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

项目摘要 帕金森病（PD）是第二常见的神经退行性疾病。典型的标志 帕金森病的发病机制是α-突触核蛋白（α-syn）的异常聚集和多巴胺能神经元的丢失。 黑质这导致PD的经典运动表现，包括震颤、运动迟缓， 僵硬和姿势不稳定Braak的理论将疾病进展描述为朊病毒样α-syn 传播，PD相关的认知障碍是由于α-syn引起的负担增加 在受影响个体的大脑皮层中扩散在疾病进展期间，50-80%的PD患者 发展为痴呆（PDD），表现出比仅患有PD的患者增加的发病率和死亡率。研究 α-Syn在PD疾病进展中的作用，我们使用PCR扩增了PD和PDD患者的α-Syn， PMCA（蛋白质错误折叠循环扩增）。我们已经确定，从PD扩增的α-syn和 PDD患者是不同的菌株，PDD菌株α-syn表现出比PDD菌株α-syn更严重的神经毒性。 PD菌株。这两种不同的α-Syn菌株可能用作PD疾病的生物标志物 进展和药物靶点。然而，进一步研究这两种α-syn菌株在 由于缺乏直接识别这两种α-syn菌株的试剂，PD进展受到阻碍。 作为回应，我们设计了一个新的合成纳米抗体变体库，这些变体是氧化还原稳定的， 去除二硫键，并确定了许多识别α-syn预制的纳米抗体， 原纤维（PFF），但不是单体形式。我们鉴定的五个纳米抗体中有两个显示 与野生型PFF相比，菌株对丝氨酸129 PFF处磷酸化α-syn的选择性。总体目标 该提案的一个重要目的是设计和表征α-syn应变特异性纳米抗体， PD-或PDD-菌株α-syn，并测试PFF特异性纳米抗体在预防细胞间干扰中的功效。 α-syn PFF的传输。实现这一目标将有助于实现我们的长期目标， α-syn菌株在PD向PDD进展中的作用，这对α-syn的发展至关重要 菌株作为PD进展的生物标志物，并开发PD的新疗法。这些纳米抗体 可能通过阻止α-syn PFF扩散为PD提供新的治疗方法。最后， 识别不同菌株的纳米抗体也可用于研究其他α-突触核蛋白病，包括 路易体痴呆和多系统萎缩。

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.