Introduction: Mitochondrial diseases comprise the largest group of inherited metabolic disorders. Neurological symptoms include epilepsy, ataxia, and cognitive impairment. Previous post-mortem neuropathological studies implicated severe oxidative phosphorylation (OXPHOS) deficiencies in GABAergic inhibitory neurons accompanied by neurodegeneration in mitochondrial disease, including Purkinje neurons of the cerebellum. This study aims to test the hypothesis that underlying hyperexcitability may arise due to neuronal network disinhibition and that metabolically demanding fast-spiking parvalbumin-expressing (PV+) neurons are highly susceptible to mitochondrial dysfunction.
Methods: A novel mouse model of mitochondrial DNA (mtDNA) depletion selectively within the PV+ cells was generated by a conditional knockout of mitochondrial transcription factor A (Tfam) gene via cre-loxP system. Mice were characterised at behavioural, electrophysiological, neuropathological, and molecular levels. A battery of behavioural tests was used to phenotype the mice, including open field, rotarod, novel object recognition, elevated plus-maze, and visual cliff tests. In vitro electrophysiology was performed in acute hippocampal slices CA3 by inducing gamma oscillations via carbachol (cholinergic agonist) and kainate (glutamatergic kainate receptor agonist).
Approach for statistical analysis: Shapiro-Wilk normality test was carried out on all data. Unpaired Student’s t-test was selected for parametric data and Mann-Whitney U test for non-parametric data. For repeated measures measurements, repeated measures ANOVA test was chosen. A minimum of n = 3 biological replicates was used per group.
Results and conclusions: Mutant mice exhibited a progressive phenotype, initiating at 8 weeks of age with tremor, cognitive impairment (in the novel object recognition test) and anxiety-like behaviour (in the elevated plus-maze test). Hyper-locomotion and stargazing (absence-like seizures) were detected at 10 weeks, with severe ataxia observed by 12 weeks. Hippocampal electrophysiology demonstrated a deficit in gamma oscillations in the knockout group upon cholinergic agonism, and aberrantly high area power of gamma oscillations upon stimulation with glutamatergic agonist kainate. Concomitantly with these data, a loss of calbindin-expressing inhibitory interneurons and a relative preservation of PV+ interneurons was detected in hippocampal CA3 region. Taken together, hyperexcitability is implicated as a feature of this mouse model. OXPHOS complexes I and IV within the PV+ cells of the knockout mice had differential deficiency levels which were brain region dependent. Purkinje neurons showed a reduction in mtDNA copy number, most severe combined complex I and IV deficiencies and modest cell loss. Metabolic rewiring was discovered in Purkinje neurons via an upregulation of aldolase C glycolytic enzyme, Krebs cycle enzyme aconitase and anaplerosis enzyme pyruvate carboxylase. The cerebellum and brainstem exhibited secondary reactive microgliosis and astrogliosis. Thalamic reticular nucleus was the second most affected brain region, which is implicated in stargazing behaviour in mice and exhibited combined OXPHOS defects, loss of GAD65-67 and protective perineuronal nets. Knockout mice had reduced weight and severely shortened lifespan. The novel mouse model recapitulates key features of neurological phenotype associated with mitochondrial dysfunction and could be used as a powerful translational preclinical model.