Fibulin-2 is an extracellular matrix inhibitor of oligodendrocytes relevant to multiple sclerosis.

Impairment of oligodendrocytes and myelin contributes to neurological disorders including multiple sclerosis (MS), stroke, and Alzheimer's disease. Regeneration of myelin (remyelination) decreases the vulnerability of demyelinated axons, but this repair process commonly fails with disease progression. A contributor to inefficient remyelination is the altered extracellular matrix (ECM) in lesions, which remains to be better defined. We have identified fibulin-2 (FBLN2) as a highly upregulated ECM component in lesions of MS and stroke and in proteome databases of Alzheimer's disease and traumatic brain injury. Focusing on MS, the inhibitory role of FBLN2 was suggested in the experimental autoimmune encephalomyelitis (EAE) model, in which genetic FBLN2 deficiency improved behavioral recovery by promoting the maturation of oligodendrocytes and enhancing remyelination. Mechanistically, when oligodendrocyte progenitors were cultured in differentiation medium, FBLN2 impeded their maturation into oligodendrocytes by engaging the Notch pathway, leading to cell death. Adeno-associated virus deletion of FBLN2 in astrocytes improved oligodendrocyte numbers and functional recovery in EAE and generated new myelin profiles after lysolecithin-induced demyelination. Collectively, our findings implicate FBLN2 as a hitherto unrecognized injury-elevated ECM, and a therapeutic target, that impairs oligodendrocyte maturation and myelin repair.

Biology of neurofibrosis with focus on multiple sclerosis.

Tissue damage elicits a wound healing response of inflammation and remodeling aimed at restoring homeostasis. Dysregulation of wound healing leads to accumulation of effector cells and extracellular matrix (ECM) components, collectively termed fibrosis, which impairs organ functions. Fibrosis of the central nervous system, neurofibrosis, is a major contributor to the lack of neural regeneration and it involves fibroblasts, microglia/macrophages and astrocytes, and their deposited ECM. Neurofibrosis occurs commonly across neurological conditions. This review describes processes of wound healing and fibrosis in tissues in general, and in multiple sclerosis in particular, and considers approaches to ameliorate neurofibrosis to enhance neural recovery.

Emerging role of galectin 3 in neuroinflammation and neurodegeneration.

Neuroinflammation and neurodegeneration are key processes that mediate the development and progression of neurological diseases. However, the mechanisms modulating these processes in different diseases remain incompletely understood. Advances in single cell based multi-omic analyses have helped to identify distinct molecular signatures such as Lgals3 that is associated with neuroinflammation and neurodegeneration in the central nervous system (CNS). Lgals3 encodes galectin-3 (Gal3), a ß-galactoside and glycan binding glycoprotein that is frequently upregulated by reactive microglia/macrophages in the CNS during various neurological diseases. While Gal3 has previously been associated with non-CNS inflammatory and fibrotic diseases, recent studies highlight Gal3 as a prominent regulator of inflammation and neuroaxonal damage in the CNS during diseases such as multiple sclerosis, Alzheimer's disease, and Parkinson's disease. In this review, we summarize the pleiotropic functions of Gal3 and discuss evidence that demonstrates its detrimental role in neuroinflammation and neurodegeneration during different neurological diseases. We also consider the challenges of translating preclinical observations into targeting Gal3 in the human CNS.

Elevated Galectin-3 Is Associated with Aging, Multiple Sclerosis, and Oxidized Phosphatidylcholine-Induced Neurodegeneration.

Aging is a significant risk factor associated with the progression of CNS neurodegenerative diseases including multiple sclerosis (MS). Microglia, the resident macrophages of the CNS parenchyma, are a major population of immune cells that accumulate in MS lesions. While they normally regulate tissue homeostasis and facilitate the clearance of neurotoxic molecules including oxidized phosphatidylcholines (OxPCs), their transcriptome and neuroprotective functions are reprogrammed by aging. Thus, determining the factors that instigate aging associated microglia dysfunction can lead to new insights for promoting CNS repair and for halting MS disease progression. Through single-cell RNA sequencing (scRNAseq), we identified Lgals3, which encodes for galectin-3 (Gal3), as an age upregulated gene by microglia responding to OxPC. Consistently, excess Gal3 accumulated in OxPC and lysolecithin-induced focal spinal cord white matter (SCWM) lesions of middle-aged mice compared with young mice. Gal3 was also elevated in mouse experimental autoimmune encephalomyelitis (EAE) lesions and more importantly in MS brain lesions from two male and one female individuals. While Gal3 delivery alone into the mouse spinal cord did not induce damage, its co-delivery with OxPC increased cleaved caspase 3 and IL-1ß within white matter lesions and exacerbated OxPC-induced injury. Conversely, OxPC-mediated neurodegeneration was reduced in Gal3-/- mice compared with Gal3+/+ mice. Thus, Gal3 is associated with increased neuroinflammation and neurodegeneration and its overexpression by microglia/macrophages may be detrimental for lesions within the aging CNS.SIGNIFICANCE STATEMENT Aging accelerates the progression of neurodegenerative diseases such as multiple sclerosis (MS). Understanding the molecular mechanisms of aging that increases the susceptibility of the CNS to damage could lead to new strategies to manage MS progression. Here, we highlight that microglia/macrophage-associated galectin-3 (Gal3) was upregulated with age exacerbated neurodegeneration in the mouse spinal cord white matter (SCWM) and in MS lesions. More importantly, co-injection of Gal3 with oxidized phosphatidylcholines (OxPCs), which are neurotoxic lipids found in MS lesions, caused greater neurodegeneration compared with injection of OxPC alone, whereas genetic loss of Gal3 reduced OxPC damage. These results demonstrate that Gal3 overexpression is detrimental to CNS lesions and suggest its deposition in MS lesions may contribute to neurodegeneration.

Enhanced liver X receptor signalling reduces brain injury and promotes tissue regeneration following experimental intracerebral haemorrhage: roles of microglia/macrophages.

BACKGROUND: Inflammation-exacerbated secondary brain injury and limited tissue regeneration are barriers to favourable prognosis after intracerebral haemorrhage (ICH). As a regulator of inflammation and lipid metabolism, Liver X receptor (LXR) has the potential to alter microglia/macrophage (M/M) phenotype, and assist tissue repair by promoting cholesterol efflux and recycling from phagocytes. To support potential clinical translation, the benefits of enhanced LXR signalling are examined in experimental ICH. METHODS: Collagenase-induced ICH mice were treated with the LXR agonist GW3965 or vehicle. Behavioural tests were conducted at multiple time points. Lesion and haematoma volume, and other brain parameters were assessed using multimodal MRI with T2-weighted, diffusion tensor imaging and dynamic contrast-enhanced MRI sequences. The fixed brain cryosections were stained and confocal microscopy was applied to detect LXR downstream genes, M/M phenotype, lipid/cholesterol-laden phagocytes, oligodendrocyte lineage cells and neural stem cells. Western blot and real-time qPCR were also used. CX3CR1CreER: Rosa26iDTR mice were employed for M/M-depletion experiments. RESULTS: GW3965 treatment reduced lesion volume and white matter injury, and promoted haematoma clearance. Treated mice upregulated LXR downstream genes including ABCA1 and Apolipoprotein E, and had reduced density of M/M that apparently shifted from proinflammatory interleukin-1ß+ to Arginase1+CD206+ regulatory phenotype. Fewer cholesterol crystal or myelin debris-laden phagocytes were observed in GW3965 mice. LXR activation increased the number of Olig2+PDGFRα+ precursors and Olig2+CC1+ mature oligodendrocytes in perihaematomal regions, and elevated SOX2+ or nestin+ neural stem cells in lesion and subventricular zone. MRI results supported better lesion recovery by GW3965, and this was corroborated by return to pre-ICH values of functional rotarod activity. The therapeutic effects of GW3965 were abrogated by M/M depletion in CX3CR1CreER: Rosa26iDTR mice. CONCLUSIONS: LXR agonism using GW3965 reduced brain injury, promoted beneficial properties of M/M and facilitated tissue repair correspondent with enhanced cholesterol recycling.

Expression of antioxidant enzymes in lesions of multiple sclerosis and its models.

Oxidative stress promotes tissue injury in the central nervous system in neurological disorders such as multiple sclerosis (MS). To protect against this, antioxidant enzymes including superoxide dismutase-1 (SOD1), heme oxygenase-1 (HO-1), peroxiredoxin-5 (PRDX5) and glutathione peroxidase-4 (GPX4) may be upregulated. However, whether antioxidant enzyme elevation in mouse models of neurodegeneration corresponds to their expression in human diseases such as MS requires investigation. Here, we analyzed and compared the expression of SOD1, HO-1, PRDX5 and GPX4 in the murine spinal cord of three models of MS: focal lesions induced by (1) oxidized phosphatidylcholine or (2) lysophosphatidylcholine (lysolecithin), and (3) diffuse lesions of experimental autoimmune encephalomyelitis. Notably, CD68+ microglia/macrophages were the predominant cellular populations that expressed the highest levels of the detected antioxidant enzymes. Overall, the expression patterns of antioxidant enzymes across the models were similar. The increase of these antioxidant enzymes was corroborated in MS brain tissue using spatial RNA sequencing. Collectively, these results show that antioxidant capacity is relatively conserved between mouse models and MS lesions, and suggest a need to investigate whether the antioxidant elevation in microglia/macrophages is a protective response during oxidative injury, neurodegeneration, and MS.

Versican promotes T helper 17 cytotoxic inflammation and impedes oligodendrocyte precursor cell remyelination.

Remyelination failure in multiple sclerosis (MS) contributes to progression of disability. The deficient repair results from neuroinflammation and deposition of inhibitors including chondroitin sulfate proteoglycans (CSPGs). Which CSPG member is repair-inhibitory or alters local inflammation to exacerbate injury is unknown. Here, we correlate high versican-V1 expression in MS lesions with deficient premyelinating oligodendrocytes, and highlight its selective upregulation amongst CSPG members in experimental autoimmune encephalomyelitis (EAE) lesions modeling MS. In culture, purified versican-V1 inhibits oligodendrocyte precursor cells (OPCs) and promotes T helper 17 (Th17) polarization. Versican-V1-exposed Th17 cells are particularly toxic to OPCs. In NG2CreER:MAPTmGFP mice illuminating newly formed GFP+ oligodendrocytes/myelin, difluorosamine (peracetylated,4,4-difluoro-N-acetylglucosamine) treatment from peak EAE reduces lesional versican-V1 and Th17 frequency, while enhancing GFP+ profiles. We suggest that lesion-elevated versican-V1 directly impedes OPCs while it indirectly inhibits remyelination through elevating local Th17 cytotoxic neuroinflammation. We propose CSPG-lowering drugs as potential dual pronged repair and immunomodulatory therapeutics for MS.

Single-cell and spatial RNA sequencing identify perturbators of microglial functions with aging.

Microglia are the immune sentinels of the central nervous system with protective roles such as the removal of neurotoxic oxidized phosphatidylcholines (OxPCs). As aging alters microglial function and elevates neurological disability in diseases such as multiple sclerosis, defining aging-associated factors that cause microglia to lose their custodial properties or even become injurious can help to restore their homeostasis. We used single-cell and spatial RNA sequencing in the spinal cord of young (6-week-old) and middle-aged (52-week-old) mice to determine aging-driven microglial reprogramming at homeostasis or after OxPC injury. We identified numerous aging-associated microglial transcripts including osteopontin elevated in OxPC-treated 52-week-old mice, which correlated with greater neurodegeneration. Osteopontin delivery into the spinal cords of 6-week-old mice worsened OxPC lesions, while its knockdown in 52-week-old lesions attenuated microglial inflammation and axon loss. Thus, elevation of osteopontin and other transcripts in aging disorders including multiple sclerosis perturbs microglial functions contributing to aging-associated neurodegeneration.

Exercise and the brain in multiple sclerosis.

While people with multiple sclerosis (PwMS) historically were advised to avoid physical activity to reduce symptoms such as fatigue, they are now encouraged to remain active and to enlist in programs of exercise. However, despite an extensive current literature that exercise not only increases physical well-being but also their cognition and mental health, many PwMS are not meeting recommended levels of exercise. Here, we emphasize the impact and mechanisms of exercise on functional and structural changes to the brain, including improved connectome, neuroprotection, neurogenesis, oligodendrogenesis, and remyelination. We review evidence from animal models of multiple sclerosis (MS) that exercise protects and repairs the brain, and provide supportive data from clinical studies of PwMS. We introduce the concept of MedXercise, where exercise provides a brain milieu particularly conducive for a brain regenerative medication to act upon. The emphasis on exercise improving brain functions and repair should incentivize PwMS to remain physically active.

Studying the microglia response to oxidized phosphatidylcholine in primary mouse neuron culture and mouse spinal cord.

Oxidized phosphatidylcholine (OxPC) found in multiple sclerosis brain lesions mediates neurodegeneration. Microglia are prominent responders to the OxPC insult, and thus, studying their protective or noxious functions is important to help halt neurodegeneration. Here, we present protocols including cell isolation and culture, animal surgeries, as well as tissue processing and isolation to study the microglia response to OxPC-mediated neurodegeneration in vitro and in vivo. For complete details on the use and execution of this protocol, please refer to Dong et al. (2021).

Oxidized phosphatidylcholines found in multiple sclerosis lesions mediate neurodegeneration and are neutralized by microglia.

Neurodegeneration occurring in multiple sclerosis (MS) contributes to the progression of disability. It is therefore important to identify and neutralize the mechanisms that promote neurodegeneration in MS. Here, we report that oxidized phosphatidylcholines (OxPCs) found in MS lesions, previously identified as end-product markers of oxidative stress, are potent drivers of neurodegeneration. Cultured neurons and oligodendrocytes were killed by OxPCs, and this was ameliorated by microglia. After OxPC injection, mouse spinal cords developed focal demyelinating lesions with prominent axonal loss. The depletion of microglia that accumulated in OxPC lesions exacerbated neurodegeneration. Single-cell RNA sequencing of lesioned spinal cords identified unique subsets of TREM2high mouse microglia responding to OxPC deposition. TREM2 was detected in human MS lesions, and TREM2-/- mice exhibited worsened OxPC lesions. These results identify OxPCs as potent neurotoxins and suggest that enhancing microglia-mediated OxPC clearance via TREM2 could help prevent neurodegeneration in MS.