Microwave & Magnetic (M2) Proteomics Reveals CNS-Specific Protein Expression Waves that Precede Clinical Symptoms of Experimental Autoimmune Encephalomyelitis

Abstract

Central nervous system-specific proteins (CSPs), transported across the damaged blood-brain-barrier (BBB) to cerebrospinal fluid (CSF) and blood (serum), might be promising diagnostic, prognostic and predictive protein biomarkers of disease in individual multiple sclerosis (MS) patients because they are not expected to be present at appreciable levels in the circulation of healthy subjects. We hypothesized that microwave & magnetic (M2) proteomics of CSPs in brain tissue might be an effective means to prioritize putative CSP biomarkers for future immunoassays in serum. To test this hypothesis, we used M2 proteomics to longitudinally assess CSP expression in brain tissue from mice during experimental autoimmune encephalomyelitis (EAE), a mouse model of MS. Confirmation of central nervous system (CNS)-infiltrating inflammatory cell response and CSP expression in serum was achieved with cytokine ELISPOT and ELISA immunoassays, respectively, for selected CSPs. M2 proteomics (and ELISA) revealed characteristic CSP expression waves, including synapsin-1 and α-II-spectrin, which peaked at day 7 in brain tissue (and serum) and preceded clinical EAE symptoms that began at day 10 and peaked at day 20. Moreover, M2 proteomics supports the concept that relatively few CNS-infiltrating inflammatory cells can have a disproportionally large impact on CSP expression prior to clinical manifestation of EAE.

Serum Neuroinflammatory Disease-Induced Central Nervous System Proteins Predict Clinical Onset of Experimental Autoimmune Encephalomyelitis

“…In line with previously published results (15, 19), large frequencies of MOG-reactive IFN-γ and IL-17 producing Th1 and Th17 cells could be detected in secondary lymphatic tissues as early as day 5 after immunization and the numbers peaked after the onset of clinical disease (Figures 1D,E). Frequencies of pathogenic Th17 cells in the CNS mirrored the disease trajectory (Figure 1F).…”

“…Confirming previous studies, statistical analysis of the CNS proteome over the course of EAE by M2 proteomics revealed two subsets of protein expression waves with a statistically significant expression change specific for pre-onset (onset-leading wave; black curve) and acute disease phase (onset-lagging wave; gray curve) of EAE (Figure 1B) (14, 15). Statistical analysis revealed proteins that were significantly up- or downregulated within each time point at the onset-leading wave (Figure 1C).…”

Cytotoxicity and Genotoxicity Assessment of Sandalwood Essential Oil in Human Breast Cell Lines MCF‐7 and MCF‐10A

“…Sample preparation for isobaric labeling was performed as described by Raphael et al (2014) [ 24 ]. C8 magnetic beads (BcMg, Bioclone Inc., CA) were suspended in 1 mL of 50% methanol.…”

Body fluid biomarkers in multiple sclerosis: how far we have come and how they could affect the clinic now and in the future

“…Animal models such as EAE allow investigations under controlled conditions with much less biological variation than humans, including large-scale discovery and evaluation of potential biomarkers and a better understanding of their involvement in disease mechanisms. Along these lines, using a novel proteomic approach (M 2 Proteomics), our laboratory has demonstrated expression changes of several different CNS-specific proteins in EAE which can be detected in mouse serum [233,277]. A number of these proteins, such as 14-3-3, GFAP and MBP, have also been reported to be differentially expressed in MS patients [233,234].…”

“…Improvements in technology may facilitate the investigation of lipids, glycosylation, or metabolic intermediates as biomarkers for different aspects of MS. Since MS is considered a neuroinflammatory disease, markers that reflect CNS-specific changes, including axonal and myelin damage, may be most useful if detected in body fluids such as serum or CSF [105,277]. …”