Mutations in presenilins are closely linked to familial Alzheimer's Disease (AD). It is well established that presenilin expression is essential for """"""""y-secretase"""""""" processing of many proteins of diverse functions, including the amyloid precursor protein and cell adhesion protein cadherins. This laboratory cloned a human delta-catenin that associates with cadherins and glutamate receptors of synaptic junction, and it redistributes upon glutamate stimulation. Our recent studies found that delta-catenin not only binds directly to presenilin-1 (PS-1), but it is also cleaved by a S-1/y-secretase-dependent activity. This RO3 small grant application proposes a research project to test the hypothesis that delta-catenin cleavage products interact with glutamate receptor complexes and facilitate postsynaptic responses to excitatory stimuli, such as amyloid beta (AB) peptides and N-methyl-D-aspartate, which increase neuronal vulnerability to excitotoxicity in AD. To test this hypothesis, Specific Aim 1 will employ PC12 cells stably expressing a tetracycline-inducible delta-catenin to determine the amino acid sequence of delta-catenin cleavage site generated by PS-1/y-secretase dependent activity. Delta-Catenin cleavage sequence will be determined using protein sequencing, site-directed mutagenesis, and cDNA transfection.
Specific Aim 2 will determine the distribution and effects of delta-catenin proteolytic fragments in the postsynaptic responses to excitatory stimuli, such as AB peptides and NMDA. Protein co-immunoprecipitation and Western Blotting will determine the interaction of delta-catenin fragments with glutamate receptor complexes. Time-lapse fluorescent microscopic imaging will determine their redistribution upon AB and NMDA stimulation. In addition, Fura-2 ratiometric imaging will be used to determine the effects of delta-catenin fragments on the alterations of intracellular calcium level, a central component in the excitotoxic neuronal death cascade. This RO3 small grant project will lead to new insights into mechanisms of synaptic plasticity regulated by PS-1 interaction with delta-catenin. It will also lay the foundation for future R01 investigation of the roles of delta-catenin proteolytic fragments in AD pathogenesis.
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