Leigh syndrome is a progressive neurodegenerative mitochondrial disease stemming from genetic defects in the mitochondrial electron transport chain, such as Complex I, which leads to lactic acidosis, bilateral necrotizing brain lesions, respiratory failure, and early death. The NADH dehydrogenase [ubiquinone] iron‐sulfur protein 4 (NDUFS4, a Complex I assembly factor) knockout (KO) mouse is an established mouse model of Leigh Syndrome. The Ndufs4 KO mouse manifests many of the biochemical and clinical aspects of Leigh syndrome, including microglial accumulation in lesioned brain regions. However, the specific contribution of altered microglial metabolism in the neuropathology of Leigh Syndrome remains understudied. The inhibition of Complex I function following loss of Ndufs4 reduces the electron transport chain activity leading to mitochondrial dysfunction and increased reductive stress, manifest as an elevation of NADH:NAD+ levels. Reductive stress and the consequent inhibition of NAD+‐dependent Krebs cycle enzymes facilitates the accumulation of intermediary metabolites, including the dicarboxylate fumarate. Fumarate can irreversibly modify protein thiols, a modification known as protein succination, and we have previously demonstrated increased protein succination in the pathologically lesioned regions of the Ndufs4 KO brain. Activated immune cells produce another dicarboxylic acid, itaconate, through the action of immunoresponsive gene 1 (Irg1). Itaconate acts as part of a negative feedback loop to suppress pro‐inflammatory activity. I propose that altered levels of Krebs cycle intermediates may interfere with the capacity to mount a normal anti‐inflammatory response in Complex I deficient Ndufs4 KO microglia; specifically as a result of impaired itaconate production. This impairment is predicted to limit microglial phagocytic function and may alter the capacity to respond to infections. Real time PCR confirms increased expression of microglial genes (Iba1, Trem2, CD68), a ~2 fold increase in pro‐ (IL‐1β) and anti‐inflammatory (IL‐10 and HO‐1) factors, but a decrease in Irg1 in late stage Ndufs4 KO olfactory bulb samples. Ndufs4 KO immune cells show decreased itaconate levels, particularly in response to lipopolysaccharide (LPS, a pro‐inflammatory stimulus). LPS stimulation enhanced glycolytic metabolism, but with a lower metabolic capacity in Ndufs4 KO vs. WT cells. Inflammatory challenge also resulted in an impaired induction of several pro‐ and anti‐inflammatory cytokines signaling factors and phagocytic uptake. KO of Ndufs4 increased metabolite‐induced protein modification. Treatment of Ndufs4 KO mice with 4‐octyl itaconate, a derivative of itaconate, enhanced the expression of pro‐phagocytic TREM2 in Ndufs4 KO brain, but did not improve fine motor behavior. Overall, our data suggests that altered mitochondrial metabolism and reduced itaconate production may impair microglial ability to resolve inflammation in the Ndufs4 KO model of Leigh Syndrome.

The NDUFS4 knockout (KO) mouse phenotype resembles the human Complex I deficiency Leigh Syndrome. The irreversible succination of protein thiols by fumarate is increased in regions of the NDUFS4 KO brain affected by neurodegeneration, suggesting a mechanistic role in neurodegenerative decline. We report the identification of a novel succinated protein, dihydrolipoyllysine-residue succinyltransferase (DLST), a component of the α-ketoglutarate dehydrogenase complex (KGDHC) of the tricarboxylic acid (TCA) cycle. Succination of DLST reduced KGDHC activity in the brainstem (BS) and olfactory bulb (OB) of KO mice. We further observed decreased mitochondrial substrate level phosphorylation, a TCA cycle reaction dependent on KGDHC derived succinyl-CoA, further aggravating the OXPHOS ATP deficit. Protein succinylation, an acylation modification that requires succinyl-CoA, was reduced in the KO mice. Our data demonstrate that the biochemical deficit extends beyond the Complex I assembly and energy defect, and functionally impairs multiple mitochondrial parameters to accelerate neuronal dysfunction.

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