Abstract
AimsCerebellar ataxia is common in patients with mitochondrial disease, and despite previous neuropathological investigations demonstrating vulnerability of the olivocerebellar pathway in patients with mitochondrial disease, the exact neurodegenerative mechanisms are still not clear. We use quantitative quadruple immunofluorescence to enable precise quantification of mitochondrial respiratory chain protein expression in Purkinje cell bodies and their synaptic terminals in the dentate nucleus.MethodsWe investigated NADH dehydrogenase [ubiquinone] 1 alpha subcomplex subunit 13 protein expression in 12 clinically and genetically defined patients with mitochondrial disease and ataxia and 10 age‐matched controls. Molecular genetic analysis was performed to determine heteroplasmy levels of mutated mitochondrial DNA in Purkinje cell bodies and inhibitory synapses.ResultsOur data reveal that complex I deficiency is present in both Purkinje cell bodies and their inhibitory synapses which surround dentate nucleus neurons. Inhibitory synapses are fewer and enlarged in patients which could represent a compensatory mechanism. Mitochondrial DNA heteroplasmy demonstrated similarly high levels of mutated mitochondrial DNA in cell bodies and synapses.ConclusionsThis is the first study to use a validated quantitative immunofluorescence technique to determine complex I expression in neurons and presynaptic terminals, evaluating the distribution of respiratory chain deficiencies and assessing the degree of morphological abnormalities affecting synapses. Respiratory chain deficiencies detected in Purkinje cell bodies and their synapses and structural synaptic changes are likely to contribute to altered cerebellar circuitry and progression of ataxia.
Highlights
Mitochondrial diseases are a clinically heterogeneous group of genetic disorders underpinned by mitochondrial respiratory chain dysfunction
Recent studies indicate that approximately 70% of patients recruited to the UK MRC Mitochondrial Disease Patient Cohort are affected by cerebellar ataxia which is debilitating and progressive
The current assay uses antibodies which are fluorescently labelled to allow visualization and quantification of COX4, NDUFA13, SY-38 and GAD-65/67 protein expression to look at mitochondrial mass, complex I, synaptophysin, and GABAergic cell bodies (Figure 1) and nerve terminals (Figure 2) simultaneously
Summary
Mitochondrial diseases are a clinically heterogeneous group of genetic disorders underpinned by mitochondrial respiratory chain dysfunction. The mitochondrial respiratory chain is responsible for the generation of ATP via Neurons are exquisitely sensitive to fluctuations in mitochondrial function and ATP generation. They possess a highly specialized architecture with long processes extending from the cell body to the distal synapses projecting onto other neuronal cells. Mitochondria regulate numerous processes including synaptic vesicle exocytosis and recycling, and calcium buffering [5,6,7] It is, not surprising that neurological deficits are widespread in patients with mitochondrial disease, and symptoms may be manifold including seizures, cognitive decline and cerebellar ataxia [8]. The cerebellum is vulnerable to mitochondrial dysfunction, and neuropathological investigation of patients often reveals cerebellar atrophy, Purkinje cell loss and mitochondrial respiratory chain deficiencies of complex I in remaining cells [9,10,11,12,13,14]
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