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Abstract Oligodendrocyte (OL) injury and subsequent loss is a pathologic hallmark of multiple sclerosis (MS). Stress granules (SGs) are membrane-less organelles containing mRNAs stalled in translation and considered as participants of the cellular response to stress. Here we show SGs in OLs in active and inactive areas of MS lesions as well as in normal-appearing white matter. In cultures of primary human adult brain derived OLs, metabolic stress conditions induce transient SG formation in these cells. Combining pro-inflammatory cytokines, which alone do not induce SG formation, with metabolic stress results in persistence of SGs. Unlike sodium arsenite, metabolic stress induced SG formation is not blocked by the integrated stress response inhibitor. Glycolytic inhibition also induces persistent SGs indicating the dependence of SG formation and disassembly on the energetic glycolytic properties of human OLs. We conclude that SG persistence in OLs in MS reflects their response to a combination of metabolic stress and pro-inflammatory conditions.

Abstract Neurodegeneration is the primary driver of disease progression in multiple sclerosis (MS) resulting in permanent disability, creating an urgent need to discover its underlying mechanisms. Herein, we establish that dysfunction of the RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) results in differential of binding to RNA targets causing alternative RNA splicing, which contributes to neurodegeneration in MS and its models. Using RNAseq of MS brains, we discovered differential expression and aberrant splicing of hnRNP A1 target RNAs involved in neuronal function and RNA homeostasis. We confirmed this in vivo in experimental autoimmune encephalomyelitis employing CLIPseq specific for hnRNP A1, where hnRNP A1 differentially binds and regulates RNA, including aberrantly spliced targets identified in human samples. Additionally, dysfunctional hnRNP A1 expression in neurons caused neurite loss and identical changes in splicing, corroborating hnRNP A1 dysfunction as a cause of neurodegeneration. Collectively, these data indicate hnRNP A1 dysfunction causes altered neuronal RNA splicing, resulting in neurodegeneration in MS.

Abstract Background Multiple sclerosis (MS) is a degenerative disease of the central nervous system (CNS) that disrupts walking function and results in other debilitating symptoms. This study compares the effects of ‘task-oriented exercise’ against ‘generalized resistance and aerobic exercise’ and a ‘stretching control’ on walking and CNS function in people with MS (PwMS). We hypothesize that task-oriented exercise will enhance walking speed and related neural changes to a greater extent than other exercise approaches. Methods This study is a single-blinded, three-arm randomized controlled trial conducted in Saskatchewan, Canada. Eligible participants are those older than 18 years of age with a diagnosis of MS and an expanded Patient-Determined Disease Steps (PDDS) score between 3 (‘gait disability’) and 6 (‘bilateral support’). Exercise interventions are delivered for 12 weeks (3 × 60-min per week) in-person under the supervision of a qualified exercise professional. Interventions differ in exercise approach, such that task-oriented exercise involves weight-bearing, walking-specific activities, while generalized resistance and aerobic exercise uses seated machine-based resistance training of major upper and lower body muscle groups and recumbent cycling, and the stretching control exercise involves seated flexibility and relaxation activities. Participants are allocated to interventions using blocked randomization that stratifies by PDDS (mild: 3–4; moderate: 5–6). Assessments are conducted at baseline, post-intervention, and at a six-week retention time point. The primary and secondary outcome measures are the Timed 25-Foot Walk Test and corticospinal excitability for the tibialis anterior muscles determined using transcranial magnetic stimulation (TMS), respectively. Tertiary outcomes include assessments of balance, additional TMS measures, blood biomarkers of neural health and inflammation, and measures of cardiorespiratory and musculoskeletal fitness. Discussion A paradigm shift in MS healthcare towards the use of “exercise as medicine” was recently proposed to improve outcomes and alleviate the economic burden of MS. Findings will support this shift by informing the development of specialized exercise programming that targets walking and changes in corticospinal excitability in PwMS. Trial registration ClinicalTrials.gov, NCT05496881, Registered August 11, 2022. https://classic.clinicaltrials.gov/ct2/show/NCT05496881 . Protocol amendment number: 01; Issue date: August 1, 2023; Primary reason for amendment: Expand eligibility to include people with all forms of MS rather than progressive forms of MS only.

Background/Objectives: Changes in nuclear morphology, alterations to the nuclear pore complex (NPC), including loss, aggregation, and dysfunction of nucleoporins (Nups), and nucleocytoplasmic transport (NCT) abnormalities have become hallmarks of neurodegenerative diseases. Previous RNA sequencing data utilizing knockdown of heterogeneous nuclear ribonucleoprotein A1 (hnRNP A1) identified enrichment for pathways and changes in RNAs related to nuclear morphology and showed differential expression of key nuclear targets. This suggests that dysfunction of hnRNP A1, which is observed in neurodegenerative diseases, may contribute to abnormalities in nuclear morphology, NPC, and NCT. Methods: We performed knockdown of hnRNP A1 in Neuro-2A cells, a neuronal cell line, to examine nuclear morphology, NPC, and NCT. Results: First, we examined nuclear morphology using Lamin B, wherein we observed increased nuclear envelope abnormalities in cells with hnRNP A1 knockdown as compared to control. To quantify changes in Lamin B, we designed and validated an automated computer-based model, which quantitatively confirmed our observations. Next, we investigated the impact of hnRNP A1 knockdown on components of the NPC and NCT. In line with the previous literature, we found changes in Nups, including altered distribution and reduced protein expression, as well as disrupted NCT. Finally, we validated our findings in multiple sclerosis (MS) brains, a disease with a significant neurodegenerative component caused by hnRNP A1 dysfunction, where neuronal nuclear envelope alterations were significantly increased as compared to controls. Conclusions: Together, these data implicate hnRNP A1 as an important contributor to nuclear morphology, Nup expression and distribution, and NCT and suggest that hnRNP A1 dysfunction may lead to defects in these processes in neurodegenerative diseases.

The RNA binding protein heterogeneous nuclear ribonucleoprotein A1 (A1) regulates RNA metabolism, which is crucial to maintaining cellular homeostasis. A1 dysfunction mechanistically contributes to reduced cell viability and loss, but molecular mechanisms of how A1 dysfunction affects cell viability and loss, and methodologies to attenuate its dysfunction, are lacking. Utilizing in silico molecular modeling and an in vitro optogenetic system, this study examined the consequences of RNA oligonucleotide (RNAO) treatment on attenuating A1 dysfunction and its downstream cellular effects. In silico and thermal shift experiments revealed that binding of RNAOs to the RNA Recognition Motif 1 of A1 is stabilized by sequence- and structure-specific RNAO-A1 interactions. Using optogenetics to model A1 cellular dysfunction, we show that sequence- and structure-specific RNAOs significantly attenuated abnormal cytoplasmic A1 self-association kinetics and A1 cytoplasmic clustering. Downstream of A1 dysfunction, we demonstrate that A1 clustering affects the formation of stress granules, activates cell stress, and inhibits protein translation. With RNAO treatment, we show that stress granule formation is attenuated, cell stress is inhibited, and protein translation is restored. This study provides evidence that sequence- and structure-specific RNAO treatment attenuates A1 dysfunction and its downstream effects, thus allowing for the development of A1-specific therapies that attenuate A1 dysfunction and restore cellular homeostasis.

Neurodegeneration, the progressive loss or damage to neurons and axons, underlies permanent disability in multiple sclerosis (MS); yet its mechanisms are incompletely understood. Recent data indicates autoimmunity to several intraneuronal antigens, including the RNA binding protein (RBP) heterogenous nuclear ribonucleoprotein A1 (hnRNP A1), as contributors to neurodegeneration. We previously showed that addition of anti-hnRNP A1 antibodies, which target the same immunodominant domain of MS IgG, to mice with experimental autoimmune encephalomyelitis (EAE) worsened disease and resulted in an exacerbation of hnRNP A1 dysfunction including cytoplasmic mislocalization of hnRNP A1, stress granule (SG) formation and neurodegeneration in the chronic stages of disease. Because this previous study focused on a singular timepoint during EAE, it is unclear whether anti-hnRNP A1 antibody induced hnRNP A1 dysfunction caused neurodegeneration or was result of it. In the present study, we analyzed in vivo and in vitro models of anti-hnRNP A1 antibody-mediated autoimmunity for markers of hnRNP A1 dysfunction and neurodegeneration over a time course to gain a better understanding of the connection between hnRNP A1 dysfunction and neurodegeneration. Anti-hnRNP A1 antibody treatment resulted in increased neuronal hnRNP A1 mislocalization and nuclear depletion temporally followed by altered RNA expression and SG formation, and lastly an increase in necroptotic signalling and neuronal cell death. Treatment with necrostatin-1s inhibited necroptosis and partially rescued anti-hnRNP A1 antibody-mediated neurodegeneration while clathrin knockdown specifically inhibited anti-hnRNP A1 antibody uptake into neurons. This data identifies a novel antibody-mediated mechanism of neurodegeneration, which may be targeted to inhibit neurodegeneration and prevent permanent neurological decline in persons living with MS.