Centenarian hippocampus displays high levels of astrocytic metallothioneins.

The hippocampus is a brain area linked to cognition. The mechanisms that maintain cognitive activity in humans are poorly understood. Centenarians display extreme longevity which is generally accompanied by better quality of life, lower cognitive impairment, and reduced incidence of pathologies including neurodegenerative diseases. We performed transcriptomic studies in hippocampus samples from individuals of different ages (centenarians [≥97 years], old, and young) and identified a differential gene expression pattern in centenarians compared to the other two groups. In particular, several isoforms of metallothioneins (MTs) were highly expressed in centenarians. Moreover, we identified that MTs were mainly expressed in astrocytes. Functional studies in human primary astrocytes revealed that MT1 and MT3 are necessary for their homeostasis maintenance. Overall, these results indicate that the expression of MTs specifically in astrocytes is a mechanism for protection during aging.

Bumetanide induces post-traumatic microglia-interneuron contact to promote neurogenesis and recovery.

Although the Na-K-Cl cotransporter (NKCC1) inhibitor bumetanide has prominent positive effects on the pathophysiology of many neurological disorders, the mechanism of action is obscure. Attention paid to elucidating the role of Nkcc1 has mainly been focused on neurons, but recent single cell mRNA sequencing analysis has demonstrated that the major cellular populations expressing NKCC1 in the cortex are non-neuronal. We used a combination of conditional transgenic animals, in vivo electrophysiology, two-photon imaging, cognitive behavioural tests and flow cytometry to investigate the role of Nkcc1 inhibition by bumetanide in a mouse model of controlled cortical impact (CCI). Here, we found that bumetanide rescues parvalbumin-positive interneurons by increasing interneuron-microglia contacts shortly after injury. The longitudinal phenotypic changes in microglia were significantly modified by bumetanide, including an increase in the expression of microglial-derived BDNF. These effects were accompanied by the prevention of CCI-induced decrease in hippocampal neurogenesis. Treatment with bumetanide during the first week post-CCI resulted in significant recovery of working and episodic memory as well as changes in theta band oscillations 1 month later. These results disclose a novel mechanism for the neuroprotective action of bumetanide mediated by an acceleration of microglial activation dynamics that leads to an increase in parvalbumin interneuron survival following CCI, possibly resulting from increased microglial BDNF expression and contact with interneurons. Salvage of interneurons may normalize ambient GABA, resulting in the preservation of adult neurogenesis processes as well as contributing to bumetanide-mediated improvement of cognitive performance.

Pioneer factor ASCL1 cooperates with the mSWI/SNF complex at distal regulatory elements to regulate human neural differentiation.

Pioneer transcription factors are thought to play pivotal roles in developmental processes by binding nucleosomal DNA to activate gene expression, though mechanisms through which pioneer transcription factors remodel chromatin remain unclear. Here, using single-cell transcriptomics, we show that endogenous expression of neurogenic transcription factor ASCL1, considered a classical pioneer factor, defines a transient population of progenitors in human neural differentiation. Testing ASCL1's pioneer function using a knockout model to define the unbound state, we found that endogenous expression of ASCL1 drives progenitor differentiation by cis-regulation both as a classical pioneer factor and as a nonpioneer remodeler, where ASCL1 binds permissive chromatin to induce chromatin conformation changes. ASCL1 interacts with BAF SWI/SNF chromatin remodeling complexes, primarily at targets where it acts as a nonpioneer factor, and we provide evidence for codependent DNA binding and remodeling at a subset of ASCL1 and SWI/SNF cotargets. Our findings provide new insights into ASCL1 function regulating activation of long-range regulatory elements in human neurogenesis and uncover a novel mechanism of its chromatin remodeling function codependent on partner ATPase activity.

Fluorescence-Activated Nuclei Negative Sorting of Neurons Combined with Single Nuclei RNA Sequencing to Study the Hippocampal Neurogenic Niche.

Adult Hippocampal Neurogenesis (AHN), which consists of a lifelong maintenance of proliferative and quiescent neural stem cells (NSCs) within the sub-granular zone (SGZ) of the dentate gyrus (DG) and their differentiation from newly born neurons into granule cells in the granule cell layer, is well validated across numerous studies. Using genetically modified animals, particularly rodents, is a valuable tool to investigate signaling pathways regulating AHN and to study the role of each cell type that compose the hippocampal neurogenic niche. To address the latter, methods combining single nuclei isolation with next generation sequencing have had a significant impact in the field of AHN to identify gene signatures for each cell population. Further refinement of these techniques is however needed to phenotypically profile rarer cell populations within the DG. Here, we present a method that utilizes Fluorescence Activated Nuclei Sorting (FANS) to exclude most neuronal populations from a single nuclei suspension isolated from freshly dissected DG, by selecting unstained nuclei for the NeuN antigen, in order to perform single nuclei RNA sequencing (snRNA-seq). This method is a potential steppingstone to further investigate intercellular regulation of the AHN and to uncover novel cellular markers and mechanisms across species.

DNGR-1-tracing marks an ependymal cell subset with damage-responsive neural stem cell potential.

Cells with latent stem ability can contribute to mammalian tissue regeneration after damage. Whether the central nervous system (CNS) harbors such cells remains controversial. Here, we report that DNGR-1 lineage tracing in mice identifies an ependymal cell subset, wherein resides latent regenerative potential. We demonstrate that DNGR-1-lineage-traced ependymal cells arise early in embryogenesis (E11.5) and subsequently spread across the lining of cerebrospinal fluid (CSF)-filled compartments to form a contiguous sheet from the brain to the end of the spinal cord. In the steady state, these DNGR-1-traced cells are quiescent, committed to their ependymal cell fate, and do not contribute to neuronal or glial lineages. However, trans-differentiation can be induced in adult mice by CNS injury or in vitro by culture with suitable factors. Our findings highlight previously unappreciated ependymal cell heterogeneity and identify across the entire CNS an ependymal cell subset wherein resides damage-responsive neural stem cell potential.

Understanding astrocyte differentiation: Clinical relevance, technical challenges, and new opportunities in the omics era.

Astrocytes are a major type of glial cells that have essential functions in development and homeostasis of the central nervous system (CNS). Immature astrocytes in the developing CNS support neuronal maturation and possess neural-stem-cell-like properties. Mature astrocytes partially lose these functions but gain new functions essential for adult CNS homeostasis. In pathological conditions, astrocytes become "reactive", which disrupts their mature homeostatic functions and reactivates some immature astrocyte-like properties, suggesting a partial reversal of astrocyte maturation. The loss of homeostatic astrocyte functions contributes to the pathogenesis of various neurological conditions, and therefore activating maturation-promoting mechanisms may be a promising therapeutic strategy to restore homeostasis. Manipulating the mechanisms underlying astrocyte maturation might also allow to facilitate CNS regeneration by enhancing developmental functions of adult astrocytes. However, such therapeutic strategies are still some distance away because of our limited understanding of astrocyte differentiation and maturation, due to biological and technical challenges, including the high degree of similarity of astrocytes with neural stem cells and the shortcomings of astrocyte markers. Current advances in systems biology have a huge potential to overcome these challenges. Recent transcriptomic analyses have already revealed new astrocyte markers and new regulators of astrocyte differentiation. However, the epigenomic changes that presumably occur during astrocyte differentiation remain an important, largely unexplored area for future research. Emerging technologies such as CRISPR/Cas9-based functional screens will further improve our understanding of the mechanisms underlying astrocyte differentiation. This may open up new clinical approaches to restore homeostasis in neurological disorders and/or promote CNS regeneration. This article is categorized under: Neurological Diseases > Genetics/Genomics/Epigenetics Neurological Diseases > Stem Cells and Development Neurological Diseases > Molecular and Cellular Physiology.

Wnt/ß-catenin signalling is dispensable for adult neural stem cell homeostasis and activation.

Adult mouse hippocampal neural stem cells (NSCs) generate new neurons that integrate into existing hippocampal networks and modulate mood and memory. These NSCs are largely quiescent and are stimulated by niche signals to activate and produce neurons. Wnt/ß-catenin signalling acts at different steps along the hippocampal neurogenic lineage, but whether it has a direct role in the regulation of NSCs remains unclear. Here, we used Wnt/ß-catenin reporters and transcriptomic data from in vivo and in vitro models to show that adult NSCs respond to Wnt/ß-catenin signalling. Wnt/ß-catenin stimulation instructed the neuronal differentiation of proliferating NSCs and promoted the activation or differentiation of quiescent NSCs in a dose-dependent manner. However, deletion of ß-catenin in NSCs did not affect either their activation or maintenance of their stem cell characteristics. Together, these results indicate that, although NSCs do respond to Wnt/ß-catenin stimulation in a dose-dependent and state-specific manner, Wnt/ß-catenin signalling is not cell-autonomously required to maintain NSC homeostasis, which reconciles some of the contradictions in the literature as to the role of Wnt/ß-catenin signalling in adult hippocampal NSCs.

The atypical Rho GTPase Rnd2 is critical for dentate granule neuron development and anxiety-like behavior during adult but not neonatal neurogenesis.

Despite the central role of Rho GTPases in neuronal development, their functions in adult hippocampal neurogenesis remain poorly explored. Here, by using a retrovirus-based loss-of-function approach in vivo, we show that the atypical Rho GTPase Rnd2 is crucial for survival, positioning, somatodendritic morphogenesis, and functional maturation of adult-born dentate granule neurons. Interestingly, most of these functions are specific to granule neurons generated during adulthood since the deletion of Rnd2 in neonatally-born granule neurons only affects dendritogenesis. In addition, suppression of Rnd2 in adult-born dentate granule neurons increases anxiety-like behavior whereas its deletion in pups has no such effect, a finding supporting the adult neurogenesis hypothesis of anxiety disorders. Thus, our results are in line with the view that adult neurogenesis is not a simple continuation of earlier processes from development, and establish a causal relationship between Rnd2 expression and anxiety.

Extensive transcriptional and chromatin changes underlie astrocyte maturation in vivo and in culture.

Astrocytes have essential functions in brain homeostasis that are established late in differentiation, but the mechanisms underlying the functional maturation of astrocytes are not well understood. Here we identify extensive transcriptional changes that occur during murine astrocyte maturation in vivo that are accompanied by chromatin remodelling at enhancer elements. Investigating astrocyte maturation in a cell culture model revealed that in vitro-differentiated astrocytes lack expression of many mature astrocyte-specific genes, including genes for the transcription factors Rorb, Dbx2, Lhx2 and Fezf2. Forced expression of these factors in vitro induces distinct sets of mature astrocyte-specific transcripts. Culturing astrocytes in a three-dimensional matrix containing FGF2 induces expression of Rorb, Dbx2 and Lhx2 and improves astrocyte maturity based on transcriptional and chromatin profiles. Therefore, extrinsic signals orchestrate the expression of multiple intrinsic regulators, which in turn induce in a modular manner the transcriptional and chromatin changes underlying astrocyte maturation.

Transient developmental imbalance of cortical interneuron subtypes presages long-term changes in behavior.

Cortical GABAergic interneurons are generated in large numbers in the ganglionic eminences and migrate into the cerebral cortex during embryogenesis. At early postnatal stages, during neuronal circuit maturation, autonomous and activity-dependent mechanisms operate within the cortex to adjust cell numbers by eliminating naturally occurring neuron excess. Here, we show that when cortical interneurons are generated in aberrantly high numbers-due to a defect in precursor cell proliferation during embryogenesis-extra parvalbumin interneurons persist in the postnatal mouse cortex during critical periods of cortical network maturation. Even though cell numbers are subsequently normalized, behavioral abnormalities remain in adulthood. This suggests that timely clearance of excess cortical interneurons is critical for correct functional maturation of circuits that drive adult behavior.

Dysfunction of the proteoglycan Tsukushi causes hydrocephalus through altered neurogenesis in the subventricular zone in mice.

The lateral ventricle (LV) is flanked by the subventricular zone (SVZ), a neural stem cell (NSC) niche rich in extrinsic growth factors regulating NSC maintenance, proliferation, and neuronal differentiation. Dysregulation of the SVZ niche causes LV expansion, a condition known as hydrocephalus; however, the underlying pathological mechanisms are unclear. We show that deficiency of the proteoglycan Tsukushi (TSK) in ependymal cells at the LV surface and in the cerebrospinal fluid results in hydrocephalus with neurodevelopmental disorder-like symptoms in mice. These symptoms are accompanied by altered differentiation and survival of the NSC lineage, disrupted ependymal structure, and dysregulated Wnt signaling. Multiple TSK variants found in patients with hydrocephalus exhibit reduced physiological activity in mice in vivo and in vitro. Administration of wild-type TSK protein or Wnt antagonists, but not of hydrocephalus-related TSK variants, in the LV of TSK knockout mice prevented hydrocephalus and preserved SVZ neurogenesis. These observations suggest that TSK plays a crucial role as a niche molecule modulating the fate of SVZ NSCs and point to TSK as a candidate for the diagnosis and therapy of hydrocephalus.

Identifying cellular signalling molecules in developmental disorders of the brain: Evidence from focal cortical dysplasia and tuberous sclerosis.

AIMS: We understand little of the pathogenesis of developmental cortical lesions, because we understand little of the diversity of the cell types that contribute to the diseases or how those cells interact. We tested the hypothesis that cellular diversity and cell-cell interactions play an important role in these disorders by investigating the signalling molecules in the commonest cortical malformations that lead to childhood epilepsy, focal cortical dysplasia (FCD) and tuberous sclerosis (TS). METHODS: Transcriptional profiling clustered cases into molecularly distinct groups. Using gene expression data, we identified the secretory signalling molecules in FCD/TS and characterised the cell types expressing these molecules. We developed a functional model using organotypic cultures. RESULTS: We identified 113 up-regulated secretory molecules in FCDIIB/TS. The top 12 differentially expressed genes (DEGs) were validated by immunohistochemistry. This highlighted two molecules, Chitinase 3-like protein 1 (CHI3L1) and C-C motif chemokine ligand 2 (CCL2) (MCP1) that were expressed in a unique population of small cells in close proximity to balloon cells (BC). We then characterised these cells and developed a functional model in organotypic slice cultures. We found that the number of CHI3L1 and CCL2 expressing cells decreased following inhibition of mTOR, the main aberrant signalling pathway in TS and FCD. CONCLUSIONS: Our findings highlight previously uncharacterised small cell populations in FCD and TS which express specific signalling molecules. These findings indicate a new level of diversity and cellular interactions in cortical malformations and provide a generalisable approach to understanding cell-cell interactions and cellular heterogeneity in developmental neuropathology.

Generation of an iPSC line (CRICKi001-A) from an individual with a germline SMARCA4 missense mutation and autism spectrum disorder.

Germline missense mutations in the BAF swi/snf chromatin remodeling subunit SMARCA4 are associated with neurodevelopmental disorders, including Coffin Siris Syndrome (CSS). Here, we generated an induced pluripotent stem cell line from a male patient with atypical CSS features and a de novo heterozygous missense mutation in the SMARCA4 gene (c.3607C>T, p.(Arg1203Cys)). Hair root derived keratinocytes were reprogrammed using non-integrative Sendai virus vector delivery of pluripotency factors. iPSCs generated display normal morphology and molecular karyotype, express pluripotency markers and are able to differentiate into the three germ layers.

Coordinated changes in cellular behavior ensure the lifelong maintenance of the hippocampal stem cell population.

Neural stem cell numbers fall rapidly in the hippocampus of juvenile mice but stabilize during adulthood, ensuring lifelong hippocampal neurogenesis. We show that this stabilization of stem cell numbers in young adults is the result of coordinated changes in stem cell behavior. Although proliferating neural stem cells in juveniles differentiate rapidly, they increasingly return to a resting state of shallow quiescence and progress through additional self-renewing divisions in adulthood. Single-cell transcriptomics, modeling, and label retention analyses indicate that resting cells have a higher activation rate and greater contribution to neurogenesis than dormant cells, which have not left quiescence. These changes in stem cell behavior result from a progressive reduction in expression of the pro-activation protein ASCL1 because of increased post-translational degradation. These cellular mechanisms help reconcile current contradictory models of hippocampal neural stem cell (NSC) dynamics and may contribute to the different rates of decline of hippocampal neurogenesis in mammalian species, including humans.

Long-term self-renewing stem cells in the adult mouse hippocampus identified by intravital imaging.

Neural stem cells (NSCs) generate neurons throughout life in the mammalian hippocampus. However, the potential for long-term self-renewal of individual NSCs within the adult brain remains unclear. We used two-photon microscopy and followed NSCs that were genetically labeled through conditional recombination driven by the regulatory elements of the stem cell-expressed genes GLI family zinc finger 1 (Gli1) or achaete-scute homolog 1 (Ascl1). Through intravital imaging of NSCs and their progeny, we identify a population of Gli1-targeted NSCs showing long-term self-renewal in the adult hippocampus. In contrast, once activated, Ascl1-targeted NSCs undergo limited proliferative activity before they become exhausted. Using single-cell RNA sequencing, we show that Gli1- and Ascl1-targeted cells have highly similar yet distinct transcriptional profiles, supporting the existence of heterogeneous NSC populations with diverse behavioral properties. Thus, we here identify long-term self-renewing NSCs that contribute to the generation of new neurons in the adult hippocampus.

Canonical Notch signaling controls the early thymic epithelial progenitor cell state and emergence of the medullary epithelial lineage in fetal thymus development.

Thymus function depends on the epithelial compartment of the thymic stroma. Cortical thymic epithelial cells (cTECs) regulate T cell lineage commitment and positive selection, while medullary (m) TECs impose central tolerance on the T cell repertoire. During thymus organogenesis, these functionally distinct sub-lineages are thought to arise from a common thymic epithelial progenitor cell (TEPC). However, the mechanisms controlling cTEC and mTEC production from the common TEPC are not understood. Here, we show that emergence of the earliest mTEC lineage-restricted progenitors requires active NOTCH signaling in progenitor TEC and that, once specified, further mTEC development is NOTCH independent. In addition, we demonstrate that persistent NOTCH activity favors maintenance of undifferentiated TEPCs at the expense of cTEC differentiation. Finally, we uncover a cross-regulatory relationship between NOTCH and FOXN1, a master regulator of TEC differentiation. These data establish NOTCH as a potent regulator of TEPC and mTEC fate during fetal thymus development, and are thus of high relevance to strategies aimed at generating/regenerating functional thymic tissue in vitro and in vivo.

In Utero Electroporation to Study Mouse Brain Development.

In utero electroporation is a rapid and powerful technique to study the development of many brain regions. This approach presents several advantages over other methods to study specific steps of brain development in vivo, from proliferation to synaptic integration. Here, we describe in detail the individual steps necessary to carry out the technique. We also highlight the variations that can be implemented to target different cerebral structures and to study specific steps of development.

Quiescence of Adult Mammalian Neural Stem Cells: A Highly Regulated Rest.

Neural stem cells in the adult mammalian brain are the source of new neurons that contribute to complex sensory and cognitive functions. Most adult neural stem cells are maintained in a state of reversible cell cycle arrest, also called quiescence. Quiescent neural stem cells present a low rate of metabolic activity and a high sensitivity to their local signaling environment, and they can be activated by diverse physiological stimuli. The balance between stem cell quiescence and activity determines not only the rate of neurogenesis but also the long-term maintenance of the stem cell pool and the neurogenic capacity of the aging brain. In recent years, significant progress has been made in characterizing quiescent stem cells thanks to the introduction of new genomic and imaging techniques. We discuss in this review our current understanding of neural stem cell quiescence and its regulation by intrinsic and systemic factors.

Id4 promotes the elimination of the pro-activation factor Ascl1 to maintain quiescence of adult hippocampal stem cells.

Quiescence is essential for the long-term maintenance of adult stem cells but how stem cells maintain quiescence is poorly understood. Here, we show that neural stem cells (NSCs) in the adult mouse hippocampus actively transcribe the pro-activation factor Ascl1 regardless of their activated or quiescent states. We found that the inhibitor of DNA binding protein Id4 is enriched in quiescent NSCs and that elimination of Id4 results in abnormal accumulation of Ascl1 protein and premature stem cell activation. Accordingly, Id4 and other Id proteins promote elimination of Ascl1 protein in NSC cultures. Id4 sequesters Ascl1 heterodimerization partner E47, promoting Ascl1 protein degradation and stem cell quiescence. Our results highlight the importance of non-transcriptional mechanisms for the maintenance of NSC quiescence and reveal a role for Id4 as a quiescence-inducing factor, in contrast with its role of promoting the proliferation of embryonic neural progenitors.

Cortical Neurogenesis Requires Bcl6-Mediated Transcriptional Repression of Multiple Self-Renewal-Promoting Extrinsic Pathways.

During neurogenesis, progenitors switch from self-renewal to differentiation through the interplay of intrinsic and extrinsic cues, but how these are integrated remains poorly understood. Here, we combine whole-genome transcriptional and epigenetic analyses with in vivo functional studies to demonstrate that Bcl6, a transcriptional repressor previously reported to promote cortical neurogenesis, acts as a driver of the neurogenic transition through direct silencing of a selective repertoire of genes belonging to multiple extrinsic pathways promoting self-renewal, most strikingly the Wnt pathway. At the molecular level, Bcl6 represses its targets through Sirt1 recruitment followed by histone deacetylation. Our data identify a molecular logic by which a single cell-intrinsic factor represses multiple extrinsic pathways that favor self-renewal, thereby ensuring robustness of neuronal fate transition.