Regulation of BACE1 transcytosis in hippocampal neurons

海马神经元 BACE1 转胞吞作用的调节

• 批准号: 9125717
• 负责人: GOPAL THINAKARAN
• 金额: $ 23.7万
• 依托单位: UNIVERSITY OF CHICAGO
• 依托单位国家: 美国
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-08-15 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9125717
• 关键词: Abeta synthesis; Affect; Age; Alzheimer' s Disease; Amyloid; Amyloid beta-Protein; Amyloid beta-Protein Precursor; Aspartic Endopeptidases; Axon; Axonal Transport; Biological Assay; Biology; Brain; Cell membrane; Cleaved cell; Coupled; Dendrites; Dendritic Spines; Endocytosis; Endosomes; Frequencies; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Hippocampus (Brain); Individual; Integral Membrane Protein; Intercellular Fluid; Mediating; Methods; Microfluidics; Modification; Molecular; Mutation; Neurodegenerative Disorders; Neurofibrillary Tangles; Neurons; Neurotransmitter Receptor; Pathogenesis; Peptides; Phosphorylation; Physiological; Play; Post-Translational Protein Processing; Presynaptic Terminals; Process; Production; Protein Family; Proteins; Publishing; Recycling; Regulation; Reporting; Senile Plaques; Site; Slice; Synapses; System; Tail; Testing; Transgenes; Vertebral column; Vesicle; abeta deposition; amyloid precursor protein processing; beta secretase; beta-site APP cleaving enzyme 1; cell motility; design; hyperphosphorylated tau; in vivo; innovation; insight; live cell imaging; neuronal cell body; novel; palmitoylation; presynaptic; prevent; protein transport; rab11 protein; response; retrograde transport; targeted treatment; trafficking; transcytosis

 DESCRIPTION (provided by applicant): BACE1 is a type I transmembrane protein that initiates Alzheimer's disease Aß production by cleavage of amyloid precursor protein (APP). Accumulating evidence demonstrates that synaptic activity dynamically regulates Aß release production near synapses. The molecular and cellular mechanisms underlying activity- dependent increase of Aß production remain largely unknown. In addition to APP, BACE1 cleaves a number of synaptic transmembrane substrates. Recently, we discovered unidirectional dendritic retrograde transport of internalized BACE1 in hippocampal neurons and found evidence that BACE1 undergoes long-range transport from somatodendritic compartment to axon, in a process termed transcytosis. Very few studies have been published on the regulation of neuronal BACE1 trafficking, and also on protein transcytosis in neurons. BACE1 undergoes post-translational S-palmitoylation, phosphorylation, and ubiquitylation. These modifications on synapse-associated proteins occur in response to synaptic activity, which, in turn, regulate dynamic protein trafficking. We hypothesize that synaptic activity modulates dynamic trafficking at the synapse and transcytosis of internalized BACE1, thus providing a crucial mechanism by which synaptic activity could promote amyloidogenic processing of APP at or near synaptic sites. The Specific Aims of this proposal are: Aim 1) To test the hypothesis that synaptic activity regulates BACE1 trafficking and transcytosis. We will use advanced live-cell imaging and FRAP methods to characterize how synaptic activity affects BACE1 localization in dendritic spines and presynaptic terminals and the local dynamics of BACE1 internalization and recycling at the synapse. In parallel, we will assay transcytosis of internalized BACE1 using microfluidic culture system. Aim 2) To test the hypothesis that synaptic activity dynamically modulates BACE1 post-translational modifications. We will investigate how synaptic activity modulates dynamic modifications within the cytosolic tail of endogenous BACE1. We will confirm and extend the findings by performing trafficking studies (as in Aim 1) in neurons expressing BACE1 bearing mutations within the sites of S-palmitoylation, phosphorylation, and ubiquitylation. Our proposal is timely, unique, and highly innovative because we were the first to describe BACE1 transcytosis in recycling endosomes. We now propose to extend our findings to study how synaptic activity modulates this highly unusual mode of protein trafficking. Our goal to investigate synaptic regulation of BACE1 is also highly significant because endogenous BACE1 and transgene expressed BACE1-YFP predominantly localize to presynaptic terminals in vivo, and BACE1 accumulates in dystrophic presynaptic terminals near senile plaques in the brains of individuals with AD. Thus, investigating how synaptic activity is coupled to BACE1 trafficking and axonal targeting is highly relevant to mechanisms underlying Alzheimer's disease pathogenesis as well as for BACE1 processing of its multiple neuronal substrates under physiological and pathological conditions.

 描述(申请人提供)：BACE1是一种I型跨膜蛋白，通过切割淀粉样前体蛋白(APP)启动阿尔茨海默病A？越来越多的证据表明，突触活动动态地调节突触附近A？释放的产生。依赖活性的Aç产量增加背后的分子和细胞机制在很大程度上仍不清楚。除了APP，BACE1还切割一些突触跨膜底物。最近，我们在海马神经元中发现了内化的BACE1的单向树突状逆行转运，并发现BACE1经历了从躯体树突间到轴突的长距离运输，这一过程被称为跨细胞作用。关于神经元BACE1转运的调节以及神经元中蛋白质的跨细胞转运的研究发表得很少。BACE1经历了S翻译后的棕榈酰化、磷酸化和泛素化。突触相关蛋白的这些修饰是对突触活动的反应，突触活动反过来又调节动态的蛋白质运输。我们假设突触活动调节突触的动态运输和内化的BACE1的跨细胞作用，从而提供了一个重要的机制，突触活动可以促进突触部位或附近APP的淀粉样蛋白形成过程。这一提议的具体目的是：目的1)检验突触活动调节BACE1转运和跨细胞作用的假设。我们将使用先进的活细胞成像和FRAP方法来表征突触活动如何影响BACE1在树突和突触前终末的定位，以及BACE1在突触的局部内化和循环动力学。同时，我们将使用微流控培养系统检测内化的BACE1的转胞作用。目的2)验证突触活动动态调节BACE1翻译后修饰的假说。我们将研究突触活动如何调节内源性BACE1胞浆尾部的动态修改。我们将通过对表达BACE1的神经元进行运输研究(如目标1)来证实和推广这一发现，这些神经元携带S棕榈酸化、磷酸化和泛素化位点的突变。我们的建议是及时的、独特的和高度创新的，因为我们是第一个描述BACE1在内体循环中的转胞作用的。我们现在建议将我们的发现扩展到研究突触活动如何调节这种非常不寻常的蛋白质运输模式。我们研究BACE1的突触调控也具有非常重要的意义，因为内源性BACE1和转基因表达的BACE1-YFP主要定位于体内的突触前终末，而BACE1积聚在AD患者大脑中靠近衰老斑块的营养不良的突触前终末。因此，研究突触活动与BACE1转运和轴突靶向是如何耦合的，对于阿尔茨海默病发病机制以及BACE1在生理和病理条件下对其多个神经元底物的处理具有高度的相关性。