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（由申请人提供）：BACE 1是一种I型跨膜蛋白，其通过切割淀粉样前体蛋白（APP）启动阿尔茨海默病A β产生。越来越多的证据表明，突触活动动态地调节突触附近的腺苷酸释放。活性依赖性的Abladium产生增加的分子和细胞机制仍然很大程度上未知。除了APP之外，BACE 1还切割许多突触跨膜底物。最近，我们发现海马神经元中内化的BACE 1的单向树枝状逆行运输，并发现BACE 1在称为转胞吞的过程中从体树隔室到轴突进行远程运输的证据。关于神经元BACE 1运输的调节以及神经元中蛋白质胞吞转运的研究很少。BACE 1经历翻译后S-棕榈酰化、磷酸化和泛素化。突触相关蛋白的这些修饰响应于突触活动而发生，这反过来又调节动态蛋白质运输。我们假设，突触活动调节动态贩运在突触和转胞吞的内化BACE 1，从而提供了一个重要的机制，突触活动可以促进淀粉样蛋白的加工APP或附近的突触网站。本提案的具体目的是：目的1）检验突触活性调节BACE 1运输和转胞吞的假设。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BACE 1在树突棘和突触前末梢中的定位以及BACE 1在突触处的内化和再循环的局部动态。同时，我们将使用微流控培养系统测定内化的BACE 1的转胞吞作用。目的2）验证突触活动动态调节BACE 1翻译后修饰的假说。我们将研究突触活动如何调节内源性BACE 1的胞质尾内的动态修饰。我们将通过在表达BACE 1的神经元中进行运输研究（如Aim 1）来证实和扩展这些发现，BACE 1在S-棕榈酰化、磷酸化和泛素化位点内具有突变。我们的建议是及时的，独特的，高度创新的，因为我们是第一个描述BACE 1转胞吞在回收内体。我们现在建议扩展我们的研究结果，研究突触活动如何调节这种极不寻常的蛋白质运输模式。我们研究BACE 1的突触调节的目标也是非常重要的，因为内源性BACE 1和转基因表达的BACE 1-YFP在体内主要定位于突触前末梢，并且BACE 1在AD个体的脑中的老年斑附近的营养不良的突触前末梢中积累。因此，研究突触活动如何与BACE 1运输和轴突靶向相结合，与阿尔茨海默病发病机制以及BACE 1在生理和病理条件下对其多种神经元底物的加工机制高度相关。