Proteomic profiling of progressive multiple sclerosis patient brains reveals regional and cell-type protein differences related to disease processes

Introduction: Multiple sclerosis (MS) is a chronic autoimmune disease of the central nervous system (CNS) characterized by the loss of protective myelin surrounding nerve fibers. Patients with progressive MS exhibit gradual declining neurologic function and increasing disability over time, in contrast to relapsing-remitting MS where patients experience defined relapsing attacks of neurologic symptoms. MS pathogenesis is marked by disruption of the blood-brain barrier and inflammatory damage to the brain and spinal cord; however, the responsible molecular mechanisms remain poorly understood. Here, we use mass spectrometry-based proteomics to study region-distinct brain tissue from progressive MS patients to gain insights into disrupted biological pathways at the protein level.

Methods: We profiled the proteomes of 8 post-mortem progressive MS patient brains in different regions (normal appearing white matter, white matter lesions, and cerebral cortex) against 8 other disease control patient region-matched brains. We also profiled the proteomes of isolated primary fetal human cultures of CNS cell types (astrocytes, neurons, microglia) and MO3.13 (oligodendrocyte-like) cells. Brain tissue and cell-type lysate samples underwent protein precipitation, digestion, and desalting before being analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) using data-independent acquisition (DIA) mode. Label-free protein identification and quantification were done using Spectronaut software to calculate the statistical significance of protein fold changes associated with MS.

Results: We found 518 significantly changed proteins (306 increased, 212 decreased) in the white matter of MS patients and 340 proteins (60 increased, 280 decreased) in the cortex of MS patients compared to controls. Cell-type profiling revealed significantly changed proteins primarily derived from astrocytes and microglia. Gene ontology analysis of these proteins revealed biological processes related to MS, such as the upregulation of proteins in the white matter involved in immune cell migration and the downregulation of mitochondrial proteins in the cortex. Similar trends in protein changes were also observed in our previous proteomic analysis of mouse brains in a demyelinating MS model (cuprizone).

Conclusion: We will be performing future work using targeting drugs, recombinant protein delivery, and gene knockouts in MS mouse models to investigate the potential role of these proteins in MS. Altogether, we present datasets that provide insight into possible molecular determinants of MS pathogenesis and potential targets for preclinical drug testing.