To elucidate the molecular mechanisms for the decrease in activity of the pyruvate dehydrogenase complex (PDHC) in Alzheimer brain, a series of studies will be carried out. To test stability post mortem, enzyme activities will be compared in neurosurgical waste tissue and autopsy brain. ELISAs will test whether each of the PDHC peptide antigens is present in normal or reduced amount and binds the antibody normally (ie has epitopes comparable to those of control peptides). The functional normality of the mRNAs coding for these peptides will be tested in in vitro translation assays. In vitro translation assays in the presence of mitochondria will test for the normality of post- translational processing of these peptides. Molecular genetic studies will be done if abnormalities are found in specific peptide(s) and will concentrate on those peptide(s). These studies will include preparation of cDNA clones from a human/gt11 library; characterization of the relevant mRNAs by Northern blots and S1 nuclease mapping; localization of the relevant gene(s) on human chromosomes; RFLP analyses to test the linkage of the abnormality in DNA to the disease; and ultimately cloning and sequencing of the relevant normal and Alzheimer gene(s). Immunochemical studies will concentrate on brain, where abnormalities have already been demonstrated; any immunochemical aberrations found in brain will, however, be tested for as well in cultured fibroblasts. The translation and genetic studies will utilize chiefly cultured cells, to minimize potential agonal and post-mortem artifacts.
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