Molecular dissection of ALS-associated toxicity of SOD1 in transgenic mice using an exon-fusion approach

Abstract

Mutations in Cu,Zn superoxide dismutase (SOD1) are associated with amyotrophic lateral sclerosis (ALS). Among more than 100 ALS-associated SOD1 mutations, premature termination codon (PTC) mutations exclusively occur in exon 5, the last exon of SOD1. The molecular basis of ALS-associated toxicity of the mutant SOD1 is not fully understood. Here, we show that nonsense-mediated mRNA decay (NMD) underlies clearance of mutant mRNA with a PTC in the non-terminal exons. To further define the crucial ALS-associated SOD1 fragments, we designed and tested an exon-fusion approach using an artificial transgene SOD1T116X that harbors a PTC in exon 4. We found that the SOD1T116X transgene with a fused exon could escape NMD in cellular models. We generated a transgenic mouse model that overexpresses SOD1T116X. This mouse model developed ALS-like phenotype and pathology. Thus, our data have demonstrated that a ‘mini-SOD1’ of only 115 amino acids is sufficient to cause ALS. This is the smallest ALS-causing SOD1 molecule currently defined. This proof of principle result suggests that the exon-fusion approach may have potential not only to further define a shorter ALS-associated SOD1 fragment, thus providing a molecular target for designing rational therapy, but also to dissect toxicities of other proteins encoded by genes of multiple exons through a ‘gain of function’ mechanism.

FUS‐immunoreactive inclusions are a common feature in sporadic and non‐SOD1 familial amyotrophic lateral sclerosis

“…In our previous studies of SOD1 inclusions in spinal cord of SOD1-linked FALS patients and SOD1 transgenic mice, we observed that antigen retrieval methodology played an essential role in detection of SOD1 inclusions in lower motor neurons 21, 22 , especially in some human cases (unpublished data). We compared three antigen retrieval protocols: boiling and microwave that are most commonly used, and high pressure decloaking chamber.…”

“…2 and supplementary figure 1), but not in the four FALS cases with SOD1 mutations. Previous studies have demonstrated that SOD1/ubiqutin-positive inclusions in spinal motor neurons are a pathological hallmark in ALS patients with SOD1 mutations and in the transgenic mice overexpressing mutant SOD1 18, 22 . Supporting the notion that the FUS-positive inclusions are absent in SOD1-linked ALS, we found that the ubiquitin-positive inclusions in spinal neurons were FUS-negative in patients with SOD1 G85R or SOD1 A4V mutation and in the transgenic mice overexpressing SOD1 G93A and SOD1 L126Z 21, 23 (Supplementary figure 2).…”

“…We compared three antigen retrieval protocols: boiling and microwave that are most commonly used, and high pressure decloaking chamber. We found that antigen retrieval using the high pressure decloaking chamber was the most efficient in detection of SOD1 inclusions, and have therefore adopted this protocol as the routine method for antigen retrieval 21, 22 . Because other studies failed to show FUS pathology in SALS using microwave protocol for antigen retrieval 8, 25, 26 , we assumed that in addition to the sensitivity of the antibodies, antigen retrieval methodology may be an important factor for influencing the detestability of FUS-positive inclusions.…”

A mechanism for low penetrance in an ALS family with a novel SOD1 deletion

“…It was reported that nonsense-mediated mRNA decay underlies the clearance of SOD1 mRNA with a premature termination codon in exons 1-4, but not in exon 5. 16 This is why among the reported SOD1 ALS mutations, stop codon mutations exclusively occur in exon 5. It is possible that nonsense-mediated decay could be involved in the degradation of the alternatively spliced SOD1 mRNA, which could be more efficient in spinal cord tissue than in blood cells, since the level of SOD1 transcript without exon 2 was lower in the spinal cord (figure 3).…”

Atypical Levels of Heavy Metals, Creatinine, Haptoglobin, and Ferritin Found in Amyotrophic Lateral Sclerosis Outpatients vs. Healthy Controls: Possible Utility as a Diagnostic Biomarker

“…Some transition metals such as iron, cobalt, zinc, selenium, molybdenum, magnesium, chromium, manganese and copper are essential for life. Others, such as mercury, lead, nickel, cadmium, palladium, and arsenic are widely used in industry and in various implants but are toxic at certain levels in humans [7][8][9].…”