Superoxide Dismutase-1 (SOD1) & Amyotrophic Lateral Sclerosis (ALS)

Amyotrophic lateral sclerosis (ALS, Lou Gehrig’s disease) is a relatively common adult-onset neurodegenerative disease, having a worldwide prevalence of ca. 5 per 100,000. ALS is characterized by the selective degeneration of spinal cord motor neurons, leading to rapid and progressive atrophy of skeletal muscles. Death typically occurs by asphyxia, almost always within 5 years of diagnosis. There is no effective therapy. Approximately 10% of ALS cases are autosomal-dominantly inherited (FALS). Of these, ca. 20% involve missense mutations in the gene encoding superoxide dismutase-1 (SOD1), a housekeeping metalloprotein responsible for dismutation of superoxide. Over 90 FALS-linked SOD1 mutations have been characterized. Animal modeling studies indicate that the pathogenicity of the SOD1 mutations does not involve loss of its normal function, but rather the gain of a toxic function. The mechanism of pathogenicity is unknown. One hypothesis holds that mutant forms of SOD1 have abnormal and toxic enzymatic activity. Degenerating populations of motor neurons in postmortem FALS brain are characterized by abnormal proteinaceous cytoplasmic inclusions. These inclusions contain mutant SOD1. Since several FALS mutations affect the stability or unfolding of SOD1 or both, it has been suggested that aggregation of mutant SOD1 produces a pathogenic species. However, the identity of the pathogenic aggregate and the mechanism linking aggregation and neurotoxicity remain elusive. Although fibrillar substructure has not been conclusively detected in FALS inclusions, parallels between FALS and other familial neurodegenerative diseases suggest that the process of SOD1 aggregation, if not the product, may resemble aggregation of the proteins linked to those diseases.

SOD1 Monomerization is Required for Aggregation

The FALS mutations are distributed throughout the SOD1 primary and tertiary structures. Some, but not all of the mutations are known to affect SOD1 stability. Others affect metal binding or enzymatic activity or both. In collaboration with Peter Lansbury (Brigham and Women’s Hospital), we have demonstrated that the FALS mutant A4V, which is linked to a common early onset and rapidly progressing (typically 1 year between diagnosis and death) form of FALS, spontaneously aggregates in vitro under conditions at which the WT dimer is stable. At low protein concentrations A4V, but not WT, populates a monomeric form. To determine whether the reduced stability of the A4V dimer was wholly or partly responsible for its rapid aggregation, we engineered an intersubunit disulfide bridge across the A4V dimer interface to produce an A4V/V148C covalent dimer that could not monomerize. This mutant SOD1 did not aggregate in vitro, suggesting a novel therapeutic strategy against FALS.

A4V was more prone to aggregate than WT. When incubated at 37°, WT dimer was stable for days, whereas A4V oligomers were detected within one hour. The A4V oligomers that accumulated after 80 hours were fractionated using superdex 200 size exclusion chromatography and analyzed by electron microscopy. The fraction of highest molecular weight contained large (diameter of ~50 nm) sphere-like and irregular oligomeric structures (bottom panels). Smaller structures, including pore-like structures that resembled “amyloid pores” (middle panels) were found in fractions of lower molecular weight. No fibrillar structures were detected. The fraction containing the A4V dimer contained structures consistent with the dimensions of the WT dimer crystal structure (top panels).
Stabilization of the A4V dimer was achieved by insertion of an intersubunit disulfide bond. Position 148 was chosen for insertion of Cys, since the resulting disulfide bond was predicted to be virtually strain-free. Purification and SDS PAGE analysis of the A4V/L148C double mutant under non-reducing conditions confirmed that intermolecular disulfide bond formation had occurred. The A4V/L148C dimer was stable over a 80 h period at 37°C, whereas A4V began to aggregate within one hour. This finding demonstrated that dimer dissociation is required for A4V aggregation.
The potentially pathogenic A4V aggregation requires SOD1 dimer dissociation and, probably, monomer unfolding. Many examples of mutations affecting a dimer-monomer equilibrium have been reported. Multimeric proteins are typically labile at interfaces and their dissociation is often linked to unfolding. Dissociation and aggregation can be promoted by disease-associated mutations. The most relevant example of disease-promoting mutations affecting protein dissociation, unfolding, and aggregation are the transthyretin (TTR) mutations that are linked to familial amyloid polyneuropathy (FAP).


» Ray et al. (2004) Biochemistry 43: 4899-4905.