The presence of pathogenic mutations in the gene coding for the copper- zinc-containing superoxide dismutase (CuZnSOD) on chromosome 21 in a subset of patients with amyotrophic lateral sclerosis (ALS) has now been firmly established. Over the last year, it has become increasingly clear that the role of mutant CuZnSOD in this disease is not directly related to its function as a superoxide dismutator. Data garnered from the expression of the cDNA for mutant SODs in various cell lines and transgenic mice support the theory that a gain of an as yet unidentified adverse function triggers motor neuron cell death. Although very recent work suggests that alterations in the tertiary structure of the enzyme play a critical role in targeted cell death the salient biochemical properties of the mutant SODs have not yet been well characterized. In this research proposal 7 mutant and the wild-type human CuZnSOD cDNA clones are overexpressed in a baculovirus/Sf21 insect cell system. In Part I, various aspects of their thermodynamic stability and chemical reactivity are defined, i.e.tendency to a) denature in the face of changing pH, temperature guanidine hydrochloride or SDS. b) lose active site copper and c) dismutate superoxide. In Part II, mechanisms for the relative instability of these enzymes are probed. Rates and extent of auto-oxidation of histidine. and 3-nitrotyrosine and dityrosine formation on exposure to hydrogen peroxide or peroxynitrite are determined. This information is compared to the relative susceptibility of these proteins to proteolytic digestion before and after oxidative modification, anti to their half-lives. In Part III patterns of oxidative damage are discerned in vitro in target molecules (DNA albumin neurofilaments) and in vivo in transgenic mice overexpressing mutant CuZnSO. This is accomplished by quantitating well accepted markers of oxidative damage (3-nitrotyrosine dityrosine, protein carbonyls. and 2-oxo-histidine). These studies will be among the first to link known mutations in CuZnSOD with defined changes in the physical and chemical properties, combined with detailed analysis of associated patterns of oxidative stress in the brain and spinal cord, and clinical information such as age of onset severity of disease, time to death.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Clinical Investigator Award (CIA) (K08)
Project #
5K08NS001895-02
Application #
2416242
Study Section
NST-2 Subcommittee (NST)
Program Officer
Kerza-Kwiatecki, a P
Project Start
1996-08-01
Project End
2001-04-30
Budget Start
1997-05-01
Budget End
1998-04-30
Support Year
2
Fiscal Year
1997
Total Cost
Indirect Cost
Name
Massachusetts General Hospital
Department
Type
DUNS #
City
Boston
State
MA
Country
United States
Zip Code
02199