Abstracts

Rationale: Deficiency in the function of the protein complex I of the mitochondrial respiratory chain is the most common biochemical cause of Leigh syndrome (LS), the most common mitochondria disorder in children. Several loss-of-function mutations in the gene that encodes the NADH dehydrogenase (ubiquinone) iron sulfur protein 4 (NDUFS4), a subunit of the protein complex I, have been causally associated with LS. We recently uncovered that KO of Ndufs4 in GABAergic neurons, not in excitatory neurons, is sufficient to reproduce the epilepsy phenotype observed in the Ndufs4-related model of LS, thus indicating the key role of GABAergic neurons in the development of LS-related epilepsy (Bolea et al., 2019).Given that cell death is a common pathology of an epileptic brain, we investigated whether Ndufs4 gene KO in GABAergic neurons causes interneuron loss in the hippocampus, a brain region with a high propensity to generate and propagate seizure activity. Methods: We generated experimental mutant and control mice with TdTomato fluorescent GABAergic neurons ofthe following genotypes: Ndufs4 flx/flx: Ai14flx/+ : Gad2cre+/- for mutants and Ndufs4+/+:Ai14flx/+ : Gad2cre+/- for controls. These mice were obtained by crossing the double floxed Ndufs4flx/+: Ai14flx/flx mice and the Ndufs4 flx/+: Gad2cre+/+ mice maintained on a congenic C57Bl6 background. Serial 50 um coronal brain sections were obtained from P45 mice, mounted on slides, and imaged on the Olympus BX61VS scanning microscope using Olympus OlyVIA 2.9 software as well as on the Zeiss LSM 710 Imager Z2 laser scanning confocal microscope (Carl Zeiss Inc.) using Zen 2009 software for analysis across or within regions respectively. Obtained images were processed in ImageJ/FIJI software (NIH, Bethesda, Maryland, USA) to obtain the number of fluorescent GABAergic neurons in mutant and control mice. Statistical significance of the quantification was assessed using 2-tailed unpaired t-tests in GraphPad Prism v5.01 (GraphPad Software Inc., San Diego, USA). Results: Knockout the both alleles of Ndufs4 only GABAergic interneurons led to cell loss within two epileptogenic subregions of the hippocampus. In the rostral CA1 region of the hippocampus, there was a 25% reduction of the number of interneuronsin mutant (131.5  14.03)(N=4) compared to control (173.0  11.84) (N=6) mice (p<0.05). In the caudal CA1 region, there was a 35% loss of interneurons in the mutant (107.5  18.45) compared to control (164.2  9.48)mice (p<0.05). Finally, in the CA3 region of the hippocampus there was a 33% loss of interneurons in mutant (99.5  12.31)compared to control (147.0  16.33)mice (p<0.05).   Conclusions: The observed interneuron cell loss in epileptogenic areas during the peak of the symptomatic stage of epilepsy (P45) suggests that the loss of interneurons may contribute to the mechanism of epilepsy in LS syndrome in our mice. Impaired mitochondrial function may also disrupt normal migration of interneurons during development (Lin-Hendel et al., 2016), as recent studies have indicated that GABAergic neurons are disproportionally vulnerable to mitochondrial defects, particularly those due to complex I deficiency. Future studies will investigate whether this interneuron loss is due to apoptosis, necrosis, and/or migration impairments in order to understand the seizure pathology.  Funding: NIH/NINDS R01NS10279602UW Neuroscience T32 Training Grant (T32NS099578)