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

基本信息

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
项目类别：
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&apos 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

项目摘要

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）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（Aβ）的积累。多年来，多种生理功能被归于 APP。有人提出，APP 可以通过其细胞粘附特性或通过其介导细胞内信号传导的假定受体样功能的双重作用来影响突触功能。通过 ? 切割全长 APP和β-分泌酶释放整个胞外域，留下能够介导细胞内信号传导的膜结合C端片段(CTF)，直到它们被β-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、GSK3? 抑制以及初级皮层神经元中树突和轴突树枝化的增强。我们鉴定了 APP 胞内结构域和异源三聚体 G 蛋白亚基 G?S 之间的新型相互作用，并通过 APP 内相互作用基序的诱变以及显性失活 G?S 突变体的表达，证明了与 G?S 和异源三聚体 G 蛋白亚基 G?S 的相互作用随后的 G?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至关重要。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关联在调节神经突生长方面发挥非转录作用。而且此前有报道称APP还可以与G?O进行交互。众所周知，cAMP/PKA 途径的激活会影响多种大脑功能，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产生 Aβ。基于这些发现，我们假设 APP 胞质结构域与 G 蛋白的功能耦合可能影响神经突生长、突触形成和 Aβ 产生。为了研究这一假设，我们建议开发过度表达 mAICD 或缺乏 G 蛋白结合位点的 mAICD 突变体的小鼠模型。我们将采用重组腺相关病毒（AAV）递送并生成转基因小鼠模型来评估大脑中mAICD表达的病理生理学相关性及其在基因治疗中的推定价值。在 AAV 注射或转基因杂交育种后，将在 APP 缺失小鼠、共表达 APP 和早老素家族性 AD 连锁突变体的小鼠及其对照同窝小鼠中监测淀粉样蛋白沉积、Aβ 产生、神经突生长和突触形成的刺激。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 Aβ 产生的细胞机制提供重要的见解，这可能会影响 AD 发病机制。