Our long-term goal is to identify and characterize the mechanisms that lead to the destruction of myelin in inflammatory demyelinating disorders. Nitric oxide (NO) has been implicated in the pathophysiology of both multiple sclerosis (MS) and experimental allergic encephalomyelitis (EAE), as high levels of this gas are released by macrophages/microglia following induction of nitric oxide synthetase by various proinflammatory cytokines. However, the mechanism by which NO leads to myelin breakdown is far from clear. Based on recent investigations regarding the molecular and cellular consequences of nitrosative damage in other systems, and a number of important findings from our laboratory, we hypothesize that NO can spread at considerable distances from inflammatory lesions to cause extensive myelin decompaction. We propose that underlying this process is the S-nitrosylation of important myelin proteins by N203, a strong nitrosylating agent that can be readily formed by NO autooxidation in a lipid-rich environment like the myelin sheath. The experiments described in this proposal will characterize in detail the mechanism of S-nitrosylation of myelin proteins in rat spinal cord slices incubated with a non-permeable NO-donor, which generates levels of NO comparable to those found in EAE and MS. These experiments will also (1) examine the existence of other NO-induced thiol-related modifications (S-thiolation and formation of protein disulfides), (2) identify the major S-nitrosylated myelin proteins by mass-spectrometry, and (3) test several compounds for their ability to prevent/revert these deleterious protein modifications. In addition, the occurrence of S-nitrosylated proteins in spinal cord during the course of EAE will be investigated and the identity of these species determined using a proteomic approach. Finally, the distribution of S-nitrosylated proteins in the affected tissue will be assessed by immunocytochemistry to directly test the hypothesis that nitrosative protein damage induced by free NO can take place at some distance from the inflammatory lesions and independently of peroxynitrite generation. The proposed studies will generate crucial information that could support a new pathway for the NO-mediated destruction of myelin during inflammatory demyelination. The elucidation of the mechanism of protein modification, the identification of the molecules in myelin that are affected by NO, and the occurrence of these modifications in the CNS of a widely-used animal model of MS are essential for understanding the pathophysiology of this devastating disorder.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
1R01NS047448-01
Application #
6709773
Study Section
Special Emphasis Panel (ZRG1-BDCN-2 (02))
Program Officer
Utz, Ursula
Project Start
2004-01-01
Project End
2007-12-31
Budget Start
2004-01-01
Budget End
2004-12-31
Support Year
1
Fiscal Year
2004
Total Cost
$312,188
Indirect Cost
Name
University of New Mexico
Department
Physiology
Type
Schools of Medicine
DUNS #
868853094
City
Albuquerque
State
NM
Country
United States
Zip Code
87131
Romero, Jorge M; Bizzozero, Oscar A (2009) Intracellular glutathione mediates the denitrosylation of protein nitrosothiols in the rat spinal cord. J Neurosci Res 87:701-9
Bizzozero, Oscar A; Zheng, Jianzheng (2009) Identification of major S-nitrosylated proteins in murine experimental autoimmune encephalomyelitis. J Neurosci Res 87:2881-9
Smerjac, Suzanne M; Bizzozero, Oscar A (2008) Cytoskeletal protein carbonylation and degradation in experimental autoimmune encephalomyelitis. J Neurochem 105:763-72
Bizzozero, Oscar A; Reyes, Savanna; Ziegler, Jennifer et al. (2007) Lipid peroxidation scavengers prevent the carbonylation of cytoskeletal brain proteins induced by glutathione depletion. Neurochem Res 32:2114-22
Bizzozero, Oscar A; Ziegler, Jennifer L; De Jesus, Gisela et al. (2006) Acute depletion of reduced glutathione causes extensive carbonylation of rat brain proteins. J Neurosci Res 83:656-67
Romero, Jorge M; Bizzozero, Oscar A (2006) Extracellular S-nitrosoglutathione, but not S-nitrosocysteine or N(2)O(3), mediates protein S-nitrosation in rat spinal cord slices. J Neurochem 99:1299-310
Bizzozero, Oscar A; DeJesus, Gisela; Bixler, Heather A et al. (2005) Evidence of nitrosative damage in the brain white matter of patients with multiple sclerosis. Neurochem Res 30:139-49
Bizzozero, Oscar A; DeJesus, Gisela; Callahan, Kelly et al. (2005) Elevated protein carbonylation in the brain white matter and gray matter of patients with multiple sclerosis. J Neurosci Res 81:687-95
Bizzozero, Oscar A; DeJesus, Gisela; Howard, Tamara A (2004) Exposure of rat optic nerves to nitric oxide causes protein S-nitrosation and myelin decompaction. Neurochem Res 29:1675-85