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Carbonylation of neuronal cytoskeletal proteins and their proteolytic degradation in acute experimental autoimmune encephalomyelitis

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Please use this identifier to cite or link to this item: http://hdl.handle.net/1928/13001

Carbonylation of neuronal cytoskeletal proteins and their proteolytic degradation in acute experimental autoimmune encephalomyelitis

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Title: Carbonylation of neuronal cytoskeletal proteins and their proteolytic degradation in acute experimental autoimmune encephalomyelitis
Author: Smerjac, Suzanne
Advisor(s): Bizzozero, Oscar
Committee Member(s): Perrone-Bizzozero, Nora
Caldwell, Kevin
Shuttleworth, C. William
Department: University of New Mexico. Biomedical Sciences Graduate Program
Subject(s): Protein Carbonyls
Oxidative Stress
Neurofilaments
Neuroinflammation
Calpain
Experimental Autoimmune Encephalomyelitis
LC Subject(s): Multiple sclerosis--Pathophysiology.
Allergic encephalomyelitis--Pathophysiology.
Oxidative stress.
Cytoskeletal proteins.
Carbonyl compounds.
Calpain.
Degree Level: Doctoral
Abstract: Oxidative stress and its damage resulting in carbonylation of proteins is known to take place in the CNS tissue of multiple sclerosis patients and of mice with experimental autoimmune encephalomyelitis (EAE), and has been implicated in the pathophysiology of these diseases. In order to investigate the possible consequences of this protein damage during acute myelin basic protein (MBP) -directed autoimmune neuroinflammation the first goal of this thesis was to characterize oxidative stress and protein carbonylation, including identification of the major oxidized protein species, in the Lewis rat model of EAE. I discovered that there is significant oxidative stress in the spinal cord of EAE rats characterized by reduced levels of glutathione, increased lipid peroxidation products, and accumulation of protein carbonyls during the inflammatory stages of the disease. Using two-dimensional oxyblot I was able to identify various CNS cytoskeletal proteins, including the neurofilaments (NFH, NFM, and NFL) and β-tubulin, as major targets of this oxidative damage. There are many possible fates for carbonylated proteins including accumulation, formation of high molecular weight aggregates and proteolysis. The specific fate likely depends not only on the particular species being oxidized but also on the level of damage it sustains. Western blotting for the neurofilaments and β-tubulin revealed that there is significant degradation of these cytoskeletal proteins during the disease and that the level of specific oxidative damage to NFL changes during the disease course. The second major objective of this thesis was to investigate the role of calpain and the proteasome, which have been proposed to selectively degrade carbonylated proteins, in the removal of these cytoskeletal proteins during neuroinflammation and to examine whether oxidative modification of the proteins made them more susceptible to degradation. This idea was tested by inhibiting calpain or the proteasome in EAE Lewis rats by intrathecal injection of calpeptin or epoxomicin during the peak of clinical disease. After inhibiting calpain, I found significant increases in total protein carbonylation and in the amount of neurofilament proteins and β-tubulin that are spared from degradation, but no changes are seen in the specific oxidation of any of these protein species. Inhibition of the proteasome did not affect total protein carbonylation or degradation, but did change the specific oxidation of NFM. These results suggest that (1) calpain is the primary clearance mechanism of neurofilament and β-tubulin in acute EAE, but that carbonylation of these proteins does not enhance their susceptibility to calpain-mediated proteolysis and (2) the proteasome may contribute to removal of carbonylated cytoskeleton, though it does not play a significant role in the axonal damage observed in acute EAE. This is the first study to investigate the relationship between protein carbonylation and targeted degradation in vivo, and provides insight into the relationship between oxidative stress and axonal damage during neuroinflammation.
Graduation Date: May 2011
URI: http://hdl.handle.net/1928/13001

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