Role of presynaptic targeting of alpha-synuclein in the pathogenesis of Parkinson's Disease and Dementia with Lewy Bodies

α-突触核蛋白突触前靶向在帕金森病和路易体痴呆发病机制中的作用

• 批准号: 9520559
• 负责人: Laura A. Volpicelli-Daley
• 金额: $ 37.13万
• 依托单位: UNIVERSITY OF ALABAMA AT BIRMINGHAM
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-05-01 至 2023-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/9520559
• 关键词: Adult; Amygdaloid structure; Antisense Oligonucleotides; Biochemistry; Brain; Brain region; Cell membrane; Cells; Confocal Microscopy; Corpus striatum structure; Cytoplasmic Inclusion; Data; Development; Disease; Disease Progression; Dominant-Negative Mutation; Family; Fluorescence Recovery After Photobleaching; Genes; Goals; Idiopathic Parkinson Disease; Immunohistochemistry; Immunoprecipitation; Injections; Knock-in Mouse; LRRK2 gene; Leucine-Rich Repeat; Lewy Bodies; Lewy Body Dementia; MYO5A gene; Membrane; Methods; Modeling; Motor; Mus; Mutation; Myosin ATPase; Myosin Type V; Neurites; Neurons; Organelles; Parkinson Disease; Parkinson' s Dementia; Pathogenesis; Pathologic; Pathway interactions; Patients; Peptides; Phosphotransferases; Physiological; Play; Presynaptic Terminals; Protein Isoforms; Proteins; Research; Risk Factors; Role; Specific qualifier value; Specificity; Subfamily lentivirinae; Substantia nigra structure; Synapses; Synapsins; Synaptic Vesicles; Testing; Toxic effect; Ventricular; Vesicle; adeno-associated viral vector; alpha synuclein; cell type; dopaminergic neuron; in vivo; inhibitor/antagonist; kinase inhibitor; member; mutant; new therapeutic target; novel; overexpression; pars compacta; preclinical development; presynaptic; prevent; rab GTP-Binding Proteins; small hairpin RNA; therapeutic target; trafficking; vector

Parkinson’s Disease (PD) and Dementia with Lewy Bodies (DLB) are characterized by cytoplasmic inclusions called Lewy Bodies and Lewy Neurites which are composed primarily of α-synuclein (α-syn). Multiple lines of evidence suggest that these inclusions contribute to disease pathogenesis. Thus, preventing α-syn aggregation is a critical therapeutic target for preventing disease progression. Normally, α-syn resides at the presynaptic terminal, preferentially associates with synaptic vesicles, and interaction of α-syn with membranes inhibit its aggregation. Our proposed study seeks to determine how presynaptic targeting of α-syn prevents it from converting to a pathologic, aggregation prone form and how PD risk factors disrupt the normal localization of α-syn and increase its propensity to forming inclusions. Mutations in leucine rich repeat kinase (LRRK2) are the most common cause of familial Parkinson’s disease. We showed that the G2019S-LRRK2 mutations increase the formation of α-syn inclusions and that LRRK2 kinase inhibitors reduce inclusion formation, but the mechanisms by which LRRK2 controls the propensity of α-syn to form aggregates is unknown. Although current research focuses on the role of LRRK2 in degradative organelles, LRRK2 has also been functionally implicated at the presynaptic terminal and may play a role in α-syn presynaptic membrane targeting. Recently, a subset of Rab GTPases was identified as LRRK2 substrates. Rabs control distinct steps in membrane trafficking pathways in the cell. Ten years ago, the Lindquist lab identified the same Rabs (that were recently shown to be phosphorylated by LRRK2) as protective in models of α-syn toxicity. We will determine if Rabs phosphorylated by LRRK2 enhance presynaptic targeting of α-syn and prevent α-syn inclusion formation. Because inclusions localize to multiple brain regions in PD and DLB, we will inject fibrils into the mouse striatum which produces inclusions in the substantia nigra pars compacta (SNpc), cortex, and amygdala. In Aim 1, we will determine the extent to which select Rab isoforms influence α-syn presynaptic targeting using novel AAV vectors to increase Rab expression in the brain or use antisense oligonucleotides (ASOs) to reduce Rab levels. Immunohistochemistry and biochemistry will be used to determine if expression of these Rabs in vivo prevents formation of fibril-induced inclusions in multiple brain regions, and loss of dopamine neurons in the SNpc. In Aim 2, we will identify the Rab effectors in neurons that specify targeting of vesicles containing α- syn cargo to the presynaptic terminal. Finally, in Aim 3, we will determine if LRRK2 kinase activity prevents presynaptic targeting of α-syn and if the mislocalization of α-syn in the cell increases its propensity to form inclusions. We will also determine the extent to which LRRK2 kinase inhibitors and ASOs in preclinical development enhance presynaptic targeting of α-syn. These studies will open up new research avenues for understanding how factors and genes that cause PD and DLB increase the propensity of α-syn to aggregate, and how to therapeutically target LRRK2 and downstream effectors to prevent the progression of PD.

帕金森病 (PD) 和路易体痴呆 (DLB) 的特征是细胞质内含物 称为路易体和路易神经突，主要由 α-突触核蛋白 (α-syn) 组成。多行 有证据表明这些内含物有助于疾病的发病机制。因此，防止α-syn 聚集是预防疾病进展的关键治疗靶点。通常，α-syn 位于 突触前末端，优先与突触小泡结合，以及 α-syn 与膜的相互作用 抑制其聚集。我们提出的研究旨在确定 α-syn 的突触前靶向如何预防它 从转变为病理性、易聚集的形式以及 PD 风险因素如何破坏正常定位 α-syn 并增加其形成夹杂物的倾向。富含亮氨酸重复激酶 (LRRK2) 的突变 家族性帕金森病的最常见原因。我们发现 G2019S-LRRK2 突变 增加 α-syn 包涵体的形成，LRRK2 激酶抑制剂减少包涵体形成，但 LRRK2 控制 α-syn 形成聚集体倾向的机制尚不清楚。虽然 目前的研究重点是LRRK2在降解细胞器中的作用，LRRK2也已被功能性地 涉及突触前末端，可能在 α-syn 突触前膜靶向中发挥作用。最近， Rab GTPases 的一个子集被鉴定为 LRRK2 底物。兔子控制膜中的不同步骤 细胞内的运输途径。十年前，林德奎斯特实验室发现了相同的拉布（最近被发现） 显示被 LRRK2 磷酸化）在 α-syn 毒性模型中具有保护作用。我们将确定拉布是否 LRRK2 磷酸化可增强 α-syn 的突触前靶向并防止 α-syn 包涵体形成。 由于包涵体位于 PD 和 DLB 的多个大脑区域，因此我们将原纤维注射到小鼠体内 纹状体在黑质致密部 (SNpc)、皮质和杏仁核中产生内含物。在 目标 1，我们将使用以下方法确定选择的 Rab 同工型影响 α-syn 突触前靶向的程度 新型 AAV 载体可增加大脑中 Rab 的表达，或使用反义寡核苷酸 (ASO) 来减少 拉布水平。将使用免疫组织化学和生物化学来确定这些 Rab 是否在 体内可防止多个脑区域中原纤维诱导的包涵体的形成，以及多巴胺神经元的损失 国家石油公司。在目标 2 中，我们将鉴定神经元中的 Rab 效应器，这些效应器指定靶向含有 α- 的囊泡 syn 货物到突触前终端。最后，在目标 3 中，我们将确定 LRRK2 激酶活性是否会阻止 α-syn 的突触前靶向，如果 α-syn 在细胞中的错误定位会增加其形成的倾向 夹杂物。我们还将确定 LRRK2 激酶抑制剂和 ASO 在临床前研究中的应用程度 发育增强 α-syn 的突触前靶向。这些研究将为以下领域开辟新的研究途径 了解导致 PD 和 DLB 的因素和基因如何增加 α-syn 聚集的倾向， 以及如何治疗靶向 LRRK2 和下游效应器以预防 PD 的进展。