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

膜靶向 APP 胞内结构域的动物模型 - 重新提交 01

• 批准号: 8665364
• 负责人: ANGELE PARENT
• 金额: $ 23.7万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-01 至 2016-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8665364
• 关键词: Accounting; Address; Adenylate Cyclase; Adhesives; Affect; Alzheimer' s Disease; Amyloid beta-Protein Precursor; Amyloid deposition; Animal Model; Binding; Binding Sites; Brain; Breeding; C-terminal; CREB1 gene; Cell physiology; Cells; Complex; Conserved Sequence; Coupling; Cultured Cells; Cyclic AMP; Cyclic AMP-Dependent Protein Kinases; Cytoplasmic Tail; Dendritic Spines; Deposition; Dominant-Negative Mutation; Drosophila genus; Elements; Etiology; Event; G-Protein-Coupled Receptors; GTP-Binding Proteins; Generations; Genes; Heterotrimeric G Protein Subunit; Imagery; Impairment; Injection of therapeutic agent; Investigation; Knock-out; Knockout Mice; Lead; Learning; Left; Length; Link; Literature; Mediating; Membrane; Memory; Messenger RNA; Monitor; Morphology; Mus; Mutagenesis; Neurites; Neurons; Pathogenesis; Pathway interactions; Pharmacology; Phosphorylation; Physiological; Presynaptic Terminals; Prions; Process; Production; Property; Protein Binding; Protein Binding Domain; Protein C; Protein S; Proteins; Recombinant adeno-associated virus (rAAV); Recombinants; Regulation; Reporting; Role; Signal Pathway; Signal Transduction; Site; Site-Directed Mutagenesis; Synapses; Synaptic plasticity; Tail; Testing; Therapeutic; Transgenic Mice; Transgenic Organisms; Variant; Viral; amyloid peptide; amyloid precursor protein processing; base; design; familial Alzheimer disease; gene therapy; in vivo; insight; mouse model; mutant; novel; novel strategies; overexpression; peptide A; polypeptide; postsynaptic; presenilin; presynaptic; protein expression; protein protein interaction; public health relevance; receptor; response; secretase; sequential proteolysis; synaptic function; synaptogenesis

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.

描述（由申请人提供）：阿尔茨海默氏病（AD）在病理上的特征是通过淀粉样蛋白前体蛋白（APP）的顺序蛋白水解产生的？ - 淀粉样蛋白肽的积累（A？）。多年来，几种生理功能已归因于应用。已经提出，应用程序可以通过其双重作用通过其细胞粘附特性或通过介导细胞内信号传导的假定受体样功能来影响突触函数。全长应用程序的分解？并且？ - 分泌酶释放整个胞外域，留下膜结合的C末端片段（CTF），能够介导细胞内信号传导，直到通过？ - 分解酶进一步处理。为了以与APP-CTF相关的构型信号传导激活，我们设计了一个膜束缚的APP细胞质结构域（MAICD）。我们发现，通过处理APP或MAICD（而不是AICD）产生的APP-CTF的积累会导致腺苷酸环化酶依赖性PKA的激活，抑制GSK3？以及增强的原代皮质神经元中的树突状和轴突含量。我们确定了APP细胞内结构域和异三个蛋白的G蛋白亚基G？s之间的新型相互作用，并且通过APP中的相互作用基序的诱变以及表达显性的负G突变体，这表明了与G？s的相互作用，与G？s以及随后的G？S辅助为腺苷酸化型环磷酸酯构成型膜脉络脉络脉冲 - 对膜酸盐的基本原状 - 均具有指标。我们的研究提供了明确的证据，表明APP细胞内结构域在通过其膜关联调节神经突的生长中可能具有非转录作用。此外，以前报道说，应用程序也可以与G？o相互作用。众所周知，CAMP/PKA途径的激活会影响几种大脑功能，例如突触可塑性和记忆形成，以及通过非淀粉样蛋白生成途径的产生。基于这些发现，我们假设APP细胞质结构域与G蛋白的功能耦合可能会影响神经突生长，突触形成和A ??产生。为了研究这一假设，我们建议开发出过表达MAICD或MAICD突变体缺乏G蛋白结合位点（S）的模型。我们将采用重组腺相关的病毒（AAV）递送，还产生转基因小鼠模型来评估MAICD在大脑中MAICD表达的生理病理学相关性及其在基因治疗中的推定价值。淀粉样蛋白的沉积，一种产生，神经突生长的刺激和突触形成将在App-Null小鼠中监测，在AAV注射或转基因间杂交繁殖后，将APP和Presenilin的家族性AD链接突变体及其对照同胞进行了表达的小鼠。我们的调查将解决与APP-CTF相关的先前未识别的细胞内信号通路的重要性。对APP-CTF及其相关的信号伙伴的更好理解可能会提供有关APP-CTF影响突触功能和A''的细胞机制的重要见解，这可能会对AD发病机理产生可能影响。