An initial top-down proteomic analysis of the standard cuprizone mouse model of multiple sclerosis

Abstract

An initial proteomic analysis of the cuprizone mouse model to characterise the breadth of toxicity by assessing cortex, skeletal muscle, spleen and peripheral blood mononuclear cells. Cuprizone treated vs. control mice for an initial characterisation. Select tissues from each group were pooled, analysed in triplicate using two-dimensional gel electrophoresis (2DE) and deep imaging and altered protein species identified using liquid chromatography tandem mass spectrometry (LC/MS/MS). Forty-three proteins were found to be uniquely detectable or undetectable in the cuprizone treatment group across the tissues analysed. Protein species identified in the cortex may potentially be linked to axonal damage in this model, and those in the spleen and peripheral blood mononuclear cells to the minimal peripheral immune cell infiltration into the central nervous system during cuprizone mediated demyelination. Primary oligodendrocytosis has been observed in type III lesions in multiple sclerosis. However, the underlying mechanisms are poorly understood. Cuprizone treatment results in oligodendrocyte apoptosis and secondary demyelination. This initial analysis identified proteins likely related to axonal damage; these may link primary oligodendrocytosis and secondary axonal damage. Furthermore, this appears to be the first study of the cuprizone model to also identify alterations in the proteomes of skeletal muscle, spleen and peripheral blood mononuclear cells. Notably, protein disulphide isomerase was not detected in the cuprizone cohort; its absence has been linked to reduced major histocompatibility class I assembly and reduced antigen presentation. Overall, the results suggest that, like experimental autoimmune encephalomyelitis, results from the standard cuprizone model should be carefully considered relative to clinical multiple sclerosis.

CD8 T-cell Recruitment Into the Central Nervous System of Cuprizone-Fed Mice: Relevance to Modeling the Etiology of Multiple Sclerosis

“…Notably, the CD8 signal was also detected in brain and spinal cord tissue by western blot analysis when Cx was combined with 0.1% CPZ and PT. This preferential increase in CD8 levels was not observed in previous CPZ-feeding studies that did not include strategies to protect the peripheral immune system (Remington et al, 2007;Partridge et al, 2016;Traka et al, 2016;Tejedor et al, 2017;Sen et al, 2019a) or which the focus of analysis was on a pan T-cell marker (CD3; Caprariello et al, 2018). While it has been argued that ''cellular sources of CD8 were reactive macrophages/microglia'' (Zhang et al, 2009), here, double labeling of the brain and spinal cord sections with IBA 1 and CD8 showed distinct microglia and CD8 cell populations.…”

“…As in the thymus, CPZ-feeding reduced splenic wet weight, the size of its white pulp (a site of B and T-cell production) and CD4/8 T-cell levels, effects that have been attributed to CPZ-induced mitochondrial dysfunction and oxidative stress (Martin et al, 2018). Furthermore, a highly sensitive top-down proteomic analysis of spleens from CPZ-fed Gi male mice identified a significant increase in arginase-I abundance as well as decreased levels of protein disulfide isomerase (Partridge et al, 2016). The increased level of arginase-I, expressed by myeloidderived suppressor cells, may have contributed to the decreased T-cell levels in the CPZ-fed mice.…”

“…While there have been numerous reports of CPZ being used to study demyelination, these have not established the involvement of peripheral T-cells at the sites of CPZ-induced demyelination in the CNS (Remington et al, 2007;Partridge et al, 2016;Traka et al, 2016;Tejedor et al, 2017;Sen et al, 2019a). It was thought that as CPZ does not induce a breach of the blood-brain barrier (BBB), T-cells have no access to the CNS (Remington et al, 2007;Skripuletz et al, 2011;Tejedor et al, 2017).…”

“…Studies have indicated that the apparent failure to trigger a T-cell-mediated CNS immune response is due to CPZ-induced atrophy of the thymus (the organ responsible for T-cell maturation and differentiation) and spleen (the organ of T and B lymphocyte production; Solti et al, 2015;Martin et al, 2018;Sui et al, 2019;Sen et al, 2019a). Two additional effects of CPZ on the integrity and function of the peripheral immune system include an increase in the abundance of splenic arginase-I (a protein expressed by myeloid-derived suppressor cells in spleen) and a decreased abundance of protein disulfide isomerize (a protein required for assembly of the major histocompatibility complex-I; Partridge et al, 2016). A change in the abundance of these proteins results in suppressed T-cell function (Kang et al, 2009).…”

Coomassie staining provides routine (sub)femtomole in‐gel detection of intact proteoforms: Expanding opportunities for genuine Top‐down Proteomics

“…Fluorescence Deep Imaging of SR‐stained 2DE gels improves proteome coverage, achieved by increasing signal from low‐abundance proteoforms following the removal of saturated gel regions and reimaging . Here, we aimed to quantify the DS of cCBB nIRFD Deep Imaging from narrow‐well 1DE models, and maximise 2DE DS by coupling Deep Imaging with the nIRFD imaging conditions assessed.…”