Delta-Catenin Cleavage by Presenilin and Synaptic Remodeling

描述(由申请人提供)：早老素的突变与家族性阿尔茨海默病(AD)密切相关。众所周知，早老素的表达对许多具有不同功能的蛋白质的“y-分泌酶”的处理是必不可少的，包括淀粉样前体蛋白和细胞黏附蛋白钙粘蛋白。本实验室克隆了一个与突触连接的钙粘附素和谷氨酸受体相关的人β-连环素，并在谷氨酸刺激下重新分布。我们最近的研究发现，β-连环蛋白不仅直接与早老素-1(PS-1)结合，而且还被依赖于S-1/y-分泌酶的活性所切割。这项RO小额拨款申请提出了一项研究项目，旨在测试以下假设：Delta-Catenin裂解产物与谷氨酸受体复合体相互作用，并促进突触后对兴奋性刺激的反应，如淀粉样β(AB)肽和N-甲基-D-天冬氨酸，这些刺激增加了AD患者神经元对兴奋性毒性的易感性。为了验证这一假设，特定目标1将使用稳定表达四环素诱导的Delta-catenin的PC12细胞来确定PS-1/y-分泌酶依赖活性产生的Delta-catenin裂解位点的氨基酸序列。Delta-Catenin裂解序列将通过蛋白质测序、定点突变和cDNA转染来确定。特异性目标2将确定β-连环蛋白降解片段在突触后对兴奋性刺激的反应中的分布和作用，如AB肽和NMDA。蛋白质免疫共沉淀和Western blotting将确定Delta-Catenin片段与谷氨酸受体复合体的相互作用。时间推移荧光显微成像将确定它们在AB和NMDA刺激下的重新分布。此外，Fura-2比率成像将被用来确定Delta-Catenin片段对细胞内钙水平变化的影响，细胞内钙水平是兴奋性毒性神经元死亡级联反应的中心成分。这一R03小额赠款项目将导致对PS-1与Delta-catenin相互作用调节突触可塑性机制的新见解。这也为未来R01研究Delta-Catenin蛋白水解物片段在AD发病中的作用奠定了基础。

DESCRIPTION (provided by applicant): 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.