Axodendritic signaling of APP-CTF

APP-CTF 的轴突信号传导

• 批准号: 8622896
• 负责人: ANGELE PARENT
• 金额: $ 19.75万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-05-01 至 2016-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8622896
• 关键词: Address; Adenylate Cyclase; Affect; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Axon; Binding; C-terminal; CREB1 gene; Cell physiology; Chimera organism; Chimeric Proteins; Complex; Conserved Sequence; Coupled; Coupling; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Tail; Dendrites; Development; Dominant-Negative Mutation; Drosophila genus; Event; Fluorescence Resonance Energy Transfer; Functional disorder; G Protein-Coupled Receptor Signaling; G-Protein-Coupled Receptors; GTP-Binding Proteins; Health; Heterotrimeric G Protein Subunit; Image; Impairment; Integral Membrane Protein; Investigation; Knockout Mice; Lead; Learning; Left; Length; Life; Literature; Mediating; Membrane; Memory; Monitor; Morphology; Mutagenesis; Mutation; Neurites; Neuronal Dysfunction; Neurons; Pathogenesis; Pathology; Pathway interactions; Pharmacology; Phosphorylation; Physiological; Presynaptic Terminals; Process; Production; Protein Binding; Protein C; Proteins; Regulation; Reporting; Research; Role; S-(2-chloro-1,1,2-trifluoroethyl)glutathione; Series; Signal Pathway; Signal Transduction; Site-Directed Mutagenesis; Synaptic plasticity; Testing; amyloid peptide; amyloid precursor protein processing; amyloidogenesis; base; design; human LTB4R protein; insight; mutant; novel; overexpression; protein expression; protein function; protein protein interaction; receptor; response; secretase; sequential proteolysis; spatiotemporal; synaptic function; synaptogenesis; tau Proteins; trafficking

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.

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 ß-淀粉样肽 (Aß) 的积累。 α- 和 β- 分泌酶裂解 APP 会释放整个胞外域，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-分泌酶进一步处理。为了以组成型方式激活与 APP-CTF 相关的推定信号传导，我们设计了膜束缚的 APP 胞质结构域 (mAICD)。我们发现，通过处理 APP 或表达 mAICD（但不是 AICD）产生的 APP-CTF 的积累会导致腺苷酸环化酶依赖性 PKA 激活、GSK3ß 抑制以及原代皮质神经元轴突树突状树枝化增强。我们发现了 APP 胞内结构域和异源三聚体 G 蛋白亚基 GαS 之间的新相互作用。通过对 APP 内确定的相互作用基序进行诱变以及显性失活 G�S 突变体的表达，我们证明与 GαS 的相互作用以及随后的 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至关重要。此外，我们的初步结果表明，先前描述的 APP-CTF 的 GαO 结合基序的突变也会减少树突生长。因此，通过与其他 G 蛋白偶联受体类比，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 分子上随机或同时结合，从而动态调节 APP 在神经元形态和神经元功能障碍中的功能。有趣的是，APP 加工和 Aß 生产是一个受到许多磷酸化事件控制的高度调控的过程，这些磷酸化事件可能受到 G 蛋白偶联受体信号级联的影响。基于这些发现，我们假设 APP 细胞质结构域与 GαS/GαO 蛋白的时空信号传导可以选择性地影响轴突发育并影响 AD 发病机制。为了研究这一假设，我们建议检查（1）β-CTF 的轴突定位是否受到 G 蛋白相互作用的调节； (2) 表征ß-CTFG-蛋白质相互作用如何选择性影响轴突信号传导； (3) 阐明 APP-CTF 的时空定位如何影响全长 APP 加工和 Aß 生产。总而言之，我们的研究提供了明确的证据，表明 APP 胞内结构域通过 cAMP 依赖性信号传导和 GSK3ß 抑制（已知这两个过程在记忆巩固、Aß 产生和 Tau 相关病理学中发挥作用）的膜关联，在调节神经突生长方面具有非转录作用。我们的研究将解决以前未被认识的与 APP-CTF 相关的细胞内信号通路的重要性。更好地了解 APP-CTF 及其相关信号传导伙伴可能会为 APP-CTF 影响突触功能和 Aß 产生的细胞机制提供重要见解，从而对 AD 发病机制产生影响。