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内显着增加。神经退行性变的机制还不清楚，没有治疗明显减缓PD的神经退行性变过程。α-突触核蛋白（α-syn）是一种对PD发病机制至关重要的蛋白质，最近的研究表明，α-syn在不同细胞群之间的传递是其引起毒性的关键。β-syn的释放是传播的第一个关键成分，并通过外泌体和非外泌体介导的途径发生。朊病毒样β-syn扩散的第二个关键特征是靶神经元的摄取，导致内源性β-syn随后的错误折叠。是什么机制调节了β-syn病理的释放和传播尚不清楚。14-3-3蛋白是分子伴侣样蛋白，可以减少蛋白质聚集，调节蛋白质分泌，促进细胞存活。我们先前已经表明，14-3-3s在几种PD模型中具有保护作用，并且可以调节与PD有关的关键蛋白LRRK 2的外泌体释放。在这项建议中，我们提出了初步的数据，过度表达的14-3-3？β-syn产生细胞中的同种型降低了释放的β-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β-syn物种的传播来防止β-syn毒性。在目标1和2中，我们将研究14-3-3s是否可以通过外泌体或替代的非外泌体途径调节β-syn释放，并评估释放的任何变化如何影响旁分泌β-syn毒性。在这些研究中，我们将使用旁分泌诱导的β-syn培养系统，其中释放的β-syn在单独培养的原代神经元中诱导细胞死亡。在目标1中，我们将使用生物化学和成像方法来确定14-3-3s是否改变了外泌体中β-syn的数量和构象。我们还将评估外泌体突触的改变如何影响旁分泌突触的毒性。在目标2中，我们将使用类似的技术来测试14-3-3s是否通过抑制内体再循环途径来减少β-syn的释放和毒性。在目标3中，我们将关注14-3-3s在暴露于细胞外β-syn的靶细胞中的作用。具体来说，我们将使用体外和体内的合成纤维模型， 测试14-3-3s是否可以减少这些模型中的syn摄取，聚集和毒性。如果我们能够确定14-3-3s调节β-syn的病理性传播，这将证明探索针对14-3-3s的潜在PD疗法是合理的。

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.