Abstract TMP109: Labeling And Isolating Cell Specific Neuronal Mitochondria And Their Functional Analysis In Mice Post Stroke

Introduction: In vivo neuronal mitochondria dysfunction post stroke has not been well investigated. Methods: Using Mito-tag mice in combine with local infection of AAV9 produced from plasmids carrying Cre-recombinase controlled by neuronal promoters, we generated reporter mice expressing a hemagglutinin (HA)-tagged EGFP that localizes to mitochondrial outer membranes in synaptophysin (SYN) and calmodulin-kinase II (CaMKII) glutamatergic neurons localized to cerebral cortex. These mice were subjected to permanent middle cerebral artery occlusion and sacrificed 14 days post stroke. Fresh brain tissues were excised from the ischemic core (IC), ischemic boundary zone (IBZ) as well as from the homologous contralateral hemisphere, and the crude mitochondrial fractions (CMF) were extracted. Neuronal mitochondria were then isolated from the CMF by anti-HA magnetic beads (Thermo Scientific) and their specificity and oxidative phosphorylation (OXPHOS) complexes were confirmed by means of Western-blot. Seahorse assay was employed to measure oxygen consumption rate (OCR) of the isolated mitochondria as an indicator of mitochondrial function. Results: The bead pulled components were co-precipitated with GFP and enriched with mitochondrial markers, e.g. voltage-dependent anion channel, cytochrome C, and COX IV, but lacked the Golgi protein RCAS1 as well as endoplasmic reticulum markers: Heme-oxygenase 1 and Calnexin, indicating that specific neuronal mitochondria have been selectively isolated. Western-blot data showed that OXPHOS components in SYN + and CAMKII + neuronal mitochondria were significantly downregulated in the IBZ and further decreased in the IC compared to the contralateral tissue, which was associated with the significant reductions of mitochondrial function measured by OCR (p<0.05, respectively, for both neuron types). These data suggesting dysfunction of neuronal mitochondria post stroke. Conclusions: We, for the first time, measure mitochondrial function in 2 types of neurons post stroke. By introducing specific neuronal promoter controlled Cre recombinase to Mito-tag mouse, mitochondria from individual neuronal linage cells can be efficiently labeled and isolated to investigate functional change post stroke.