We have previously explored the metabolism of B-amyloid precursor (APP) in a variety of cultured cells models and sought to produce insoluble Beta-amyloid deposits in cultured human neuronal cells. Mutations flanking the Beta-protein domain migrate with familial Alzheimer's Disease (FAD) supporting the hypothesis that accelerate B-amyloid deposition is sufficient to cause AD. APP670/671 mutations markedly accelerate B-protein production, but now FAD APP717 mutations effect APP and Beta-protein metabolism remains controversial; it may result in a greater percentage of longer forms of B-protein. Because it is a critical determinant in aggregation, we will investigate the C-terminus of secreted B. We are developing specific antibody probes to the C- terminus of B to explore this region in experiments with cultured cells with and without the APP717 mutations, and in AD and control CSF and brains available through the ADRC. Unfortunately, APP-like molecules of similar size are detected by many N-terminal APP antibodies and all C- terminal APP antibodies confusing results in most cultured cell systems even if transection are used. Hence, we are using CNS-derived neuroblastoma which lack detectable endogenous APP or APP-like cross- reacting molecules to examine the FAD APP mutations effects and APP metabolism. Secretion of B protein by cultured cells raise a number of issues concerning the metabolism of B-protein in vitro and the relationship of normal B-protein secretion to pathological B-protein deposition. We propose experiments to analyze the cellular pathway involved in B- production using a unique collection of processing mutants and treatment. Because B-degradation may be just as relevant to amyloidosis as production rates, we also design experiments to define the mechanism of B-degradation which is completely unknown. We have detected and propose to identify unknown proteins associated with APP, and may be involved in APP metabolism. Differences in the transport and metabolism of alternatively spliced APP are suggested from experiments with brain and CSF, but have not been shown in cultured cells. APP751, but not APP695 transgenics are reported to make diffuse B deposits. Thus, we also propose to look at B-protein and APP isoform metabolism in polarized APP transfected and parent NT2/D1 derived human neurons. These experiments should have a direct impact on understanding AD pathogenesis.
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