Axodendritic signaling of APP-CTF

阿尔茨海默病（AD）的病理特征是淀粉样蛋白前体蛋白（APP）的顺序蛋白水解产生ß-淀粉样肽（ß）的积累。α-和β分泌酶对APP的切割释放整个外畴，留下能够介导细胞内信号传导的膜结合c端片段（CTF），直到它们被γ分泌酶进一步处理。为了以一种推定的方式激活与APP- ctf相关的信号，我们设计了一个膜系住的APP细胞质结构域（mAICD）。我们发现，由APP加工或mAICD（而非AICD）表达产生的APP- ctfs的积累导致PKA的腺苷酸环化酶依赖性激活，GSK3ß的抑制，以及初级皮质神经元轴突树突的增强。我们发现了APP胞内结构域与异三聚体g蛋白亚基GαS之间的一种新的相互作用。通过诱变APP中发现的相互作用基序以及显性GαS负突变体的表达，我们证明了与GαS的相互作用以及随后GαS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的生长。因此，与其他G蛋白偶联受体类似，G蛋白通过APP- ctf介导的信号传导可能涉及Gα o和Gα s在同一APP分子上的随机或同时结合，从而动态调节APP功能对神经元形态和功能障碍的影响。有趣的是，APP加工和asas的产生是一个高度调控的过程，受g蛋白偶联受体信号级联反应影响的磷酸化事件的控制。基于这些发现，我们推测APP细胞质域的GαS/ g α o蛋白时空信号可能选择性地影响树突发育并影响AD发病机制。为了验证这一假说，我们提出检验(1)ß-CTF的轴突定位是否受G-蛋白相互作用的调节；(2)表征ß- ctfg蛋白相互作用如何选择性地影响轴突信号传导；(3)阐明APP- ctf的时空定位对全长APP加工和ass产生的影响。总之，我们的研究提供了明确的证据，表明APP胞内结构域在调节神经突生长中具有非转录作用，通过camp依赖的信号传导和GSK3ß抑制来调节其膜关联，这两个过程已知在记忆巩固、asβ产生和tau相关病理中起作用。我们的研究将解决与APP-CTF相关的先前未被识别的细胞内信号通路的重要性。更好地了解APP-CTF及其相关信号伙伴可能为APP-CTF影响突触功能和β生成的细胞机制提供重要见解，从而对AD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.