Detection the mutated protein aggregation and mitochondrial function in fibroblasts from amyotrophic lateral sclerosis patients with SOD1 gene mutations

Abstract

To explore mutant superoxide dismutase (SOD)1 protein expression and mitochondrial function in amyotrophic lateral sclerosis (ALS) patients' fibroblasts carrying different SOD1 mutations. SOD1 gene mutation was detected using PCR and direct sequencing. Skin fibroblasts of three familial ALS patients with mutations and age/gender matched controls obtained by a punch skin biopsy were cultured. We performed immunofluorescence staining and quantitative detection of SOD1 proteins and mitochondrial membrane potential. Also, we detected the intracellular ROS by flow cytometry. We found that fibroblasts from familial ALS patients carried SOD1-V14M, SOD1-G16A, SOD1-C111Y mutation, respectively. The cytoplasm abnormal SOD1 protein aggregates appeared in ALS patients carrying SOD1 mutations. And the cytoplasmic/nuclear ratio of SOD1 aggregates increased 2.54, 2.80, 3.25-fold for each mutations, respectively, compared to the control group. Three SOD1 mutant groups showed loss of mitochondrial membrane potential and the ratio of red / green fluorescence intensity decreased by 36%, 124%, 142%, respectively, compared to the control group. The intracellular ROS levels also increased 3.33, 3.65, and 6.87-fold respectively. This work highlights that ALS alters SOD1 protein expression, mitochondrial function, and increases the ROS level even in peripheral tissues outside the central nervous system. Fibroblasts might therefore represent a powerful and minimally invasive tool to investigate ALS pathogenic mechanisms, which might translate into considerable advances in clinical management of the disease.