Cytoskeletal structure in cells harboring two mutations: R133C in NOTCH3 and 5650G>A in mitochondrial DNA

Area of Science:

• Cell Biology
• Neurogenetics
• Mitochondrial Medicine

Background:

• Cerebral autosomal-dominant arteriopathy with subcortical infarcts and leucoencephalopathy (CADASIL) is a genetic disorder affecting blood vessels in the brain.
• A patient presented with CADASIL due to NOTCH3 mutation and a concurrent myopathy linked to a mitochondrial DNA (mtDNA) mutation (5650G>A).

Purpose of the Study:

• To investigate the morphological changes in cytoskeletal components and mitochondria in fibroblasts and myoblasts from the patient.
• To determine the pathogenic role of the identified mtDNA mutation in the patient's myopathy.

Main Methods:

• Immunolabeling of cytoskeletal proteins (tubulin, actin, vimentin) in patient-derived cells.
• Mitochondrial morphology and distribution analysis.
• Muscle histochemical analysis, including ragged red fibres (RRFs) and cytochrome c oxidase (COX) staining.
• Quantification of the proportion of mutated mtDNA in different muscle fiber types.

Main Results:

• Patient fibroblasts and myoblasts exhibited a sparse tubulin network forming asters, with reduced mitochondrial abundance and branching.
• Muscle histochemistry revealed ragged red fibres (RRFs) and cytochrome c oxidase (COX)-negative fibres.
• The proportion of mutated mtDNA (5650G>A) was higher in COX-negative fibres and RRFs compared to normal muscle fibres, supporting its pathogenicity.

Conclusions:

• The 5650G>A mtDNA mutation is likely pathogenic, contributing to the observed myopathy.
• Observed alterations in the tubulin network and mitochondrial distribution in patient cells may not be solely explained by the mtDNA mutation, suggesting potential interplay with the CADASIL-related genetic defect.