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Alzheimer's disease (AD) is becoming increasingly prevalent worldwide. It represents one of the greatest medical challenges as no pharmacologic treatments are available to prevent disease progression. Astrocytes play crucial functions within neuronal circuits by providing metabolic and functional support, regulating interstitial solute composition, and modulating synaptic transmission. In addition to these physiological functions, growing evidence points to an essential role of astrocytes in neurodegenerative diseases like AD. Early-stage AD is associated with hypometabolism and oxidative stress. Contrary to neurons that are vulnerable to oxidative stress, astrocytes are particularly resistant to mitochondrial dysfunction and are therefore more resilient cells. In our study, we leveraged astrocytic mitochondrial uncoupling and examined neuronal function in the 3xTg AD mouse model. We overexpressed the mitochondrial uncoupling protein 4 (UCP4), which has been shown to improve neuronal survival in vitro. We found that this treatment efficiently prevented alterations of hippocampal metabolite levels observed in AD mice, along with hippocampal atrophy and reduction of basal dendrite arborization of subicular neurons. This approach also averted aberrant neuronal excitability observed in AD subicular neurons and preserved episodic-like memory in AD mice assessed in a spatial recognition task. These findings show that targeting astrocytes and their mitochondria is an effective strategy to prevent the decline of neurons facing AD-related stress at the early stages of the disease.

Retinal astrocytes are vital for neuronal homeostasis in the retina. Together with Müller glia, they provide retinal cells with neurotrophic factors, antioxidative support, and defense mechanisms such as the formation of the blood-retinal barrier. Substantial heterogeneity of astrocyte morphology and function represents a challenge for identification of distinct subtypes which may be potential targets for therapeutic purposes. Hence, identification of novel markers of astrocyte subpopulations is highly relevant to better understand the molecular mechanisms involved in retinal development, homeostasis, and pathology. In this study, we observed that the cell cycle regulator, p16INK4a, is expressed in immature astrocytes in the mouse retina. Immunohistochemical analysis showed p16INK4a expression in the optic nerve of wild-type mice from 3 days to 3 months of age and in the nerve fiber layer of the adult mouse retina. Colocalization of p16INK4a expression and glial fibrillary acidic protein (immature/mature astrocyte marker) tends to decrease with age. However, colocalization of p16INK4a expression and vimentin (immature astrocyte marker) remains high in the optic nerve from the early postnatal period to adulthood. The observations from this study provide a valuable tool for further investigations of ocular astrocytes in the developing retina as well as in degenerative retinopathies.

Background Astrocytes play a crucial, yet not fully elucidated role in the selective motor neuron pathology in amyotrophic lateral sclerosis (ALS). Among other responsibilities, astrocytes provide important neuronal homeostatic support, however this function is highly compromised in ALS. The establishment of fully human coculture systems can be used to further study the underlying mechanisms of the dysfunctional intercellular interplay, and has the potential to provide a platform for revealing novel therapeutic entry points. Methods In this study, we characterised human induced pluripotent stem cell (hiPSC)-derived astrocytes from FUS-ALS patients, and incorporated these cells into a human motor unit microfluidics model to investigate the astrocytic effect on hiPSC-derived motor neuron network and functional neuromuscular junctions (NMJs) using immunocytochemistry and live-cell recordings. FUS-ALS cocultures were systematically compared to their CRISPR-Cas9 gene-edited isogenic control systems. Results We observed a dysregulation of astrocyte homeostasis, which resulted in a FUS-ALS-mediated increase in reactivity and secretion of inflammatory cytokines. Upon coculture with motor neurons and myotubes, we detected a cytotoxic effect on motor neuron-neurite outgrowth, NMJ formation and functionality, which was improved or fully rescued by isogenic control astrocytes. We demonstrate that ALS astrocytes have both a gain-of-toxicity and loss-of-support function involving the WNT/β-catenin pathway, ultimately contributing to the disruption of motor neuron homeostasis, intercellular networks and NMJs. Conclusions Our findings shine light on a complex, yet highly important role of astrocytes in ALS, and provides further insight in to their pathological mechanisms. Graphical Abstract

Background In response to brain injury or inflammation, astrocytes undergo hypertrophy, proliferate, and migrate to the damaged zone. These changes, collectively known as "astrogliosis", initially protect the brain; however, astrogliosis can also cause neuronal dysfunction. Additionally, these astrocytes undergo intracellular changes involving alterations in the expression and localization of many proteins, including αvβ3 integrin. Our previous reports indicate that Thy-1, a neuronal glycoprotein, binds to this integrin inducing Connexin43 (Cx43) hemichannel (HC) opening, ATP release, and astrocyte migration. Despite such insight, important links and molecular events leading to astrogliosis remain to be defined. Methods Using bioinformatics approaches, we analyzed different Gene Expression Omnibus datasets to identify changes occurring in reactive astrocytes as compared to astrocytes from the normal mouse brain. In silico analysis was validated by both qRT-PCR and immunoblotting using reactive astrocyte cultures from the normal rat brain treated with TNF and from the brain of a hSOD1G93A transgenic mouse model. We evaluated the phosphorylation of Cx43 serine residue 373 (S373) by AKT and ATP release as a functional assay for HC opening. In vivo experiments were also performed with an AKT inhibitor (AKTi). Results The bioinformatics analysis revealed that genes of the PI3K/AKT signaling pathway were among the most significantly altered in reactive astrocytes. mRNA and protein levels of PI3K, AKT, as well as Cx43, were elevated in reactive astrocytes from normal rats and from hSOD1G93A transgenic mice, as compared to controls. In vitro, reactive astrocytes stimulated with Thy-1 responded by activating AKT, which phosphorylated S373Cx43. Increased pS373Cx43 augmented the release of ATP to the extracellular medium and AKTi inhibited these Thy-1-induced responses. Furthermore, in an in vivo model of inflammation (brain damage), AKTi decreased the levels of astrocyte reactivity markers and S373Cx43 phosphorylation. Conclusions Here, we identify changes in the PI3K/AKT molecular signaling network and show how they participate in astrogliosis by regulating the HC protein Cx43. Moreover, because HC opening and ATP release are important in astrocyte reactivity, the phosphorylation of Cx43 by AKT and the associated increase in ATP release identify a potential therapeutic window of opportunity to limit the adverse effects of astrogliosis.

We describe a subset of glioblastoma, the most prevalent malignant adult brain tumour, harbouring a bias towards hypomethylation at defined differentially methylated regions. This epigenetic signature correlates with an enrichment for an astrocytic gene signature, which together with the identification of enriched predicted binding sites of transcription factors known to cause demethylation and to be involved in astrocytic/glial lineage specification, point to a shared ontogeny between these glioblastomas and astroglial progenitors. At functional level, increased invasiveness, at least in part mediated by SRPX2, and macrophage infiltration characterise this subset of glioblastoma.

Vanishing white matter (VWM) is classified as a leukodystrophy with astrocytes as primary drivers in its pathogenesis. Magnetic resonance imaging has documented the progressive thinning of cortices in long-surviving patients. Routine histopathological analyses, however, have not yet pointed to cortical involvement in VWM. Here, we provide a comprehensive analysis of the VWM cortex. We employed high-resolution-mass-spectrometry-based proteomics and immunohistochemistry to gain insight into possible molecular disease mechanisms in the cortices of VWM patients. The proteome analysis revealed 268 differentially expressed proteins in the VWM cortices compared to the controls. A majority of these proteins formed a major protein interaction network. A subsequent gene ontology analysis identified enrichment for terms such as cellular metabolism, particularly mitochondrial activity. Importantly, some of the proteins with the most prominent changes in expression were found in astrocytes, indicating cortical astrocytic involvement. Indeed, we confirmed that VWM cortical astrocytes exhibit morphological changes and are less complex in structure than control cells. Our findings also suggest that these astrocytes are immature and not reactive. Taken together, we provide insights into cortical involvement in VWM, which has to be taken into account when developing therapeutic strategies.

Tauopathies are neurodegenerative disorders involving the accumulation of tau isoforms in cell subpopulations such as astrocytes. The origins of the 3R and 4R isoforms of tau that accumulate in astrocytes remain unclear. Extracellular vesicles (EVs) were isolated from primary neurons overexpressing 1N3R or 1N4R tau or from human brain extracts (progressive supranuclear palsy or Pick disease patients or controls) and characterized (electron microscopy, nanoparticle tracking analysis (NTA), proteomics). After the isolated EVs were added to primary astrocytes or human iPSC-derived astrocytes, tau transfer and mitochondrial system function were evaluated (ELISA, immunofluorescence, MitoTracker staining). We demonstrated that neurons in which 3R or 4R tau accumulated had the capacity to transfer tau to astrocytes and that EVs were essential for the propagation of both isoforms of tau. Treatment with tau-containing EVs disrupted the astrocytic mitochondrial system, altering mitochondrial morphology, dynamics, and redox state. Although similar levels of 3R and 4R tau were transferred, 3R tau-containing EVs were significantly more damaging to astrocytes than 4R tau-containing EVs. Moreover, EVs isolated from the brain fluid of patients with different tauopathies affected mitochondrial function in astrocytes derived from human iPSCs. Our data indicate that tau pathology spreads to surrounding astrocytes via EVs-mediated transfer and modifies their function.

Brain lactate formation, intercellular exchange and utilization has been implicated in memory formation. However, the individual role of either neuronal or astroglial monocarboxylate transporters for the acquisition and consolidation of information remains incomplete. Using novel transgenic mice and a viral vector approach to decrease the expression of each transporter in a cell-specific manner within the dorsal hippocampus, we show that both neuronal MCT2 and astroglial MCT4 are required for spatial information acquisition and retention (at 24 h post-training) in distinct hippocampus-dependent tasks. Intracerebral infusion of lactate rescued spatial learning in mice with reduced levels of astroglial MCT4 but not of neuronal MCT2, suggesting that lactate transfer from astrocytes and utilization in neurons contribute to hippocampal-dependent learning. In contrast, only neuronal MCT2 was shown to be required for long-term (7 days post training) memory formation. Interestingly, reduced MCT2 expression levels in mature neurons result in a heterologous effect as it blunts hippocampal neurogenesis associated with memory consolidation. These results suggest important but distinct contributions of both neuronal MCT2 and astroglial MCT4 in learning and memory processes, going beyond a simple passive role as alternative energy substrate suppliers or in waste product disposal.

Glioblastoma (GBM) ranks among the most lethal cancers, with current therapies offering only palliation. Inter- and intrapatient heterogeneity is a hallmark of GBM, with epigenetically distinct cancer stem-like cells (CSCs) at the apex. Targeting GSCs remains a challenging task because of their unique biology, resemblance to normal neural stem/progenitor cells, and resistance to standard cytotoxic therapy. Here, we find that the chromatin regulator, JmjC domain histone H3K36me2/me1 demethylase KDM2B, is highly expressed in glioblastoma surgical specimens compared to normal brain. Targeting KDM2B function genetically or pharmacologically impaired the survival of patient-derived primary glioblastoma cells through the induction of DNA damage and apoptosis, sensitizing them to chemotherapy. KDM2B loss decreased the GSC pool, which was potentiated by coadministration of chemotherapy. Collectively, our results demonstrate KDM2B is crucial for glioblastoma maintenance, with inhibition causing loss of GSC survival, genomic stability, and chemoresistance.

Background Autoreactive Th1 and Th17 cells are believed to mediate the pathology of multiple sclerosis in the central nervous system (CNS). Their interaction with microglia and astrocytes in the CNS is crucial for the regulation of the neuroinflammation. Previously, we have shown that only Th1 but not Th17 effectors activate microglia. However, it is not clear which cells are targets of Th17 effectors in the CNS. Methods To understand the effects driven by Th17 cells in the CNS, we induced experimental autoimmune encephalomyelitis in wild-type mice and CD4+ T cell-specific integrin α4-deficient mice where trafficking of Th1 cells into the CNS was affected. We compared microglial and astrocyte response in the brain and spinal cord of these mice. We further treated astrocytes with supernatants from highly pure Th1 and Th17 cultures and assessed the messenger RNA expression of neurotrophic factors, cytokines and chemokines, using real-time PCR. Data obtained was analyzed using the Kruskal-Wallis test. Results We observed in α4-deficient mice weak microglial activation but comparable astrogliosis to that of wild-type mice in the regions of the brain populated with Th17 infiltrates, suggesting that Th17 cells target astrocytes and not microglia. In vitro, in response to supernatants from Th1 and Th17 cultures, astrocytes showed altered expression of neurotrophic factors, pro-inflammatory cytokines and chemokines. Furthermore, increased expression of chemokines in Th1- and Th17-treated astrocytes enhanced recruitment of microglia and transendothelial migration of Th17 cells in vitro. Conclusion Our results demonstrate the delicate interaction between T cell subsets and glial cells and how they communicate to mediate their effects. Effectors of Th1 act on both microglia and astrocytes whereas Th17 effectors preferentially target astrocytes to promote neuroinflammation. Electronic supplementary material The online version of this article (10.1186/s12974-017-0978-3) contains supplementary material, which is available to authorized users.

Genome-wide association studies (GWAS) have identified genetic variants associated with age-related macular degeneration (AMD), one of the leading causes of blindness in the elderly. However, it has been challenging to identify the cell types associated with AMD given the genetic complexity of the disease. Here we perform massively parallel single-cell RNA sequencing (scRNA-seq) of human retinas using two independent platforms, and report the first single-cell transcriptomic atlas of the human retina. Using a multi-resolution network-based analysis, we identify all major retinal cell types, and their corresponding gene expression signatures. Heterogeneity is observed within macroglia, suggesting that human retinal glia are more diverse than previously thought. Finally, GWAS-based enrichment analysis identifies glia, vascular cells, and cone photoreceptors to be associated with the risk of AMD. These data provide a detailed analysis of the human retina, and show how scRNA-seq can provide insight into cell types involved in complex, inflammatory genetic diseases., “Genome-wide association studies have identified variants associated with age-related macular degeneration (AMD); however, other than identifying this as a complement mediated inflammatory disease, little biology has emerged. Here, authors used novel computational tools from the Broad Institute to examine the relationship of single-cell transcriptomics and genome-wide association studies (GWAS) in the human retina and demonstrate that GWAS-associated risk alleles associated with AMD are enriched in glia and vascular cells and that human retinal glia are more diverse than previously thought

International audience; OBJECTIVE:The two related tumor necrosis factor members a proliferation-inducing ligand (APRIL) and B-cell activation factor (BAFF) are currently targeted in autoimmune diseases as B-cell regulators. In multiple sclerosis (MS), combined APRIL/BAFF blockade led to unexpected exacerbated inflammation in the central nervous system (CNS) of patients. Here, we investigate the role of the APRIL/BAFF axis in the CNS.METHODS:APRIL expression was analyzed in MS lesions by immunohistochemistry. The in vivo role of APRIL was assessed in the murine MS model, experimental autoimmune encephalitis (EAE). Functional in vitro studies were performed with human and mouse astrocytes.RESULTS:APRIL was expressed in lesions from EAE. In its absence, the disease was worst. Lesions from MS patients also showed APRIL expression upon infiltration of macrophages. Notably, all the APRIL secreted by these macrophages specifically targeted astrocytes. The upregulation of chondroitin sulfate proteoglycan, sometimes bearing chondroitin sulfate of type E sugar moieties, binding APRIL, in reactive astrocytes explained the latter selectivity. Astrocytes responded to APRIL by producing a sufficient amount of IL-10 to dampen antigen-specific T-cell proliferation and pathogenic cytokine secretion. Finally, an intraspinal delivery of recombinant APRIL before disease onset, shortly reduced EAE symptoms. Repeated intravenous injections of recombinant APRIL before and even at disease onset also had an effect.INTERPRETATION:Our data show that APRIL mediates an anti-inflammatory response from astrocytes in MS lesions. This protective activity is not shared with BAFF. ANN NEUROL 2019;85:406-420.© 2019 American Neurological Association.

© 2015 Elsevier Inc. All rights reserved. The occurrence of cognitive disturbances upon CNS inflammation or infection has been correlated with increased levels of the cytokine tumor necrosis factor a (TNFa). To date however no specific mechanism via which this cytokine could alter cognitive circuits has been demonstrated. Here we show that local increase of TNFa in the hippocampal dentate gyrus activates astrocyte TNF receptor type 1 (TNFR1) which in turn triggers an astrocyte neuron signaling cascade that results in persistent functional modification of hippocampal excitatory synapses. Astrocytic TNFR1 signaling is necessary for the hippocampal synaptic alteration and contextual learning memory impairment observed in experimental autoimmune encephalitis (EAE) an animal model of multiple sclerosis (MS). This process may contribute to the pathogenesis of cognitive disturbances in MS as well as in other CNS conditions accompanied by inflammatory states or infections.

Mitochondria vary in morphology and function in different tissues; however, little is known about their molecular diversity among cell types. Here we engineered MitoTag mice, which express a Cre recombinase-dependent green fluorescent protein targeted to the outer mitochondrial membrane, and developed an isolation approach to profile tagged mitochondria from defined cell types. We determined the mitochondrial proteome of the three major cerebellar cell types (Purkinje cells, granule cells and astrocytes) and identified hundreds of mitochondrial proteins that are differentially regulated. Thus, we provide markers of cell-type-specific mitochondria for the healthy and diseased mouse and human central nervous systems, including in amyotrophic lateral sclerosis and Alzheimer's disease. Based on proteomic predictions, we demonstrate that astrocytic mitochondria metabolize long-chain fatty acids more efficiently than neuronal mitochondria. We also characterize cell-type differences in mitochondrial calcium buffering via the mitochondrial calcium uniporter (Mcu) and identify regulator of microtubule dynamics protein 3 (Rmdn3) as a determinant of endoplasmic reticulum-mitochondria proximity in Purkinje cells. Our approach enables exploring mitochondrial diversity in many in vivo contexts.

Astrocytes clear glutamate and potassium, both of which are released into the extracellular space during neuronal activity. These processes are intimately linked with energy metabolism. Whereas astrocyte glutamate uptake causes cytosolic and mitochondrial acidification, extracellular potassium induces bicarbonate-dependent cellular alkalinization. This study aimed at quantifying the combined impact of glutamate and extracellular potassium on mitochondrial parameters of primary cultured astrocytes. Glutamate in 3 mM potassium caused a stronger acidification of mitochondria compared to cytosol. 15 mM potassium caused alkalinization that was stronger in the cytosol than in mitochondria. While the combined application of 15 mM potassium and glutamate led to a marked cytosolic alkalinization, pH only marginally increased in mitochondria. Thus, potassium and glutamate effects cannot be arithmetically summed, which also applies to their effects on mitochondrial potential and respiration. The data implies that, because of the nonlinear interaction between the effects of potassium and glutamate, astrocytic energy metabolism will be differentially regulated.

Astrocytes, the largest glial population in human and murine brains, are crucial to the regulation of synaptic connectivity. During the first three weeks of postnatal development, immature astrocytes express mGlu5 and expands several fold while undergoing a transition towards their mature phase. Although mGlu5-mediated signalling in astrocyte functions has been extensively studied in the last decades, whether this signalling is implicated in the mechanisms governing their development, as well as the effects of dysregulated astrocytic development on neurodevelopmental disorders, are still unclear. The aim of this review is to examine what is known about the mGlu5-mediated signalling in the developing astrocytes and its possible contribution to the pathophysiology of autism spectrum disorders.

International audience; Astrocytes, microglia, and tanycytes play active roles in the regulation of hypothalamic feeding circuits. These non-neuronal cells are crucial in determining the functional interactions of specific neuronal subpopulations involved in the control of metabolism. Recent advances in biology, optics, genetics, and pharmacology have resulted in the emergence of novel and highly sophisticated approaches for studying hypothalamic neuronal-glial networks. Here we summarize the progress in the field and argue that glial-neuronal interactions provide a core hub integrating food-related cues, interoceptive signals, and internal states to adapt a complex set of physiological responses operating on different timescales to finely tune behavior and metabolism according to metabolic status. This expanding knowledge helps to redefine our understanding of the physiology of food intake and energy metabolism.

The communication between the immune and central nervous system (CNS) is affected in many neurological disorders. Peripheral injections of the endotoxin lipopolysaccharide (LPS) are widely used to study this communication: an LPS challenge leads to a biphasic syndrome that starts with acute sickness and is followed by persistent brain inflammation and chronic behavioral alterations such as depressive-like symptoms. In vitro, the response to LPS treatment has been shown to involve enhanced expression of system x c - . This cystine-glutamate antiporter, with xCT as specific subunit, represents the main glial provider of extracellular glutamate in mouse hippocampus. Here we injected male xCT knockout and wildtype mice with a single intraperitoneal dose of 5 mg/kg LPS. LPS-injection increased hippocampal xCT expression but did not alter the mainly astroglial localization of the xCT protein. Peripheral and central inflammation (as defined by cytokine levels and morphological activation of microglia) as well as LPS-induced sickness and depressive-like behavior were significantly attenuated in xCT-deficient mice compared with wildtype mice. Our study is the first to demonstrate the involvement of system x c - in peripheral and central inflammation in vivo and the potential therapeutic relevance of its inhibition in brain disorders characterized by peripheral and central inflammation, such as depression.

Emerging as an important correlate of neurological dysfunction in Multiple Sclerosis (MS), extended focal and diffuse gray matter abnormalities have been found and linked to clinical manifestations such as seizures, fatigue and cognitive dysfunction. To investigate possible underlying mechanisms we analyzed the molecular alterations in histopathological normal appearing cortical gray matter (NAGM) in MS. By performing a differential gene expression analysis of NAGM of control and MS cases we identified reduced transcription of astrocyte specific genes involved in the astrocyte–neuron lactate shuttle (ANLS) and the glutamate–glutamine cycle (GGC). Additional quantitative immunohistochemical analysis demonstrating a CX43 loss in MS NAGM confirmed a crucial involvement of astrocytes and emphasizes their importance in MS pathogenesis. Concurrently, a Toll-like/IL-1β signaling expression signature was detected in MS NAGM, indicating that immune-related signaling might be responsible for the downregulation of ANLS and GGC gene expression in MS NAGM. Indeed, challenging astrocytes with immune stimuli such as IL-1β and LPS reduced their ANLS and GGC gene expression in vitro. The detected upregulation of IL1B in MS NAGM suggests inflammasome priming. For this reason, astrocyte cultures were treated with ATP and ATP/LPS as for inflammasome activation. This treatment led to a reduction of ANLS and GGC gene expression in a comparable manner. To investigate potential sources for ANLS and GGC downregulation in MS NAGM, we first performed an adjuvant-driven stimulation of the peripheral immune system in C57Bl/6 mice in vivo. This led to similar gene expression changes in spinal cord demonstrating that peripheral immune signals might be one source for astrocytic gene expression changes in the brain. IL1B upregulation in MS NAGM itself points to a possible endogenous signaling process leading to ANLS and GGC downregulation. This is supported by our findings that, among others, MS NAGM astrocytes express inflammasome components and that astrocytes are capable to release Il-1β in-vitro. Altogether, our data suggests that immune signaling of immune- and/or central nervous system origin drives alterations in astrocytic ANLS and GGC gene regulation in the MS NAGM. Such a mechanism might underlie cortical brain dysfunctions frequently encountered in MS patients.

Traumatic brain injury (TBI) is one of the major causes of death and disability in pediatrics, and results in a complex cascade of events including the disruption of the blood-brain barrier (BBB). A controlled-cortical impact on post-natal 17-day-old rats induced BBB disruption by IgG extravasation from 1 to 3 days after injury and returned to normal at day 7. In parallel, we characterized the expression of three caveolin isoforms, caveolin 1 (cav-1), caveolin 2 (cav-2) and caveolin 3 (cav-3). While cav-1 and cav-2 are expressed on endothelial cells, both cav-1 and cav-3 were found to be present on reactive astrocytes, in vivo and in vitro. Following TBI, cav-1 expression was increased in blood vessels at 1 and 7 days in the perilesional cortex. An increase of vascular cav-2 expression was observed 7 days after TBI. In contrast, astrocytic cav-3 expression decreased 3 and 7 days after TBI. Activation of endothelial nitric oxide synthase (eNOS) (via its phosphorylation) was detected 1 day after TBI and phospho-eNOS was detected both in association with blood vessels and with astrocytes. The molecular changes involving caveolins occurring in endothelial cells following juvenile-TBI might participate, independently of eNOS activation, to a mechanism of BBB repair while, they might subserve other undefined roles in astrocytes.

Background Multiple sclerosis (MS) is an autoimmune disorder characterized by chronic inflammation, demyelination, and neuronal damage. During autoimmunity, cytokines are important mediators of the inflammation. In this line, interleukin-27 (IL-27) modulates inflammation and can be produced directly at inflammatory sites such as in the joints during rheumatoid arthritis or in the central nervous system (CNS) during MS. While in animal models of MS, treatment with IL-27 decreases the disease severity, its role in humans is not clearly established and it is not known if IL-27 could be detected in the cerebrospinal fluid (CSF) of MS patients. Methods In this study, we measured IL-27 levels using a quantitative enzyme-linked immunosorbent assay in CSF of patients with relapsing remitting multiple sclerosis (RRMS), isolated optic neuritis (ON) and non-inflammatory neurological disease (NIND) as well as in the sera of healthy donors (HD) and RRMS patients undergoing different disease modifying treatments. We further confirmed by immunohistology of patient biopsies the identity of IL-27 producing cells in the brain of active MS lesions. Results We observed that IL-27 levels are increased in the CSF but not in the sera of RRMS compared to HD. We confirmed that IL-27 is expressed in active MS plaques by astrocytes of MS patients. Conclusions Our results point toward a local secretion of IL-27 in the CNS that is increased during autoimmune processes. We propose that local production of IL-27 could sign the induction of a regulatory response that promotes inflammation’s resolution. The effect of new immunomodulatory therapies on cerebral IL-27 production could be used to understand the biology of IL-27 in MS disease.

The homeodomain transcription factor Nkx2.1 (NK2 homeobox 1) controls cell differentiation of telencephalic GABAergic interneurons and oligodendrocytes. Here we show that Nkx2.1 also regulates astrogliogenesis of the telencephalon from embryonic day (E) 14.5 to E16.5. Moreover we identify the different mechanisms by which Nkx2.1 controls the telencephalic astrogliogenesis. In Nkx2.1 knockout (Nkx2.1−/−) mice a drastic loss of astrocytes is observed that is not related to cell death. Further, in vivo analysis using BrdU incorporation reveals that Nkx2.1 affects the proliferation of the ventral neural stem cells that generate early astrocytes. Also, in vitro neurosphere assays showed reduced generation of astroglia upon loss of Nkx2.1, which could be due to decreased precursor proliferation and possibly defects in glial specification/differentiation. Chromatin immunoprecipitation analysis and in vitro co-transfection studies with an Nkx2.1-expressing plasmid indicate that Nkx2.1 binds to the promoter of glial fibrillary acidic protein (GFAP), primarily expressed in astrocytes, to regulate its expression. Hence, Nkx2.1 controls astroglial production spatiotemporally in embryos by regulating proliferation of the contributing Nkx2.1-positive precursors.

Contributions of glial cells to neuroenergetics have been the focus of extensive debate. Here we provide positron emission tomography evidence that activation of astrocytic glutamate transport via the excitatory amino acid transporter GLT-1 triggers widespread but graded glucose uptake in the rodent brain. Our results highlight the need for a reevaluation of the interpretation of [(18)F]FDG positron emission tomography data, whereby astrocytes would be recognized as contributing to the [(18)F]FDG signal.

Neurodegenerative disorders are a major public health problem because of the high frequency of these diseases. Genome editing with the CRISPR/Cas9 system is making it possible to modify the sequence of genes linked to these disorders. We designed the KamiCas9 self-inactivating editing system to achieve transient expression of the Cas9 protein and high editing efficiency. In the first application, the gene responsible for Huntington’s disease (HD) was targeted in adult mouse neuronal and glial cells. Mutant huntingtin (HTT) was efficiently inactivated in mouse models of HD, leading to an improvement in key markers of the disease. Sequencing of potential off-targets with the constitutive Cas9 system in differentiated human iPSC revealed a very low incidence with only one site above background level. This off-target frequency was significantly reduced with the KamiCas9 system. These results demonstrate the potential of the self-inactivating CRISPR/Cas9 editing for applications in the context of neurodegenerative diseases.

Over the last thirty years, a growing number of studies showed that astrocytes play a pivotal role in the energy support to synapses. More precisely, astrocytes adjust energy production to neuronal energy needs through different mechanisms grouped under the term "neurometabolic coupling" (NMC). In this review we describe these mechanisms of coupling and how they involve astrocytes. From a physiological point of view, these mechanisms of coupling are particularly important to ensure normal synaptic functioning when neurons undergo rapid and repetitive changes in the firing rate such as during the sleep/wake transitions. Investigations into brain energy metabolism during the sleep/wake cycle have been mainly focused on glucose (Gluc) consumption and on glycogen metabolism. However, the recent development of substrate-specific biosensors allowed measurements of the variation in extracellular levels of glutamate, Gluc and lactate (Lac) with a time resolution compatible with sleep stage duration. Together with gene expression data these experiments allowed to better define the variations of energy metabolite regulation across the sleep/wake cycle. The aim of this review is to bring into perspective the role of astrocytes and NMC in the regulation of the sleep/wake cycle. The data reviewed also suggest an important role of the astrocytic network. In addition, the role of astrocytes in NMC mechanisms is consistent with the "local and use dependent" sleep hypothesis.

Huntington's disease (HD) is a fatal neurodegenerative disease in which an early and selective vulnerability of striatal Spiny Projection Neurons is observed. However, several studies have highlighted the implication of glial cells, and in particular astrocytes, in the pathophysiological mechanisms of this disease. A better understanding of the respective contributions of neurons and astrocytes in HD is needed and would be important for the development of new therapeutic approaches. Today, no comparable in vivo models expressing the mutant HTT selectively in astrocytes or in neurons are available. In this study, we developed comparable cell-type specific mouse models expressing a fragment of Huntingtin specifically in neurons, astrocytes, or in both cell populations of the adult mouse basal ganglia circuit. This approach allowed us to characterize behavioral alterations occurring as soon as 4 weeks postinjection. Interestingly, less severe but significant behavioral alterations were also observed in the two cell-type specific models. We further showed that astrocytes are less affected by mHTT compared to neurons, in particular concerning mHTT aggregation. Additionally, a more indirect contribution of astrocytes compared to neurons was observed in several pathophysiological mechanisms such as astrogliosis and neuronal dysfunction. Finally, we showed that direct and indirect transcriptional alterations within the glial glutamatergic clearing system are caused by astrocytic and neuronal expression of mHTT, respectively. We anticipate that our study will help to better understand the contributions of astrocytes to HD and guide future therapeutic efforts. GLIA 2016;64:1841-1856.

Vanishing white matter (VWM) is a fatal leukodystrophy that is caused by mutations in genes encoding subunits of eukaryotic translation initiation factor 2B (eIF2B). Disease onset and severity are codetermined by genotype. White matter astrocytes and oligodendrocytes are almost exclusively affected; however, the mechanisms of VWM development remain unclear. Here, we used VWM mouse models, patients' tissue, and cell cultures to investigate whether astrocytes or oligodendrocytes are the primary affected cell type. We generated 2 mouse models with mutations (Eif2b5Arg191His/Arg191His and Eif2b4Arg484Trp/Arg484Trp) that cause severe VWM in humans and then crossed these strains to develop mice with various mutation combinations. Phenotypic severity was highly variable and dependent on genotype, reproducing the clinical spectrum of human VWM. In all mutant strains, impaired maturation of white matter astrocytes preceded onset and paralleled disease severity and progression. Bergmann glia and retinal Müller cells, nonforebrain astrocytes that have not been associated with VWM, were also affected, and involvement of these cells was confirmed in VWM patients. In coculture, VWM astrocytes secreted factors that inhibited oligodendrocyte maturation, whereas WT astrocytes allowed normal maturation of VWM oligodendrocytes. These studies demonstrate that astrocytes are central in VWM pathomechanisms and constitute potential therapeutic targets. Importantly, astrocytes should also be considered in the pathophysiology of other white matter disorders.

INTRODUCTION Astrocytes translate incoming information and generate functional outputs via Ca 2+ signaling. Thereby, they respond to neuronal activity, producing downstream modulation of synaptic functions, and may participate in hemodynamics regulation. Deciphering the “Ca 2+ language” of astrocytes is therefore essential for defining their roles in brain physiology and pathology. However, the specifics of astrocytic Ca 2+ signaling are still poorly understood, and recent studies producing inconsistent or contradictory results have fostered debate on the actual role of astrocytes in synaptic and vascular functions. RATIONALE A neglected potential source of inconsistencies lies in the way astrocytic Ca 2+ signaling has been studied to date, mostly by conventional two-dimensional (2D) imaging, which assumes that sampling a single (~1 μm) focal plane is representative of the entire astrocytic cell. This is, however, dubious given that astrocytes are highly 3D cells, entertain heterogeneous 3D relations with neighboring structures, and display Ca 2+ signals on a local scale. Therefore, we developed a new method to three-dimensionally scan entire astrocytes and observe full-cell Ca 2+ dynamics. RESULTS With our 3D approach, we sampled astrocytes at a sufficient rate to detect events with durations of >1.5 s throughout the cell, and faster ones in selected substructures. We found that Ca 2+ activity in an individual astrocyte is heterogeneously scattered throughout the cell, largely compartmented within each region, and preponderantly local. The majority resides in the “gliapil,” the peripheral region composed of fine (optically subresolved) structures occupying ∼75% of the astrocyte volume. Within the central (resolvable) “core,” the soma is mostly inactive, whereas processes are frequently active yet show widely different activity between them. Even in individual processes, activity distributes heterogeneously, with alternating “hot” and “cold” spots. We performed 3D imaging in awake mice and in adult brain slices. Activity in vivo was faster and more frequent, particularly in endfeet, yet similar in properties and cellular distribution to slices, except for the presence of cell-wide “global” Ca 2+ events mainly associated with mouse movement. Contrary to current beliefs, global events were not sweeping waves, but rather consisted of multifocal Ca 2+ elevations that started at multiple gliapil loci and then spread to the core. At the vascular interface, astrocytic Ca 2+ activity was mostly restricted to individual endfeet, even to their fractions, and only occasionally coordinated with the endfoot process or the rest of the astrocyte. Two or more endfeet were mainly asynchronous, even when enwrapping the same vessel. Astrocytic structures and axons intersected three-dimensionally, and minimal axonal activity (individual action potentials) produced time-correlated astrocytic Ca 2+ elevations in small spots ( CONCLUSION We provide the first comprehensive 3D map of Ca 2+ activity in an individual astrocyte. Its widespread, heterogeneous, local, and mostly 3D nature confirms the appropriateness of our whole-cell imaging approach. Past 2D studies, often focusing on somatic Ca 2+ dynamics, inadequately described the emerging richness and complexity of the astrocyte activity, notably at astrocyte-synapse and astrocyte-vascular interfaces, where activity is small, fast, and frequent. In this context, we can foresee future challenges in extending studies to the gliapil, whose structures fall below current optical resolution, and in reporting the complete gamut of astrocyte Ca 2+ signals at the whole-cell scale, both requiring technical advances. Nonetheless, the technique demonstrated here promises to make 3D Ca 2+ imaging the state-of-the-art approach for Ca 2+ studies addressing the role of astrocytes in brain function.

Neuroinflammation is the local reaction of the brain to infection, trauma, toxic molecules or protein aggregates. The brain resident macrophages, microglia, are able to trigger an appropriate response involving secretion of cytokines and chemokines, resulting in the activation of astrocytes and recruitment of peripheral immune cells. IL-1β plays an important role in this response; yet its production and mode of action in the brain are not fully understood and its precise implication in neurodegenerative diseases needs further characterization. Our results indicate that the capacity to form a functional NLRP3 inflammasome and secretion of IL-1β is limited to the microglial compartment in the mouse brain. We were not able to observe IL-1β secretion from astrocytes, nor do they express all NLRP3 inflammasome components. Microglia were able to produce IL-1β in response to different classical inflammasome activators, such as ATP, Nigericin or Alum. Similarly, microglia secreted IL-18 and IL-1α, two other inflammasome-linked pro-inflammatory factors. Cell stimulation with α-synuclein, a neurodegenerative disease-related peptide, did not result in the release of active IL-1β by microglia, despite a weak pro-inflammatory effect. Amyloid-β peptides were able to activate the NLRP3 inflammasome in microglia and IL-1β secretion occurred in a P2X7 receptor-independent manner. Thus microglia-dependent inflammasome activation can play an important role in the brain and especially in neuroinflammatory conditions.