An impaired 20S proteasome contributes to the accumulation of oxidized proteins in multiple sclerosis and its animal model.

摘要

Carbonylated (oxidized) proteins are known to accumulate in the brain of patients with multiple sclerosis (MS) and in the spinal cord of rats with acute experimental autoimmune encephalomyelitis (EAE). Yet, our knowledge regarding mechanism(s) underlying the build-up of protein carbonyls in these inflammatory demyelinating disorders is quite limited. The objectives of this dissertation were (1) to measure the changes in protein carbonylation during disease progression, and to identify the target cells and modified proteins in the cerebellum of EAE animals, prepared by active immunization of C57/BL6 mice with MOG35-55 peptide, (2) to determine if the accumulation of carbonylated proteins in the CNS of these animals is due to a defect in the degradation of the modified proteins and (3) to establish if a similar mechanism underlies the buildup of carbonylated proteins in the cerebral white matter (WM) and gray matter (GM) of MS patients. Initial studies using double immunofluorescence microscopy showed that carbonyls accumulate mostly in white matter astrocytes of EAE mice, both in the acute and chronic phase. Two-dimensional oxyblot and mass spectrometry analysis identified beta-actin, beta-tubulin, GFAP and HSC-71 as the major carbonylation species throughout disease. Using a pull-down/western blot method I also discovered that the proportion of carbonylated cytoskeletal proteins is elevated in chronic EAE, suggesting that as disease progresses from the inflammatory to the neurodegenerative phase there may be an inappropriate removal of these species. This idea was subsequently tested by identifying the 20S proteasome as the proteolytic system responsible for the elimination of oxidized cytoskeletal proteins in cultured astrocytes and by demonstrating that the proteasomal activities were reduced in chronic EAE. These findings were finally extended to the human disease, where I found a profound decrease in proteasomal activity both in the normal-appearing GM and WM of MS patients. Collectively, the studies presented in this dissertation demonstrate that an impaired 20S proteasome in the central nervous system of chronic EAE mice and MS patients significantly contributes to the accumulation of carbonylated (and potentially toxic) proteins. This work may provide the foundation for future studies aimed at developing new approaches to treat MS.