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（αsyn）是PD发病机制的核心蛋白质，最近的研究表明，αsyn在不同细胞群之间的传递是其引起毒性的关键。α-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。αsyn的朊病毒样扩散的第二个关键特征是靶神经元的摄取，导致内源性αsyn随后的错误折叠。什么机制调节αsyn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。在这个建议中，我们提出了初步的数据，14-3-3θ异构体在α-syn产生细胞中的过表达降低了释放的α-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α-syn物质的传播来保护免受α-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αsyn释放，并评估释放的任何变化如何影响旁分泌αsyn毒性。对于这些研究，我们将使用旁分泌诱导的α-syn培养系统，其中释放的α-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αsyn的数量和构象。我们还将评估外泌体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是否通过抑制再循环内体途径来减少αsyn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αsyn的靶细胞中的作用。具体来说，我们将使用体外和体内α-syn纤维模型， 在这些模型中测试14-3-3s是否可以降低α-syn摄取、聚集和毒性。如果我们能够确定14-3-3s调节αsyn的病理传递，这将证明探索针对14-3-3s的潜在PD治疗是合理的。

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 (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.