Astrocytic TIMP-1 regulates production of Anastellin, an inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. Astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout (Timp1KO) mice do not efficiently remyelinate following a demyelinating injury. Here, we performed an unbiased proteomic analysis and identified a fibronectin-derived peptide called Anastellin (Ana) that was unique to the Timp1KO astrocyte secretome. Ana was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Ana is known to act upon the sphingosine-1-phosphate receptor 1, and we determined that Ana also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro. Administration of FTY720 to wild-type C57BL/6 mice during MOG35-55-experimental autoimmune encephalomyelitis ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 (Timp1KO) had no effect. Analysis of Timp1 and fibronectin (FN1) transcripts from primary human astrocytes from healthy and multiple sclerosis (MS) donors revealed lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Last, analyses of proteomic databases of MS samples identified Ana peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high disease activity. A role for Ana in MS as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and innate remyelination potential in the MS brain.

The role of neural stem cells in regulating glial scar formation and repair.

Glial scars are a common pathological occurrence in a variety of central nervous system (CNS) diseases and injuries. They are caused after severe damage and consist of reactive glia that form a barrier around the damaged tissue that leads to a non-permissive microenvironment which prevents proper endogenous regeneration. While there are a number of therapies that are able to address some components of disease, there are none that provide regenerative properties. Within the past decade, neural stem cells (NSCs) have been heavily studied due to their potent anti-inflammatory and reparative capabilities in disease and injury. Exogenously applied NSCs have been found to aid in glial scar healing by reducing inflammation and providing cell replacement. However, endogenous NSCs have also been found to contribute to the reactive environment by different means. Further understanding how NSCs can be leveraged to aid in the resolution of the glial scar is imperative in the use of these cells as regenerative therapies. To do so, humanised 3D model systems have been developed to study the development and maintenance of the glial scar. Herein, we explore the current work on endogenous and exogenous NSCs in the glial scar as well as the novel 3D stem cell-based technologies being used to model this pathology in a dish.

Cellular senescence in progenitor cells contributes to diminished remyelination potential in progressive multiple sclerosis.

Cellular senescence is a form of adaptive cellular physiology associated with aging. Cellular senescence causes a proinflammatory cellular phenotype that impairs tissue regeneration, has been linked to stress, and is implicated in several human neurodegenerative diseases. We had previously determined that neural progenitor cells (NPCs) derived from induced pluripotent stem cell (iPSC) lines from patients with primary progressive multiple sclerosis (PPMS) failed to promote oligodendrocyte progenitor cell (OPC) maturation, whereas NPCs from age-matched control cell lines did so efficiently. Herein, we report that expression of hallmarks of cellular senescence were identified in SOX2+ progenitor cells within white matter lesions of human progressive MS (PMS) autopsy brain tissues and iPS-derived NPCs from patients with PPMS. Expression of cellular senescence genes in PPMS NPCs was found to be reversible by treatment with rapamycin, which then enhanced PPMS NPC support for oligodendrocyte (OL) differentiation. A proteomic analysis of the PPMS NPC secretome identified high-mobility group box-1 (HMGB1), which was found to be a senescence-associated inhibitor of OL differentiation. Transcriptome analysis of OPCs revealed that senescent NPCs induced expression of epigenetic regulators mediated by extracellular HMGB1. Lastly, we determined that progenitor cells are a source of elevated HMGB1 in human white matter lesions. Based on these data, we conclude that cellular senescence contributes to altered progenitor cell functions in demyelinated lesions in MS. Moreover, these data implicate cellular aging and senescence as a process that contributes to remyelination failure in PMS, which may impact how this disease is modeled and inform development of future myelin regeneration strategies.

Extracellular vesicle fibrinogen induces encephalitogenic CD8+ T cells in a mouse model of multiple sclerosis.

Extracellular vesicles (EVs) are emerging as potent mediators of intercellular communication with roles in inflammation and disease. In this study, we examined the role of EVs from blood plasma (pEVs) in an experimental autoimmune encephalomyelitis mouse model of central nervous system demyelination. We determined that pEVs induced a spontaneous relapsing-remitting disease phenotype in MOG35-55-immunized C57BL/6 mice. This modified disease phenotype was found to be driven by CD8+ T cells and required fibrinogen in pEVs. Analysis of pEVs from relapsing-remitting multiple sclerosis patients also identified fibrinogen as a significant portion of pEV cargo. Together, these data suggest that fibrinogen in pEVs contributes to the perpetuation of neuroinflammation and relapses in disease.

Systemic TLR2 tolerance enhances central nervous system remyelination.

BACKGROUND: Multiple sclerosis (MS) is a central nervous system (CNS) autoimmune disease characterized by both inflammatory demyelination and impaired remyelination. Studies indicate that Toll-like receptor 2 (TLR2) signaling contributes to both the inflammatory component and the defective remyelination in MS. While most MS therapeutics target adaptive immunity, we recently reported that reducing TLR2 signaling in innate immune cells by inducing TLR2 tolerance attenuates adoptively transferred experimental autoimmune encephalomyelitis. Given that previous reports suggest TLR2 signaling also inhibits myelin repair, the objective of this study was to assess how reducing TLR2 signaling through TLR2 tolerance induction affects CNS myelin repair. METHODS: Chow containing 0.2% cuprizone was fed to male and female wild-type (WT) C57BL/6 mice or TLR2-deficient (TLR2-/-) mice for 5 weeks to induce demyelination. During a 2-week remyelination period following discontinuation of cuprizone, WT mice received either low dose TLR2 ligands to induce systemic TLR2 tolerance or vehicle control (VC). Remyelination was evaluated via electron microscopy and immunohistochemical analysis of microglia and oligodendrocytes in the corpus callosum. Statistical tests included 2-way ANOVA and Mann-Whitney U analyses. RESULTS: Inducing TLR2 tolerance in WT mice during remyelination significantly enhanced myelin recovery, restoring unmyelinated axon frequency and myelin thickness to baseline levels compared to VC-treated mice. Mechanistically, enhanced remyelination in TLR2 tolerized mice was associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. This result was confirmed in vitro by inducing TLR2 tolerance in WT microglia cultures. TLR2-/- mice, without TLR2 tolerance induction, also significantly enhanced myelin recovery compared to WT mice, adding confirmation that reduced TLR2 signaling is associated with enhanced remyelination. DISCUSSION: Our results suggest that reducing TLR2 signaling in vivo by inducing TLR2 tolerance significantly enhances myelin repair. Furthermore, the enhanced remyelination resulting from TLR2 tolerance induction is associated with a shift in corpus callosum microglia from a pro-inflammatory iNOS+ phenotype to a non-inflammatory/pro-repair Arg1+ phenotype. While deletion of TLR2 would be an impractical approach in vivo, reducing innate immune signaling through TLR2 tolerance induction may represent a novel, two-pronged approach for treating both inflammatory and myelin repair components of MS.

A microglial hypothesis of globoid cell leukodystrophy pathology.

Globoid cell leukodystrophy (GLD), also known as Krabbe's disease, is a fatal demyelinating disease accompanied by the formation of giant, multinucleated cells called globoid cells. Previously believed to be a byproduct of inflammation, these cells can be found early in disease before evidence of any damage. The precise mechanism by which these globoid cells cause oligodendrocyte dysfunction is not completely understood, nor is their cell type defined. This Review outlines the idea that microglial cells are transformed into an unknown and undefined novel M3 phenotype in GLD, which is cytotoxic to oligodendrocytes, leading to disease progression. © 2016 Wiley Periodicals, Inc.

CD8+ T cell depletion prevents neuropathology in a mouse model of globoid cell leukodystrophy.

Globoid cell leukodystrophy (GLD) or Krabbe's disease is a fatal genetic demyelinating disease of the central nervous system caused by loss-of-function mutations in the galactosylceramidase (galc) gene. While the metabolic basis for disease is known, the understanding of how this results in neuropathology is not well understood. Herein, we report that the rapid and protracted elevation of CD8+ cytotoxic T lymphocytes occurs coincident with clinical disease in a mouse model of GLD. Administration of a function-blocking antibody against CD8α effectively prevented disease onset, reduced morbidity and mortality, and prevented CNS demyelination in mice. These data indicate that subsequent to the genetic cause of disease, neuropathology is driven by pathogenic CD8+ T cells, thus offering novel therapeutic potential for treatment of GLD.

Astrocytic TIMP-1 regulates production of Anastellin, a novel inhibitor of oligodendrocyte differentiation and FTY720 responses.

Astrocyte activation is associated with neuropathology and the production of tissue inhibitor of metalloproteinase-1 (TIMP1). TIMP1 is a pleiotropic extracellular protein that functions both as a protease inhibitor and as a growth factor. We have previously demonstrated that murine astrocytes that lack expression of Timp1 do not support rat oligodendrocyte progenitor cell (rOPC) differentiation, and adult global Timp1 knockout ( Timp1 KO ) mice do not efficiently remyelinate following a demyelinating injury. To better understand the basis of this, we performed unbiased proteomic analyses and identified a fibronectin-derived peptide called anastellin that is unique to the murine Timp1 KO astrocyte secretome. Anastellin was found to block rOPC differentiation in vitro and enhanced the inhibitory influence of fibronectin on rOPC differentiation. Anastellin is known to act upon the sphingosine-1-phosphate receptor 1 (S1PR1), and we determined that anastellin also blocked the pro-myelinating effect of FTY720 (or fingolimod) on rOPC differentiation in vitro . Further, administration of FTY720 to wild-type C57BL/6 mice during MOG 35-55 -EAE ameliorated clinical disability while FTY720 administered to mice lacking expression of Timp1 in astrocytes ( Timp1 cKO ) had no effect. Analysis of human TIMP1 and fibronectin ( FN1 ) transcripts from healthy and multiple sclerosis (MS) patient brain samples revealed an inverse relationship where lower TIMP1 expression was coincident with elevated FN1 in MS astrocytes. Lastly, we analyzed proteomic databases of MS samples and identified anastellin peptides to be more abundant in the cerebrospinal fluid (CSF) of human MS patients with high versus low disease activity. The prospective role for anastellin generation in association with myelin lesions as a consequence of a lack of astrocytic TIMP-1 production could influence both the efficacy of fingolimod responses and the innate remyelination potential of the the MS brain. Significance Statement: Astrocytic production of TIMP-1 prevents the protein catabolism of fibronectin. In the absence of TIMP-1, fibronectin is further digested leading to a higher abundance of anastellin peptides that can bind to sphingosine-1-phosphate receptor 1. The binding of anastellin with the sphingosine-1-phosphate receptor 1 impairs the differentiation of oligodendrocytes progenitor cells into myelinating oligodendrocytes in vitro , and negates the astrocyte-mediated therapeutic effects of FTY720 in the EAE model of chronic CNS inflammation. These data indicate that TIMP-1 production by astrocytes is important in coordinating astrocytic functions during inflammation. In the absence of astrocyte produced TIMP-1, elevated expression of anastellin may represent a prospective biomarker for FTY720 therapeutic responsiveness.

Integrative transcriptomic and metabolic analyses of the mammalian hibernating brain identifies a key role for succinate dehydrogenase in ischemic tolerance.

Ischemic stroke results in a loss of tissue homeostasis and integrity, the underlying pathobiology of which stems primarily from the depletion of cellular energy stores and perturbation of available metabolites 1 . Hibernation in thirteen-lined ground squirrels (TLGS), Ictidomys tridecemlineatus , provides a natural model of ischemic tolerance as these mammals undergo prolonged periods of critically low cerebral blood flow without evidence of central nervous system (CNS) damage 2 . Studying the complex interplay of genes and metabolites that unfolds during hibernation may provide novel insights into key regulators of cellular homeostasis during brain ischemia. Herein, we interrogated the molecular profiles of TLGS brains at different time points within the hibernation cycle via RNA sequencing coupled with untargeted metabolomics. We demonstrate that hibernation in TLGS leads to major changes in the expression of genes involved in oxidative phosphorylation and this is correlated with an accumulation of the tricarboxylic acid (TCA) cycle intermediates citrate, cis-aconitate, and α-ketoglutarate-αKG. Integration of the gene expression and metabolomics datasets led to the identification of succinate dehydrogenase (SDH) as the critical enzyme during hibernation, uncovering a break in the TCA cycle at that level. Accordingly, the SDH inhibitor dimethyl malonate (DMM) was able to rescue the effects of hypoxia on human neuronal cells in vitro and in mice subjected to permanent ischemic stroke in vivo . Our findings indicate that studying the regulation of the controlled metabolic depression that occurs in hibernating mammals may lead to novel therapeutic approaches capable of increasing ischemic tolerance in the CNS.

Soluble factors influencing the neural stem cell niche in brain physiology, inflammation, and aging.

Within the adult central nervous system (CNS) of most mammals resides a resident stem cell population, known as neural stem cells (NSCs). NSCs are located within specific niches of the CNS and maintain a self-renewal and proliferative capacity to generate new neurons, astrocytes, and oligodendrocytes throughout adulthood. The NSC niches are dynamic and active environments that are within proximity to the systemic circulation and the cerebrospinal fluid (CSF). Therefore, NSCs respond not only to factors present in the local microenvironment of the niche but also to factors present in the systemic macroenvironment. The factors can be soluble forms such as cytokines and chemokines located in the circulation or directly from local cells, such as microglia and astrocytes. Additionally, recent evidence points towards physiological aging and its association with a progressive loss of function and a decline in the self-renewal and regenerative capacities of CNS NSCs, which can be further exacerbated by changes in the local and systemic milieu. This review will highlight the main intrinsic and extrinsic regulators of neural stem cell function under homeostatic and inflammatory conditions including those trafficked within extracellular membrane vesicles. Further, discussion will center around how intrinsic and extrinsic factors impact normal homeostatic functions within the adult brain and in aging.

NSCs: Sentinel Cells of the Brain.

Adult neural stem cells (NSCs) have the ability to oscillate between activated and dormant states in response to extrinsic regulators. In this issue of Cell Stem Cell, Belenguer et al. (2020) identify a direct role for systemic TNF-α, which acts through its receptors TNFRII and TNFRI as a regulator of NSC activation and a return to quiescence, respectively.

Stem Cell Therapies for Progressive Multiple Sclerosis.

Multiple sclerosis (MS) is a chronic inflammatory disease of the central nervous system characterized by demyelination and axonal degeneration. MS patients typically present with a relapsing-remitting (RR) disease course, manifesting as sporadic attacks of neurological symptoms including ataxia, fatigue, and sensory impairment. While there are several effective disease-modifying therapies able to address the inflammatory relapses associated with RRMS, most patients will inevitably advance to a progressive disease course marked by a gradual and irreversible accrual of disabilities. Therapeutic intervention in progressive MS (PMS) suffers from a lack of well-characterized biological targets and, hence, a dearth of successful drugs. The few medications approved for the treatment of PMS are typically limited in their efficacy to active forms of the disease, have little impact on slowing degeneration, and fail to promote repair. In looking to address these unmet needs, the multifactorial therapeutic benefits of stem cell therapies are particularly compelling. Ostensibly providing neurotrophic support, immunomodulation and cell replacement, stem cell transplantation holds substantial promise in combatting the complex pathology of chronic neuroinflammation. Herein, we explore the current state of preclinical and clinical evidence supporting the use of stem cells in treating PMS and we discuss prospective hurdles impeding their translation into revolutionary regenerative medicines.

Stem Cells of the Aging Brain.

The adult central nervous system (CNS) contains resident stem cells within specific niches that maintain a self-renewal and proliferative capacity to generate new neurons, astrocytes, and oligodendrocytes throughout adulthood. Physiological aging is associated with a progressive loss of function and a decline in the self-renewal and regenerative capacities of CNS stem cells. Also, the biggest risk factor for neurodegenerative diseases is age, and current in vivo and in vitro models of neurodegenerative diseases rarely consider this. Therefore, combining both aging research and appropriate interrogation of animal disease models towards the understanding of the disease and age-related stem cell failure is imperative to the discovery of new therapies. This review article will highlight the main intrinsic and extrinsic regulators of neural stem cell (NSC) aging and discuss how these factors impact normal homeostatic functions within the adult brain. We will consider established in vivo animal and in vitro human disease model systems, and then discuss the current and future trajectories of novel senotherapeutics that target aging NSCs to ameliorate brain disease.

The neural stem cell secretome and its role in brain repair.

Compelling evidence from experimental animal disease models and early-phase clinical trials identifies the transplantation of neural progenitor/stem cells (NSCs) as a viable path towards the development of clinically applicable exogenous stem cell therapies. Building from current advances in the field of NSC biology and following the positive outcomes of NSC transplantation studies, the contemporary view is that transplanted NSCs act as local 'factories' capable of producing and secreting a wide array of immune and neurotrophic factors. This has launched a 'stem cell race' to identify the mechanisms behind stem-cell mediated repair in what has been labeled the paracrine hypothesis. This hypothesis proposes that NSC grafts act as a natural source of potent biologics capable of modulating and promoting the restoration of several key functions in the central nervous system (CNS) tissue following acute or chronic tissue damage. Investigators have been inspired to examine novel ways to harness and utilize the pro-regenerative properties of NSC therapies as an alternative approach to a more classical (small molecule based) treatment of CNS diseases. In this review, we will discuss the most recent findings of human NSC (hNSCs) transplants in experimental animal models of CNS diseases that identify of hNSC-secreted factors, including those trafficked within extracellular membrane vesicles (EVs), and the outcomes of recent clinical trials utilizing hNSC therapeutics in CNS diseases.

Harnessing the Neural Stem Cell Secretome for Regenerative Neuroimmunology.

Increasing evidence foresees the secretome of neural stem cells (NSCs) to confer superimposable beneficial properties as exogenous NSC transplants in experimental treatments of traumas and diseases of the central nervous system (CNS). Naturally produced secretome biologics include membrane-free signaling molecules and extracellular membrane vesicles (EVs) capable of regulating broad functional responses. The development of high-throughput screening pipelines for the identification and validation of NSC secretome targets is still in early development. Encouraging results from pre-clinical animal models of disease have highlighted secretome-based (acellular) therapeutics as providing significant improvements in biochemical and behavioral measurements. Most of these responses are being hypothesized to be the result of modulating and promoting the restoration of key inflammatory and regenerative programs in the CNS. Here, we will review the most recent findings regarding the identification of NSC-secreted factors capable of modulating the immune response to promote the regeneration of the CNS in animal models of CNS trauma and inflammatory disease and discuss the increased interest to refine the pro-regenerative features of the NSC secretome into a clinically available therapy in the emerging field of Regenerative Neuroimmunology.

Astrocyte Support for Oligodendrocyte Differentiation can be Conveyed via Extracellular Vesicles but Diminishes with Age.

The aging brain is associated with significant changes in physiology that alter the tissue microenvironment of the central nervous system (CNS). In the aged CNS, increased demyelination has been associated with astrocyte hypertrophy and aging has been implicated as a basis for these pathological changes. Aging tissues accumulate chronic cellular stress, which can lead to the development of a pro-inflammatory phenotype that can be associated with cellular senescence. Herein, we provide evidence that astrocytes aged in culture develop a spontaneous pro-inflammatory and senescence-like phenotype. We found that extracellular vesicles (EVs) from young astrocyte were sufficient to convey support for oligodendrocyte differentiation while this support was lost by EVs from aged astrocytes. Importantly, the negative influence of culture age on astrocytes, and their cognate EVs, could be countered by treatment with rapamycin. Comparative proteomic analysis of EVs from young and aged astrocytes revealed peptide repertoires unique to each age. Taken together, these findings provide new information on the contribution of EVs as potent mediators by which astrocytes can extert changing influence in either the disease or aged brain.

TIMP-1 Promotes Oligodendrocyte Differentiation Through Receptor-Mediated Signaling.

The extracellular protein tissue inhibitor of metalloproteinase (TIMP)-1 is both a matrix metalloproteinase (MMP) inhibitor and a trophic factor. Mice lacking TIMP-1 exhibit delayed central nervous system myelination during postnatal development and impaired remyelination following immune-mediated injury in adulthood. We have previously determined that the trophic action of TIMP-1 on oligodendrocyte progenitor cells (OPCs) to mature into oligodendrocytes is independent of its MMP inhibitory function. However, the mechanism by which TIMP-1 promotes OPC differentiation is not known. To address this gap in our understanding, herein, we report that TIMP-1 signals via a CD63/ß1-integrin receptor complex to activate Akt (protein kinase B) to promote ß-catenin signaling in OPCs. The regulation of ß-catenin by TIMP-1 to promote OPC differentiation was counteracted, but not abrogated, by canonical signaling evoked by Wnt7a. These data provide a previously uncharacterized trophic action of TIMP-1 to regulate oligodendrocyte maturation via a CD63/ß1-integrin/Akt pathway mechanism. These findings contribute to our emerging understanding on the role of TIMP-1 as a growth factor expressed to promote CNS myelination during development and induced in the adult to promote myelin repair.

A Refined Bead-Free Method to Identify Astrocytic Exosomes in Primary Glial Cultures and Blood Plasma.

Astrocytes are the most abundant glial cell type in the central nervous system (CNS) and are known to fulfill critical homeostatic functions. Dysfunction of activated astrocytes is also known to participate in the development of several neurological diseases. Astrocytes can be uniquely identified by expression of the intermediate filament protein glial acidic fibrillary protein (GFAP). Herein, we report on the development of a rigorous and sensitive methodology to identify GFAP+ exosomes in primary culture using flow cytometry. We then demonstrate that activated astrocytes release increased amounts of exosomes in response to treatment with interleukin-1ß. Using this methodology, we report the identification of GFAP+ exosomes in blood and then use a mouse model of inflammatory demyelination, experimental autoimmune encephalomyelitis (EAE), to examine whether the abundance of GFAP+ exosomes in blood circulation changes during clinical illness. We find a detectable increase in the presence of GFAP+ exosomes in EAE mice when compared with non-EAE, control mice. Our data provide a novel perspective on the presence of GFAP in blood as it identifies exosomes as potential astrocyte-derived signals within blood. These data are complementary to previous clinical studies that reported elevated GFAP protein in blood samples from multiple sclerosis (MS) patients during a clinical relapse. These data also reveal the existence of a potential systemic role for astrocyte-derived exosomes in CNS conditions involving inflammation such as multiple sclerosis.

iPS-derived neural progenitor cells from PPMS patients reveal defect in myelin injury response.

Primary progressive multiple sclerosis (PPMS) is a chronic demyelinating disease of the central nervous system (CNS) currently lacking any effective treatment. Promoting endogenous brain repair offers a potential strategy to halt and possibly restore neurologic function in PPMS. To understand how the microenvironment within white matter lesions plays a role in repair we have focused on neural progenitor cells (NPCs) since these are found in lesions in PPMS and have been found to influence oligodendrocyte progenitor cell maturation (OPCs). To better understand the cellular nature of NPCs in PPMS we developed iPS cells from blood samples of PPMS patients and age matched non-disease spouse or blood relative controls. Using these iPS cell lines we determined that the NPCs from PPMS cases provided no neuroprotection against active CNS demyelination compared to NPCs from control iPS lines which were capable of completely preventing injury. Conditioned media (CM) from PPMS NPCs provides no protection to OPCs and prevents maturation of OPCs into oligodendrocytes in vitro. We also found that CM from PPMS iPS NPCs elicited patient-specific differences in the response to compounds that should foster oligodendrocyte (OL) maturation. Together, these data establish a new model for understanding the nature of myelination defects in PPMS which may lead to novel targeted approaches for preventing demyelination in these patients.

Aberrant production of tenascin-C in globoid cell leukodystrophy alters psychosine-induced microglial functions.

Globoid cell leukodystrophy (GLD), or Krabbe disease, is a rare and often fatal demyelinating disease caused by mutations in the galactocerebrosidase (galc) gene that result in accumulation of galactosylsphingosine (psychosine). We recently reported that the extracellular matrix (ECM) protease, matrix metalloproteinase-3, is elevated in GLD and that it regulates psychosine-induced microglial activation. Here, we examined central nervous system ECM component expression in human GLD patients and in the twitcher mouse model of GLD using immunohistochemistry. The influence of ECM proteins on primary murine microglial responses to psychosine was evaluated using ECM proteins as substrates and analyzed by quantitative real-time polymerase chain reaction, immunocytochemistry, and ELISA. Functional analysis of microglial cytotoxicity was performed on oligodendrocytes in coculture, and cell death was measured by lactose dehydrogenase assay. Tenascin-C (TnC) was expressed at higher levels in human GLD and in twitcher mice versus controls. Microglial responses to psychosine were enhanced by TnC, as determined by an increase in globoid-like cell formation, matrix metalloproteinase-3 mRNA expression, and higher toxicity toward oligodendrocytes in culture. These findings were consistent with a shift toward the M1 microglial phenotype in TnC-grown microglia. Thus, elevated TnC expression in GLD modified microglial responses to psychosine. These data offer a novel perspective and enhance understanding of the microglial contribution to GLD pathogenesis.