Abstract WP337: the Ischemic Neuroprotectant Protein Kinase C Epsilon, Phosphorylates Enzymes in Neuronal NADH Shuttle and Regulates Mitochondrial Respiration and Glycolysis

Background: Among PKCε-mediated potential protective mechanisms in ischemic preconditioning, one target that we surmise is crucial for neuroprotection is the malate-aspartate shuttle (MAS). MAS is the most important NAD + /NADH shuttle importing reducing equivalents (NADH) generated by glycolysis from the cytosol into the mitochondria, and thus, tightly controls neuronal respiration. We hypothesized that PKCε regulates MAS in the brain and mediates neuronal mitochondrial respiration. Method: Phosphoproteomics analysis and immunoprecipitation (IP) were used to determine PKCε-induced phosphorylation of MAS components. Synaptosomes from cortices of naïve SD rats were treated with Tat-conjugated ΨεRACK (selective PKCε activator) or control Tat peptide and sent for phosphoproteomics analysis. To confirm the predicted phosphorylation, IP was performed. SD rats were treated with ΨεRACK (i.p.) or Tat and sacrificed 48hrs later. Proteins from rat cortical synaptosomes were immunoprecipitated with phosphoserine/threonine/tyrosine and immunoblotted for predicted phosphorylated proteins. Once phosphorylation was determined, we evaluated energy metabolism in primary neurons. Neuronal cultures were obtained from embryonic pups cortices and treated with ΨεRACK or Tat on DIV 7-10. Cellular respiration and glycolytic function were evaluated 48hrs after treatment using Seahorse XF Analyzer. Result: Phosphoproteomics analysis and IP results revealed increased phosphorylation of key MAS components: the mitochondrial aspartate aminotransferase (GOT2) and the oxoglutarate carrier (OGC). After PKCε activation, GOT2 and OGC exhibited serine phosphorylation increased by 2.14 fold (P < 0.05, n=4-5), and 1.74 fold (P < 0.01, n=4-5), respectively. In addition, in primary neurons, ΨεRACK treatment increased the basal and maximum cellular respiration by 0.13 (P < 0.05, n=4), and 0.22 fold (P < 0.01, n=4), respectively. The basal and maximum glycolysis were increased by 0.14, and 0.21 fold (P<0.05, n=4), respectively. Conclusion: Our data strongly suggest that PKCε regulates MAS, and increases neuronal metabolism by promoting cellular respiration and glycolysis. These changes may be linked to our observed PKCε-induced ischemic tolerance in the brain.