Abstracts

Rationale: In this work we investigated large deletions of mitochondrial DNA as potential molecular cause for mitochondrial dysfunction in hippocampal subfields of patients with hippocampal sclerosis, one of the most common forms of therapy-resistant temporal lobe epilepsy. These mitochondrial DNA rearrangements are the accepted cause for mitochondrial dysfunction in various neurological disorders and aging. Methods: Applying long-range PCR, single molecule PCR and multiplex PCR we identified, quantified and mapped mitochondrial DNA deletions in hippocampal subfields of 63 patients with hippocampal sclerosis and of 20 patients with lesional temporal lobe epilepsy, who underwent epilepsy surgery. Using a next generation sequencing approach we performed in a limited number of patients deep sequencing of the mitochondrial genome to determine the complete deletional spectrum. Results: The deletion loads approached in the CA3 hippocampal subfield and the dentate gyrus of patients with hippocampal sclerosis 2-8 % of total mitochondrial DNA content. A major fraction of the detected deletions had a prominent 3' breakpoint hot spot at the end of the D-loop (around np 16,070) (Fig. 1, cf. red vertical bar at 16,000). That location of 3' breakpoints strongly implies reactive oxygen species-caused mitochondrial DNA double strand breaks as potential mechanism of deletion formation. High levels of deleted mitochondrial DNA molecules appear to correlate with the presence of cytochrome c oxidase-negative succinate dehydrogenate positive neurons in the CA3 and CA4 hippocampal subfields of patients with hippocampal sclerosis. Hippocampal samples from age-matched patients with epilepsy due to lesions in the temporal lobe did contain substantially lower levels of deleted mitochondrial DNA (Fig. 2, please note the approximately 10-fold lower amouts of deletion species). Additionally, these deletions observed in lesional epilepsy showed the standard distribution of deletional breakpoints (Fig. 2, cf. low vertical bar at 16,000), which has been reported for the aging brain (Guo X., Popadin K.Y., Markuzon N., Orlov Y.L., Kraytsberg Y., Krishnan K.J., Zsurka G., Turnbull D.M., Kunz W.S., Khrapko K. Repeats, longevity and the sources of mtDNA deletions: evidence from 'deletional spectra'. Trends Genet. 2010; 26:340-343). Conclusions: Dysfunction of mitochondrial oxidative phosphorylation in the epileptic focus of patients with Ammon's horn sclerosis appears to be related to clonal expansion of specific types of deleted molecules of mitochondrial DNA generated by increased oxidative stress. This mechanism is proposed to be relevant for epileptogenesis and seizure generation in the hippocampus of patients with temporal lobe epilepsy and hippocampal sclerosis.