CXCL10/IgG1 Axis in Multiple Sclerosis as a Potential Predictive Biomarker of Disease Activity.

BACKGROUND AND OBJECTIVES: Multiple sclerosis (MS) is a heterogeneous disease, and its course is difficult to predict. Prediction models can be established by measuring intrathecally synthesized proteins involved in inflammation, glial activation, and CNS injury. METHODS: To determine how these intrathecal proteins relate to the short-term, i.e., 12 months, disease activity in relapsing-remitting MS (RRMS), we measured the intrathecal synthesis of 46 inflammatory mediators and 14 CNS injury or glial activation markers in matched serum and CSF samples from 47 patients with MS (pwMS), i.e., 23 RRMS and 24 clinically isolated syndrome (CIS), undergoing diagnostic lumbar puncture. Subsequently, all pwMS were followed for ≥12 months in a retrospective follow-up study and ultimately classified into "active", i.e., developing clinical and/or radiologic disease activity, n = 18) or "nonactive", i.e., not having disease activity, n = 29. Disease activity in patients with CIS corresponded to conversion to RRMS. Thus, patients with CIS were subclassified as "converters" or "nonconverters" based on their conversion status at the end of a 12-month follow-up. Twenty-seven patients with noninflammatory neurologic diseases were included as negative controls. Data were subjected to differential expression analysis and modeling techniques to define the connectivity arrangement (network) between neuroinflammation and CNS injury relevant to short-term disease activity in RRMS. RESULTS: Lower age and/or higher CXCL13 levels positively distinguished active/converting vs nonactive/nonconverting patients. Network analysis significantly improved the prediction of short-term disease activity because active/converting patients featured a stronger positive connection between IgG1 and CXCL10. Accordingly, analysis of disease activity-free survival demonstrated that pwMS, both RRMS and CIS, with a lower or negative IgG1-CXCL10 correlation, have a higher probability of activity-free survival than the patients with a significant correlation (p < 0.0001, HR ≥ 2.87). DISCUSSION: Findings indicate that a significant IgG1-CXCL10 positive correlation predicts the risk of short-term disease activity in patients with RRMS and CIS. Thus, the present results can be used to develop a predictive model for MS activity and conversion to RRMS.

TDP-43-stratified single-cell proteomics of postmortem human spinal motor neurons reveals protein dynamics in amyotrophic lateral sclerosis.

A limitation of conventional bulk-tissue proteome studies in amyotrophic lateral sclerosis (ALS) is the confounding of motor neuron (MN) signals by admixed non-MN proteins. Here, we leverage laser capture microdissection and nanoPOTS single-cell mass spectrometry-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control tissues. In a follow-up analysis, we examine the impact of stratification of MNs based on cytoplasmic transactive response DNA-binding protein 43 (TDP-43)+ inclusion pathology on the profiles of 2,238 proteins. We report extensive overlap in differentially abundant proteins identified in ALS MNs with or without overt TDP-43 pathology, suggesting early and sustained dysregulation of cellular respiration, mRNA splicing, translation, and vesicular transport in ALS. Together, these data provide insights into proteome-level changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein dynamics in human neurologic diseases.

High throughput method for detecting murine brain atrophy using a clinical 3T MRI.

BACKGROUND: There is a lack of understanding of the mechanisms by which the CNS is injured in multiple sclerosis (MS). Since Theiler's murine encephalomyelitis virus (TMEV) infection in SJL/J mice is an established model of progressive disability in MS, and CNS atrophy correlates with progressive disability in MS, we used in vivo MRI to quantify total ventricular volume in TMEV infection. We then sought to identify immunological and virological biomarkers that correlated with increased ventricular size. METHODS: Mice, both infected and control, were followed for 6 months. Cerebral ventricular volumes were determined by MRI, and disability was assessed by Rotarod. A range of immunological and virological measures was obtained using standard techniques. RESULTS: Disability was present in infected mice with enlarged ventricles, while infected mice without enlarged ventricles had Rotarod performance similar to sham mice. Ventricular enlargement was detected as soon as 1 month after infection. None of the immunological and virological measures correlated with the development of ventricular enlargement. CONCLUSIONS: These results support TMEV infection with brain MRI monitoring as a useful model for exploring the biology of disability progression in MS, but they did not identify an immunological or virological correlate with ventricular enlargement.

TDP-43-stratified single-cell proteomic profiling of postmortem human spinal motor neurons reveals protein dynamics in amyotrophic lateral sclerosis.

Unbiased proteomics has been employed to interrogate central nervous system (CNS) tissues (brain, spinal cord) and fluid matrices (CSF, plasma) from amyotrophic lateral sclerosis (ALS) patients; yet, a limitation of conventional bulk tissue studies is that motor neuron (MN) proteome signals may be confounded by admixed non-MN proteins. Recent advances in trace sample proteomics have enabled quantitative protein abundance datasets from single human MNs (Cong et al., 2020b). In this study, we leveraged laser capture microdissection (LCM) and nanoPOTS (Zhu et al., 2018c) single-cell mass spectrometry (MS)-based proteomics to query changes in protein expression in single MNs from postmortem ALS and control donor spinal cord tissues, leading to the identification of 2515 proteins across MNs samples (>900 per single MN) and quantitative comparison of 1870 proteins between disease groups. Furthermore, we studied the impact of enriching/stratifying MN proteome samples based on the presence and extent of immunoreactive, cytoplasmic TDP-43 inclusions, allowing identification of 3368 proteins across MNs samples and profiling of 2238 proteins across TDP-43 strata. We found extensive overlap in differential protein abundance profiles between MNs with or without obvious TDP-43 cytoplasmic inclusions that together point to early and sustained dysregulation of oxidative phosphorylation, mRNA splicing and translation, and retromer-mediated vesicular transport in ALS. Our data are the first unbiased quantification of single MN protein abundance changes associated with TDP-43 proteinopathy and begin to demonstrate the utility of pathology-stratified trace sample proteomics for understanding single-cell protein abundance changes in human neurologic diseases.

Theiler's virus-induced demyelinating disease as an infectious model of progressive multiple sclerosis.

Multiple sclerosis (MS) is a neuroinflammatory and neurodegenerative disease of unknown etiology. However, several studies suggest that infectious agents, e.g., Human Herpes Viruses (HHV), may be involved in triggering the disease. Molecular mimicry, bystander effect, and epitope spreading are three mechanisms that can initiate immunoreactivity leading to CNS autoimmunity in MS. Theiler's murine encephalomyelitis virus (TMEV)-induced demyelinating disease (TMEV-IDD) is a pre-clinical model of MS in which intracerebral inoculation of TMEV results in a CNS autoimmune disease that causes demyelination, neuroaxonal damage, and progressive clinical disability. Given the spectra of different murine models used to study MS, this review highlights why TMEV-IDD represents a valuable tool for testing the viral hypotheses of MS. We initially describe how the main mechanisms of CNS autoimmunity have been identified across both MS and TMEV-IDD etiology. Next, we discuss how adaptive, innate, and CNS resident immune cells contribute to TMEV-IDD immunopathology and how this relates to MS. Lastly, we highlight the sexual dimorphism observed in TMEV-IDD and MS and how this may be tied to sexually dimorphic responses to viral infections. In summary, TMEV-IDD is an underutilized murine model that recapitulates many unique aspects of MS; as we learn more about the nature of viral infections in MS, TMEV-IDD will be critical in testing the future therapeutics that aim to intervene with disease onset and progression.

Divergent complement system activation in two clinically distinct murine models of multiple sclerosis.

Multiple sclerosis (MS) is a neurological disease featuring neuroinflammation and neurodegeneration in young adults. So far, most research has focused on the peripheral immune system, which appears to be the driver of acute relapses. Concurrently, the mechanisms underlying neurodegeneration in the progressive forms of the disease remain unclear. The complement system, a molecular component of the innate immunity, has been recently implicated in several neurological disorders, including MS. However, it is still unknown if the complement proteins detected in the central nervous system (CNS) are actively involved in perpetuating chronic inflammation and neurodegeneration. To address this knowledge gap, we compared two clinically distinct mouse models of MS: 1) proteolipid protein (PLP)-induced experimental autoimmune encephalomyelitis (rEAE) resembling a relapsing-remitting disease course, and 2) Theiler's murine encephalomyelitis virus-induced demyelinating disease (TMEV-IDD) resembling a progressive disease. Real-time PCR was performed in the spinal cord of rEAE mice, TMEV-IDD mice and age-matched sham controls to quantify gene expression for a broad range of complement components. In both experimental models, we found significantly increased expression of complement factors, such as C1q, C3, CfB, and C3aR. We showed that the complement system, specifically the classical complement pathway, was associated with TMEV-IDD pathogenesis, as the expression of C1q, C3 and C3aR1 were all significantly correlated to a worse disease outcome (all P≤0.0168). In line with this finding, C1q and C3 deposition was observed in the spinal cord of TMEV-IDD mice. Furthermore, C1q deposition was detected in spinal cord regions characterized by inflammation, demyelination, and axonal damage. Conversely, activation of the classical complement cascade seemed to result in protection from rEAE (C1q: P=0.0307). Interestingly, the alternative pathway related to a worse disease outcome in rEAE (CFb: P=0.0006). Overall, these results indicate potential divergent roles for the complement system in MS. The chronic-progressive disease form is more reliant on the activation of the classic complement pathway, while protecting from acute relapses. Conversely, relapsing MS appears more likely affected by the alternative pathway. Understanding the functions of the complement system in MS is critical and can lead to better, more targeted therapies in the future.

Cell-Specific Transcriptional Responses to Heat Shock in the Mouse Utricle Epithelium.

Sensory epithelia of the inner ear contain mechanosensory hair cells (HCs) and glia-like supporting cells (SCs), both of which are required for hearing and balance functions. Each of these cell types has unique responses to ototoxic and cytoprotective stimuli. Non-lethal heat stress in the mammalian utricle induces heat shock proteins (HSPs) and protects against ototoxic drug-induced hair cell death. Induction of HSPs in the utricle demonstrates cell-type specificity at the protein level, with HSP70 induction occurring primarily in SCs, while HSP32 (also known as heme oxygenase 1, HMOX1) is induced primarily in resident macrophages. Neither of these HSPs are robustly induced in HCs, suggesting that HCs may have little capacity for induction of stress-induced protective responses. To determine the transcriptional responses to heat shock of these different cell types, we performed cell-type-specific transcriptional profiling using the RiboTag method, which allows for immunoprecipitation (IP) of actively translating mRNAs from specific cell types. RNA-Seq differential gene expression analyses demonstrated that the RiboTag method identified known cell type-specific markers as well as new markers for HCs and SCs. Gene expression differences suggest that HCs and SCs exhibit differential transcriptional heat shock responses. The chaperonin family member Cct8 was significantly enriched only in heat-shocked HCs, while Hspa1l (HSP70 family), and Hspb1 and Cryab (HSP27 and HSP20 families, respectively) were enriched only in SCs. Together our data indicate that HCs exhibit a limited but unique heat shock response, and SCs exhibit a broader and more robust transcriptional response to protective heat stress.

Isolating Central Nervous System Tissues and Associated Meninges for the Downstream Analysis of Immune cells.

The central nervous system (CNS) is comprised of the brain and spinal cord and is enveloped by the meninges, membranous layers serving as a barrier between the periphery and the CNS. The CNS is an immunologically specialized site, and in steady state conditions, immune privilege is most evident in the CNS parenchyma. In contrast, the meninges harbor a diverse array of resident cells, including innate and adaptive immune cells. During inflammatory conditions triggered by CNS injury, autoimmunity, infection, or even neurodegeneration, peripherally derived immune cells may enter the parenchyma and take up residence within the meninges. These cells are thought to perform both beneficial and detrimental actions during CNS disease pathogenesis. Despite this knowledge, the meninges are often overlooked when analyzing the CNS compartment, because conventional CNS tissue extraction methods omit the meningeal layers. This protocol presents two distinct methods for the rapid isolation of murine CNS tissues (i.e., brain, spinal cord, and meninges) that are suitable for downstream analysis via single-cell techniques, immunohistochemistry, and in situ hybridization methods. The described methods provide a comprehensive analysis of CNS tissues, ideal for assessing the phenotype, function, and localization of cells occupying the CNS compartment under homeostatic conditions and during disease pathogenesis.

Exosomes mediate sensory hair cell protection in the inner ear.

Hair cells, the mechanosensory receptors of the inner ear, are responsible for hearing and balance. Hair cell death and consequent hearing loss are common results of treatment with ototoxic drugs, including the widely used aminoglycoside antibiotics. Induction of heat shock proteins (HSPs) confers protection against aminoglycoside-induced hair cell death via paracrine signaling that requires extracellular heat shock 70-kDa protein (HSP70). We investigated the mechanisms underlying this non-cell-autonomous protective signaling in the inner ear. In response to heat stress, inner ear tissue releases exosomes that carry HSP70 in addition to canonical exosome markers and other proteins. Isolated exosomes from heat-shocked utricles were sufficient to improve survival of hair cells exposed to the aminoglycoside antibiotic neomycin, whereas inhibition or depletion of exosomes from the extracellular environment abolished the protective effect of heat shock. Hair cell-specific expression of the known HSP70 receptor TLR4 was required for the protective effect of exosomes, and exosomal HSP70 interacted with TLR4 on hair cells. Our results indicate that exosomes are a previously undescribed mechanism of intercellular communication in the inner ear that can mediate nonautonomous hair cell survival. Exosomes may hold potential as nanocarriers for delivery of therapeutics against hearing loss.

Quantitative Measurement of Intrathecally Synthesized Proteins in Mice.

Cerebrospinal fluid (CSF), a fluid found in the brain and the spinal cord, is of great importance to both basic and clinical science. The analysis of the CSF protein composition delivers crucial information in basic neuroscience research as well as neurological diseases. One caveat is that proteins measured in CSF may derive from both intrathecal synthesis and transudation from serum, and protein analysis of CSF can only determine the sum of these two components. To discriminate between protein transudation from the blood and intrathecally produced proteins in animal models as well as in humans, CSF protein profiling measurements using conventional protein analysis tools must include the calculation of the albumin CSF/serum quotient (Qalbumin), a marker of the integrity of the blood-brain interface (BBI), and the protein index (Qprotein/Qalbumin), an estimate of intrathecal protein synthesis. This protocol illustrates the entire procedure, from CSF and blood collection to quotients and indices calculations, for the quantitative measurement of intrathecal protein synthesis and BBI impairment in mouse models of neurological disorders.

Inflammation alters AMPA-stimulated calcium responses in dorsal striatal D2 but not D1 spiny projection neurons.

Neuroinflammation precedes neuronal loss in striatal neurodegenerative diseases and can be exacerbated by the release of proinflammatory molecules by microglia. These molecules can affect trafficking of AMPARs. The preferential trafficking of calcium-permeable versus impermeable AMPARs can result in disruptions of [Ca2+ ]i and alter cellular functions. In striatal neurodegenerative diseases, changes in [Ca2+ ]i and L-type voltage-gated calcium channels (VGCCs) have been reported. Therefore, this study sought to determine whether a proinflammatory environment alters AMPA-stimulated [Ca2+ ]i through calcium-permeable AMPARs and/or L-type VGCCs in dopamine-2- and dopamine-1-expressing striatal spiny projection neurons (D2 and D1 SPNs) in the dorsal striatum. Mice expressing the calcium indicator protein, GCaMP in D2 or D1 SPNs, were utilized for calcium imaging. Microglial activation was assessed by morphology analyses. To induce inflammation, acute mouse striatal slices were incubated with lipopolysaccharide (LPS). Here we report that LPS treatment potentiated AMPA responses only in D2 SPNs. When a nonspecific VGCC blocker was included, we observed a decrease of AMPA-stimulated calcium fluorescence in D2 but not D1 SPNs. The remaining agonist-induced [Ca2+ ]i was mediated by calcium-permeable AMPARs because the responses were completely blocked by a selective calcium-permeable AMPAR antagonist. We used isradipine, the highly selective L-type VGCC antagonist to determine the role of L-type VGCCs in SPNs treated with LPS. Isradipine decreased AMPA-stimulated responses selectively in D2 SPNs after LPS treatment. Our findings suggest that dorsal striatal D2 SPNs are specifically targeted in proinflammatory conditions and that L-type VGCCs and calcium-permeable AMPARs are important mediators of this effect.