The brain energy metabolism is met primarily in mitochondria by oxidative metabolism of pyruvate, which is regulated by pyruvate dehydrogenase complex (PDHC). The PDHC is controlled by a kinase-phosphatase mediated phosphorylation (inactivation) and dephosphorylation (activation) process. Deficits of PDHC have been found in an increasing number of neurological disorders, including Alzheimer's disease. Changes of phosphorylation or activation state of PDHC have also been found to accompany many physiological, pharmacological and behavioral manipulations that give rise to altered brain functions. These changes of PDHC may reflect changes of pyruvate oxidation or Ca sequestration by mitochondria. The molecular events underlying these changes are unclear, however. To this end, the relationship among PDHC regulatory behavior, pyruvate ovidation and mitochondrial Ca sequestration will be delineated in this proposition. The mitochondrial Ca-uptake process is distinct from the effect of intramitochondrially accumulated Ca. The effects of these two processes on pyruvate oxidation and PDHC phosphorylation will be evaluated, using mitochondria treated with ionophore A23187 in Ca-EGTA buffer and mitochonria pre-loaded or depleted with Ca, respectively. With respect to enzyme protein, PDHC may be regulated by phosphorylation at multiple sites and by protein translation during postnatal development. The significance of multiple phosphorylation will be evaluated by comparing the phosphate profile of the phosphopeptide generated by immunoprecipitation and 2-dimensional gel electrophoresis. The enzyme induction mechanism will be tested by radioimmunoassays and in vivo translation assays of the PDHC-peptides. Aging decreases pyruvate oxidation and Ca metabolism. To further test the hypothesis that mitochondrial Ca sequestration is also imparied in aging, the Ca buffering capacity, and Ca uptake and its coupling to pyruvate oxidation and possible link to PDHC phosphorylation will be comared in mitochondria from different age groups.
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