Repression of developmental transcription factor networks triggers aging-associated gene expression in human glial progenitor cells.

Human glial progenitor cells (hGPCs) exhibit diminished expansion competence with age, as well as after recurrent demyelination. Using RNA-sequencing to compare the gene expression of fetal and adult hGPCs, we identify age-related changes in transcription consistent with the repression of genes enabling mitotic expansion, concurrent with the onset of aging-associated transcriptional programs. Adult hGPCs develop a repressive transcription factor network centered on MYC, and regulated by ZNF274, MAX, IKZF3, and E2F6. Individual over-expression of these factors in iPSC-derived hGPCs lead to a loss of proliferative gene expression and an induction of mitotic senescence, replicating the transcriptional changes incurred during glial aging. miRNA profiling identifies the appearance of an adult-selective miRNA signature, imposing further constraints on the expansion competence of aged GPCs. hGPC aging is thus associated with acquisition of a MYC-repressive environment, suggesting that suppression of these repressors of glial expansion may permit the rejuvenation of aged hGPCs.

A conserved molecular logic for neurogenesis to gliogenesis switch in the cerebral cortex.

During development, neural stem cells in the cerebral cortex, also known as radial glial cells (RGCs), generate excitatory neurons, followed by production of cortical macroglia and inhibitory neurons that migrate to the olfactory bulb (OB). Understanding the mechanisms for this lineage switch is fundamental for unraveling how proper numbers of diverse neuronal and glial cell types are controlled. We and others recently showed that Sonic Hedgehog (Shh) signaling promotes the cortical RGC lineage switch to generate cortical oligodendrocytes and OB interneurons. During this process, cortical RGCs generate intermediate progenitor cells that express critical gliogenesis genes Ascl1, Egfr, and Olig2. The increased Ascl1 expression and appearance of Egfr+ and Olig2+ cortical progenitors are concurrent with the switch from excitatory neurogenesis to gliogenesis and OB interneuron neurogenesis in the cortex. While Shh signaling promotes Olig2 expression in the developing spinal cord, the exact mechanism for this transcriptional regulation is not known. Furthermore, the transcriptional regulation of Olig2 and Egfr has not been explored. Here, we show that in cortical progenitor cells, multiple regulatory programs, including Pax6 and Gli3, prevent precocious expression of Olig2, a gene essential for production of cortical oligodendrocytes and astrocytes. We identify multiple enhancers that control Olig2 expression in cortical progenitors and show that the mechanisms for regulating Olig2 expression are conserved between the mouse and human. Our study reveals evolutionarily conserved regulatory logic controlling the lineage switch of cortical neural stem cells.

Melatonin Enhances Neural Differentiation of Adipose-Derived Mesenchymal Stem Cells.

Adipose-derived mesenchymal stem cells (ASCs) are adult multipotent stem cells, able to differentiate toward neural elements other than cells of mesodermal lineage. The aim of this research was to test ASC neural differentiation using melatonin combined with conditioned media (CM) from glial cells. Isolated from the lipoaspirate of healthy donors, ASCs were expanded in a basal growth medium before undergoing neural differentiation procedures. For this purpose, CM obtained from olfactory ensheathing cells and from Schwann cells were used. In some samples, 1 µM of melatonin was added. After 1 and 7 days of culture, cells were studied using immunocytochemistry and flow cytometry to evaluate neural marker expression (Nestin, MAP2, Synapsin I, GFAP) under different conditions. The results confirmed that a successful neural differentiation was achieved by glial CM, whereas the addition of melatonin alone did not induce appreciable changes. When melatonin was combined with CM, ASC neural differentiation was enhanced, as demonstrated by a further improvement of neuronal marker expression, whereas glial differentiation was attenuated. A dynamic modulation was also observed, testing the expression of melatonin receptors. In conclusion, our data suggest that melatonin's neurogenic differentiation ability can be usefully exploited to obtain neuronal-like differentiated ASCs for potential therapeutic strategies.

scRNA-seq data from the larval Drosophila ventral cord provides a resource for studying motor systems function and development.

The Drosophila larval ventral nerve cord (VNC) shares many similarities with the spinal cord of vertebrates and has emerged as a major model for understanding the development and function of motor systems. Here, we use high-quality scRNA-seq, validated by anatomical identification, to create a comprehensive census of larval VNC cell types. We show that the neural lineages that comprise the adult VNC are already defined, but quiescent, at the larval stage. Using fluorescence-activated cell sorting (FACS)-enriched populations, we separate all motor neuron bundles and link individual neuron clusters to morphologically characterized known subtypes. We discovered a glutamate receptor subunit required for basal neurotransmission and homeostasis at the larval neuromuscular junction. We describe larval glia and endorse the general view that glia perform consistent activities throughout development. This census represents an extensive resource and a powerful platform for future discoveries of cellular and molecular mechanisms in repair, regeneration, plasticity, homeostasis, and behavioral coordination.

Generation of human cerebral organoids with a structured outer subventricular zone.

Outer radial glia (oRG) emerge as cortical progenitor cells that support the development of an enlarged outer subventricular zone (oSVZ) and the expansion of the neocortex. The in vitro generation of oRG is essential to investigate the underlying mechanisms of human neocortical development and expansion. By activating the STAT3 signaling pathway using leukemia inhibitory factor (LIF), which is not expressed in guided cortical organoids, we define a cortical organoid differentiation method from human pluripotent stem cells (hPSCs) that recapitulates the expansion of a progenitor pool into the oSVZ. The oSVZ comprises progenitor cells expressing specific oRG markers such as GFAP, LIFR, and HOPX, closely matching human fetal oRG. Finally, incorporating neural crest-derived LIF-producing cortical pericytes into cortical organoids recapitulates the effects of LIF treatment. These data indicate that increasing the cellular complexity of the organoid microenvironment promotes the emergence of oRG and supports a platform to study oRG in hPSC-derived brain organoids routinely.

A model of human neural networks reveals NPTX2 pathology in ALS and FTLD.

Human cellular models of neurodegeneration require reproducibility and longevity, which is necessary for simulating age-dependent diseases. Such systems are particularly needed for TDP-43 proteinopathies1, which involve human-specific mechanisms2-5 that cannot be directly studied in animal models. Here, to explore the emergence and consequences of TDP-43 pathologies, we generated induced pluripotent stem cell-derived, colony morphology neural stem cells (iCoMoNSCs) via manual selection of neural precursors6. Single-cell transcriptomics and comparison to independent neural stem cells7 showed that iCoMoNSCs are uniquely homogenous and self-renewing. Differentiated iCoMoNSCs formed a self-organized multicellular system consisting of synaptically connected and electrophysiologically active neurons, which matured into long-lived functional networks (which we designate iNets). Neuronal and glial maturation in iNets was similar to that of cortical organoids8. Overexpression of wild-type TDP-43 in a minority of neurons within iNets led to progressive fragmentation and aggregation of the protein, resulting in a partial loss of function and neurotoxicity. Single-cell transcriptomics revealed a novel set of misregulated RNA targets in TDP-43-overexpressing neurons and in patients with TDP-43 proteinopathies exhibiting a loss of nuclear TDP-43. The strongest misregulated target encoded the synaptic protein NPTX2, the levels of which are controlled by TDP-43 binding on its 3' untranslated region. When NPTX2 was overexpressed in iNets, it exhibited neurotoxicity, whereas correcting NPTX2 misregulation partially rescued neurons from TDP-43-induced neurodegeneration. Notably, NPTX2 was consistently misaccumulated in neurons from patients with amyotrophic lateral sclerosis and frontotemporal lobar degeneration with TDP-43 pathology. Our work directly links TDP-43 misregulation and NPTX2 accumulation, thereby revealing a TDP-43-dependent pathway of neurotoxicity.

Nestin Regulates Müller Glia Proliferation After Retinal Injury.

Purpose: The proliferative and neurogenic potential of retinal Müller glia after injury varies widely across species. To identify the endogenous mechanisms regulating the proliferative response of mammalian Müller glia, we comparatively analyzed the expression and function of nestin, an intermediate filament protein established as a neural stem cell marker, in the mouse and rat retinas after injury. Methods: Nestin expression in the retinas of C57BL/6 mice and Wistar rats after methyl methanesulfonate (MMS)-induced photoreceptor injury was examined by immunofluorescence and Western blotting. Adeno-associated virus (AAV)-delivered control and nestin short hairpin RNA (shRNA) were intravitreally injected to rats and Müller glia proliferation after MMS-induced injury was analyzed by BrdU incorporation and immunofluorescence. Photoreceptor removal and microglia/macrophage infiltration were also analyzed by immunofluorescence. Results: Rat Müller glia re-entered the cell cycle and robustly upregulated nestin after injury whereas Müller glia proliferation and nestin upregulation were not observed in mice. In vivo knockdown of nestin in the rat retinas inhibited Müller glia proliferation while transiently stimulating microglia/macrophage infiltration and phagocytic removal of dead photoreceptors. Conclusions: Our findings suggest a critical role for nestin in the regulation of Müller glia proliferation after retinal injury and highlight the importance of cross species analysis to identify the molecular mechanisms regulating the injury responses of the mammalian retina.

Spatially resolved multiomics of human cardiac niches.

The function of a cell is defined by its intrinsic characteristics and its niche: the tissue microenvironment in which it dwells. Here we combine single-cell and spatial transcriptomics data to discover cellular niches within eight regions of the human heart. We map cells to microanatomical locations and integrate knowledge-based and unsupervised structural annotations. We also profile the cells of the human cardiac conduction system1. The results revealed their distinctive repertoire of ion channels, G-protein-coupled receptors (GPCRs) and regulatory networks, and implicated FOXP2 in the pacemaker phenotype. We show that the sinoatrial node is compartmentalized, with a core of pacemaker cells, fibroblasts and glial cells supporting glutamatergic signalling. Using a custom CellPhoneDB.org module, we identify trans-synaptic pacemaker cell interactions with glia. We introduce a druggable target prediction tool, drug2cell, which leverages single-cell profiles and drug-target interactions to provide mechanistic insights into the chronotropic effects of drugs, including GLP-1 analogues. In the epicardium, we show enrichment of both IgG+ and IgA+ plasma cells forming immune niches that may contribute to infection defence. Overall, we provide new clarity to cardiac electro-anatomy and immunology, and our suite of computational approaches can be applied to other tissues and organs.

Role of Lipids in Regulation of Neuroglial Interactions.

Lipids comprise an extremely heterogeneous group of compounds that perform a wide variety of biological functions. Traditional view of lipids as important structural components of the cell and compounds playing a trophic role is currently being supplemented by information on the possible participation of lipids in signaling, not only intracellular, but also intercellular. The review article discusses current data on the role of lipids and their metabolites formed in glial cells (astrocytes, oligodendrocytes, microglia) in communication of these cells with neurons. In addition to metabolic transformations of lipids in each type of glial cells, special attention is paid to the lipid signal molecules (phosphatidic acid, arachidonic acid and its metabolites, cholesterol, etc.) and the possibility of their participation in realization of synaptic plasticity, as well as in other possible mechanisms associated with neuroplasticity. All these new data can significantly expand our knowledge about the regulatory functions of lipids in neuroglial relationships.

Isolation and Differentiation of Neurons and Glial Cells from Olfactory Epithelium in Living Subjects.

The study of psychiatric and neurological diseases requires the substrate in which the disorders occur, that is, the nervous tissue. Currently, several types of human bio-specimens are being used for research, including postmortem brains, cerebrospinal fluid, induced pluripotent stem (iPS) cells, and induced neuronal (iN) cells. However, these samples are far from providing a useful predictive, diagnostic, or prognostic biomarker. The olfactory epithelium is a region close to the brain that has received increased interest as a research tool for the study of brain mechanisms in complex neuropsychiatric and neurological diseases. The olfactory sensory neurons are replaced by neurogenesis throughout adult life from stem cells on the basement membrane. These stem cells are multipotent and can be propagated in neurospheres, proliferated in vitro and differentiated into multiple cell types including neurons and glia. For all these reasons, olfactory epithelium provides a unique resource for investigating neuronal molecular markers of neuropsychiatric and neurological diseases. Here, we describe the isolation and culture of human differentiated neurons and glial cells from olfactory epithelium of living subjects by an easy and non-invasive exfoliation method that may serve as a useful tool for the research in brain diseases.

Single-cell multiome sequencing clarifies enteric glial diversity and identifies an intraganglionic population poised for neurogenesis.

The enteric nervous system (ENS) consists of glial cells (EGCs) and neurons derived from neural crest precursors. EGCs retain capacity for large-scale neurogenesis in culture, and in vivo lineage tracing has identified neurons derived from glial cells in response to inflammation. We thus hypothesize that EGCs possess a chromatin structure poised for neurogenesis. We use single-cell multiome sequencing to simultaneously assess transcription and chromatin accessibility in EGCs undergoing spontaneous neurogenesis in culture, as well as small intestine myenteric plexus EGCs. Cultured EGCs maintain open chromatin at genomic loci accessible in neurons, and neurogenesis from EGCs involves dynamic chromatin rearrangements with a net decrease in accessible chromatin. A subset of in vivo EGCs, highly enriched within the myenteric ganglia and that persist into adulthood, have a gene expression program and chromatin state consistent with neurogenic potential. These results clarify the mechanisms underlying EGC potential for neuronal fate transition.

A Timm-Nissl multiplane microscopic atlas of rat brain zincergic terminal fields and metal-containing glia.

The ability of Timm's sulphide silver method to stain zincergic terminal fields has made it a useful neuromorphological marker. Beyond its roles in zinc-signalling and neuromodulation, zinc is involved in the pathophysiology of ischemic stroke, epilepsy, degenerative diseases and neuropsychiatric conditions. In addition to visualising zincergic terminal fields, the method also labels transition metals in neuronal perikarya and glial cells. To provide a benchmark reference for planning and interpretation of experimental investigations of zinc-related phenomena in rat brains, we have established a comprehensive repository of serial microscopic images from a historical collection of coronally, horizontally and sagittally oriented rat brain sections stained with Timm's method. Adjacent Nissl-stained sections showing cytoarchitecture, and customised atlas overlays from a three-dimensional rat brain reference atlas registered to each section image are included for spatial reference and guiding identification of anatomical boundaries. The Timm-Nissl atlas, available from EBRAINS, enables experimental researchers to navigate normal rat brain material in three planes and investigate the spatial distribution and density of zincergic terminal fields across the entire brain.

Glial cells expressing visual cycle genes are vital for photoreceptor survival in the zebrafish pineal gland.

Photoreceptors in the vertebrate eye are dependent on the retinal pigmented epithelium for a variety of functions including retinal re-isomerization and waste disposal. The light-sensitive pineal gland of fish, birds, and amphibians is evolutionarily related to the eye but lacks a pigmented epithelium. Thus, it is unclear how these functions are performed. Here, we ask whether a subpopulation of zebrafish pineal cells, which express glial markers and visual cycle genes, is involved in maintaining photoreceptors. Selective ablation of these cells leads to a loss of pineal photoreceptors. Moreover, these cells internalize exorhodopsin that is secreted by pineal rod-like photoreceptors, and in turn release CD63-positive extracellular vesicles (EVs) that are taken up by pdgfrb-positive phagocytic cells in the forebrain meninges. These results identify a subpopulation of glial cells that is critical for pineal photoreceptor survival and indicate the existence of cells in the forebrain meninges that receive EVs released by these pineal cells and potentially function in waste disposal.

Heparin Protects Human Neural Progenitor Cells from Zika Virus-Induced Cell Death While Preserving Their Differentiation into Mature Neuroglial Cells.

Zika virus (ZIKV) is an arbovirus member of the Flaviviridae family that causes severe congenital brain anomalies in infected fetuses. The key target cells of ZIKV infection, human neural progenitor cells (hNPCs), are highly permissive to infection that causes the inhibition of cell proliferation and induces cell death. We have previously shown that pharmaceutical-grade heparin inhibits virus-induced cell death with negligible effects on in vitro virus replication in ZIKV-infected hNPCs at the "high" multiplicity of infection (MOI) of 1. Here, we show that heparin inhibits formation of ZIKV-induced intracellular vacuoles, a signature of paraptosis, and inhibits necrosis and apoptosis of hNPCs grown as neurospheres (NS). To test whether heparin preserved the differentiation of ZIKV-infected hNPCs into neuroglial cells, hNPCs were infected at the MOI of 0.001. In this experimental condition, heparin inhibited ZIKV replication by ca. 2 log10, mostly interfering with virion attachment, while maintaining its protective effect against ZIKV-induced cytopathicity. Heparin preserved differentiation into neuroglial cells of hNPCs that were obtained from either human-induced pluripotent stem cells (hiPSC) or by fetal tissue. Quite surprisingly, multiple additions of heparin to hNPCs enabled prolonged virus replication while preventing virus-induced cytopathicity. Collectively, these results highlight the potential neuroprotective effect of heparin that could serve as a lead compound to develop novel agents for preventing the damage of ZIKV infection on the developing brain. IMPORTANCE ZIKV is a neurotropic virus that invades neural progenitor cells (NPCs), causing inhibition of their proliferation and maturation into neurons and glial cells. We have shown previously that heparin, an anticoagulant also used widely during pregnancy, prevents ZIKV-induced cell death with negligible inhibition of virus replication. Here, we demonstrate that heparin also exerts antiviral activity against ZIKV replication using a much lower infectious inoculum. Moreover, heparin interferes with different modalities of virus-induced cell death. Finally, heparin-induced prevention of virus-induced NPC death allows their differentiation into neuroglial cells despite the intracellular accumulation of virions. These results highlight the potential use of heparin, or pharmacological agents derived from it, in pregnant women to prevent the devastating effects of ZIKV infection on the developing brain of their fetuses.

Murine glial progenitor cells transplantation and synthetic PreImplantation Factor (sPIF) reduces inflammation and early motor impairment in ALS mice.

Amyotrophic lateral sclerosis (ALS) is a progressive motor neuronal disorder characterized by neuronal degeneration and currently no effective cure is available to stop or delay the disease from progression. Transplantation of murine glial-restricted precursors (mGRPs) is an attractive strategy to modulate ALS development and advancements such as the use of immune modulators could potentially extend graft survival and function. Using a well-established ALS transgenic mouse model (SOD1G93A), we tested mGRPs in combination with the immune modulators synthetic PreImplantation Factor (sPIF), Tacrolimus (Tac), and Costimulatory Blockade (CB). We report that transplantation of mGRPs into the cisterna magna did not result in increased mice survival. The addition of immunomodulatory regimes again did not increase mice lifespan but improved motor functions and sPIF was superior compared to other immune modulators. Immune modulators did not affect mGRPs engraftment significantly but reduced pro-inflammatory cytokine production. Finally, sPIF and CB reduced the number of microglial cells and prevented neuronal number loss. Given the safety profile and a neuroprotective potential of sPIF, we envision its clinical application in near future.

Neuronal-glial communication perturbations in murine SOD1G93A spinal cord.

Amyotrophic lateral sclerosis (ALS) is an incurable disease characterized by proteinaceous aggregate accumulation and neuroinflammation culminating in rapidly progressive lower and upper motor neuron death. To interrogate cell-intrinsic and inter-cell type perturbations in ALS, single-nucleus RNA sequencing was performed on the lumbar spinal cord in the murine ALS model SOD1G93A transgenic and littermate control mice at peri-symptomatic onset stage of disease, age 90 days. This work uncovered perturbed tripartite synapse functions, complement activation and metabolic stress in the affected spinal cord; processes evidenced by cell death and proteolytic stress-associated gene sets. Concomitantly, these pro-damage events in the spinal cord co-existed with dysregulated reparative mechanisms. This work provides a resource of cell-specific niches in the ALS spinal cord and asserts that interwoven dysfunctional neuronal-glial communications mediating neurodegeneration are underway prior to overt disease manifestation and are recapitulated, in part, in the human post-mortem ALS spinal cord.

Reprogramming cellular identity in vivo.

Cellular identity is established through complex layers of genetic regulation, forged over a developmental lifetime. An expanding molecular toolbox is allowing us to manipulate these gene regulatory networks in specific cell types in vivo. In principle, if we found the right molecular tricks, we could rewrite cell identity and harness the rich repertoire of possible cellular functions and attributes. Recent work suggests that this rewriting of cell identity is not only possible, but that newly induced cells can mitigate disease phenotypes in animal models of major human diseases. So, is the sky the limit, or do we need to keep our feet on the ground? This Spotlight synthesises key concepts emerging from recent efforts to reprogramme cellular identity in vivo. We provide our perspectives on recent controversies in the field of glia-to-neuron reprogramming and identify important gaps in our understanding that present barriers to progress.

Regulation of stem cell identity by miR-200a during spinal cord regeneration.

Axolotls are an important model organism for multiple types of regeneration, including functional spinal cord regeneration. Remarkably, axolotls can repair their spinal cord after a small lesion injury and can also regenerate their entire tail following amputation. Several classical signaling pathways that are used during development are reactivated during regeneration, but how this is regulated remains a mystery. We have previously identified miR-200a as a key factor that promotes successful spinal cord regeneration. Here, using RNA-seq analysis, we discovered that the inhibition of miR-200a results in an upregulation of the classical mesodermal marker brachyury in spinal cord cells after injury. However, these cells still express the neural stem cell marker sox2. In vivo cell tracking allowed us to determine that these cells can give rise to cells of both the neural and mesoderm lineage. Additionally, we found that miR-200a can directly regulate brachyury via a seed sequence in the 3'UTR of the gene. Our data indicate that miR-200a represses mesodermal cell fate after a small lesion injury in the spinal cord when only glial cells and neurons need to be replaced.

Androgens increase excitatory neurogenic potential in human brain organoids.

The biological basis of male-female brain differences has been difficult to elucidate in humans. The most notable morphological difference is size, with male individuals having on average a larger brain than female individuals1,2, but a mechanistic understanding of how this difference arises remains unknown. Here we use brain organoids3 to show that although sex chromosomal complement has no observable effect on neurogenesis, sex steroids-namely androgens-lead to increased proliferation of cortical progenitors and an increased neurogenic pool. Transcriptomic analysis and functional studies demonstrate downstream effects on histone deacetylase activity and the mTOR pathway. Finally, we show that androgens specifically increase the neurogenic output of excitatory neuronal progenitors, whereas inhibitory neuronal progenitors are not increased. These findings reveal a role for androgens in regulating the number of excitatory neurons and represent a step towards understanding the origin of sex-related brain differences in humans.

Reduced mitochondria membrane potential and lysosomal acidification are associated with decreased oligomeric Aß degradation induced by hyperglycemia: A study of mixed glia cultures.

Diabetes is a risk factor for Alzheimer's disease (AD), a chronic neurodegenerative disease. We and others have shown prediabetes, including hyperglycemia and obesity induced by high fat and high sucrose diets, is associated with exacerbated amyloid beta (Aß) accumulation and cognitive impairment in AD transgenic mice. However, whether hyperglycemia reduce glial clearance of oligomeric amyloid-ß (oAß), the most neurotoxic Aß aggregate, remains unclear. Mixed glial cultures simulating the coexistence of astrocytes and microglia in the neural microenvironment were established to investigate glial clearance of oAß under normoglycemia and chronic hyperglycemia. Ramified microglia and low IL-1ß release were observed in mixed glia cultures. In contrast, amoeboid-like microglia and higher IL-1ß release were observed in primary microglia cultures. APPswe/PS1dE9 transgenic mice are a commonly used AD mouse model. Microglia close to senile plaques in APPswe/PS1dE9 transgenic mice exposed to normoglycemia or chronic hyperglycemia exhibited an amoeboid-like morphology; other microglia were ramified. Therefore, mixed glia cultures reproduce the in vivo ramified microglial morphology. To investigate the impact of sustained high-glucose conditions on glial oAß clearance, mixed glia were cultured in media containing 5.5 mM glucose (normal glucose, NG) or 25 mM glucose (high glucose, HG) for 16 days. Compared to NG, HG reduced the steady-state level of oAß puncta internalized by microglia and astrocytes and decreased oAß degradation kinetics. Furthermore, the lysosomal acidification and lysosomal hydrolysis activity of microglia and astrocytes were lower in HG with and without oAß treatment than NG. Moreover, HG reduced mitochondrial membrane potential and ATP levels in mixed glia, which can lead to reduced lysosomal function. Overall, continuous high glucose reduces microglial and astrocytic ATP production and lysosome activity which may lead to decreased glial oAß degradation. Our study reveals diabetes-induced hyperglycemia hinders glial oAß clearance and contributes to oAß accumulation in AD pathogenesis.