Abstract Mitochondrial encephalomyopathy with mitochondrial DNA (mtDNA) depletion is a class of mitochondrial disease that causes neurological dysfunction with intellectual and developmental disabilities and myopathy. Deficiency of ADP-specific succinyl-CoA synthetase (SCS), a component of the TCA cycle, is one of the causes of mitochondrial encephalomyopathy with mtDNA depletion. SCS is an important integration point of the TCA cycle, intersecting energy metabolism and posttranslational succinylation. Previous studies have demonstrated that embryonic mice homozygous for a constitutive mutant Sucla2 allele are embryonic lethal and exhibit ADP-specific SCS deficiency and mtDNA depletion with detrimental effects on energy metabolism. For this proposal, to generate adult Sucla2 deficient animals, a conditional knockout strategy of Sucla2 limited to the postnatal forebrain is employed. Initial investigations reveal that the mutant animals exhibit a defect in learning and memory and perturbations of energy metabolism and global protein succinylation in the cortex in the absence of mtDNA depletion. This proposal is based on the hypothesis that adult phenotypes of Sucla2 deficiency are caused by a combination of TCA cycle dysfunction and perturbations of global posttranslational succinylation. The Sucla2-forebrain knockout model will be analyzed by a targeted battery of behavioral assays designed to fully characterize different cognitive domains for deficits. To determine the potential differential effects of developmental timing of Sucla2 loss, a conditional knockout with prenatal onset of Sucla2 deficiency will also be generated and subjected to longitudinal broad behavioral and in vivo physiological screening, including basic motor, autonomic, sensory, and cognitive assays in both adolescent and adult stages. Cortex from the forebrain conditional knockout model will be analyzed for histopathological changes, metabolomic alterations, TCA and ETC enzyme activities, global posttranslational succinylation and mtDNA content. An innovative approach using integrated single nuclear (sn) RNA-seq and snATAC-seq to identify differential gene expression patterns and alterations in chromatin accessibility landscape in different cortical cell types defined by distinct expression clusters will be employed and the datasets used to inform histopathological and metabolomic analyses. These studies will provide insights into the pathogenic mechanisms of Sucla2/SCS deficiency and establish a preclinical model to explore and develop novel therapeutic approaches.