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

OBJECTIVES: To explore mutant superoxide dismutase (SOD)1 protein expression and mitochondrial function in amyotrophic lateral sclerosis (ALS) patients' fibroblasts carrying different SOD1 mutations.
METHODS: 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.
RESULTS: 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.
CONCLUSIONS: 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.