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

描述(申请人提供)：阿尔茨海默病(AD)的病理特征是β-淀粉样多肽(A？)通过淀粉样前体蛋白(APP)的顺序蛋白分解产生。多年来，一些生理功能被归因于APP。已有研究表明，APP可通过其细胞黏附特性或其可能的受体样功能影响突触功能，从而影响突触功能。全长应用程序的割裂被？分泌酶释放整个胞外结构域，留下能够介导细胞内信号的膜结合C末端片段(CTF)，直到它们被？-分泌酶进一步处理。为了以一种结构性的方式激活与APP-CTF相关的假定信号，我们设计了一个膜拴系APP细胞质结构域(MAICD)。我们发现，APP加工产生的APP-CTF的积累或mAICD(但不是AICD)的表达导致了PKA的腺苷环化酶依赖的激活，GSK3？的抑制，并增强了原代皮质神经元的树突和轴突树枝。我们发现了APP胞内区与异源三聚体G-蛋白亚基G？S之间的一种新的相互作用，并通过突变APP内的相互作用基序以及显性负的G？S突变体的表达，证明了与G？S的相互作用以及随后G？S与腺苷环化酶的偶联是膜结合型APP胞内区诱导轴突生长所必需的。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结合在调节轴突生长中发挥非转录作用。此外，先前报道APP还可以与G？O相互作用。已知激活cAMP/PKA通路可以影响一些脑功能，如突触可塑性和记忆形成，以及通过非淀粉样变通路产生A？？基于这些发现，我们推测APP胞浆结构域与G蛋白的功能偶联可能影响突起的生长、突触的形成和Aβ的产生。为了研究这一假说，我们建议建立过表达mAICD的小鼠模型或缺乏G蛋白结合位点的mAICD突变体(S)。我们将采用重组腺相关病毒(AAV)传递，并建立转基因小鼠模型，以评估mAICD在大脑中表达的生理病理相关性及其在基因治疗中的潜在价值。在注射AAV或转基因杂交后，将监测APP缺失型小鼠、共表达家族性AD连锁突变体APP和早老素突变的小鼠及其对照仔鼠的淀粉样蛋白沉积、Aβ产生、对轴突生长和突触形成的刺激作用。我们的研究将解决与APP-CTF相关的先前未知的细胞内信号通路的重要性。更好地了解APP-CTF及其相关的信号伙伴可能为APP-CTF影响突触功能和Aβ产生的细胞机制提供重要的见解，这可能对AD的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.