Axodendritic signaling of APP-CTF

阿尔茨海默病（AD）的病理特征是通过淀粉样前体蛋白（APP）的连续蛋白水解产生的β-淀粉样肽（AAPs）的积累。α-和β-分泌酶切割APP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γ-分泌酶进一步加工。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，积累的APP-CTF产生的APP处理或表达mAICD（但不是AICD）的结果在腺苷酸环化酶依赖性激活PKA，抑制GSK 3 β，并增强轴突树突树枝在原代皮层神经元。我们鉴定了APP胞内结构域与异源三聚体G蛋白亚基GαS之间的一种新的相互作用。通过对APP内鉴定的相互作用基序的诱变以及显性负G S突变体的表达，我们证明了与GαS的相互作用以及随后Gα S与腺苷酸环化酶的偶联对于膜结合APP胞内结构域诱导的神经突生长是必不可少的。此外，我们的初步结果表明，先前描述的APP-CTF的GαO结合基序的突变也减少了树突状生长。因此，与其他G蛋白偶联受体类似，通过APP-CTF的G蛋白介导的信号传导可能涉及GαO和GαS在同一APP分子上的随机或同时结合，从而允许在神经元形态和神经元功能障碍中动态调节APP功能。有趣的是，APP加工和AAPO 2产生是一个高度调节的过程，在许多磷酸化事件的控制下，可能受到G蛋白偶联受体信号级联的影响。基于这些发现，我们推测APP胞浆区与GαS/Gα O蛋白的时空信号传导可以选择性地影响轴树突发育并影响AD的发病机制。为了研究这一假设，我们提出检查（1）APP-CTF的轴树突定位是否受G-蛋白相互作用调节;（2）表征APP-CTF-蛋白相互作用如何选择性地影响轴树突信号传导;以及（3）阐明APP-CTF的时空定位如何影响全长APP加工和AAP 10产生。总之，我们的研究提供了明确的证据，即APP胞内结构域在通过cAMP依赖性信号传导和GSK 3 β抑制通过其膜缔合调节神经突生长中具有非转录作用，这两个过程已知在记忆巩固、AAPO 2产生和Tau相关病理中具有作用。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。对APP-CTF及其相关信号分子的深入了解，可能有助于我们深入了解APP-CTF影响突触功能和腺苷酸产生的细胞机制，从而对AD的发病机制产生影响。

Alzheimer's disease (AD) is pathologically characterized by the accumulation of ß-amyloid peptides (Aß) generated via sequential proteolysis of amyloid precursor protein (APP). Cleavage of APP by α- and ß- secretases releases the entire ectodomain, leaving behind membrane bound C-terminal fragments (CTF) capable of mediating intracellular signaling until they are further processed by γ-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 GSK3ß, and enhanced axondendritic arborization in primary cortical neurons. We identified a novel interaction between APP intracellular domain and the heterotrimeric G-protein subunit GαS. By mutagenesis of the interaction motif identified within APP as well as expression of a dominant negative G�S mutant, we demonstrated that interaction with GαS and subsequent GαS coupling to adenylate cyclase are essential for membrane-bound APP intracellular domain-induced neurite outgrowth. Moreover, our preliminary results indicate that mutation of a previously described GαO binding motif of APP-CTF also reduces dendritic outgrowth. Thus, by analogy to other G-protein coupled receptors, it is possible that G- protein-mediated signaling through APP-CTF involves stochastic or simultaneous binding of GαO and GαS on the same APP molecule allowing dynamic regulation of APP function in neuronal morphology and neuronal dysfunction. Interestingly, APP processing and Aß production is a highly regulated process under the control of a number of phosphorylation events that could be affected by G-protein coupled receptor signaling cascades. Based on these findings, we hypothesize that spatiotemporal signaling of APP cytoplasmic domain with GαS/GαO-proteins could selectively affect axodendritic development and impact on AD pathogenesis. In order to investigate this hypothesis, we propose to examine (1) if axodendritic localization of ß-CTF is regulated by G- protein interactions; (2) characterize how ß-CTFG-protein interactions selectively affect axodendritic signaling; and (3) elucidate how spatiotemporal localization of APP-CTF affects full-length APP processing and Aß production. Altogether, our study provides clear evidence that APP intracellular domain has a non- transcriptional role in regulating neurite outgrowth through its membrane association via cAMP-dependent signaling and GSK3ß inhibition - two processes that are known to have a role in memory consolidation, Aß production and Tau-associated pathology. 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 Aß production, thus have an impact on AD pathogenesis.