Elucidating a molecular pathway for synaptic vesicle maintenance and degradation

突触不仅在数月至数年的过程中支持神经元之间的稳定信号传递，而且它们还具有基于来自其他神经元的输入快速改变其响应特性的显著能力。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”。突触蛋白稳态的一个重要方面是突触前终扣内突触囊泡（SV）池的维持（SV-稳态）。SV池通过维持蛋白质的局部储库以促进囊泡再循环来支持神经递质的持续释放[3，4]。此外，在许多形式的神经变性中，SV损失先于突触变性和细胞死亡[5- 8]，这表明SV停滞的破坏引发更广泛的变性过程。因此，了解SV停滞是如何维持和调节的，将为研究SV停滞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，虽然调节SV外吞/内吞作用的分子已被广泛研究，但调节SV维持和降解的分子仍然几乎完全未知。该建议的总体目标是阐明介导哺乳动物神经元SV蛋白降解的分子途径。我们以前的研究已经确定了该途径的三个潜在组分（E3泛素连接酶Siah 1，运输所需的内体分选复合物（ESCRT）系统和小GTdR Rab 35），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激活是否破坏SV停滞并触发突触变性。在目标1中，我们将测试Siah 1是否是SV蛋白泛素化的关键介质。我们认为，它的过度激活诱导超泛素化和SV蛋白的降解，随后SV损失和突触变性，而它的抑制或敲低导致SV池的大小和稳定性增加。为了验证这一假设，我们将使用生物化学，免疫荧光显微镜，实时成像，和电子显微镜，以评估Siah 1的增益或损失的SV蛋白丰度和营业额的功能的影响。在目标2中，我们将确定是否泛素化SV蛋白通过ESCRT途径靶向溶酶体。在这里，我们将使用目标1的技术与关键ESCRT蛋白的敲低一起来检查ESCRT途径是否是SV蛋白在正常和病理条件下降解所必需的。在目标3中，我们将评估Rab 35是否介导SV蛋白降解并在ESCRT途径上游发挥作用。我们将再次使用Aim 1的技术，以及Rab 35功能的获得和丧失，以揭示Rab 35是否将SV膜蛋白分类为内体中间体，促进其进入ESCRT依赖性降解途径。总之，这些研究将提供新的见解，如何SV-停滞是维持，以及它的失调如何有助于突触变性和神经退行性疾病的病因。

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.