interneurons, can dictate the RGL choice between quiescence and activation. Furthermore, PV+ interneuron activation restores RGL quiescence after social isolation, an experience that induces RGL activation and symmetrical division. Our study identifies a niche cell­signal­receptor trio and a local circuitry mechanism that control the activation and self-renewal mode of quiescent adult neural stem cells in response to neuronal activity and experience.

Single cell transcriptomics reveals early photoreceptor states, cell-specific transcript isoforms, and cancer-predisposing features.

Human cone photoreceptors differ from rods and serve as the retinoblastoma cell-of-origin. Here, we used deep full-length single-cell RNA-sequencing to distinguish post-mitotic cone and rod developmental states and cone-specific features that contribute to retinoblastomagenesis. The analyses revealed early post-mitotic cone- and rod-directed populations characterized by higher THRB or NRL regulon activities, an immature photoreceptor precursor population with concurrent cone and rod gene and regulon expression, and distinct early and late cone and rod maturation states distinguished by maturation-associated declines in RAX regulon activity. Unexpectedly, both L/M cone and rod precursors co-expressed NRL and THRB RNAs, yet they differentially expressed functionally antagonistic NRL isoforms and prematurely terminated THRB transcripts. Early L/M cone precursors exhibited successive expression of lncRNAs along with MYCN, which composed the seventh most L/M-cone-specific regulon, and SYK, which contributed to the early cone precursors' proliferative response to RB1 loss. These findings reveal previously unrecognized photoreceptor precursor states and a role for early cone-precursor-intrinsic SYK expression in retinoblastoma initiation.

Subthreshold repetitive transcranial magnetic stimulation suppresses ketamine-induced poly population spikes in rat sensorimotor cortex.

Cortical oscillations within or across brain regions play fundamental roles in sensory, motor, and memory functions. It can be altered by neuromodulations such as repetitive transcranial magnetic stimulation (rTMS) and pharmacological manipulations such as ketamine. However, the neurobiological basis of the effects of rTMS and ketamine, as well as their interactions, on cortical oscillations is not understood. In this study, we developed and applied a rodent model that enabled simultaneous rTMS treatment, pharmacological manipulations, and invasive electrophysiological recordings, which is difficult in humans. Specifically, a miniaturized C-shaped coil was designed and fabricated to deliver focal subthreshold rTMS above the primary somatosensory (S1) and motor (M1) cortex in rats. Multi-electrode arrays (MEA) were implanted to record local field potentials (LFPs) and single unit activities. A novel form of synchronized activities, poly population spikes (PPS), was discovered as the biomarker of ketamine in LFPs. Brief subthreshold rTMS effectively and reversibly suppressed PPS while increasing the firing rates of single unit activities. These results suggest that ketamine and rTMS have convergent but opposing effects on cortical oscillations and circuits. This highly robust phenomenon has important implications to understanding the neurobiological mechanisms of rTMS and ketamine as well as developing new therapeutic strategies involving both neuromodulation and pharmacological agents.

Altered adult neurogenesis and gliogenesis in patients with mesial temporal lobe epilepsy.

The hippocampus is the most common seizure focus in people. In the hippocampus, aberrant neurogenesis plays a critical role in the initiation and progression of epilepsy in rodent models, but it is unknown whether this also holds true in humans. To address this question, we used immunofluorescence on control healthy hippocampus and surgical resections from mesial temporal lobe epilepsy (MTLE), plus neural stem-cell cultures and multi-electrode recordings of ex vivo hippocampal slices. We found that a longer duration of epilepsy is associated with a sharp decline in neuronal production and persistent numbers in astrogenesis. Further, immature neurons in MTLE are mostly inactive, and are not observed in cases with local epileptiform-like activity. However, immature astroglia are present in every MTLE case and their location and activity are dependent on epileptiform-like activity. Immature astroglia, rather than newborn neurons, therefore represent a potential target to continually modulate adult human neuronal hyperactivity.

Inhibition of BMP signaling in P-Cadherin positive hair progenitor cells leads to trichofolliculoma-like hair follicle neoplasias.

BACKGROUND: Skin stem cells contribute to all three major lineages of epidermal appendages, i.e., the epidermis, the hair follicle, and the sebaceous gland. In hair follicles, highly proliferative committed progenitor cells, called matrix cells, are located at the base of the follicle in the hair bulb. The differentiation of these early progenitor cells leads to specification of a central hair shaft surrounded by an inner root sheath (IRS) and a companion layer. Multiple signaling molecules, including bone morphogenetic proteins (BMPs), have been implicated in this process. METHODS: To further probe the contribution of BMP signaling to hair follicle development and maintenance we employed a transgenic mouse that expresses the BMP inhibitor, Noggin, to disrupt BMP signaling specifically in subset of hair follicle progenitors under the control of neuron specific enolase (Nse) promoter. We then studied the skin tumor phenotypes of the transgenic mice through histology, immunohistochemistry and Western Blotting to delineate the underlying mechanisms. Double transgenic mice expressing BMP as well as noggin under control of the Nse promoter were used to rescue the skin tumor phenotypes. RESULTS: We found that the transgene is expressed specifically in a subpopulation of P-cadherin positive progenitor cells in Nse-Noggin mice. Blocking BMP signaling in this cell population led to benign hair follicle-derived neoplasias resembling human trichofolliculomas, associated with down-regulation of E-cadherin expression and dynamic regulation of CD44. CONCLUSIONS: These observations further define a critical role for BMP signaling in maintaining the homeostasis of hair follicles, and suggest that dysregulation of BMP signaling in hair follicle progenitors may contribute to human trichofolliculoma.

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.

Gamified Dual-Task Training for Individuals with Parkinson Disease: An Exploratory Study on Feasibility, Safety, and Efficacy.

OBJECTIVES: The feasibility and safety of the use of neurorehabilitation technology (SMARTfit® Trainer system) by physical therapists in implementing a gamified physical-cognitive dual-task training (DTT) paradigm for individuals with Parkinson disease (IWPD) was examined. Additionally, the efficacy of this gamified DTT was compared to physical single-task training (STT), both of which were optimized using physio-motivational factors, on changes in motor and cognitive outcomes, and self-assessed disability in activities of daily living. METHODS: Using a cross-over study design, eight participants with mild-to-moderate idiopathic PD (including one with mild cognitive impairment) completed both training conditions (i.e., gamified DTT and STT). For each training condition, the participants attended 2-3 sessions per week over 8.8 weeks on average, with the total amount of training being equivalent to 24 1 h sessions. A washout period averaging 11.5 weeks was inserted between training conditions. STT consisted of task-oriented training involving the practice of functional tasks, whereas for gamified DTT, the same task-oriented training was implemented simultaneously with varied cognitive games using an interactive training system (SMARTfit®). Both training conditions were optimized through continual adaptation to ensure the use of challenging tasks and to provide autonomy support. Training hours, heart rate, and adverse events were measured to assess the feasibility and safety of the gamified DTT protocol. Motor and cognitive function as well as perceived disability were assessed before and after each training condition. RESULTS: Gamified DTT was feasible and safe for this cohort. Across participants, significant improvements were achieved in more outcome measures after gamified DTT than they were after STT. Individually, participants with specific demographic and clinical characteristics responded differently to the two training conditions. CONCLUSION: Physical therapists' utilization of technology with versatile hardware configurations and customizable software application selections was feasible and safe for implementing a tailor-made intervention and for adapting it in real-time to meet the individualized, evolving training needs of IWPD. Specifically in comparison to optimized STT, there was a preliminary signal of efficacy for gamified DTT in improving motor and cognitive function as well as perceived disability in IWPD.

Noggin expands neural stem cells in the adult hippocampus.

New neurons are added to the adult hippocampus throughout life and contribute to cognitive functions, including learning and memory. It remains unclear whether ongoing neurogenesis arises from self-renewing neural stem cells (NSCs) or from multipotential progenitor cells that cannot self-renew in the hippocampus. This is primarily based on observations that neural precursors derived from the subventricular zone (SVZ) can be passaged long term, whereas hippocampal subgranular zone (SGZ) precursors are rapidly depleted by passaging. We demonstrate here that high levels of bone morphogenetic protein (BMP) signaling occur in hippocampal but not SVZ precursors in vitro, and blocking BMP signaling with Noggin is sufficient to foster hippocampal cell self-renewal, proliferation, and multipotentiality using single-cell clonal analysis. Moreover, NSC maintenance requires continual Noggin exposure, which implicates BMPs as crucial regulators of NSC aging. In vivo, Noggin is expressed in the adult dentate gyrus and limits BMP signaling in proliferative cells of the SGZ. Transgenic Noggin overexpression in the SGZ increases multiple precursor cell populations but proportionally increases the glial fibrillary acidic protein-positive cell population at the expense of other precursors, suggesting that Noggin acts on NSCs in vivo. To confirm this, we used a dual thymidine analog paradigm to repeatedly label slowly dividing cells over a long duration. We find that small populations of label-retaining cells exist in the SGZ and that Noggin overexpression increases their numbers. Thus, we propose that the adult hippocampus contains a population of NSCs, which can be expanded both in vitro and in vivo by blocking BMP signaling.

Evaluation of Weng et al.: Leveraging Cell Type Correlations and Single-Cell Bioinformatics to Identify Progenitor Diversity and Lineage.

An example of the peer review process for "Single-Cell Transcriptomics Resolves Intermediate Glial Progenitors and Uncovers a Pivotal Determinant of Cell Fate and Gliomagenesis" (Weng et al., 2019) is presented here.

Diversity of Neural Precursors in the Adult Mammalian Brain.

Aided by advances in technology, recent studies of neural precursor identity and regulation have revealed various cell types as contributors to ongoing cell genesis in the adult mammalian brain. Here, we use stem-cell biology as a framework to highlight the diversity of adult neural precursor populations and emphasize their hierarchy, organization, and plasticity under physiological and pathological conditions.

Pushing and pulling on adult neural stem cells.

How plastic is adult neurogenesis? In this issue of Cell Stem Cell, Sierra et al. (2015) use epilepsy models to probe how hippocampal neural stem cells (NSCs) respond to graded pathological conditions. They uncover changes in cell fate potential upon NSC activation, but it comes at a cost.

Latent tri-lineage potential of adult hippocampal neural stem cells revealed by Nf1 inactivation.

Endogenous neural stem cells (NSCs) in the adult hippocampus are considered to be bi-potent, as they only produce neurons and astrocytes in vivo. In mouse, we found that inactivation of neurofibromin 1 (Nf1), a gene mutated in neurofibromatosis type 1, unlocked a latent oligodendrocyte lineage potential to produce all three lineages from NSCs in vivo. Our results suggest an avenue for promoting stem cell plasticity by targeting barriers of latent lineage potential.

A septo-temporal molecular gradient of sfrp3 in the dentate gyrus differentially regulates quiescent adult hippocampal neural stem cell activation.

BACKGROUND: A converging body of evidence indicates that levels of adult hippocampal neurogenesis vary along the septo-temporal axis of the dentate gyrus, but the molecular mechanisms underlying this regional heterogeneity are not known. We previously identified a niche mechanism regulating proliferation and neuronal development in the adult mouse dentate gyrus resulting from the activity-regulated expression of secreted frizzled-related protein 3 (sfrp3) by mature neurons, which suppresses activation of radial glia-like neural stem cells (RGLs) through inhibition of Wingless/INT (WNT) protein signaling. RESULTS: Here, we show that activation rates within the quiescent RGL population decrease gradually along the septo-temporal axis in the adult mouse dentate gyrus, as defined by MCM2 expression in RGLs. Using in situ hybridization and quantitative real-time PCR, we identified an inverse septal-to-temporal increase in the expression of sfrp3 that emerges during postnatal development. Elimination of sfrp3 and its molecular gradient leads to increased RGL activation, preferentially in the temporal region of the adult dentate gyrus. CONCLUSIONS: Our study identifies a niche mechanism that contributes to the graded distribution of neurogenesis in the adult dentate gyrus and has important implications for understanding functional differences associated with adult hippocampal neurogenesis along the septo-temporal axis.

Single-Cell RNA-Seq with Waterfall Reveals Molecular Cascades underlying Adult Neurogenesis.

Somatic stem cells contribute to tissue ontogenesis, homeostasis, and regeneration through sequential processes. Systematic molecular analysis of stem cell behavior is challenging because classic approaches cannot resolve cellular heterogeneity or capture developmental dynamics. Here we provide a comprehensive resource of single-cell transcriptomes of adult hippocampal quiescent neural stem cells (qNSCs) and their immediate progeny. We further developed Waterfall, a bioinformatic pipeline, to statistically quantify singe-cell gene expression along a de novo reconstructed continuous developmental trajectory. Our study reveals molecular signatures of adult qNSCs, characterized by active niche signaling integration and low protein translation capacity. Our analyses further delineate molecular cascades underlying qNSC activation and neurogenesis initiation, exemplified by decreased extrinsic signaling capacity, primed translational machinery, and regulatory switches in transcription factors, metabolism, and energy sources. Our study reveals the molecular continuum underlying adult neurogenesis and illustrates how Waterfall can be used for single-cell omics analyses of various continuous biological processes.

Secreted frizzled-related protein 3 regulates activity-dependent adult hippocampal neurogenesis.

Adult neurogenesis, the process of generating mature neurons from adult neural stem cells, proceeds concurrently with ongoing neuronal circuit activity and is modulated by various physiological and pathological stimuli. The niche mechanism underlying the activity-dependent regulation of the sequential steps of adult neurogenesis remains largely unknown. Here, we report that neuronal activity decreases the expression of secreted frizzled-related protein 3 (sFRP3), a naturally secreted Wnt inhibitor highly expressed by adult dentate gyrus granule neurons. Sfrp3 deletion activates quiescent radial neural stem cells and promotes newborn neuron maturation, dendritic growth, and dendritic spine formation in the adult mouse hippocampus. Furthermore, sfrp3 reduction is essential for activity-induced adult neural progenitor proliferation and the acceleration of new neuron development. Our study identifies sFRP3 as an inhibitory niche factor from local mature dentate granule neurons that regulates multiple phases of adult hippocampal neurogenesis and suggests an interesting activity-dependent mechanism governing adult neurogenesis via the acute release of tonic inhibition.

A unifying hypothesis on mammalian neural stem cell properties in the adult hippocampus.

Continuously generated new neurons promote circuitry plasticity within specialized regions and contribute to specific functions of the adult mammalian brain. A number of recent studies have investigated the cellular origin of adult neurogenesis in the hippocampus, yielding divergent models of neural stem cell behavior. An essential question remains whether these models are overlapping or fundamentally discrete. We review evidence that primary neural precursors in the adult hippocampus exhibit significant heterogeneity in their properties of self-renewal, multi-lineage differentiation and regulation, representing a range from unipotential committed precursors to bona fide self-renewing multipotent neural stem cells. We further present a testable unifying hypothesis of adult neural stem cell behavior in vivo to outline a common framework for future studies of molecular and cellular mechanisms regulating adult neural stem cells and how these cells may contribute to hippocampal function and repair.