Animal models of membrane-targeted APP intracellular domain - Resubmission 01

描述（由申请人提供）：阿尔茨海默病（AD）的病理特征是？-淀粉样肽（A？）通过淀粉样前体蛋白（APP）的连续蛋白水解产生。近年来，APP被认为具有多种生理功能，它可以通过其细胞粘附特性或通过其介导细胞内信号传导的受体样功能来影响突触功能。全长APP的切割？然后呢？分泌酶释放整个胞外域，留下能够介导细胞内信号传导的膜结合C末端片段（CTF），直到它们被？分泌酶为了以组成型方式激活与APP-CTF相关的推定信号传导，我们设计了膜系连的APP胞质结构域（mAICD）。我们发现，由APP加工或mAICD（但不是AICD）表达产生的APP-CTF的积累导致腺苷酸环化酶依赖的PKA激活，GSK 3？抑制，并增强原代皮层神经元中的树突和轴突分支。我们确定了一种新的APP胞内结构域和异源三聚体G蛋白亚基G？S，并通过诱变的互动基序内APP以及表达的显性负G？S突变体，表明与G？S和随后的G？S偶联腺苷酸环化酶是必不可少的膜结合APP胞内结构域诱导的突起生长。我们的研究提供了明确的证据表明，APP胞内结构域可以通过其膜缔合在调节神经突起生长中发挥非转录作用。而且，此前有消息称，APP还能与G？O.已知cAMP/PKA通路的激活影响几种脑功能，如突触可塑性和记忆形成，以及A？通过非淀粉样蛋白生成途径产生。基于这些发现，我们推测，APP胞浆结构域与G蛋白的功能耦合可能会影响神经突起的生长，突触的形成和A？？生产为了研究这一假设，我们建议开发过表达mAICD或缺乏G蛋白结合位点的mAICD突变体的小鼠模型。我们将采用重组腺相关病毒（AAV）交付，也产生转基因小鼠模型，以评估mAICD表达在大脑中的生理病理学相关性及其在基因治疗中的推定价值。淀粉样蛋白沉积，A？？在AAV注射或转基因杂交育种后，在APP-无效小鼠、共表达APP和早老素的家族性AD连锁突变体的小鼠及其对照同窝出生的小鼠中监测神经突产生、刺激神经突生长和突触形成。我们的研究将解决一个以前未被识别的细胞内信号通路与APP-CTF的重要性。更好地了解APP-CTF及其相关的信号合作伙伴可能提供重要的见解APP-CTF影响突触功能和A？生产，这可能对AD发病机制产生潜在影响。

DESCRIPTION (provided by applicant): Alzheimer's disease (AD) is pathologically characterized by the accumulation of ?-amyloid peptides (A?) generated via sequential proteolysis of amyloid precursor protein (APP). Over the years several physiological functions have been ascribed to APP. It has been proposed that APP can affect synaptic function by its dual roles via its cell-adhesive properties or through its putative receptor-like function that mediates intracellular signaling. Cleavage of full-length APP by ? and ?-secretases releases the entire ectodomain, leaving behind membrane bound C-terminal fragments (CTF) capable of mediating intracellular signaling until they are further processed by ?-secretase. In order to activate in a constitutive manner putative signaling associated with APP-CTF, we have designed a membrane-tethered APP cytoplasmic domain (mAICD). We found that accumulation of APP-CTFs generated by processing of APP or expression of mAICD (but not AICD) results in adenylate cyclase-dependent activation of PKA, inhibition of GSK3?, and enhanced dendritic and axonal arborization in primary cortical neurons. We identified a novel interaction between APP intracellular domain and the heterotrimeric G-protein subunit G?S, and by mutagenesis of the interaction motif within APP as well as expression of a dominant negative G?S mutant, demonstrate that interaction with G?S and subsequent G?S coupling to adenylate cyclase are essential for membrane-bound APP intracellular domain-induced neurite outgrowth. Our study provides clear evidence that APP intracellular domain can have a non-transcriptional role in regulating neurite outgrowth through its membrane association. Moreover, it was previously reported that APP could also interact with G?O. Activation of cAMP/PKA pathway is known to impact several brain functions such as synaptic plasticity and memory formation, as well as A??production through non-amyloidogenic pathway. Based on these findings, we hypothesize that functional coupling of APP cytoplasmic domain with G-proteins could influence neurite outgrowth, synapse formation and A??production. In order to investigate this hypothesis, we propose to develop mouse models that overexpress mAICD or mAICD mutant lacking G-protein binding site(s). We will employ recombinant adeno-associated viral (AAV) delivery and also generate transgenic mouse models to assess the physiopathological relevance of mAICD expression in the brain and its putative value in gene therapy. Amyloid deposition, A??production, stimulation of neurite outgrowth and synapse formation will be monitored in APP-null mice, mice coexpressing familial AD-linked mutant of APP and presenilin, and their control littermates, following AAV injections or transgenic intercross breeding. Our investigation will address the importance of a previously unrecognized intracellular signaling pathway associated with APP-CTF. A better understanding of APP-CTF and its associated signaling partners might provide important insights into the cellular mechanisms by which APP-CTF affects synaptic function and A??production, which could potentially impact on AD pathogenesis.