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

帕金森病(PD)和路易体痴呆(DLB)的特点是胞浆内包涵体 称为路易小体和路易神经突起，主要由α-突触核蛋白(α-SYN)组成。多行 有证据表明，这些包裹体参与了疾病的发病机制。因此，防止α-SYN 聚集是防止疾病进展的关键治疗目标。通常，α-syn驻留在 突触前末端优先与突触小泡结合，α-SYN与膜相互作用 抑制其聚集。我们提出的研究试图确定突触前靶向α-SYN是如何阻止它的 从转化为病理性的、易于聚集的形式以及PD危险因素如何破坏正常的定位 并增加了α-SYN形成夹杂物的倾向。富含亮氨酸重复序列激酶(LRRK2)的突变包括 家族性帕金森氏症最常见的原因。我们发现G2019S-LRRK2突变 增加α-SYN包涵体的形成，而LRRK2激酶抑制剂减少包涵体的形成，但 LRRK2控制α-SYN形成聚集体的倾向的机制尚不清楚。虽然 目前的研究主要集中在LRRK2在降解细胞器中的作用，LRRK2也在功能上 与突触前终末有关，可能在α-SYN突触前膜靶向中发挥作用。最近， Rab GTP酶的一部分被鉴定为LRRK2底物。RABS控制膜中不同的步骤 细胞内的运输路径。十年前，林德奎斯特实验室发现了相同的RAB(最近 在α-SYN毒性模型中显示被LRRK2磷酸化)作为保护作用。我们将确定拉布斯是否 LRRK2的磷酸化增强了α-SYN的突触前靶向性，并防止了α-SYN包涵体的形成。 由于包涵体定位于PD和DLB的多个脑区，我们将向小鼠体内注射纤维 黑质致密部(SNPC)、皮质和杏仁核内产生内含物的纹状体。在……里面 目标1，我们将确定选择RAB亚型对α-SYN突触前靶向的影响程度 新型AAV载体增加Rab在脑内的表达或使用反义寡核苷酸(ASO)减少 RAB水平。免疫组织化学和生化方法将用于确定这些RAB在 体内阻止纤维诱导的多个脑区内包涵体的形成和脑内多巴胺神经元的丢失 SNPC。在目标2中，我们将确定神经元中指定靶向含有α-的囊泡的RAB效应器。 SYN货物到突触前终端。最后，在目标3中，我们将确定LRRK2激酶活性是否可以阻止 α-SYN的突触前靶向以及α-SYN在细胞内的错误定位是否增加了其形成的倾向 包裹体。我们还将确定LRRK2激酶抑制剂和ASO在临床前的程度 开发增强α-SYN的突触前靶向性。这些研究将开辟新的研究途径 了解导致PD和DLB的因素和基因如何增加α-SYN聚集的倾向， 以及如何通过治疗靶向LRRK2和下游效应器来防止PD的进展。

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.