Labeling and isolating cell specific neuronal mitochondria and their functional analysis in mice post stroke.

Dendritic and axonal plasticity, which mediates neurobiological recovery after a stroke, critically depends on the mitochondrial function of neurons. To investigate, in vivo, neuronal mitochondrial function at the stroke recovery stage, we employed Mito-tag mice combined with cerebral cortical infection of AAV9 produced from plasmids carrying Cre-recombinase controlled by two neuronal promoters, synapsin-I (SYN1) and calmodulin-kinase IIa to induce expression of a hemagglutinin (HA)-tagged enhanced green fluorescence protein (EGFP) that localizes to mitochondrial outer membranes of SYN1 positive (SYN) and CaMKIIa positive (CaMKIIa) neurons. These mice were then subjected to permanent middle cerebral artery occlusion (MCAO) and sacrificed 14 days post stroke. Neuronal mitochondria were then selectively isolated from the fresh brain tissues excised from the ischemic core (IC), ischemic boundary zone (IBZ), as well as from the homologous contralateral hemisphere (CON) by anti-HA magnetic beads for functional analyses. We found that the bead pulled neuronal specific mitochondria 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 oxidative phosphorylation (OXPHOS) components in SYN and CAMKII neuronal mitochondria were significantly decreased in the IBZ and further decreased in the IC compared to the contralateral tissue, which was associated with the significant reductions of mitochondrial function indicated by oxygen consumption rate (OCR) (p < 0.05, respectively, for both neuron types). These data suggest dysfunction of neuronal mitochondria post stroke is present during the stroke recovery stage. Collectively, for the first time, we demonstrated that using a Mito-tag mouse line combined with AAV9 carrying Cre recombinase approach, neuronal specific mitochondria can be efficiently isolated from the mouse brain to investigate their functional changes post stroke.