Understanding the Molecular Mechanisms of Mitochondrial Muscle Disease using a Quantitative, Quadruple Immunofluorescent Assay

Abstract

Mitochondrial disease can be caused by pathogenic variants in either nuclear DNA or mitochondrial DNA (mtDNA). Affected patients present with a considerable genetic, clinical and biochemical heterogeneity, thereby complicating the diagnostic pathways. For pathogenic mtDNA variants, this is further compounded by heteroplasmy – the mixture of both wild-type and mutated mtDNA existing within a single cell. When the proportion of mutant mtDNA exceeds a critical threshold of mutated mtDNA molecules, the cell becomes biochemically dysfunctional. This dysfunction can be identified using a recently described quadruple immunofluorescent assay through the assessment of Complex I, Complex IV and porin protein expression in individual fibres within 10μm skeletal muscle sections from patients. The assessment of Complex I dysfunction is crucial, as deficiency of this particular respiratory chain complex is the most common form of mitochondrial dysfunction observed in mitochondrial disease. This thesis describes two studies that have utilised this assay with the aim of i) improving the diagnosis of mitochondrial disease and ii) understanding the molecular mechanisms underlying mitochondrial disease and the expression of a biochemical defect. Given the lack of histochemical tests to interrogate Complex I in skeletal muscle, the viability of the quadruple immunofluorescent assay in detecting Complex I deficiency was assessed in a heterogeneous cohort of patients with genetically-proven pathogenic variants within mtDNA-encoded or nuclear-encoded (structural Complex I subunits or assembly factors) genes. Clear diagnostic potential was evident, particularly for patients with nuclear-encoded gene defects whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work up of patients. The quadruple immunofluorescent assay was also used to characterise –at a single cell level - the biochemical status of skeletal muscle fibres from patients harbouring the m.3243A>G mutation, the most prevalent, pathogenic heteroplasmic mtDNA point mutation. Two groups of patients were identified: those showing a predominance of Complex I deficiency and those expressing no biochemical defect in their skeletal muscle. Isolation of single muscle fibres enabled investigation of the relationship between the biochemical deficiency and single fibre m.3243A>G heteroplasmy, identifying differences in the biochemical threshold for Complex I deficiency between patients.