Relatively little information is available concerning macromolecular changes that may occur in normal aging and Alzheimer's disease (AD) brains at the levels of DNA, RNA and protein synthesis. The overall goals of this grant proposal are to test the hypotheses that, first, human brain messenger RNA (mRNA) levels, structure, function and/or products of translation may change during normal aging; and, second, that more drastic alterations may represent pathogenetic mechanisms relevant to the development of hallmark lesions of the demented brain, the neuritic plaques and neurofibrillary tangles. The first hypothesis will be tested by a comprehensive series of investigations on normal postmortem (pm) human brains of various ages; the additional influence of the pm interval will be carefully evaluated by comparisons with human biopsy specimens and laboratory animals. The second hypothesis will be tested by studies on AD pm brains and biopsy specimens that are histopathologically rated and suitably matched to normal controls. The following experiments will be conducted: (1) Measurement of the yield, size and in vitro protein synthesis activity of mRNA. Our preliminary studies have demonstrated that mRNA yield and activity are decreased in the pm AD brain compared to normal controls. (2) Several mechanisms responsible for mRNA alterations will be evaluated. These include: an increase in the absolute levels of tissue ribonucleases; a redistribution of lysosomal ribonucleases; alterations in the 5'-terminal cap structure of mRNA; and, alterations in the 3'-terminal polyadenylic acid sequences. (3) We shall examine whether or not alterations occur in the levels of neuronal cytoskeletal proteins (neurofilament proteins (NFPs), tubulin, and actin) synthesized in vitro by mRNA from discrete regions of the AD brain and then compare these data to levels of the native proteins in tissues from the same regions. Preliminary studies have shown decreased activity of most AD brain mRNAs including tubulin mRNA, however, the actin mRNA appears to be spared. We shall use monoclonal and monospecific antibodies to quantify cytoskeletal protein levels in in vitro protein synthesis systems and in tissues. If AD brain mRNA alterations are found then monoclonal antibodies to NFPs will demonstrate whether or not the alterations are neuron-specific. (4) These investigations will also be carried out on affected and unaffected regions of the same AD brain. (5) All biochemical data will be quantitatively correlated with the degree of plaque and tangle involvement. The proposed experimentation represents the first extensive exploration of normal human brain aging and AD at the level of mRNA and protein synthesis. These studies bear upon the possibility that alterations in specific gene products are directly related to the etiology of Alzheimer's disease.
