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Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis 1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY + p300 + memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY + p300 + astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)

Cell-cell interactions in the central nervous system play important roles in neurologic diseases. However, little is known about the specific molecular pathways involved, and methods for their systematic identification are limited. Here, we developed a forward genetic screening platform that combines CRISPR-Cas9 perturbations, cell coculture in picoliter droplets, and microfluidic-based fluorescence-activated droplet sorting to identify mechanisms of cell-cell communication. We used SPEAC-seq (systematic perturbation of encapsulated associated cells followed by sequencing), in combination with in vivo genetic perturbations, to identify microglia-produced amphiregulin as a suppressor of disease-promoting astrocyte responses in multiple sclerosis preclinical models and clinical samples. Thus, SPEAC-seq enables the high-throughput systematic identification of cell-cell communication mechanisms.

Cell-cell interactions control the physiology and pathology of the central nervous system (CNS). To study astrocyte cell interactions in vivo, we developed rabies barcode interaction detection followed by sequencing (RABID-seq), which combines barcoded viral tracing and single-cell RNA sequencing (scRNA-seq). Using RABID-seq, we identified axon guidance molecules as candidate mediators of microglia-astrocyte interactions that promote CNS pathology in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis (MS). In vivo cell-specific genetic perturbation EAE studies, in vitro systems, and the analysis of MS scRNA-seq datasets and CNS tissue established that Sema4D and Ephrin-B3 expressed in microglia control astrocyte responses via PlexinB2 and EphB3, respectively. Furthermore, a CNS-penetrant EphB3 inhibitor suppressed astrocyte and microglia proinflammatory responses and ameliorated EAE. In summary, RABID-seq identified microglia-astrocyte interactions and candidate therapeutic targets., (Copyright © 2021 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.)

Multiple sclerosis is a chronic inflammatory disease of the CNS 1 . Astrocytes contribute to the pathogenesis of multiple sclerosis 2 , but little is known about the heterogeneity of astrocytes and its regulation. Here we report the analysis of astrocytes in multiple sclerosis and its preclinical model experimental autoimmune encephalomyelitis (EAE) by single-cell RNA sequencing in combination with cell-specific Ribotag RNA profiling, assay for transposase-accessible chromatin with sequencing (ATAC-seq), chromatin immunoprecipitation with sequencing (ChIP-seq), genome-wide analysis of DNA methylation and in vivo CRISPR-Cas9-based genetic perturbations. We identified astrocytes in EAE and multiple sclerosis that were characterized by decreased expression of NRF2 and increased expression of MAFG, which cooperates with MAT2α to promote DNA methylation and represses antioxidant and anti-inflammatory transcriptional programs. Granulocyte-macrophage colony-stimulating factor (GM-CSF) signalling in astrocytes drives the expression of MAFG and MAT2α and pro-inflammatory transcriptional modules, contributing to CNS pathology in EAE and, potentially, multiple sclerosis. Our results identify candidate therapeutic targets in multiple sclerosis.

Metabolism has been shown to control peripheral immunity, but little is known about its role in central nervous system (CNS) inflammation. Through a combination of proteomic, metabolomic, transcriptomic, and perturbation studies, we found that sphingolipid metabolism in astrocytes triggers the interaction of the C2 domain in cytosolic phospholipase A2 (cPLA2) with the CARD domain in mitochondrial antiviral signaling protein (MAVS), boosting NF-κB-driven transcriptional programs that promote CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, multiple sclerosis. cPLA2 recruitment to MAVS also disrupts MAVS-hexokinase 2 (HK2) interactions, decreasing HK enzymatic activity and the production of lactate involved in the metabolic support of neurons. Miglustat, a drug used to treat Gaucher and Niemann-Pick disease, suppresses astrocyte pathogenic activities and ameliorates EAE. Collectively, these findings define a novel immunometabolic mechanism that drives pro-inflammatory astrocyte activities, outlines a new role for MAVS in CNS inflammation, and identifies candidate targets for therapeutic intervention., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Astrocytes are increasingly recognized as active contributors to the disease process in multiple sclerosis (MS), rather than being merely reactive. We investigated the expression of a selected microRNA (miRNA) panel that could contribute both to the injury and to the recovery phases of the disease. Individual astrocytes were laser microdissected from brain sections. We then compared the miRNAs' expressions in MS and control brain samples at different lesional stages in white versus grey matter regions. In active MS lesions, we found upregulation of ischemia-related miRNAs in white but not grey matter, often with reversion to the normal state in inactive lesions. In contrast to our previous findings on MS macrophages, expression of 2 classical inflammatory-related miRNAs, miRNA-155 and miRNA-146a, was reduced in astrocytes from active and chronic active MS lesions in white and grey matter, suggesting a lesser direct pathogenetic role for these miRNAs in astrocytes. miRNAs within the categories regulating aquaporin4 (-100, -145, -320) and glutamate transport/apoptosis/neuroprotection (-124a, -181a, and -29a) showed some contrasting responses. The regional and lesion-stage differences of expression of these miRNAs indicate the remarkable ability of astrocytes to show a wide range of selective responses in the face of differing insults and phases of resolution., (© 2019 American Association of Neuropathologists, Inc. All rights reserved.)

During inflammatory processes of the central nervous system, helper T cells have the capacity to cross the blood-brain barrier and injure or kill neural cells through cytotoxic mechanisms. Glial fibrillary acidic protein (GFAP) is an intermediate filament protein that is part of the astrocyte cytoskeleton that can become fragmented in neuroinflammatory conditions. The mechanism of action by which helper T cells with cytotoxic properties injure astrocytes is not completely understood. Primary human astrocytes were obtained from fetal brain tissue. Human helper (CD4 + ) T cells were isolated from peripheral blood mononuclear cells and activated with the superantigen staphylococcal enterotoxin E (SEE). Granzyme B was detected by enzyme linked immunosorbent assay and intracellular flow cytometry. GFAP fragmentation was monitored by western blotting. Cell death was monitored by lactic acid dehydrogenase release and terminal biotin-dUTP nick labeling (TUNEL). Astrocyte migration was monitored by scratch assay. Adult human oligodendrocytes were cultured with sublethally injured astrocytes to determine support function. Helper T cells activated with SEE expressed granzyme B but not perforin. Helper T cells released granzyme B upon contact with astrocytes and caused GFAP fragmentation in a caspase-dependent, MHCII-independent manner. Sublethally injured astrocytes were not apoptotic; however, their processes were thin and elongated, their migration was attenuated, and their ability to support oligodendrocytes was reduced in vitro. Helper T cells can release granzyme B causing sublethal injury to astrocytes, which compromises the supportive functions of astrocytes. Blocking these pathways may lead to improved resolution of neuroinflammatory lesions., (© 2018 Wiley Periodicals, Inc.)

Genome-wide studies have identified genetic variants linked to neurologic diseases. Environmental factors also play important roles, but no methods are available for their comprehensive investigation. We developed an approach that combines genomic data, screens in a novel zebrafish model, computational modeling, perturbation studies, and multiple sclerosis (MS) patient samples to evaluate the effects of environmental exposure on CNS inflammation. We found that the herbicide linuron amplifies astrocyte pro-inflammatory activities by activating signaling via sigma receptor 1, inositol-requiring enzyme-1α (IRE1α), and X-box binding protein 1 (XBP1). Indeed, astrocyte-specific shRNA- and CRISPR/Cas9-driven gene inactivation combined with RNA-seq, ATAC-seq, ChIP-seq, and study of patient samples suggest that IRE1α-XBP1 signaling promotes CNS inflammation in experimental autoimmune encephalomyelitis (EAE) and, potentially, MS. In summary, these studies define environmental mechanisms that control astrocyte pathogenic activities and establish a multidisciplinary approach for the systematic investigation of the effects of environmental exposure in neurologic disorders., (Copyright © 2018 Elsevier Inc. All rights reserved.)

Background: The success of clinical trials of selective B cell depletion in patients with relapsing multiple sclerosis (MS) indicates B cells are important contributors to peripheral immune responses involved in the development of new relapses. Such B cell contribution to peripheral inflammation likely involves antibody-independent mechanisms. Of growing interest is the potential that B cells, within the MS central nervous system (CNS), may also contribute to the propagation of CNS-compartmentalized inflammation in progressive (non-relapsing) disease. B cells are known to persist in the inflamed MS CNS and are more recently described as concentrated in meningeal immune-cell aggregates, adjacent to the subpial cortical injury which has been associated with progressive disease. How B cells are fostered within the MS CNS and how they may contribute locally to the propagation of CNS-compartmentalized inflammation remain to be elucidated., Methods: We considered whether activated human astrocytes might contribute to B cell survival and function through soluble factors. B cells from healthy controls (HC) and untreated MS patients were exposed to primary human astrocytes that were either maintained under basal culture conditions (non-activated) or pre-activated with standard inflammatory signals. B cell exposure to astrocytes included direct co-culture, co-culture in transwells, or exposure to astrocyte-conditioned medium. Following the different exposures, B cell survival and expression of T cell co-stimulatory molecules were assessed by flow cytometry, as was the ability of differentially exposed B cells to induce activation of allogeneic T cells., Results: Secreted factors from both non-activated and activated human astrocytes robustly supported human B cell survival. Soluble products of pre-activated astrocytes also induced B cell upregulation of antigen-presenting cell machinery, and these B cells, in turn, were more efficient activators of T cells. Astrocyte-soluble factors could support survival and activation of B cell subsets implicated in MS, including memory B cells from patients with both relapsing and progressive forms of disease., Conclusions: Our findings point to a potential mechanism whereby activated astrocytes in the inflamed MS CNS not only promote a B cell fostering environment, but also actively support the ability of B cells to contribute to the propagation of CNS-compartmentalized inflammation, now thought to play key roles in progressive disease.

Recent experimental and clinical studies on astrocytes are unraveling the capabilities of these multi-functional cells in normal homeostasis, and in central nervous system (CNS) disease. This review focuses on understanding their behavior in all aspects of the initiation, evolution, and resolution of the multiple sclerosis (MS) lesion. Astrocytes display remarkable flexibility and variability of their physical structure and biochemical output, each aspect finely tuned to the specific stage and location of the disease, participating in both pathogenic and beneficial changes seen in acute and progressive forms. As examples, chemo-attractive or repulsive molecules may facilitate the entry of destructive immune cells but may also aid in the recruitment of oligodendrocyte precursors, essential for repair. Pro-inflammatory cytokines may attack pathogenic cells and also destroy normal oligodendrocytes, myelin, and axons. Protective trophic factors may also open the blood-brain barrier and modulate the extracellular matrix to favor recruitment and persistence of CNS-specific immune cells. A chronic glial scar may confer structural support following tissue loss and inhibit ingress of further noxious insults and also inhibit migration of reparative cells and molecules into the damaged tissue. Continual study into these processes offers the therapeutic opportunities to enhance the beneficial capabilities of these cells while limiting their destructive effects., (© The Author(s), 2016.)

Anatomic distribution and age are variables linked to functions of astrocytes under physiologic and pathologic conditions. We measured the relative expression of a panel of microRNAs (miRNAs) in astrocytes captured by laser micro-dissection from normal human adult white and grey matter, human fetal white matter and germinal matrix samples. Although expression of most miRNAs was comparable between adult and fetal samples, regional differences were observed. In the adult cerebral cortex, expression of miRNAs in morphologically distinct inter-laminar astrocytes underlying the glial limitans differed from those in deeper cortical layers, suggesting functional specialization possibly related to structural stability and defense from potentially harmful factors in the cerebrospinal fluid. Differences between adult white and grey matter miRNA expression included higher expression of pro-inflammatory miRNAs in the former, potentially contributing to differences in inflammation between grey and white matter plaques in multiple sclerosis. Lower expression of miRNAs in fetal versus adult white matter astrocytes likely reflects the immaturity of these migrating cells. Highly expressed miRNAs in the fetal germinal matrix are probably relevant in development and also recapitulate some responses to injury. Future studies can address regional alterations of miRNA expression in pathological conditions.

This review focuses on the effects of the agents currently approved (or in late clinical trials) as therapies for multiple sclerosis (MS) on the glial cell populations of the central nervous system (CNS). These are comprised of astrocytes, microglia, and oligodendrocytes (OLs), and their progenitors (OPCs). Although the efficacy of these agents is to date established only for the relapsing component of the disease and linked to effects on the systemic immune system, each has been examined with regard to effects on the CNS compartment. The impact of therapies on glia would include modulating these cells immune reactivity, which is considered to underlie the tissue injury process in MS and to any subsequent repair process. As reviewed, these agents can exert their effects either indirectly by modulating the constituents of the systemic immune system or directly depending on their capacity to traverse the blood brain barrier (BBB). Most available data has been derived from administration of these agents in animal models or application to glial cells in vitro. The challenge remains of translating these observations into effective means to impact on the progressive course of disease and reverse existent disabilities.

Astrocytes have complex roles in health and disease, thus it is important to study the pathways that regulate their function. Here we report that lactosylceramide (LacCer) synthesized by β-1,4-galactosyltransferase 6 (B4GALT6) is upregulated in the central nervous system (CNS) of mice during chronic experimental autoimmune encephalomyelitis (EAE), a model of multiple sclerosis (MS). LacCer acts in an autocrine manner to control astrocyte transcriptional programs that promote neurodegeneration. In addition, LacCer in astrocytes controls the recruitment and activation of microglia and CNS-infiltrating monocytes in a non-cell autonomous manner by regulating production of the chemokine CCL2 and granulocyte-macrophage colony-stimulating factor (GM-CSF), respectively. We also detected high B4GALT6 gene expression and LacCer concentrations in CNS MS lesions. Inhibition of LacCer synthesis in mice suppressed local CNS innate immunity and neurodegeneration in EAE and interfered with the activation of human astrocytes in vitro. Thus, B4GALT6 regulates astrocyte activation and is a potential therapeutic target for MS and other neuroinflammatory disorders.

Background: FTY720 (fingolimod, Gilenya) is a daily oral therapy for multiple sclerosis that readily accesses the central nervous system (CNS). FTY720 is a structural analog to the sphingolipid sphingosine-1-phosphate (S1P) and is a cognate ligand for the S1P G-protein coupled receptors (S1PR). Studies in experimental autoimmune encephalomyelitis using mice with conditionally deleted S1P1R from astrocytes indicate that one beneficial effect of FTY720 in this model is via downregulating external receptors, which inhibits responses induced by the natural ligand. Another proposed effect of FTY720 on neuroinflammation is its ability to maintain persistent signaling in cells via internalized S1P1R resulting in functional responses that include suppressing intracellular calcium release. We used human fetal astrocytes to investigate potential dual inhibitory- and function-inducing effects of daily FTY720 on responses relevant to neuroinflammation. For the inhibitory effects, we used signaling and proliferation induced by the natural ligand S1P. For the function-inducing responses, we measured inhibition of intracellular calcium release stimulated by the proinflammatory cytokine, interleukin (IL)-1β., Methods: Astrocytes derived from human fetal CNS specimens and maintained in dissociated cultures were exposed to 100 nM of the biologically active form of FTY720 over a dosing regimen that ranged from a single exposure (with or without washout after 1 h) to daily exposures up to 5 days. Responses measured include: phosphorylation of extracellular-signal-regulated kinases (pERK1/2) by Western blotting, Ki-67 immunolabeling for cell proliferation, IL-1β-induced calcium release by ratiometric fluorescence, and cytokine/chemokine (IL-6, CXCL10) secretions by ELISA., Results: We observed that a single addition of FTY720 inhibited subsequent S1PR ligand-induced pERK1/2 signaling for >24 h. Daily FTY720 treatments (3-5 days) maintained this effect together with a loss of proliferative responses to the natural ligand S1P. Repeated FTY720 dosing concurrently maintained a functional cell response as measured by the inhibition of intracellular calcium release when stimulated by the cytokine IL-1β. Recurrent FTY720 treatments did not inhibit serum- or IL-1β-induced pERK1/2. The secretions of IL-6 and CXCL10 in response to IL-1β were unaffected by FTY720 treatment(s)., Conclusion: Our results indicate that daily FTY720 exposures may regulate specific neuroinflammatory responses by desensitizing astrocytes to external S1PR stimuli while sustaining cellular influences that are independent of new surface S1PR activation.

The contribution of local factors to the activation of immune cells infiltrating the CNS of patients with multiple sclerosis (MS) remains to be defined. The cytokine IL-15 is pivotal in the maintenance and activation of CD8 T lymphocytes, a prominent lymphocyte population found in MS lesions. We investigated whether astrocytes are a functional source of IL-15 sufficient to enhance CD8 T lymphocyte responses and whether they provide IL-15 in the inflamed CNS of patients with MS. We observed that human astrocytes in primary cultures increased surface IL-15 levels upon activation with combinations of proinflammatory cytokines. Expanded human myelin autoreactive CD8 T lymphocytes cultured with such activated astrocytes displayed elevated lytic enzyme content, NKG2D expression, and Ag-specific cytotoxicity. These functional enhancements were abrogated by anti-IL-15-blocking Abs. Immunohistochemical analysis of brain tissue sections obtained from patients with MS demonstrated colocalization for IL-15 and the astrocyte marker glial fibrillary acidic protein within white matter lesions. The majority of astrocytes (80-90%) present in demyelinating MS lesions expressed IL-15, whereas few astrocytes in normal control brain sections had detectable IL-15. IL-15 could be detected in the majority of Iba-1-expressing microglia in the control sections, albeit in lower numbers when compared with microglia/macrophages in MS lesions. Furthermore, infiltrating CD8 T lymphocytes in MS lesions were in close proximity to IL-15-expressing cells. Astrocyte production of IL-15 resulting in the activation of CD8 T lymphocytes ascribes a role for these cells as contributors to the exacerbation of tissue damage during MS pathogenesis.

Autoantibody neuromyelitis optica-IgG (NMO-IgG) recognizing aquaporin-4 (AQP4) is implicated as playing a central role in the physiopathology of NMO. The aim of this in vitro-based study was to characterize functional consequences of interaction between NMO-IgG and cells of the neurovascular unit (astrocytes and brain endothelium) that would provide insight into recognized features of NMO, namely altered blood-brain barrier (BBB) permeability and granulocyte recruitment. We used sera from NMO and longitudinally extensive transverse myelitis cases shown to bind in a characteristic perivascular pattern to primate cerebellar slices. Using flow cytometry, we found that sera from NMO-IgG-positive patients reacted with CNS-derived human fetal astrocytes, whereas sera from multiple sclerosis patients did not. We demonstrated that NMO-IgG binding to astrocytes alters aquaporin-4 polarized expression and increases permeability of a human BBB endothelium/astrocyte barrier. We further demonstrated that NMO-IgG binding to human fetal astrocytes can result in NK cell degranulation, astrocyte killing by Ab-dependent cellular cytotoxicity and complement-dependent granulocyte attraction through the BBB model. Our study highlights important functional roles for NMO-IgG that could account for pathological lesions and BBB dysfunction observed in NMO.

Neuronal injury and loss are recognized features of neuroinflammatory disorders, including acute and chronic encephalitides and multiple sclerosis; destruction of astrocytes has been demonstrated in cases of Rasmussen encephalitis. Here, we show that innate immune cells (i.e. natural killer [NK] and gammadelta T cells) cause loss of neurons from primary human neuron-enriched cultures by destroying the supporting astrocytes. Interleukin 2-activated NK cells caused loss of astrocytes within 1 hour, whereas neurons were lost at 4 hours. Time-lapse imaging indicated that delayed neuron loss was due to early destruction of supporting astrocytes. Selective blocking of astrocyte death with anti-NKG2D antibodies reduced neuron loss, as did blocking of CD54 on astrocytes. gammadelta T cells also induced astrocyte cytotoxicity, leading to subsequent neuronal displacement. In astrocytes, NK cells caused caspase-dependent fragmentation of the intermediate filament proteins glial fibrillary acidic protein and vimentin, whereas anti-CD3-activated T cells produced fragmentation to a lesser extent and without measurable cytotoxicity. Glial fibrillary acidic protein fragmentation was also demonstrated in lysates from chronic multiple sclerosis plaques but not from normal control white matter. These data suggest that non-major histocompatibility complex-restricted immune effector cells may contribute to neuron loss in neuroinflammatory disorders indirectly through injury of glia.

The monoclonal antibody (mAb) neuronal nuclei (NeuN) labels the nuclei of mature neurons in vivo in vertebrates. NeuN has also been used to define post-mitotic neurons or differentiating neuronal precursors in vitro. In this study, we demonstrate that the NeuN mAb labels the nuclei of astrocytes cultured from fetal and adult human, newborn rat, and embryonic mouse brain tissue. A non-neuronal fibroblast cell line (3T3) also displayed NeuN immunoreactivity. We confirmed that NeuN labels neurons but not astrocytes in sections of P10 rat brain. Western blot analysis of NeuN immunoreactive species revealed a distribution of bands in nucleus-enriched fractions derived from the different cell lines that was similar, but not identical to adult rat brain homogenates. We then examined the hypothesis that the glial fibrillary acidic protein/NeuN-double positive population of cells might correspond to neuronal precursors. Although the NeuN-positive astrocytes were proliferating, no evidence of neurogenesis was detected. Furthermore, expression of additional neuronal precursor markers was not detected. Our results indicate that primary astrocytes derived from mouse, rat, and human brain express NeuN. Our findings are consistent with NeuN being a selective marker of neurons in vivo, but indicate that studies utilizing NeuN-immunoreactivity as a definitive marker of post-mitotic neurons in vitro should be interpreted with caution.

The specific signals mediating the activation of microglia and astrocytes as a prelude to, or consequence of, CNS inflammation continue to be defined. We investigated TLRs as novel receptors mediating innate immune responses in human glial cells. We find that microglia express mRNA for TLRs 1-9, whereas astrocytes express robust TLR3, low-level TLR 1, 4, 5, and 9, and rare-to-undetectable TLR 2, 6, 7, 8, and 10 mRNA (quantitative real-time PCR). We focused on TLRs 3 and 4, which can signal through both the MyD88-dependent and -independent pathways, and on the MyD88-restricted TLR2. By flow cytometry, we established that microglia strongly express cell surface TLR2; TLR3 is expressed at higher levels intracellularly. Astrocytes express both cell surface and intracellular TLR3. All three TLRs trigger microglial activation upon ligation. TLR3 signaling induces the strongest proinflammatory polarizing response, characterized by secretion of high levels of IL-12, TNF-alpha, IL-6, CXCL-10, and IL-10, and the expression of IFN-beta. CXCL-10 and IL-10 secretion following TLR4 ligation are comparable to that of TLR3; however, other responses were lower or absent. TLR2-mediated responses are dominated by IL-6 and IL-10 secretion. Astrocytes respond to TLR3 ligation, producing IL-6, CXCL-10, and IFN-beta, implicating these cells as contributors to proinflammatory responses. Initial TLR-mediated glial activation also regulates consequent TLR expression; while TLR2 and TLR3 are subject to positive feedback, TLR4 is down-regulated in microglia. Astrocytes up-regulate all three TLRs following TLR3 ligation. Our data indicate that activation of innate immune responses in the CNS is not homogeneous but rather tailored according to cell type and environmental signal.

Recent experimental and clinical studies on astrocytes are unraveling the capabilities of these multi-functional cells in normal homeostasis, and in central nervous system (CNS) disease. This review focuses on understanding their behavior in all aspects of the initiation, evolution, and resolution of the multiple sclerosis (MS) lesion. Astrocytes display remarkable flexibility and variability of their physical structure and biochemical output, each aspect finely tuned to the specific stage and location of the disease, participating in both pathogenic and beneficial changes seen in acute and progressive forms. As examples, chemo-attractive or repulsive molecules may facilitate the entry of destructive immune cells but may also aid in the recruitment of oligodendrocyte precursors, essential for repair. Pro-inflammatory cytokines may attack pathogenic cells and also destroy normal oligodendrocytes, myelin, and axons. Protective trophic factors may also open the blood–brain barrier and modulate the extracellular matrix to favor recruitment and persistence of CNS-specific immune cells. A chronic glial scar may confer structural support following tissue loss and inhibit ingress of further noxious insults and also inhibit migration of reparative cells and molecules into the damaged tissue. Continual study into these processes offers the therapeutic opportunities to enhance the beneficial capabilities of these cells while limiting their destructive effects. [ABSTRACT FROM AUTHOR]

NKG2D is an activating or coactivating receptor expressed on human natural killer (NK) cells, CD8+ T cells, and γ/δ T cells. NKG2D ligands have been detected on many tumor cell types and can be induced on nontransformed cells by environmental signals including DNA damage and inflammation. We investigated the contribution of NKG2D--NKG2D ligand interaction on CNS-directed immune responses. We observed that primary cultures of human adult oligodendrocytes and fetal astrocytes expressed ligands for NKG2D in vitro whereas neurons, microglia, and adult astrocytes did not. Disruption of the NKG2D--NKG2D ligand interaction using blocking antibodies significantly inhibited killing of primary human oligodendrocytes mediated by activated human NK cells, γ/δ T cells, and allo-reactive CD8+ T cells. NKG2D ligands [major histocompatibility complex class I chain-related molecules A and B (MICA/B)] were detected in groups of cells and colocalized with an oligodendrocyte marker (adenomatous polyposis coli) in white matter sections obtained from multiple sclerosis lesions but not in normal control samples. CD8+ T cells could be detected in close proximity to MICA/B+ cells within multiple sclerosis lesions, supporting an in vivo interaction between these immune effectors and stressed MICA/B-expressing oligodendrocytes. These results imply that NKG2D--NKG2D ligand interaction can potentially contribute to cytotoxic responses mediated by activated immune effector cells in the inflamed CNS, as observed in multiple sclerosis. [ABSTRACT FROM AUTHOR]

A fundamental problem in oncology is that anticancer chemotherapeutics kill both cancer and healthy cells in the surrounding tissues. Resveratrol is a natural antioxidant with intriguing and opposing biological properties: it reduces viability of some cancer cells but not of non-transformed ones (in equimolar concentrations). Therefore, we examined resveratrol in human non-transformed primary astrocytes and astrocytoma. Resveratrol reduced reactive oxygen species in astrocytes, but not in astrocytoma. Such cell-type dependent response is particularly evident with analyses at the single cell level showing clear population difference in high and low glutathione levels. Due to resveratrol's poor aqueous solubility that limits its use in clinics, we incorporated it into stimulus-responsive micelles assembled from miktoarm polymers. This could be an attractive chemotherapeutic delivery strategy in nano-oncology. As a proof of principle, we show that these formulations containing resveratrol markedly decrease astrocytoma viability, particularly in combination with temozolomide, a first line chemotherapeutic for astrocytoma. Astrocytes and astrocytoma show disparity in response to resveratrol: intracellular GSH levels are increased in human primary astrocytes, while astrocytoma cells show increase in ROS and depletion of GSH. Incorporation of resveratrol in miktoarm star-based formulations, and in combination therapy with temozolomide, synergistically reduces astrocytoma cell viability. [Display omitted] • Resveratrol reduces the abundance of ROS in astrocytes but not in astrocytoma. • Cells with low or high glutathione content respond differently to resveratrol. • Miktoarm micelles are a suitable vehicle for resveratrol and other natural compounds. • Combination treatment with resveratrol effectively reduces astrocytoma viability. • Resveratrol-loaded miktoarm micelles enhance reduction in viable astrocytoma. [ABSTRACT FROM AUTHOR]