Cellular architecture of evolving neuroinflammatory lesions and multiple sclerosis pathology.

Multiple sclerosis (MS) is a neurological disease characterized by multifocal lesions and smoldering pathology. Although single-cell analyses provided insights into cytopathology, evolving cellular processes underlying MS remain poorly understood. We investigated the cellular dynamics of MS by modeling temporal and regional rates of disease progression in mouse experimental autoimmune encephalomyelitis (EAE). By performing single-cell spatial expression profiling using in situ sequencing (ISS), we annotated disease neighborhoods and found centrifugal evolution of active lesions. We demonstrated that disease-associated (DA)-glia arise independently of lesions and are dynamically induced and resolved over the disease course. Single-cell spatial mapping of human archival MS spinal cords confirmed the differential distribution of homeostatic and DA-glia, enabled deconvolution of active and inactive lesions into sub-compartments, and identified new lesion areas. By establishing a spatial resource of mouse and human MS neuropathology at a single-cell resolution, our study unveils the intricate cellular dynamics underlying MS.

Small-molecule-induced epigenetic rejuvenation promotes SREBP condensation and overcomes barriers to CNS myelin regeneration.

Remyelination failure in diseases like multiple sclerosis (MS) was thought to involve suppressed maturation of oligodendrocyte precursors; however, oligodendrocytes are present in MS lesions yet lack myelin production. We found that oligodendrocytes in the lesions are epigenetically silenced. Developing a transgenic reporter labeling differentiated oligodendrocytes for phenotypic screening, we identified a small-molecule epigenetic-silencing-inhibitor (ESI1) that enhances myelin production and ensheathment. ESI1 promotes remyelination in animal models of demyelination and enables de novo myelinogenesis on regenerated CNS axons. ESI1 treatment lengthened myelin sheaths in human iPSC-derived organoids and augmented (re)myelination in aged mice while reversing age-related cognitive decline. Multi-omics revealed that ESI1 induces an active chromatin landscape that activates myelinogenic pathways and reprograms metabolism. Notably, ESI1 triggered nuclear condensate formation of master lipid-metabolic regulators SREBP1/2, concentrating transcriptional co-activators to drive lipid/cholesterol biosynthesis. Our study highlights the potential of targeting epigenetic silencing to enable CNS myelin regeneration in demyelinating diseases and aging.

Immunology of relapse and remission in multiple sclerosis.

Eighty percent of individuals with multiple sclerosis (MS) initially develop a clinical pattern with periodic relapses followed by remissions, called relapsing-remitting MS (RRMS). This period of fluctuating disease may last for a decade or more. Clinical relapses reflect acute inflammation in the central nervous system (CNS), composed of the brain and spinal cord. Often, different anatomic areas in the CNS are involved each time a relapse occurs, resulting in varied clinical manifestations in each instance. Relapses are nearly always followed by some degree of remission, though recovery to baseline status before the flare is often incomplete. There are nine approved drugs for treatment of RRMS. The most potent drug for inhibiting relapses, the humanized anti-α4 integrin antibody known as Natalizumab, blocks homing of mononuclear cells to the CNS. The mechanisms of action of the approved drugs for RRMS provide a strong foundation for understanding the pathobiology of the relapse. Despite substantial progress in controlling relapses with the current armamentarium of medications, there is much to learn and ever more effective and safe therapies to develop.

Cell Type-Specific Intralocus Interactions Reveal Oligodendrocyte Mechanisms in MS.

Multiple sclerosis (MS) is an autoimmune disease characterized by attack on oligodendrocytes within the central nervous system (CNS). Despite widespread use of immunomodulatory therapies, patients may still face progressive disability because of failure of myelin regeneration and loss of neurons, suggesting additional cellular pathologies. Here, we describe a general approach for identifying specific cell types in which a disease allele exerts a pathogenic effect. Applying this approach to MS risk loci, we pinpoint likely pathogenic cell types for 70%. In addition to T cell loci, we unexpectedly identified myeloid- and CNS-specific risk loci, including two sites that dysregulate transcriptional pause release in oligodendrocytes. Functional studies demonstrated inhibition of transcriptional elongation is a dominant pathway blocking oligodendrocyte maturation. Furthermore, pause release factors are frequently dysregulated in MS brain tissue. These data implicate cell-intrinsic aberrations outside of the immune system and suggest new avenues for therapeutic development. VIDEO ABSTRACT.

Propionic Acid Shapes the Multiple Sclerosis Disease Course by an Immunomodulatory Mechanism.

Short-chain fatty acids are processed from indigestible dietary fibers by gut bacteria and have immunomodulatory properties. Here, we investigate propionic acid (PA) in multiple sclerosis (MS), an autoimmune and neurodegenerative disease. Serum and feces of subjects with MS exhibited significantly reduced PA amounts compared with controls, particularly after the first relapse. In a proof-of-concept study, we supplemented PA to therapy-naive MS patients and as an add-on to MS immunotherapy. After 2 weeks of PA intake, we observed a significant and sustained increase of functionally competent regulatory T (Treg) cells, whereas Th1 and Th17 cells decreased significantly. Post-hoc analyses revealed a reduced annual relapse rate, disability stabilization, and reduced brain atrophy after 3 years of PA intake. Functional microbiome analysis revealed increased expression of Treg-cell-inducing genes in the intestine after PA intake. Furthermore, PA normalized Treg cell mitochondrial function and morphology in MS. Our findings suggest that PA can serve as a potent immunomodulatory supplement to MS drugs.

Conceptual and Experimental Tools to Understand Spatial Effects and Transport Phenomena in Nonlinear Biochemical Networks Illustrated with Patchy Switching.

Many biochemical systems are spatially heterogeneous and exhibit nonlinear behaviors, such as state switching in response to small changes in the local concentration of diffusible molecules. Systems as varied as blood clotting, intracellular calcium signaling, and tissue inflammation are all heavily influenced by the balance of rates of reaction and mass transport phenomena including flow and diffusion. Transport of signaling molecules is also affected by geometry and chemoselective confinement via matrix binding. In this review, we use a phenomenon referred to as patchy switching to illustrate the interplay of nonlinearities, transport phenomena, and spatial effects. Patchy switching describes a change in the state of a network when the local concentration of a diffusible molecule surpasses a critical threshold. Using patchy switching as an example, we describe conceptual tools from nonlinear dynamics and chemical engineering that make testable predictions and provide a unifying description of the myriad possible experimental observations. We describe experimental microfluidic and biochemical tools emerging to test conceptual predictions by controlling transport phenomena and spatial distribution of diffusible signals, and we highlight the unmet need for in vivo tools.

Intestinal epithelial dopamine receptor signaling drives sex-specific disease exacerbation in a mouse model of multiple sclerosis.

Although the gut microbiota can influence central nervous system (CNS) autoimmune diseases, the contribution of the intestinal epithelium to CNS autoimmunity is less clear. Here, we showed that intestinal epithelial dopamine D2 receptors (IEC DRD2) promoted sex-specific disease progression in an animal model of multiple sclerosis. Female mice lacking Drd2 selectively in intestinal epithelial cells showed a blunted inflammatory response in the CNS and reduced disease progression. In contrast, overexpression or activation of IEC DRD2 by phenylethylamine administration exacerbated disease severity. This was accompanied by altered lysozyme expression and gut microbiota composition, including reduced abundance of Lactobacillus species. Furthermore, treatment with N2-acetyl-L-lysine, a metabolite derived from Lactobacillus, suppressed microglial activation and neurodegeneration. Taken together, our study indicates that IEC DRD2 hyperactivity impacts gut microbial abundances and increases susceptibility to CNS autoimmune diseases in a female-biased manner, opening up future avenues for sex-specific interventions of CNS autoimmune diseases.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (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.

The neuropathobiology of multiple sclerosis.

Chronic low-grade inflammation and neuronal deregulation are two components of a smoldering disease activity that drives the progression of disability in people with multiple sclerosis (MS). Although several therapies exist to dampen the acute inflammation that drives MS relapses, therapeutic options to halt chronic disability progression are a major unmet clinical need. The development of such therapies is hindered by our limited understanding of the neuron-intrinsic determinants of resilience or vulnerability to inflammation. In this Review, we provide a neuron-centric overview of recent advances in deciphering neuronal response patterns that drive the pathology of MS. We describe the inflammatory CNS environment that initiates neurotoxicity by imposing ion imbalance, excitotoxicity and oxidative stress, and by direct neuro-immune interactions, which collectively lead to mitochondrial dysfunction and epigenetic dysregulation. The neuronal demise is further amplified by breakdown of neuronal transport, accumulation of cytosolic proteins and activation of cell death pathways. Continuous neuronal damage perpetuates CNS inflammation by activating surrounding glia cells and by directly exerting toxicity on neighbouring neurons. Further, we explore strategies to overcome neuronal deregulation in MS and compile a selection of neuronal actuators shown to impact neurodegeneration in preclinical studies. We conclude by discussing the therapeutic potential of targeting such neuronal actuators in MS, including some that have already been tested in interventional clinical trials.

Iron Drives T Helper Cell Pathogenicity by Promoting RNA-Binding Protein PCBP1-Mediated Proinflammatory Cytokine Production.

Iron deposition is frequently observed in human autoinflammatory diseases, but its functional significance is largely unknown. Here we showed that iron promoted proinflammatory cytokine expression in T cells, including GM-CSF and IL-2, via regulating the stability of an RNA-binding protein PCBP1. Iron depletion or Pcbp1 deficiency in T cells inhibited GM-CSF production by attenuating Csf2 3' untranslated region (UTR) activity and messenger RNA stability. Pcbp1 deficiency or iron uptake blockade in autoreactive T cells abolished their capacity to induce experimental autoimmune encephalomyelitis, an animal model for multiple sclerosis. Mechanistically, intracellular iron protected PCBP1 protein from caspase-mediated proteolysis, and PCBP1 promoted messenger RNA stability of Csf2 and Il2 by recognizing UC-rich elements in the 3' UTRs. Our study suggests that iron accumulation can precipitate autoimmune diseases by promoting proinflammatory cytokine production. RNA-binding protein-mediated iron sensing may represent a simple yet effective means to adjust the inflammatory response to tissue homeostatic alterations.

Exosomal miR-23a-3p derived from human umbilical cord mesenchymal stem cells promotes remyelination in central nervous system demyelinating diseases by targeting Tbr1/Wnt pathway.

Oligodendrocyte precursor cells are present in the adult central nervous system, and their impaired ability to differentiate into myelinating oligodendrocytes can lead to demyelination in patients with multiple sclerosis, accompanied by neurological deficits and cognitive impairment. Exosomes, small vesicles released by cells, are known to facilitate intercellular communication by carrying bioactive molecules. In this study, we utilized exosomes derived from human umbilical cord mesenchymal stem cells (HUMSCs-Exos). We performed sequencing and bioinformatics analysis of exosome-treated cells to demonstrate that HUMSCs-Exos can stimulate myelin gene expression in oigodendrocyte precursor cells. Functional investigations revealed that HUMSCs-Exos activate the Pi3k/Akt pathway and regulate the Tbr1/Wnt signaling molecules through the transfer of miR-23a-3p, promoting oligodendrocytes differentiation and enhancing the expression of myelin-related proteins. In an experimental autoimmune encephalomyelitis model, treatment with HUMSCs-Exos significantly improved neurological function and facilitated remyelination. This study provides cellular and molecular insights into the use of cell-free exosome therapy for central nervous system demyelination associated with multiple sclerosis, demonstrating its great potential for treating demyelinating and neurodegenerative diseases.

Autotaxin in encephalitogenic CD4 T cells as a therapeutic target for multiple sclerosis.

Multiple sclerosis (MS) is an immune-mediated inflammatory disease of the CNS. A defining characteristic of MS is the ability of autoreactive T lymphocytes to cross the blood-brain barrier and mediate inflammation within the CNS. Previous work from our lab found the gene Enpp2 to be highly upregulated in murine encephalitogenic T cells. Enpp2 encodes for the protein autotaxin, a secreted glycoprotein that catalyzes the production of lysophosphatidic acid and promotes transendothelial migration of T cells from the bloodstream into the lymphatic system. The present study sought to characterize autotaxin expression in T cells during CNS autoimmune disease and determine its potential therapeutic value. Myelin-activated CD4 T cells upregulated expression of autotaxin in vitro, and ex vivo analysis of CNS-infiltrating CD4 T cells showed significantly higher autotaxin expression compared with cells from healthy mice. In addition, inhibiting autotaxin in myelin-specific T cells reduced their encephalitogenicity in adoptive transfer studies and decreased in vitro cell motility. Importantly, using two mouse models of MS, treatment with an autotaxin inhibitor ameliorated EAE severity, decreased the number of CNS infiltrating T and B cells, and suppressed relapses, suggesting autotaxin may be a promising therapeutic target in the treatment of MS.

Defective sphingosine 1-phosphate receptor 1 (S1P1) phosphorylation exacerbates TH17-mediated autoimmune neuroinflammation.

Sphingosine 1-phosphate (S1P) signaling regulates lymphocyte egress from lymphoid organs into systemic circulation. The sphingosine phosphate receptor 1 (S1P1) agonist FTY-720 (Gilenya) arrests immune trafficking and prevents multiple sclerosis (MS) relapses. However, alternative mechanisms of S1P-S1P1 signaling have been reported. Phosphoproteomic analysis of MS brain lesions revealed S1P1 phosphorylation on S351, a residue crucial for receptor internalization. Mutant mice harboring an S1pr1 gene encoding phosphorylation-deficient receptors (S1P1(S5A)) developed severe experimental autoimmune encephalomyelitis (EAE) due to autoimmunity mediated by interleukin 17 (IL-17)-producing helper T cells (TH17 cells) in the peripheral immune and nervous system. S1P1 directly activated the Jak-STAT3 signal-transduction pathway via IL-6. Impaired S1P1 phosphorylation enhances TH17 polarization and exacerbates autoimmune neuroinflammation. These mechanisms may be pathogenic in MS.

Determinants and Biomarkers of Progression Independent of Relapses in Multiple Sclerosis.

Clinical, pathological, and imaging evidence in multiple sclerosis (MS) suggests that a smoldering inflammatory activity is present from the earliest stages of the disease and underlies the progression of disability, which proceeds relentlessly and independently of clinical and radiological relapses (PIRA). The complex system of pathological events driving "chronic" worsening is likely linked with the early accumulation of compartmentalized inflammation within the central nervous system as well as insufficient repair phenomena and mitochondrial failure. These mechanisms are partially lesion-independent and differ from those causing clinical relapses and the formation of new focal demyelinating lesions; they lead to neuroaxonal dysfunction and death, myelin loss, glia alterations, and finally, a neuronal network dysfunction outweighing central nervous system (CNS) compensatory mechanisms. This review aims to provide an overview of the state of the art of neuropathological, immunological, and imaging knowledge about the mechanisms underlying the smoldering disease activity, focusing on possible early biomarkers and their translation into clinical practice. ANN NEUROL 2024;96:1-20.

Transcript Profiles of Microglia/Macrophage Cells at the Borders of Chronic Active and Subpial Gray Matter Lesions in Multiple Sclerosis.

OBJECTIVE: Microglia/macrophages line the border of demyelinated lesions in both cerebral white matter and the cortex in the brains of multiple sclerosis patients. Microglia/macrophages associated with chronic white matter lesions are thought to be responsible for slow lesion expansion and disability progression in progressive multiple sclerosis, whereas those lining gray matter lesions are less studied. Profiling these microglia/macrophages could help to focus therapies on genes or pathways specific to lesion expansion and disease progression. METHODS: We compared the morphology and transcript profiles of microglia/macrophages associated with borders of white matter (WM line) and subpial gray matter lesions (GM line) using laser capture microscopy. We performed RNA sequencing on isolated cells followed by immunocytochemistry to determine the distribution of translational products of transcripts increased in WM line microglia. RESULTS: Cells in the WM line appear activated, with shorter processes and larger cell bodies, whereas those in the GM line appear more homeostatic, with smaller cell bodies and multiple thin processes. Transcript profiling revealed 176 genes in WM lines and 111 genes in GM lines as differentially expressed. Transcripts associated with immune activation and iron homeostasis were increased in WM line microglia, whereas genes belonging to the canonical Wnt signaling pathway were increased in GM line microglia. INTERPRETATION: We propose that the mechanisms of demyelination and dynamics of lesion expansion are responsible for differential transcript expression in WM lines and GM lines, and posit that increased expression of the Fc epsilon receptor, spleen tyrosine kinase, and Bruton's tyrosine kinase, play a key role in regulating microglia/macrophage function at the border of chronic active white matter lesions. ANN NEUROL 2024;95:907-916.

Prevention of experimental autoimmune encephalomyelitis by targeting 6-sulfo sialyl Lewis X glycans involved in lymphocyte homing.

Lymphocyte homing to peripheral lymph nodes (PLN) is critical for immune surveillance. However, autoimmune diseases such as multiple sclerosis (MS) can occur due to excessive immune responses in the PLN. Here we show that 6-sulfo sialyl Lewis X (6-sulfo sLex) glycans on high endothelial venules that function as ligands for l-selectin on lymphocytes play a critical role in the pathogenesis of experimental autoimmune encephalomyelitis (EAE), an animal model of MS. In N-acetylglucosamine-6-O-sulfotransferase (GlcNAc6ST)-1 and GlcNAc6ST-2 double-knockout mice lacking the expression of 6-sulfo sLeX glycans, the EAE symptoms and the numbers of effector Th1 and Th17 cells in the draining lymph nodes (dLN) and spinal cords (SC) were significantly reduced. To determine whether 6-sulfo sLeX could serve as a target for MS, we also examined the effects of anti-glycan monoclonal antibody (mAb) SF1 against 6-sulfo sLeX in EAE. Administration of mAb SF1 significantly reduced EAE symptoms and the numbers of antigen-specific effector T cells in the dLN and SC in association with suppression of critical genes including Il17a and Il17f that are involved in the pathogenesis of EAE. Taken together, these results suggest that 6-sulfo sLeX glycan would serve as a novel target for MS.

p38γ MAPK delays myelination and remyelination and is abundant in multiple sclerosis lesions.

Multiple sclerosis is a chronic inflammatory disease in which disability results from the disruption of myelin and axons. During the initial stages of the disease, injured myelin is replaced by mature myelinating oligodendrocytes that differentiate from oligodendrocyte precursor cells. However, myelin repair fails in secondary and chronic progressive stages of the disease and with ageing, as the environment becomes progressively more hostile. This may be attributable to inhibitory molecules in the multiple sclerosis environment including activation of the p38MAPK family of kinases. We explored oligodendrocyte precursor cell differentiation and myelin repair using animals with conditional ablation of p38MAPKγ from oligodendrocyte precursors. We found that p38γMAPK ablation accelerated oligodendrocyte precursor cell differentiation and myelination. This resulted in an increase in both the total number of oligodendrocytes and the migration of progenitors ex vivo and faster remyelination in the cuprizone model of demyelination/remyelination. Consistent with its role as an inhibitor of myelination, p38γMAPK was significantly downregulated as oligodendrocyte precursor cells matured into oligodendrocytes. Notably, p38γMAPK was enriched in multiple sclerosis lesions from patients. Oligodendrocyte progenitors expressed high levels of p38γMAPK in areas of failed remyelination but did not express detectable levels of p38γMAPK in areas where remyelination was apparent. Our data suggest that p38γ could be targeted to improve myelin repair in multiple sclerosis.

Sphingosine 1-phosphate receptor signaling.

The sphingosine 1-phosphate (S1P) receptor signaling system is a productive model system. A hydrophobic zwitterionic lysophospholipid ligand with difficult physical properties interacts with five high-affinity G protein-coupled receptors to generate multiple downstream signals. These signals modulate homeostasis and pathology on a steep agonist concentration-response curve. Ligand presence is essential for vascular development and endothelial integrity, while acute increases in ligand concentrations result in cardiac death. Understanding this integrated biochemical system has exemplified the impact of both genetics and chemistry. Developing specific tools with defined biochemical properties for the reversible modulation of signals in real time has been essential to complement insights gained from genetic approaches that may be irreversible and compensated. Despite its knife-edge between life and death, this system, based in part on receptor subtype-selectivity and in part on differential attenuation of deleterious signals, now appears to be on the cusp of meaningful therapy for multiple sclerosis.

Role of ferroptosis in neuroimmunity and neurodegeneration in multiple sclerosis revealed by multi-omics data.

Previous studies have found that ferroptosis plays an important role in a variety of neurological diseases. However, the precise role of ferroptosis in the multiple sclerosis patients remains uncertain. We defined and validated a computational metric of ferroptosis levels. The ferroptosis scores were computed using the AUCell method, which reflects the enrichment scores of ferroptosis-related genes through gene ranking. The reliability of the ferroptosis score was assessed using various methods, involving cells induced to undergo ferroptosis by six different ferroptosis inducers. Through a comprehensive approach integrating snRNA-seq, spatial transcriptomics, and spatial proteomics data, we explored the role of ferroptosis in multiple sclerosis. Our findings revealed that among seven sampling regions of different white matter lesions, the edges of active lesions exhibited the highest ferroptosis score, which was associated with activation of the phagocyte system. Remyelination lesions exhibit the lowest ferroptosis score. In the cortex, ferroptosis score were elevated in neurons, relevant to a variety of neurodegenerative disease-related pathways. Spatial transcriptomics demonstrated a significant co-localization among ferroptosis score, neurodegeneration and microglia, which was verified by spatial proteomics. Furthermore, we established a diagnostic model of multiple sclerosis based on 24 ferroptosis-related genes in the peripheral blood. Ferroptosis might exhibits a dual role in the context of multiple sclerosis, relevant to both neuroimmunity and neurodegeneration, thereby presenting a promising and novel therapeutic target. Ferroptosis-related genes in the blood that could potentially serve as diagnostic and prognostic markers for multiple sclerosis.

Citrullinated human and murine MOG35-55 display distinct biophysical and biochemical behavior.

The peptide spanning residues 35 to 55 of the protein myelin oligodendrocyte glycoprotein (MOG) has been studied extensively in its role as a key autoantigen in the neuroinflammatory autoimmune disease multiple sclerosis. Rodents and nonhuman primate species immunized with this peptide develop a neuroinflammatory condition called experimental autoimmune encephalomyelitis, often used as a model for multiple sclerosis. Over the last decade, the role of citrullination of this antigen in the disease onset and progression has come under increased scrutiny. We recently reported on the ability of these citrullinated MOG35-55 peptides to aggregate in an amyloid-like fashion, suggesting a new potential pathogenic mechanism underlying this disease. The immunodominant region of MOG is highly conserved between species, with the only difference between the murine and human protein, a polymorphism on position 42, which is serine in mice and proline for humans. Here, we show that the biophysical and biochemical behavior we previously observed for citrullinated murine MOG35-55 is fundamentally different for human and mouse MOG35-55. The citrullinated human peptides do not show amyloid-like behavior under the conditions where the murine peptides do. Moreover, we tested the ability of these peptides to stimulate lymphocytes derived from MOG immunized marmoset monkeys. While the citrullinated murine peptides did not produce a proliferative response, one of the citrullinated human peptides did. We postulate that this unexpected difference is caused by disparate antigen processing. Taken together, our results suggest that further study on the role of citrullination in MOG-induced experimental autoimmune encephalomyelitis is necessary.