Leigh Syndrome (LS) is a neurodegenerative disease due to the dysfunction of mitochondria. It usually begins in infancy and its incidence is around 1 in 40,000 individuals. Children with LS experience a progressive decline in their cognitive and motor functions often accompanied by severe treatment-resistant epileptic seizures. Mutations in Ndufs4, the gene that encodes a subunit of mitochondrial complex have been linked to LS. Mice carrying Ndufs4 recapitulate several key characteristic clinical manifestations of LS. Using these mouse models, our lab has demonstrated that GABAergic interneurons play an important role in the pathophysiology of LS. Mice with Ndufs4 knockouts (KO) restricted to GABAergic neurons located across all brain regions exhibit seizures. However, seizures in epilepsy patients and animal models typically originate from forebrain structures. Therefore, in this project, we examined whether the inactivation of Ndufs4 in GABAergic neurons of the forebrain alone is sufficient to cause seizures in mice. Homozygotes floxed Ndfus4 mice were crossed with Dlx56Cre+ or Gly2TCre+ mice to KO the gene specifically in interneurons of the forebrain or brainstem. We hypothesized that only mice with KO in the former region will exhibit seizures. Conditional KO mice from these two lines were tested for thermal seizure susceptibility. Surprisingly, both Dlx56creKO and Gly2TCre KO mice exhibited thermally induced myoclonic and generalized tonic clinic seizures. These findings indicate that GABAergic interneurons regions outside of the forebrain are critically involved in the pathogenesis of epilepsy in LS.

Dravet Syndrome (DS) is a severe form of childhood onset epilepsy occurring in about 1 out of 16,000 births. The disease is characterized by treatment-resistant seizures, ataxia (or loss of muscle coordination), developmental delay, cognitive impairment, and increased rate of premature mortality mostly due to sudden unexpected death in epilepsy (SUDEP). DS is predominantly caused by a heterozygous loss-of-function mutation in the SCN1A gene, which codes for the pore-forming alpha subunit of the NaV1.1 voltage-gated sodium channel. Our lab has previously shown that selectively introducing these mutations into neurons expressing the neurotransmitter GABA, specifically GABAergic interneurons of the forebrain, is sufficient to cause DS phenotypes in mice. In this study, we investigated whether an SCN1A gene replacement therapy precisely targeted to this interneuronal population can rescue epilepsy and SUDEP. Our lab, in collaboration with the Allen Institute, has developed a novel dual SCN1A-intein-AAV with forebrain GABAergic interneuron targeting capability using a Dlx56-based enhancer. Mice treated with this vector at postnatal day (P) 0 via intracerebroventricular injection were monitored for spontaneous mortality up to P70 and tested for susceptibility to thermally induced seizures. All untreated mice (n=31/31) died by postnatal week 6. In addition, 86.5% (n=13/15) of them exhibited thermally induced myoclonic seizures (MCS) and 100% (n=15/15) of them showed generalized tonic clonic seizures (GTCS). In striking contrast, none of the treated mice died (n=9/9, p=3.3e-14, Fisher’s exact test) nor exhibited MCS (n=0/9, p=7.1e-4, Fisher’s exact test) or GTCS (n=0/9, p=1.1e-5, Fisher’s exact test). These findings suggest that precision therapy targeting the very site of disease etiology can completely protect against epilepsy and related mortality in DS.

Leigh syndrome (LS) is the most common form of mitochondrial disease in children. It affects 1 in every 40,000 births and its clinical manifestations include ataxia, seizures, failure to thrive and premature death. Genetic mutations in more than 75 different genes have been associated with LS. Among them is NDUFS4, the gene that codes for a subunit of the protein complex I of the mitochondria. Mice carrying a whole-body knockout (KO) of this gene greatly model this illness; they recapitulate multiple phenotypes of LS in patients. Prior studies in the lab have shown that the KO of Ndufs4 in GABAergic neurons, not in excitatory neurons, across all brain regions, reproduce the epilepsy phenotype seen in the global KO mice. Surprisingly, new KO mice with Ndufs4 inactivation restricted to GABAergic neurons of the brainstem and cerebellum interneurons, mediated by GlycineT2Cre, also have epilepsy. In this study, we sought to uncover the brain regions that house neurons involved in seizure activity in these mice. Brain regions experiencing neuronal hyperactivity during seizures in this new model of LS were examined. A thermal seizure was induced in the Ndufs4 GlycineT2Cre KO mice. For control condition, mice were exposed to a sham experiment. Forty-five minutes after the seizures or sham procedure, the mice were anaesthetized, and their brains were fixed and harvested. Brain slices were prepared and stained with a c-Fos antibody and finally imaged on the confocal microscope. Interestingly, high c-Fos immunoactivity was observed in the cerebellum alone and not in forebrain brain regions generally known to be involved in seizure generation. These findings indicate the participation of the cerebellum in seizure generation in Leigh syndrome epilepsy. In future studies we plan to increase the sample size and confirm the results with statistical methods.