Search

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 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.

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.

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.

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.

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.

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.

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.

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.

During synaptic activity, the clearance of neuronally released glutamate leads to an intracellular sodium concentration increase in astrocytes that is associated with significant metabolic cost. The proximity of mitochondria at glutamate uptake sites in astrocytes raises the question of the ability of mitochondria to respond to these energy demands. We used dynamic fluorescence imaging to investigate the impact of glutamatergic transmission on mitochondria in intact astrocytes. Neuronal release of glutamate induced an intracellular acidification in astrocytes, via glutamate transporters, that spread over the mitochondrial matrix. The glutamate-induced mitochondrial matrix acidification exceeded cytosolic acidification and abrogated cytosol-to-mitochondrial matrix pH gradient. By decoupling glutamate uptake from cellular acidification, we found that glutamate induced a pH-mediated decrease in mitochondrial metabolism that surpasses the Ca2+-mediated stimulatory effects. These findings suggest a model in which excitatory neurotransmission dynamically regulates astrocyte energy metabolism by limiting the contribution of mitochondria to the metabolic response, thereby increasing the local oxygen availability and preventing excessive mitochondrial reactive oxygen species production.

Changes in intracellular Na(+) concentration underlie essential neurobiological processes, but few reliable tools exist for their measurement. Here we characterize a new synthetic Na(+)-sensitive fluorescent dye, Asante Natrium Green (ANG), with unique properties. This indicator was excitable in the visible spectrum and by two-photon illumination, suffered little photobleaching and located to the cytosol were it remained for long durations without noticeable unwanted effects on basic cell properties. When used in brain tissue, ANG yielded a bright fluorescent signal during physiological Na(+) responses both in neurons and astrocytes. Synchronous electrophysiological and fluorometric recordings showed that ANG produced accurate Na(+) measurement in situ. This new Na(+) indicator opens innovative ways of probing neuronal circuits.

The presenilin-1 gene is mutated in early-onset familial Alzheimer’s disease. The mutation Pro117Leu is implicated in a very severe form of the disease, with an onset of less than thirty years. The consequences of this mutation on neurogenesis in the hippocampus of adult transgenic mice have already been studied in situ. The survival of neural progenitor cells was impaired resulting in decreased neurogenesis in the dentate gyrus. Our intention was to verify if similar alterations could occur in vitro in progenitor cells from the murine ganglionic eminences isolated from embryos of this same transgenic mouse model. These cells were grown in culture as neurospheres and after differentiation the percentage of neurons generated as well as their morphology were analysed. The mutation results in a significant decrease in neurogenesis compared to the wild type mice and the neurons grow longer and more ramified neurites. A shift of differentiation towards gliogenesis was observed that could explain decreased neurogenesis despite increased proliferation of neural precursors in transgenic neurospheres. A diminished survival of the newly generated mutant neurons is also proposed. Our data raise the possibility that these alterations in embryonic development might contribute to increase the severity of the Alzheimer’s disease phenotype later in adulthood.

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.

Transmissible spongiform encephalopathies are characterized by spongiosis, astrocytosis and accumulation of PrPSc, an isoform of the normal host protein PrPC. The exact cell types responsible for agent propagation and pathogenesis are still uncertain. To determine the possible role of astrocytes, we generated mice devoid of murine PrP but expressing hamster PrP transgenes driven by the astrocyte-specific GFAP promoter. After inoculation with hamster scrapie, these mice accumulated infectivity and PrPSc to high levels, developed severe disease after 227 +/- 5 days and died 7 +/- 4 days later. Therefore, astrocytes could play an important role in scrapie pathogenesis, possibly by an indirect toxic effect on neurons. Interestingly, mice expressing the same transgenes but also endogenous murine PrP genes propagated infectivity without developing disease.

Inflammation is a highly regulated process with common but also specific characteristics in each tissue affected. Recruitment of leukocytes from the blood to the injured tissue is an important early step in the inflammatory cascade. This review highlights the role of connexins (Cxs) in the regulation of both acute and chronic inflammatory processes. Cxs form gap junction channels that provide a cytoplasmic continuity between adjacent cells allowing the intercellular exchange of ions and metabolites. Their structural halves form connexons or hemichannels. Each of them consists of 6 Cx proteins and hemichannels not taking part in gap junction formation but facilitating the release of small molecules such as ATP. Based on the differential distribution of various Cxs in different tissues such as the brain, lung capillaries and large blood vessels, our aim was to analyze the specific roles of Cxs in the inflammatory process in these tissues. Three typical sites of inflammation were chosen to shed light on similarities and differences in several types of responses: (1) atherosclerosis as a model for chronic inflammation, (2) the lung as an example of acute inflammation and (3) the ‘immune-privileged’ environment of the brain to highlight specific reactions of the vasculature to ischemic damage and inflammation at this site.

BACKGROUND AND PURPOSE: The chemokine receptor CCR5 is well known for its function in immune cells; however, it is also expressed in the brain, where its specific role remains to be elucidated. Because genetic factors may influence the risk of developing cerebral ischaemia or affect its clinical outcome, we have analysed the role of CCR5 in experimental stroke. EXPERIMENTAL APPROACH: Permanent cerebral ischaemia was performed by occlusion of the middle cerebral artery in wild-type and CCR5-deficient mice. Locomotor behaviour, infarct size and histochemical alterations were analysed at different time points after occlusion. KEY RESULTS: The cerebral vasculature was comparable in wild-type and CCR5-deficient mice. However, the size of the infarct and the motor deficits after occlusion were markedly increased in CCR5-deficient mice as compared with wild type. No differences between wild-type and CCR5-deficient mice were elicited by occlusion with respect to the morphology and abundance of astrocytes and microglia. Seven days after occlusion the majority of CCR5-deficient mice displayed neutrophil invasion in the infarct region, which was not observed in wild type. As compared with wild type, the infarct regions of CCR5-deficient mice were characterized by increased neuronal death. CONCLUSIONS AND IMPLICATIONS: Lack of CCR5 increased the severity of brain injury following occlusion of the middle cerebral artery. This is of particular interest with respect to the relatively frequent occurrence of CCR5 deficiency in the human population (1-2% of the Caucasian population) and the advent of CCR5 inhibitors as novel drugs.

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.

Accumulating evidence suggests that changes in the metabolic signature of astrocytes underlie their response to neuroinflammation, but how proinflammatory stimuli induce these changes is poorly understood. By monitoring astrocytes following acute cortical injury, we identified a differential and region-specific remodeling of their mitochondrial network: while astrocytes within the penumbra of the lesion undergo mitochondrial elongation, those located in the core-the area invaded by proinflammatory cells-experience transient mitochondrial fragmentation. In brain slices, proinflammatory stimuli reproduced localized changes in mitochondrial dynamics, favoring fission over fusion. This effect was triggered by Drp1 phosphorylation and ultimately resulted in reduced respiratory capacity. Furthermore, maintenance of the mitochondrial architecture critically depended on the induction of autophagy. Deletion of Atg7, required for autophagosome formation, prevented the reestablishment of tubular mitochondria, leading to marked reactive oxygen species accumulation and cell death. Thus, our data reveal autophagy to be essential for regenerating astrocyte mitochondrial networks during inflammation.

Amyloid-β (Aβ) peptides play a key role in the pathogenesis of Alzheimer's disease and exert various toxic effects on neurons; however, relatively little is known about their influence on glial cells. Astrocytes play a pivotal role in brain homeostasis, contributing to the regulation of local energy metabolism and oxidative stress defense, two aspects of importance for neuronal viability and function. In the present study, we explored the effects of Aβ peptides on glucose metabolism in cultured astrocytes. Following Aβ25-35exposure, we observed an increase in glucose uptake and its various metabolic fates, i.e., glycolysis (coupled to lactate release), tricarboxylic acid cycle, pentose phosphate pathway, and incorporation into glycogen. Aβ increased hydrogen peroxide production as well as glutathione release into the extracellular space without affecting intracellular glutathione content. A causal link between the effects of Aβ on glucose metabolism and its aggregation and internalization into astrocytes through binding to members of the class A scavenger receptor family could be demonstrated. Using astrocyte-neuron cocultures, we observed that the overall modifications of astrocyte metabolism induced by Aβ impair neuronal viability. The effects of the Aβ25-35fragment were reproduced by Aβ1-42but not by Aβ1-40. Finally, the phosphoinositide 3-kinase (PI3-kinase) pathway appears to be crucial in these events since both the changes in glucose utilization and the decrease in neuronal viability are prevented by LY294002, a PI3-kinase inhibitor. This set of observations indicates that Aβ aggregation and internalization into astrocytes profoundly alter their metabolic phenotype with deleterious consequences for neuronal viability.