Orthologous proteins of experimental de- and remyelination are differentially regulated in the CSF proteome of multiple sclerosis subtypes

Abstract

ObjectiveHere, we applied a multi-omics approach (i) to examine molecular pathways related to de- and remyelination in multiple sclerosis (MS) lesions; and (ii) to translate these findings to the CSF proteome in order to identify molecules that are differentially expressed among MS subtypes.MethodsTo relate differentially expressed genes in MS lesions to de- and remyelination, we compared transcriptome of MS lesions to transcriptome of cuprizone (CPZ)-induced de- and remyelination. Protein products of the overlapping orthologous genes were measured within the CSF by quantitative proteomics, parallel reaction monitoring (PRM). Differentially regulated proteins were correlated with molecular markers of inflammation by using MesoScale multiplex immunoassay. Expression kinetics of differentially regulated orthologous genes and proteins were examined in the CPZ model.ResultsIn the demyelinated and remyelinated corpus callosum, we detected 1239 differentially expressed genes; 91 orthologues were also differentially expressed in MS lesions. Pathway analysis of these orthologues suggested that the TYROBP (DAP12)-TREM2 pathway, TNF-receptor 1, CYBA and the proteasome subunit PSMB9 were related to de- and remyelination. We designed 129 peptides representing 51 orthologous proteins, measured them by PRM in 97 individual CSF, and compared their levels between relapsing (n = 40) and progressive MS (n = 57). Four proteins were differentially regulated among relapsing and progressive MS: tyrosine protein kinase receptor UFO (UFO), TIMP-1, apolipoprotein C-II (APOC2), and beta-2-microglobulin (B2M). The orthologous genes/proteins in the mouse brain peaked during acute remyelination. UFO, TIMP-1 and B2M levels correlated inversely with inflammation in the CSF (IL-6, MCP-1/CCL2, TARC/CCL17). APOC2 showed positive correlation with IL-2, IL-16 and eotaxin-3/CCL26.ConclusionsPathology-based multi-omics identified four CSF markers that were differentially expressed in MS subtypes. Upregulated TIMP-1, UFO and B2M orthologues in relapsing MS were associated with reduced inflammation and reflected reparatory processes, in contrast to the upregulated orthologue APOC2 in progressive MS that reflected changes in lipid metabolism associated with increased inflammation.

Proteomic changes during experimental de- and remyelination in the corpus callosum

“…As we demonstrated previously [15], CPZ induced demyelination increases in extent rapidly during the first 4 weeks, then at a much slower rate for the following 2 weeks before the time window for regeneration closes [13]. Accordingly, we followed the same protocol as previously [14,21], namely, 4 weeks of CPZ treatment (demyelination) followed by 2 (early remyelination) and 14 (late remyelination) days of recovery after termination of the CPZ treatment. Proteomic analysis of the corpus callosums resulted in altogether 4886 proteins, however,…”

“…In an attempt to identify key elements regulating de-and remyelination, we isolated and homogenized the corpus callosum from mice exposed to CPZ, and performed liquid-chromatography mass-specrometry analysis from the homogenates. Following the same protocol we have used for studying transcriptome changes and the effect of microRNA-146a on CPZinduced demyelination [14,21], we induced demyelination in all mice except the controls (C) for 4 weeks with 0.2% CPZ (4wD), then allowed remyelination by terminating CPZ supplementation for 2 days (2dR) and 2 weeks (2wR). From the 20 samples (5 mice per group) we could identify altogether 3183 unmodified proteins.…”

Molecular markers in the CSF proteome differentiate neuroinflammatory diseases

“…TIMP-1 is produced by astrocytes in both homeostasis and early/acute inflammatory events [29]. We have previously found TIMP-1 peak during acute remyelination in the cuprizone model and to be associated with reduced inflammation in the CSF of MS [12]. Induction of TIMP-1 in neurons and astrocytes was also related to early cellular events triggered by seizures and with long-lasting changes in tissue reorganization and/or neuroprotection [30].…”

“…CSF from each patient was precipitated with ethanol/acetone, dissolved in urea buffer containing DTT, as described in a previous paper with parallel reaction monitoring (PRM) [12].Total protein content was estimated by AAA, and 10 ug of proteins were digested with trypsin. After digestion, Stable Isotope Standards (SIS) mix was added in equal volume to every sample (both previously prepared [12] and additional ones). Peptides in each sample were labelled with one of the TMT 11plex label.…”

Absence of miRNA-146a Differentially Alters Microglia Function and Proteome

“…Activated microglia within the demyelinating areas of CPZ lesions express various cytokines, chemokines and growth factors (21,22), and inefficient clearance of myelin debris by microglia impairs remyelinating processes in the CPZ model (23). During the acute phase of remyelination in the CPZ model, a large number of genes are transcribed that suggest microglia activation, and among them are orthologs of differentially expressed genes also in MS lesions (24).…”