Regulation of cellular release of proteins in Parkinson neurodegeneration

描述(由申请人提供)：帕金森氏病(PD)的个人和社会成本预计在未来20年内显著增加。神经退行性变的机制尚不清楚，没有任何治疗方法可以明显减缓帕金森病患者的神经退行性变过程。α-突触核蛋白(α-syn)是一种在帕金森病发病机制中起核心作用的蛋白质，最近的研究表明，α-syn在不同细胞群体之间的传递是其毒性的关键。α-SYN的释放是传递的第一个关键成分，通过胞外体和非胞外体介导的途径发生。α突触蛋白的第二个关键特征是靶神经元摄取，从而导致内源性α突触蛋白的错误折叠。目前尚不清楚是什么机制调控αSYN病理的释放和扩散。14-3-3蛋白是一种伴侣样蛋白，可以减少蛋白质聚集，调节蛋白质分泌，促进细胞存活。我们之前已经证明，14-3-3S在几种帕金森病模型中具有保护作用，并可以调节LRRK2的外体释放，LRRK2是与帕金森病有关的关键蛋白。在这项建议中，我们提供了初步的数据，在θ合成产生细胞中过表达14-3-3α亚型可以降低释放的α合成的毒性。我们的中心假设是，14-3-3蛋白可以通过减少有毒α同种病毒的传播来保护α同种病毒的毒性。在目标1和目标2中，我们将研究14-3-3S是否可以通过外体或其他非外体途径调节αSYN的释放，并评估释放的任何变化如何影响旁分泌αSYN的毒性。在这些研究中，我们将使用旁分泌诱导的αSYN培养系统，在该培养系统中，释放αSYN诱导单独培养的原代神经元细胞死亡。在目标1中，我们将使用生化和成像方法来确定14-3-3S是否改变了外体中αSYN的数量和构象。我们还将评估外体αSYN的改变如何影响旁分泌αSYN毒性。在目标2中，我们将使用类似的技术来测试14-3-3s是否通过抑制循环内体途径来减少α合成酶的释放和毒性。在目标3中，我们将重点研究14-3-3S对细胞外αSYN暴露的靶细胞的影响。具体地说，我们将使用体外和体内的α融合纤维模型来 测试14-3-3S是否能减少α的摄取、聚集和毒性。如果我们能够确定14-3-3s调控αSYN的病理传递，这将证明有理由探索针对14-3-3s的潜在PD治疗方法。

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 (αsyn) is a protein that is central to PD pathogenesis, and recent studies show that the transmission of αsyn between different cell populations is key to its ability to cause toxicity. Release of αsyn 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 αsyn is the uptake in target neurons, leading to consequent misfolding of endogenous αsyn. What mechanisms regulate the release and spread of αsyn 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 14-3-3θ isoform in αsyn-producing cells reduces the toxicity of released αsyn. Our central hypothesis is that 14-3-3 proteins can protect against αsyn toxicity by reducing the transmission of toxic αsyn species. In Aims 1 and 2, we will investigate whether 14-3-3s can regulate αsyn release through exosomes or alternative non-exosomal pathways and assess how any changes in release impacts paracrine αsyn toxicity. For these studies, we will use a paracrine inducible αsyn culture system in which released αsyn 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 αsyn in exosomes. We will also assess how alterations in exosomal αsyn impact paracrine αsyn toxicity. In Aim 2, we will use similar techniques to test if 14-3-3s reduce αsyn 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 αsyn. Specifically, we will use in vitro and in vivo αsyn fibril models to test whether 14-3-3s can reduce αsyn uptake, aggregation, and toxicity in these models. If we can establish that 14-3-3s regulate the pathological transmission of αsyn, this would justify exploration of potential PD therapies targeting the 14-3-3s.