The Oligomerization of Mutant Superoxide Dismutase-1 into Toxic Detergent Insoluble Species Observed in Famililal Amyotrophic Lateral Sclerosis
Karch, Celeste M.
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Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease resulting in paralysis and death due to motor neuron degeneration in the brainstem and spinal cord. The genetically linked familial form of ALS (fALS) enables study through molecular genetics with the key enzyme superoxide dismutase (SOD). Two competing hypotheses attempt to explain the gain of function property. The "Copper Hypothesis" proposes that mutations reduce the shielding of the copper cofactor in the enzyme's binding site, allowing for the production of toxic radical species H202 and ONOO. The aggregation hypothesis, however, speculates that mutations in the encoding region of SOD 1 produces errors that destabilize hydrophobic bonds, producing misfolded dimers that contain intermolecular bonds instead of intramolecular bonds, thus forming SOD 1 aggregates. To investigate the aggregation hypothesis as an explanation for SOD1 toxicity in fALS, mutant SOD1 protein was metabolically radiolabeled with 35 S-cyestine in order to trace oligomerization into detergent insoluble species. Analysis of SOD 1 conversion revealed that only soluble SODI could be detected in the supernatant fraction, decreasing in concentration over time. Control experiments in HEK293 cells confirmed these results. Mouse sciatic nerves were then assessed in a physiological pulse chase experiment to further investigate the aggregation hypothesis in vivo. Insoluble species were produced in the SOD1 wild type and 58 2 (H46R/H48Q) sick lines, indicating aggregation in the sciatic nerve. Mice expressing a truncated SODI mutant, line 171 (126z) sick, demonstrated an absence of soluble and insoluble SOD 1, demonstrating that aggregates in the spinal cord may be sufficient to cause disease. In vitro and in vivo models support the correlation between aggregation in SOD 1 mutations causing fALS toxicity proposed in the aggregation hypothesis; however, evidence from in vivo models provided insight to the location of aggregates that cause disease. In demonstrating aggregation propensity leads to toxicity in SOD 1, fALS shares a key feature of all neurodegenerative diseases.