Extensive respiratory chain defects in inhibitory interneurones in patients with mitochondrial disease

Aims: Mitochondrial disorders are among the most frequently inherited cause of neurological disease and arise due to mutations in mitochondrial or nuclear DNA. Currently, we do not understand the specific involvement of certain brain regions or selective neuronal vulnerability in mitochondrial disease. Recent studies suggest γ-aminobutyric acid (GABA)-ergic interneurones are particularly susceptible to respiratory chain dysfunction. In this neuropathological study, we assess the impact of mitochondrial DNA defects on inhibitory interneurones in patients with mitochondrial disease. Methods: Histochemical, immunohistochemical and immunofluorescent assays were performed on post-mortem brain tissue from 10 patients and 10 age-matched control individuals. We applied a quantitative immunofluorescent method to interrogate complex I and IV protein expression in mitochondria within GABAergic interneurone populations in the frontal, temporal and occipital cortices. We also evaluated the density of inhibitory interneurones in serial sections to determine if cell loss was occurring. Results: We observed significant, global reductions in complex I expression within GABAergic interneurones in frontal, temporal and occipital cortices in the majority of patients. While complex IV expression is more variable, there is reduced expression in patients harbouring m.8344A>G point mutations and POLG mutations. In addition to the severe respiratory chain deficiencies observed in remaining interneurones, quantification of GABAergic cell density showed a dramatic reduction in cell density suggesting interneurone loss. Conclusions: We propose that the combined loss of interneurones and severe respiratory deficiency in remaining interneurones contributes to impaired neuronal network oscillations and could underlie development of neurological deficits, such as cognitive impairment and epilepsy, in mitochondrial disease

MT-ND5 Mutation Exhibits Highly Variable Neurological Manifestations at Low Mutant Load.

Mutations in the m.13094T>C MT-ND5 gene have been previously described in three cases of Leigh Syndrome (LS). In this retrospective, international cohort study we identified 20 clinically affected individuals (13 families) and four asymptomatic carriers. Ten patients were deceased at the time of analysis (median age of death was 10years (range: 5·4months-37years, IQR=17·9years). Nine patients manifested with LS, one with mitochondrial encephalomyopathy, lactic acidosis and stroke-like episodes (MELAS), and one with Leber hereditary optic neuropathy. The remaining nine patients presented with either overlapping syndromes or isolated neurological symptoms. Mitochondrial respiratory chain activity analysis was normal in five out of ten muscle biopsies. We confirmed maternal inheritance in six families, and demonstrated marked variability in tissue segregation, and phenotypic expression at relatively low blood mutant loads. Neuropathological studies of two patients manifesting with LS/MELAS showed prominent capillary proliferation, microvacuolation and severe neuronal cell loss in the brainstem and cerebellum, with conspicuous absence of basal ganglia involvement. These findings suggest that whole mtDNA genome sequencing should be considered in patients with suspected mitochondrial disease presenting with complex neurological manifestations, which would identify over 300 known pathogenic variants including the m.13094T>C

Microangiopathy in the cerebellum of patients with mitochondrial DNA disease

Neuropathological findings in mitochondrial DNA disease vary and are often dependent on the type of mitochondrial DNA defect. Many reports document neuronal cell loss, demyelination, gliosis and necrotic lesions in post-mortem material. However, previous studies highlight vascular abnormalities in patients harbouring mitochondrial DNA defects, particularly in those with the m.3243A>G mutation in whom stroke-like events are part of the mitochondrial encephalopathy lactic acidosis and stroke-like episodes syndrome. We investigated microangiopathic changes in the cerebellum of 16 genetically and clinically well-defined patients. Respiratory chain deficiency, high levels of mutated mitochondrial DNA and increased mitochondrial mass were present within the smooth muscle cells and endothelial cells comprising the vessel wall in patients. These changes were not limited to those harbouring the m.3243A>G mutation frequently associated with mitochondrial encephalopathy, lactic acidosis and stroke-like episodes, but were documented in patients harbouring m.8344A>G and autosomal recessive polymerase (DNA directed), gamma (POLG) mutations. In 8 of the 16 patients, multiple ischaemic-like lesions occurred in the cerebellar cortex suggestive of vascular smooth muscle cell dysfunction. Indeed, changes in vascular smooth muscle and endothelium distribution and cell size are indicative of vascular cell loss. We found evidence of blood–brain barrier breakdown characterized by plasma protein extravasation following fibrinogen and IgG immunohistochemistry. Reduced immunofluorescence was also observed using markers for endothelial tight junctions providing further evidence in support of blood–brain barrier breakdown. Understanding the structural and functional changes occurring in central nervous system microvessels in patients harbouring mitochondrial DNA defects will provide an important insight into mechanisms of neurodegeneration in mitochondrial DNA disease. Since therapeutic strategies targeting the central nervous system are limited, modulating vascular function presents an exciting opportunity to lessen the burden of disease in these patients

Mitochondrial DNA depletion in respiratory chain-deficient Parkinson disease neurons.

OBJECTIVE: To determine the extent of respiratory chain abnormalities and investigate the contribution of mitochondrial DNA (mtDNA) to the loss of respiratory chain complexes (CI-IV) in the substantia nigra (SN) of idiopathic Parkinson disease (IPD) patients at the single-neuron level. METHODS: Multiple-label immunofluorescence was applied to postmortem sections of 10 IPD patients and 10 controls to quantify the abundance of CI-IV subunits (NDUFB8 or NDUFA13, SDHA, UQCRC2 and COXI), and mitochondrial transcription factors (TFAM and TFB2M) relative to mitochondrial mass (Porin and GRP75) in dopaminergic neurons. To assess the involvement of mtDNA in respiratory chain deficiency in IPD, SN neurons, isolated with laser-capture microdissection, were assayed for mtDNA deletions, copy number and presence of transcription/replication-associated 7S DNA employing a triplex real-time PCR assay. RESULTS: While mitochondrial mass was unchanged in single SN neurons from IPD patients, we observed a significant reduction in the abundances of CI and II subunits. At single-cell level, CI and II deficiencies were correlated in patients. The CI deficit concomitantly occurred with low abundances of the mtDNA transcription factors TFAM and TFB2M, which also initiate transcription-primed mtDNA replication. Consistent with this, real-time PCR analysis revealed fewer transcription/replication-associated mtDNA molecules and an overall reduction in mtDNA copy number in patients. This effect was more pronounced in single IPD neurons with severe complex I deficiency. INTERPRETATION: Respiratory chain dysfunction in IPD neurons not only involves CI, but also extends to CII. These deficiencies are possibly a consequence of the interplay between nDNA and mtDNA-encoded factors mechanistically connected via TFAM. This article is protected by copyright. All rights reserved

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

BACKGROUND: Respiratory chain (RC) deficiencies are found in primary mtDNA diseases. Focal RC defects are also associated with ageing and neurodegenerative disorders, e.g. in substantia nigra (SN) neurons from Parkinson's disease patients. In mitochondrial disease and ageing, mtDNA mutational loads vary considerably between neurons necessitating single cell-based assessment of RC deficiencies. Evaluating the full extent of RC deficiency within SN neurons is challenging because their size precludes investigations in serial sections. We developed an assay to measure RC abnormalities in individual SN neurons using quadruple immunofluorescence. NEW METHOD: Using antibodies against subunits of complex I (CI) and IV, porin and tyrosine hydroxylase together with IgG subtype-specific fluorescent labelled secondary antibodies, we quantified the expression of CI and CIV compared to mitochondrial mass in dopaminergic neurons. CI:porin and CIV:porin ratios were determined relative to a standard control. RESULTS: Quantification of expression of complex subunits in midbrain sections from patients with mtDNA disease and known RC deficiencies consistently showed reduced CI:porin and/or CIV:porin ratios. COMPARISON WITH EXISTING METHOD(S): The standard histochemical method to investigate mitochondrial dysfunction, the cytochrome c oxidase/succinate dehydrogenase assay, measures CIV and CII activities. To also study CI in a patient, immunohistology in additional sections, i.e. in different neurons, is required. Our method allows correlation of the expression of CI, CIV and mitochondrial mass at a single cell level. CONCLUSION: Quantitative quadruple-label immunofluorescence is a reliable tool to measure RC deficiencies in individual neurons that will enable new insights in the molecular mechanisms underlying inherited and acquired mitochondrial dysfunction