Amyotrophic lateral sclerosis (ALS) is a devastating disease caused by a progressive loss of motor neurons. The production of nitric oxide by interneurons, reactive astrocytes or skeletal muscle may indirectly contribute to motor neuron destruction in ALS. Sixteen missense mutations modifying thirteen different amino acid positions of Cu,Zn superoxide dismutase (SOD) have been implicated with the autosomal dominant familial ALS associated with chromosome 21. The dominant action and the fact that none of the mutations are nonsense mutations indicate that the mutant proteins catalyze another reaction rather than simply diminish superoxide scavenging. The superoxide scavenging activity in blood samples from familial ALS patients is depressed by about 50%, which will only double the concentration of superoxide. Superoxide reacts with nitric oxide at a rate of 6.7 X 10(9)M(-1s-1), three times faster than with native SOD, to form the powerful oxidant peroxynitrite (ONOO-). Peroxynitrite reacts with superoxide dismutase, resulting in the nitration of tyrosines in proteins. The nitrating reaction is among the fastest observed for peroxynitrite, but is limited to a 9% yield by the narrow active site pocket of SOD. We propose that the mutations result in a subtle opening of the active site to expose the catalytic copper, which increases peroxynitrite-mediated nitration. We further propose that nitration of critical tyrosine residues, such as the substrates for the trkB tyrosine kinase receptor for growth factors in the neuromuscular junction, contributes to the demise of motor neurons. We have found that 3-5% of the tyrosine residues in plasma proteins from familial ALS patients are nitrated. Furthermore, an antibody that recognizes peroxynitrite-modified proteins stains altered astrocytes surrounding degenerating motor neurons in ALS patients as well as around amyloid deposits in Alzheimer's brains.
The Specific Aims are to express the mutant Sods in E. coli and characterize their reactions with superoxide, nitric oxide and peroxynitrite, develop immunological and fluorescent assays for protein nitration, assay protein nitration in blood, muscle and spinal cord from human sporadic and familial ALS patients, and determine the effects of intrathecal and intramuscular injection of SIN-1, a compound that decomposes to generate peroxynitrite, into transgenic mice expressing mutant SODs. There is considerable interest in treating ALS patients with SOD due to the decreased superoxide activity of the mutations. The nitration hypothesis predicts that native SOD may accelerate injury, but does offer other therapeutic approaches involving manipulation of nitric oxide synthesis.
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