Elucidating a molecular pathway for synaptic vesicle maintenance and degradation

阐明突触小泡维持和降解的分子途径

• 批准号: 8899649
• 负责人: Clarissa Leigh Waites
• 金额: $ 34.83万
• 依托单位: COLUMBIA UNIVERSITY HEALTH SCIENCES
• 依托单位国家: 美国
• 财政年份: 2013
• 资助国家: 美国
• 起止时间: 2013-06-15 至 2016-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8899649
• 关键词: Affect; Alzheimer' s Disease; American; Biochemistry; Caring; Carrier Proteins; Cell Death; Cell physiology; Communication; Complex; Data; Degradation Pathway; Disease; Drosophila genus; Elderly; Electron Microscopy; Endocytosis; Etiology; Eukaryotic Cell; Event; Exhibits; Future; Gene Expression Profiling; Glutamates; Goals; Health; Hippocampus (Brain); Homeostasis; Huntington Disease; Image; Immunofluorescence Microscopy; Life; Lysosomes; Maintenance; Mediating; Mediator of activation protein; Membrane Protein Traffic; Membrane Proteins; Molecular; Monitor; Monomeric GTP-Binding Proteins; Multivesicular Body; Nerve Degeneration; Nervous system structure; Neurodegenerative Disorders; Neuromuscular Junction; Neurons; Parkinson Disease; Pathway interactions; Phenotype; Physiological; Process; Productivity; Property; Protein Biosynthesis; Proteins; RNA Interference; Recycling; Regulation; Role; Signal Transduction; Site; Sorting - Cell Movement; Structure; Synapses; Synaptic Vesicles; System; Techniques; Testing; Ubiquitination; Vesicle; Work; age related; base; cost; insight; loss of function; neuronal cell body; neurotransmitter release; novel; presynaptic; protein degradation; protein function; rab GTP-Binding Proteins; research study; response; trafficking; ubiquitin-protein ligase

DESCRIPTION (provided by applicant): Synapses not only support stable signaling between neurons over the course of months to years, but they also have the remarkable capacity to rapidly change their response properties based on inputs from other neurons. These functions depend upon the exquisite regulation of protein synthesis, trafficking, and degradation, collectively termed 'protein homeostasis' or 'proteostasis'. A critical aspect of synaptic proteostasis is the maintenance of synaptic vesicle (SV) pools within presynaptic boutons ('SV-stasis'). SV pools support the sustained release of neurotransmitter by maintaining a local reservoir of proteins to facilitate vesicle recycling [3, 4]. Moreover, SV loss precedes synapse degeneration and cell death in many forms of neurodegeneration [5- 8], suggesting that disruption of SV-stasis triggers more widespread degenerative processes. Understanding how SV-stasis is maintained and regulated will therefore provide critical insights into the etiology of neurodegenerative diseases such as Alzheimer's and Parkinson's. However, while the molecules that regulate SV exo/endocytosis have been extensively studied, those that regulate SV maintenance and degradation remain almost entirely unknown. The overall goal of this proposal is to elucidate the molecular pathway that mediates SV protein degradation in mammalian glutamatergic neurons. Our previous studies have identified three potential components of this pathway (the E3 ubiquitin ligase Siah1, the endosomal sorting complex required for transport (ESCRT) system, and the small GTPase Rab35), and this project will evaluate their roles in facilitating SV protein degradation. We will further assess whether pathological activation of this pathway disrupts SV-stasis and triggers synapse degeneration. In Aim 1, we will test whether Siah1 is a key mediator of SV protein ubiquitination. We propose that its over-activation induces hyper-ubiquitination and degradation of SV proteins, followed by SV loss and synapse degeneration, while its inhibition or knockdown leads to increased SV pool size and stability. To test this hypothesis, we will use biochemistry, immunofluorescence microscopy, live imaging, and electron microscopy to assess effects of Siah1 gain- or loss-of-function on SV protein abundance and turnover. In Aim 2, we will determine whether ubiquitinated SV proteins are targeted to lysosomes via the ESCRT pathway. Here, we will use the techniques from Aim 1 together with knockdown of key ESCRT proteins to examine whether the ESCRT pathway is essential for SV protein degradation under normal and pathological conditions. In Aim 3, we will evaluate whether Rab35 mediates SV protein degradation and functions upstream of the ESCRT pathway. We will again use techniques from Aim 1, together with Rab35 gain- and loss-of-function, to reveal whether Rab35 sorts SV membrane proteins into endosomal intermediates, promoting their entry into an ESCRT-dependent degradative pathway. Together, these studies will provide novel insights into how SV-stasis is maintained, and how its dysregulation contributes to synapse degeneration and the etiology of neurodegenerative disease.

描述(申请人提供)：突触不仅支持神经元之间在几个月到几年的过程中稳定的信号传递，而且它们还具有基于其他神经元的输入快速改变其反应特性的非凡能力。这些功能依赖于蛋白质合成、运输和降解的精细调节，统称为“蛋白质稳态”或“蛋白质稳态”。突触蛋白平衡的一个关键方面是维持突触前突触内的突触小泡(SV)池(SV-STESTATE)。SV池支持神经递质的持续释放，通过维持局部蛋白质储存库来促进囊泡循环[3，4]。此外，在许多形式的神经变性中，SV丢失先于突触退化和细胞死亡[5-8]，这表明SV停滞的破坏触发了更广泛的退化过程。因此，了解SV停滞是如何维持和调节的，将为了解心力衰竭的病因提供重要的见解。 神经退行性疾病，如阿尔茨海默氏症和帕金森氏症。然而，尽管调控SV外吞/内吞的分子已经被广泛研究，但那些调控SV维持和降解的分子仍然几乎完全未知。这项建议的总体目标是阐明哺乳动物谷氨酸能神经元中SV蛋白降解的分子途径。我们之前的研究已经确定了这一途径的三个潜在组成部分(E3泛素连接酶Siah1，运输所需的内体分类复合体(ESCRT)系统，以及小GTP酶Rab35)，本项目将评估它们在促进SV蛋白降解中的作用。我们将进一步评估该通路的病理性激活是否会破坏SV停滞并触发突触变性。在目标1中，我们将测试Siah1是否是SV蛋白泛素化的关键介体。我们认为，它的过度激活诱导了SV蛋白的超泛素化和降解，继而导致SV丢失和突触退化，而它的抑制或敲除则导致SV池大小和稳定性的增加。为了验证这一假设，我们将使用生化、免疫荧光显微镜、实时成像和电子显微镜来评估Siah1功能获得或丧失对SV蛋白丰度和周转的影响。在目标2中，我们将确定泛素化的SV蛋白是否通过ESCRT途径靶向溶酶体。在这里，我们将使用AIM 1中的技术和关键ESCRT蛋白的敲除来研究ESCRT途径在正常和病理条件下是否对SV蛋白的降解是必不可少的。在目标3中，我们将评估Rab35是否介导了SV蛋白的降解和ESCRT途径上游的功能。我们将再次使用Aim 1中的技术，以及Rab35的功能获得和丧失，来揭示Rab35是否将SV膜蛋白分类为内体中间体，促进它们进入ESCRT依赖的降解途径。总之，这些研究将为SV停滞是如何维持的，以及它的失调如何导致突触退化和神经退行性疾病的病因提供新的见解。