Delta-Catenin Cleavage by Presenilin and Synaptic Remodeling

描述（由申请人提供）：早老素突变与家族性阿尔茨海默病（AD）密切相关。已经确定，早老素表达对于许多不同功能的蛋白质的“γ-分泌酶”加工是必不可少的，包括淀粉样前体蛋白和细胞粘附蛋白钙粘蛋白。本实验室克隆了一种人δ-连环蛋白，它与突触连接处的钙粘蛋白和谷氨酸受体相关，并在谷氨酸刺激后重新分布。我们最近的研究发现，δ-连环蛋白不仅直接结合早老素-1（PS-1），但它也被S-1/γ-分泌酶依赖性活性切割。该RO 3小额资助申请提出了一个研究项目，以测试以下假设：δ-连环蛋白裂解产物与谷氨酸受体复合物相互作用，并促进对兴奋性刺激的突触后反应，如淀粉样蛋白β（AB）肽和N-甲基-D-天冬氨酸，这增加了AD中神经元对兴奋性毒性的脆弱性。为了检验这一假设，Specific Aim 1将采用稳定表达四环素诱导的δ-连环蛋白的PC 12细胞来确定由PS-1/γ-分泌酶依赖性活性产生的δ-连环蛋白切割位点的氨基酸序列。将使用蛋白质测序、定点诱变和cDNA转染来确定δ-连环蛋白切割序列。具体目标2将确定δ-连环蛋白蛋白水解片段在兴奋性刺激（如AB肽和NMDA）的突触后反应中的分布和作用。蛋白质免疫共沉淀和蛋白质印迹将确定δ-连环蛋白片段与谷氨酸受体复合物的相互作用。延时荧光显微成像将确定它们在AB和NMDA刺激后的重新分布。此外，Fura-2比率成像将用于确定δ-连环蛋白片段对细胞内钙水平改变的影响，细胞内钙水平是兴奋性毒性神经元死亡级联反应的中心组分。这个RO 3小额资助项目将导致对PS-1与δ-连环蛋白相互作用调节的突触可塑性机制的新见解。这也将为未来的R 01研究delta-catenin蛋白水解片段在AD发病机制中的作用奠定基础。

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.