Regulation of cellular release of proteins in Parkinson neurodegeneration

描述（由申请人提供）：帕金森病（PD）的个人和社会成本预计将在未来二十年显著增加。神经退行性变的机制尚不清楚，也没有任何治疗能明显减缓PD的神经退行性过程。α -突触核蛋白（-syn）是PD发病机制的核心蛋白，最近的研究表明-syn在不同细胞群之间的传递是其引起毒性的关键。syn的释放是传播的第一个关键因素，并通过外泌体和非外泌体介导的途径发生。朊病毒样扩散的第二个关键特征是靶神经元的摄取，从而导致内源性- syn的错误折叠。调控- syn病理释放和扩散的机制尚不清楚。14-3-3蛋白是类伴侣蛋白，可减少蛋白质聚集，调节蛋白质分泌，促进细胞存活。我们之前已经证明14-3-3在几种PD模型中具有保护作用，并且可以调节LRRK2的外泌体释放，LRRK2是PD的关键蛋白。在本提案中，我们提出了14-3-3？产生syn的细胞中的异构体降低了释放的syn的毒性。我们的中心假设是14-3-3蛋白可以通过减少有毒syn物种的传播来防止syn毒性。在目的1和2中，我们将研究14-3-3是否可以通过外泌体或其他非外泌体途径调节- syn释放，并评估释放的任何变化如何影响旁分泌- syn毒性。在这些研究中，我们将使用旁分泌诱导的- syn培养系统，在该系统中释放- syn诱导分离培养的原代神经元细胞死亡。在目的1中，我们将使用生化和成像方法来确定14-3-3是否会改变外泌体中- syn的数量和构象。我们还将评估外泌体syn的改变如何影响旁分泌syn毒性。在目标2中，我们将使用类似的技术来测试14-3-3是否通过抑制循环内体途径减少syn释放和毒性。在目标3中，我们将重点关注14-3-3对暴露于细胞外同步蛋白的靶细胞的影响。具体来说，我们将使用体外和体内syn纤维模型来

DESCRIPTION (provided by applicant): The personal and societal costs of Parkinson's disease (PD) are expected to increase significantly in the next two decades. The mechanisms of neurodegeneration are not well understood, and no treatment clearly slows the neurodegenerative process in PD. Alpha-synuclein (�syn) is a protein that is central to PD pathogenesis, and recent studies show that the transmission of �syn between different cell populations is key to its ability to cause toxicity. Release of �syn is the first critical componen of transmission, and occurs through exosomal and non-exosomal mediated pathways. A second key feature of prion-like spread of �syn is the uptake in target neurons, leading to consequent misfolding of endogenous �syn. What mechanisms regulate the release and spread of �syn pathology are not known. The 14-3-3 proteins are chaperone-like proteins that can reduce protein aggregation, regulate protein secretion, and promote cell survival. We have previously shown that 14-3-3s are protective in several models of PD and can regulate the exosomal release of LRRK2, a key protein implicated in PD. In this proposal, we present preliminary data that overexpression of the 14-3-3? isoform in �syn-producing cells reduces the toxicity of released �syn. Our central hypothesis is that 14-3-3 proteins can protect against �syn toxicity by reducing the transmission of toxic �syn species. In Aims 1 and 2, we will investigate whether 14-3-3s can regulate �syn release through exosomes or alternative non-exosomal pathways and assess how any changes in release impacts paracrine �syn toxicity. For these studies, we will use a paracrine inducible �syn culture system in which released �syn induced cell death in separately culture primary neurons. In Aim 1, we will use biochemical and imaging approaches to determine if 14-3-3s alter the amount and conformation of �syn in exosomes. We will also assess how alterations in exosomal �syn impact paracrine �syn toxicity. In Aim 2, we will use similar techniques to test if 14-3-3s reduce �syn release and toxicity through inhibition of the recycling endosomal pathway. In Aim 3, we will focus on the effects of 14-3-3s in target cells exposed to extracellular �syn. Specifically, we will use in vitro and in vivo �syn fibril models to test whether 14-3-3s can reduce �syn uptake, aggregation, and toxicity in these models. If we can establish that 14-3-3s regulate the pathological transmission of �syn, this would justify exploration of potential PD therapies targeting the 14-3-3s.