Mariana C. Rocha

The genetics and pathology of mitochondrial disease.

Mitochondria are double‐membrane‐bound organelles that are present in all nucleated eukaryotic cells and are responsible for the production of cellular energy in the form of ATP. Mitochondrial function is under dual genetic control – the 16.6‐kb mitochondrial genome, with only 37 genes, and the nuclear genome, which encodes the remaining ∼1300 proteins of the mitoproteome. Mitochondrial dysfunction can arise because of defects in either mitochondrial DNA or nuclear mitochondrial genes, and can present in childhood or adulthood in association with vast clinical heterogeneity, with symptoms affecting a single organ or tissue, or multisystem involvement. There is no cure for mitochondrial disease for the vast majority of mitochondrial disease patients, and a genetic diagnosis is therefore crucial for genetic counselling and recurrence risk calculation, and can impact on the clinical management of affected patients. Next‐generation sequencing strategies are proving pivotal in the discovery of new disease genes and the diagnosis of clinically affected patients; mutations in >250 genes have now been shown to cause mitochondrial disease, and the biochemical, histochemical, immunocytochemical and neuropathological characterization of these patients has led to improved diagnostic testing strategies and novel diagnostic techniques. This review focuses on the current genetic landscape associated with mitochondrial disease, before focusing on advances in studying associated mitochondrial pathology in two, clinically relevant organs – skeletal muscle and brain.

Mitochondrial and inflammatory changes in sporadic inclusion body myositis

Sporadic inclusion body myositis (sIBM) is the most common late onset muscle disease causing progressive weakness. In light of the lack of effective treatment, we investigated potential causes underlying muscle wasting. We hypothesized that accumulation of mitochondrial respiratory deficiency in muscle fibres may lead to fibre atrophy and degeneration, contributing to muscle mass reduction.

A novel immunofluorescent assay to investigate oxidative phosphorylation deficiency in mitochondrial myopathy: understanding mechanisms and improving diagnosis

Oxidative phosphorylation defects in human tissues are often challenging to quantify due to a mosaic pattern of deficiency. Biochemical assays are difficult to interpret due to the varying enzyme deficiency levels found in individual cells. Histochemical analysis allows semi-quantitative assessment of complex II and complex IV activities, but there is no validated histochemical assay to assess complex I activity which is frequently affected in mitochondrial pathology. To help improve the diagnosis of mitochondrial disease and to study the mechanisms underlying mitochondrial abnormalities in disease, we have developed a quadruple immunofluorescent technique enabling the quantification of key respiratory chain subunits of complexes I and IV, together with an indicator of mitochondrial mass and a cell membrane marker. This assay gives precise and objective quantification of protein abundance in large numbers of individual muscle fibres. By assessing muscle biopsies from subjects with a range of different mitochondrial genetic defects we have demonstrated that specific genotypes exhibit distinct biochemical signatures in muscle, providing evidence for the diagnostic use of the technique, as well as insight into the underlying molecular pathology. Stringent testing for reproducibility and sensitivity confirms the potential value of the technique for mechanistic studies of disease and in the evaluation of therapeutic approaches.

Quantitative quadruple-label immunofluorescence of mitochondrial and cytoplasmic proteins in single neurons from human midbrain tissue.

Highlights • We developed an assay to quantify respiratory chain deficiencies in single neurons.• Quadruple-label immunofluorescence was combined with quantitative image analysis.• The single-cell assay was applied to tyrosine hydroxylase-positive midbrain neurons.• The expression of complexes I and IV was determined relative to mitochondrial mass.• The assay proved specific in patients with known respiratory chain deficiencies.

Mitochondrial dysfunction in myofibrillar myopathy.

Highlights • Clonally expanded mtDNA deletions were found in a small number of patient fibres.• Complex I and IV deficiency is higher than in control muscle.• Mitochondrial mass is significantly reduced in patients relative to controls.• No relationship between MFM protein aggregates and reduced mitochondrial mass.• Negative correlations was detected between mitochondrial mass and muscle fibre area.

Using a quantitative quadruple immunofluorescent assay to diagnose isolated mitochondrial Complex I deficiency

Isolated Complex I (CI) deficiency is the most commonly observed mitochondrial respiratory chain biochemical defect, affecting the largest OXPHOS component. CI is genetically heterogeneous; pathogenic variants affect one of 38 nuclear-encoded subunits, 7 mitochondrial DNA (mtDNA)-encoded subunits or 14 known CI assembly factors. The laboratory diagnosis relies on the spectrophotometric assay of enzyme activity in mitochondrially-enriched tissue homogenates, requiring at least 50 mg skeletal muscle, as there is no reliable histochemical method for assessing CI activity directly in tissue cryosections. We have assessed a validated quadruple immunofluorescent OXPHOS (IHC) assay to detect CI deficiency in the diagnostic setting, using 10 µm transverse muscle sections from 25 patients with genetically-proven pathogenic CI variants. We observed loss of NDUFB8 immunoreactivity in all patients with mutations affecting nuclear-encoding structural subunits and assembly factors, whilst only 3 of the 10 patients with mutations affecting mtDNA-encoded structural subunits showed loss of NDUFB8, confirmed by BN-PAGE analysis of CI assembly and IHC using an alternative, commercially-available CI (NDUFS3) antibody. The IHC assay has clear diagnostic potential to identify patients with a CI defect of Mendelian origins, whilst highlighting the necessity of complete mitochondrial genome sequencing in the diagnostic work-up of patients with suspected mitochondrial disease.

Dysferlin mutations and mitochondrial dysfunction.

Highlights • Complex I deficiency is higher in patients with DYSF mutations than controls.• Complex IV deficiency is higher in patients with DYSF mutations than controls.• DYSF mutations may alter Ca++ buffering causing respiratory chain deficiency.• No evidence of mitochondrial DNA deletions detected in dysferlin patients.

Unique quadruple immunofluorescence assay demonstrates mitochondrial respiratory chain dysfunction in osteoblasts of aged and PolgA −/− mice

Fragility fractures caused by osteoporosis affect millions of people worldwide every year with significant levels of associated morbidity, mortality and costs to the healthcare economy. The pathogenesis of declining bone mineral density is poorly understood but it is inherently related to increasing age. Growing evidence in recent years, especially that provided by mouse models, suggest that accumulating somatic mitochondrial DNA mutations may cause the phenotypic changes associated with the ageing process including osteoporosis. Methods to study mitochondrial abnormalities in individual osteoblasts, osteoclasts and osteocytes are limited and impair our ability to assess the changes seen with age and in animal models of ageing. To enable the assessment of mitochondrial protein levels, we have developed a quadruple immunofluorescence method to accurately quantify the presence of mitochondrial respiratory chain components within individual bone cells. We have applied this technique to a well-established mouse model of ageing and osteoporosis and show respiratory chain deficiency.

Pathogenic mtDNA mutations causing mitochondrial myopathy: The need for muscle biopsy

Pathogenic mitochondrial tRNA (mt-tRNA) gene mutations represent a prominent cause of primary mitochondrial DNA (mtDNA)-related disease despite accounting for only 5%–10% of the mitochondrial genome.1,2 Although some common mt-tRNA mutations, such as the m.3243A>G mutation, exist, the majority are rare and have been reported in only a small number of cases.3 The MT-TP gene, encoding mt-tRNAPro, is one of the less polymorphic mt-tRNA genes, and only 5 MT-TP mutations have been reported as a cause of mitochondrial muscle disease to date (table e-1 at Neurology.org/ng, P6–10). We report 5 patients with myopathic phenotypes, each harboring different pathogenic mutations in the MT-TP gene, highlighting the importance of MT-TP mutations as a cause of mitochondrial muscle disease and the requirement to study clinically relevant tissue.

Pathological mechanisms underlying single large-scale mitochondrial DNA deletions

Single, large‐scale deletions in mitochondrial DNA (mtDNA) are a common cause of mitochondrial disease. This study aimed to investigate the relationship between the genetic defect and molecular phenotype to improve understanding of pathogenic mechanisms associated with single, large‐scale mtDNA deletions in skeletal muscle.