Design and characterization of Nanobodies to dementia-related α-synuclein strains in Parkinson’s disease

项目摘要 帕金森病(PD)是第二种最常见的神经退行性疾病。典型的特点是 帕金森病的病因是错折叠α-突触核蛋白(α-SYN)的异常堆积和多巴胺能神经元的丢失。 黑质。这会导致帕金森病的典型运动表现，包括震颤、运动迟缓、 僵硬，姿势不稳。布拉克的理论描述了疾病的进展是类似于普恩的α-syn 弥漫性，帕金森病相关的认知障碍是由于α-SYN导致的负担增加 在患病个体的大脑皮层中扩散。在疾病发展过程中，50%-80%的帕金森病患者 罹患痴呆症(PDD)，显示出比单纯PD患者更高的发病率和死亡率。学习 α-SYN在PD疾病进展中的作用，我们从PD和PDD患者中扩增α-SYN 蛋白质错折叠循环扩增(PMCA)。我们已经确定从PD和Pd扩增的α-SYN PDD患者有不同的菌株，PDD菌株α-SYN表现出比 PD菌株。这两株不同的α-syn毒株有可能作为帕金森病的生物标志物 进展和药物靶点。然而，对这两个α-SYN菌株在致病过程中作用的进一步研究 由于缺乏直接识别这两个α-SYN毒株的试剂，PD的进展一直受到阻碍。 作为回应，我们设计了一个新的合成纳米体变异体文库，它是氧化还原稳定的，通过 去除了二硫键，并鉴定了一些识别预制α-SYN的纳米体 原纤维(PFF)，但不是单体形式。我们表征的五个纳米实体中有两个显示 丝氨酸129pff处的磷酸化α-SYN对野生型pff的菌株选择性。总体目标 这项建议的目的是设计和表征α-SYN菌株特异的纳米体，这些纳米体可以识别 PD或pdd株α-SYN，并测试pFf特异性纳米抗体在防止细胞到细胞方面的效果 α-SYN PFF的传输。实现这一目标将有助于我们理解长期目标 α-SYN株在PD向PDD发展过程中的作用，这对α-SYN的发展至关重要 菌株作为帕金森病进展的生物标志物，并开发治疗帕金森病的新疗法。这些纳米小体 通过防止α-syn pff扩散，可能为帕金森病提供新的治疗方法。最后， 识别不同菌株的纳米体也可以用于研究其他α-突触核病，包括 伴有路易体和多系统萎缩的痴呆。

Project Summary Parkinson's disease (PD) is the second most common neurodegenerative disorder. The typical hallmark of PD is the abnormal accumulation of misfolded α-synuclein (α-syn) and dopaminergic neuron loss in the substantia nigra. This results in the classic motor manifestations of PD, including tremor, bradykinesia, rigidity, and postural instability. Braak's theory described the disease progression as prion-like α-syn spreading, and the PD-related cognitive impairment is due to the increased burden caused by α-syn spreading in the cortex of afflicted individuals. During the disease progression, 50-80% of PD patients develop dementia (PDD), showing increased morbidity and mortality above those with PD alone. To study the roles of α-syn in PD disease progression, we have amplified α-Syn from PD and PDD patients using PMCA (protein misfolding cyclic amplification). We have determined that the amplified α-syn from PD and PDD patients are different strains and the PDD strain α-syn exhibits more severe neurotoxicity than the PD strain. These two distinct α-Syn strains could potentially be used as biomarkers for PD disease progression and drug targets. However, further studies on the roles of these two α-syn strains during the PD progression have been impeded by a lack of reagents for direct recognition of these two α-syn strains. In response, we have designed a new library of synthetic nanobody variants that are redox stable, by removal of the disulfide bond, and have identified a number of nanobodies that recognize α-syn preformed fibrils (PFF), but not the monomeric form. Two out of the five nanobodies that we characterized showed strain selectivity for the phosphorylated α-syn at serine 129 PFF over wild type PFF. The overall objective of this proposal is to design and characterize α-syn strain-specific nanobodies that can recognize either PD- or PDD-strain α-syn, and to test the efficacy of PFF-specific nanobodies in preventing the cell-to-cell transmission of α-syn PFF. Accomplishing this objective will contribute to our long-term goal to understand the roles of α-syn strains in the progression from PD to PDD, which is critical for the development of α-syn strains as biomarkers for PD progression, and to develop new therapeutics for PD. These nanobodies could potentially provide new therapeutic treatment for PD by preventing α-syn PFF spreading. Lastly, nanobodies recognizing distinct strains can also be applied to study other α-synucleinopathies, including dementia with Lewy body and multiple system atrophy.