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.

Inactivation of a non-canonical gp130 signaling arm attenuates chronic systemic inflammation and multimorbidity induced by a high-fat diet.

Interleukin-6 (IL-6) is a major pro-inflammatory cytokine for which the levels in plasma demonstrate a robust correlation with age and body mass index (BMI) as part of the senescence-associated secretory phenotype. IL-6 cytokines also play a crucial role in metabolic homeostasis and regenerative processes, primarily via the canonical STAT3 pathway. Thus, selective modulation of IL-6 signaling may offer a unique opportunity for therapeutic interventions. Recently, we discovered that a non-canonical signaling pathway downstream of tyrosine (Y) 814 within the intracellular domain of gp130, the IL-6 co-receptor, is responsible for the recruitment and activation of SRC family of kinases (SFK). Mice with constitutive genetic inactivation of gp130 Y814 (F814 mice) show accelerated resolution of inflammatory response and superior regenerative outcomes in skin wound healing and posttraumatic models of osteoarthritis. The current study was designed to explore if selective genetic or pharmacological inhibition of the non-canonical gp130-Y814/SFK signaling reduces systemic chronic inflammation and multimorbidity in a high-fat diet (HFD)-induced model of accelerated aging. F814 mice showed significantly reduced inflammatory response to HFD in adipose and liver tissue, with significantly reduced levels of systemic inflammation compared to wild type mice. F814 mice were also protected from HFD-induced bone loss and cartilage degeneration. Pharmacological inhibition of gp130-Y814/SFK in mice on HFD mirrored the effects observed in F814 mice on HFD; furthermore, this pharmacological treatment also demonstrated a marked increase in physical activity levels and protective effects against inflammation-associated suppression of neurogenesis in the brain tissue compared to the control group. These findings suggest that selective inhibition of SFK signaling downstream of gp130 receptor represents a promising strategy to alleviate systemic chronic inflammation. Increased degenerative changes and tissue senescence are inevitable in obese and aged organisms, but we demonstrated that the systemic response and inflammation-associated multi-morbidity can be therapeutically mitigated.

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.

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.

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.

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.

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.

The Presence of Neural Stem Cells and Changes in Stem Cell-Like Activity With Age in Mouse Spiral Ganglion Cells In Vivo and In Vitro.

OBJECTIVES: Spiral ganglion neurons (SGNs) include potential endogenous progenitor populations for the regeneration of the peripheral auditory system. However, whether these populations are present in adult mice is largely unknown. We examined the presence and characteristics of SGN-neural stem cells (NSCs) in mice as a function of age. METHODS: The expression of Nestin and Ki67 was examined in sequentially dissected cochlear modiolar tissues from mice of different ages (from postnatal day to 24 weeks) and the sphere-forming populations from the SGNs were isolated and differentiated into different cell types. RESULTS: There were significant decreases in Nestin and Ki67 double-positive mitotic progenitor cells in vivo with increasing mouse age. The SGNs formed spheres exhibiting self-renewing activity and multipotent capacity, which were seen in NSCs and were capable of differentiating into neuron and glial cell types. The SGN spheres derived from mice at an early age (postnatal day or 2 weeks) contained more mitotic stem cells than those from mice at a late age. CONCLUSION: Our findings showed the presence of self-renewing and proliferative subtypes of SGN-NSCs which might serve as a promising source for the regeneration of auditory neurons even in adult mice.

Mapping molecular landmarks of human skeletal ontogeny and pluripotent stem cell-derived articular chondrocytes.

Tissue-specific gene expression defines cellular identity and function, but knowledge of early human development is limited, hampering application of cell-based therapies. Here we profiled 5 distinct cell types at a single fetal stage, as well as chondrocytes at 4 stages in vivo and 2 stages during in vitro differentiation. Network analysis delineated five tissue-specific gene modules; these modules and chromatin state analysis defined broad similarities in gene expression during cartilage specification and maturation in vitro and in vivo, including early expression and progressive silencing of muscle- and bone-specific genes. Finally, ontogenetic analysis of freshly isolated and pluripotent stem cell-derived articular chondrocytes identified that integrin alpha 4 defines 2 subsets of functionally and molecularly distinct chondrocytes characterized by their gene expression, osteochondral potential in vitro and proliferative signature in vivo. These analyses provide new insight into human musculoskeletal development and provide an essential comparative resource for disease modeling and regenerative medicine.

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.

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.

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.

Heterogeneity of Radial Glia-Like Cells in the Adult Hippocampus.

Adult neurogenesis is tightly regulated by the neurogenic niche. Cellular contacts between niche cells and neural stem cells are hypothesized to regulate stem cell proliferation or lineage choice. However, the structure of adult neural stem cells and the contact they form with niche cells are poorly described. Here, we characterized the morphology of radial glia-like (RGL) cells, their molecular identity, proliferative activity, and fate determination in the adult mouse hippocampus. We found the coexistence of two morphotypes of cells with prototypical morphological characteristics of RGL stem cells: Type α cells, which represented 76% of all RGL cells, displayed a long primary process modestly branching into the molecular layer and type ß cells, which represented 24% of all RGL cells, with a shorter radial process highly branching into the outer granule cell layer-inner molecular layer border. Stem cell markers were expressed in type α cells and coexpressed with astrocytic markers in type ß cells. Consistently, in vivo lineage tracing indicated that type α cells can give rise to neurons, astrocytes, and type ß cells, whereas type ß cells do not proliferate. Our results reveal that the adult subgranular zone of the dentate gyrus harbors two functionally different RGL cells, which can be distinguished by simple morphological criteria, supporting a morphofunctional role of their thin cellular processes. Type ß cells may represent an intermediate state in the transformation of type α, RGL stem cells, into astrocytes.

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.

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.

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.

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.