Trafficking and endosomal sorting of APP and BACE-1

描述（由申请人提供）：淀粉样前体蛋白（APP）依次被β-和γ-分泌酶裂解，在脑中产生淀粉样β-肽（AAPs）-阿尔茨海默病的核心参与者。β-分泌酶-1（BACE-1）对APP的裂解是产生APP的限速步骤。据信，在作为不溶性原纤维沉积在脑中之前，在可溶性和低聚状态下，黄芪对神经元发挥其毒性。因此，由于与bth病理生理学和治疗学相关的原因，了解APP产生Ablation的机制和途径是许多实验室的主要焦点。一个有趣的方面是，它的释放依赖于神经元的活动-增强的突触活动导致更多的腺苷酸释放。虽然参与APP在神经元中的运输和裂解的途径是明显重要的，但绝大多数先前关于APP/BACE-1运输的研究都是在非神经元细胞中进行的。这些发现可能并不总是适用于高度极化的神经元，并且已知具有非常不同的运输机制。此外，关于神经元活动如何通过BACE-1调节APP加工的推断需要在神经元中进行工作。流行的观点是，在突触前末梢，伴随高突触活性的突触囊泡再循环增加，导致APP内体的内化增加，其中分泌酶发生蛋白水解。然而，我们最近使用活体神经元成像的研究显示了相当令人惊讶的结果，APP和BACE-1通常在不同的囊泡中运输-可能阻止不减弱的切割-但在活性诱导后在树突中会聚。这使我们提出了一种新的模型，即神经元活动将APP和BACE-1聚集在树突中，这两种分子相互作用。只有随后这两种分子才被分类到轴突的远端末端。本提案中的实验将检查来自该工作模型的许多预测，并剖析APP和BACE-1的运输途径;揭示它们与淀粉样蛋白生成和神经元活动的关系。本研究的目的有四：1）验证APP和BACE-1在生物合成后首先以不同的载体进入树突的假说。2)确定特定神经元 APP/BACE-1相互作用的亚细胞位点和腺苷酸释放。3)确定轴突APP/BACE-1携带细胞器的生物起源和分子组成。4)可视化APP/BACE-1在大脑和人类诱导多能干细胞（iPSC）中的相关性。总的来说，这些研究的结果将为APP和BACE-1的运输途径提供新的见解，并证明神经元活动如何调节这些途径以增强APP裂解和AACE释放。

DESCRIPTION (provided by applicant): The amyloid precursor protein (APP) is sequentially cleaved by ß- and γ-secretases to generate amyloid ß-peptide (Aß) in the brain - a central player in Alzheimer's disease. APP cleavage by ß-secretase-1 (BACE-1) is the rate-limiting step for production of Aß. Aß is believed to exert its toxicity on neurons while in a soluble and oligomeric state, prior to deposition as insoluble fibrils in brain. Thus, for reasons related to bth pathophysiology and therapeutics, understanding mechanisms and pathways of Aß generation from APP is a major focus of many laboratories. An intriguing aspect of Aß production is that its release is dependent upon neuronal activity - enhanced synaptic activity results in more Aß release. Though pathways involved in trafficking and cleavage of APP in neurons are of obvious importance, the vast majority of previous studies on APP/BACE-1 trafficking have been carried out in non-neuronal cells. These findings may not always be applicable to neurons, which are highly polarized and are known to have very different trafficking mechanisms. Furthermore, inferences on how neuronal activity modulates APP processing by BACE-1 require work in neurons. The prevailing view is that at presynaptic terminals, heightened synaptic vesicle recycling that accompanies high synaptic activity results in increased internalization into endosomes of APP where proteolysis by secretases take place. However, our recent studies using live neuronal imaging showed rather surprising results in that APP and BACE-1 normally traffic in distinct vesicles - perhaps preventing unabated cleavage - but converge in dendrites upon activity-induction. This led us to propose a new model whereby neuronal activity brings together APP and BACE-1 in dendrites where the two molecules interact. Only subsequently are these two molecules sorted into axons to distal terminals. Experiments in this proposal will examine a number of predictions that emanate from this working model and dissect the trafficking pathways of APP and BACE-1; revealing their relationship to amyloidogenesis and neuronal activity. Four Aims are proposed: 1) Test the hypothesis that APP and BACE-1 are first conveyed into dendrites in distinct carriers after biogenesis. 2) Determine specific neuronal subcellular site(s) of APP/BACE-1 interaction and Aß release. 3) Determine the biogenesis and molecular composition of the axonal APP/BACE-1-carrying organelle. 4) Visualize APP/BACE-1 associations in brains and in human induced pluripotent stem cells (iPSCs). Collectively, results from these studies will provide new insights into the trafficking pathways of APP and BACE-1 and demonstrate how neuronal activity modulates these pathways to enhance APP cleavage and Aß release.