Regulation of cellular release of proteins in Parkinson neurodegeneration

帕金森神经变性中蛋白质细胞释放的调节

• 批准号: 8764591
• 负责人: Talene Alene Yacoubian
• 金额: $ 32.57万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-08-01 至 2019-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8764591
• 关键词: 14-3-3 Proteins; Accounting; Affect; Biochemical; Biological Assay; Brain region; Cell Death; Cell Survival; Cells; Data; Disease; Disease Progression; Disease model; Endosomes; Goals; Guanosine Triphosphate Phosphohydrolases; Health; Image; In Vitro; Intervention; LRRK2 gene; Mediating; Microglia; Modeling; Molecular Chaperones; Molecular Conformation; Nature; Nerve Degeneration; Neurodegenerative Disorders; Neurons; Neurotoxins; Parkinson Disease; Pathogenesis; Pathology; Pathway interactions; Pattern; Population; Prevalence; Process; Property; Protein Isoforms; Protein Secretion; Proteins; Recombinants; Recycling; Regulation; Role; Staging; Symptoms; System; Techniques; Testing; Therapeutic Intervention; Toxic effect; alpha synuclein; base; c-Myc Staining Method; cell growth regulation; cost; disability; extracellular; in vivo; link protein; neuron loss; neurotoxicity; overexpression; paracrine; prion-like; protein aggregation; therapeutic target; transmission process; uptake

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.

描述（由适用提供）：预计帕金森氏病的个人和社会成本预计在未来二十年内将显着增加。神经退行性的机制尚不清楚，没有治疗清楚地减慢了PD中的神经退行性过程。 α-突触核蛋白（αSyn）是一种对PD发病机理至关重要的蛋白质，最近的研究表明，不同细胞种群之间αSyn的传播是其引起毒性能力的关键。 αSyn的释放是传播的第一个关键组成部分，并通过外泌体和非外胞体介导的途径发生。 αSyn的prion样蔓延的第二个关键特征是靶神经元中的摄取，导致内源性αSyn的折叠错误。哪些机制调节αSyn病理的释放和扩散尚不清楚。 14-3-3蛋白是伴侣样蛋白，可以减少蛋白质聚集，调节蛋白质分泌并促进细胞存活。我们先前已经表明，在几种PD模型中受到14-3-3的保护，可以调节LRRK2的外泌体释放，LRRK2是PD中植入的关键蛋白。在此提案中，我们提供了初步数据，即产生αSyn的细胞中14-3-3θ同工型过度表达降低了释放αSyn的毒性。我们的中心假设是14-3-3蛋白可以通过减少有毒αSyn物种的传播来预防αSyn毒性。在AIMS 1和2中，我们将研究14-3-3s是否可以通过外泌体或替代性的非外神体途径调节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，其中释放的αSyn诱导了单独培养的原代神经元的细胞死亡。在AIM 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αSyn的量和构象。我们还将评估外泌体αSyn的变化如何影响旁分泌αSyn毒性。在AIM 2中，我们将使用类似的技术来测试14-3-3s是否通过抑制回收内体途径来降低αSyn释放和毒性。在AIM 3中，我们将专注于暴露于细胞外αSyn的靶细胞中14-3-3的影响。特别是，我们将使用体外和体内和体内αsyn原纤维模型对 测试在这些模型中14-3-3s是否可以减少αSyn的吸收，聚集和毒性。如果我们可以确定14-3-3S调节αSyn的病理传播，这将证明探索针对14-3-3S的潜在PD疗法。