Oligomeric intermediates in the aggregation of Cu, Zn superoxide dismutase

Mutations in superoxide dismutase (CuZnSOD) that give rise to familial amyotrophic lateral sclerosis (FALS) lead to deposition of insoluble aggregates in motor neurons. Considerable evidence suggests that soluble oligomers are the toxic species in protein aggregation diseases. Therefore, we have studied the in vitro formation of soluble aggregation intermediates from various mutant CuZnSODs. Wild type and mutant CuZnSODs were agitated under a variety of conditions which lead to amyloid formation. The time course of the formation of soluble intermediate oligomeric forms of both wild-type and mutant CuZnSOD was investigated by dynamic light scattering (DLS) combined with electron microscopy, circular dichroism and size exclusion chromatography. Wild type, apo and some mutant CuZnSODs form soluble oligomeric intermediates whose diameters ranged from 15 nm to 35 nm. Further incubation of these oligomers led to the formation of insoluble fibrillar or network-like aggregates. For mutant H46R in the presence of guanidine-HCl and Tris(2-carboxyethyl)phosphine, the oligomer had a secondary structure similar to that of the starting material, and a major change in secondary structure occurred upon conversion of the oligomer to network-like aggregates. The oligomers observed in this study might be the actual toxic species in the aggregation of CuZnSOD that leads to amyotrophic lateral sclerosis.