Chemical Fingerprints of Cognitive Impairment-related alpha-Synuclein Strains using 3D Small Molecule Microarray and Related Therapeutic Application

项目总结 就直接的医疗和社会护理费用而言，痴呆症具有重大的社会和经济影响。刘易斯 躯体痴呆症(LBD)是痴呆症最常见的原因之一，包括帕金森氏症和 痴呆(PDD)和路易体痴呆(DLB)。大约30%的阿尔茨海默病(AD)患者 也患有LBD，导致比单独使用AD更快、更严重的认知能力下降。LBD关联 大脑中一种名为α-突触核蛋白(α-SYN)的蛋白质异常沉积。大量尸检研究由 Braak等人。显示α-SYN病理在PDD中以一种刻板印象的方式扩散，运动的开始 症状表现为黑质(SN)多巴胺能神经元丢失，约80%的患者最终 大脑皮层α-SYN病理的PDD研究进展。总之，致病性α-SYN的传播是一种主要的 LBD中认知障碍(CI)的驱动因素。最近的研究支持致病的α-SYN可能表现为 以一种类似于菌株特有的普恩病毒的方式显示出不同的生化和病理表型。连 重组α-SYN聚集体(一个菌株)经过循环聚集后可以转化为另一个菌株，而这两个 菌株经蛋白酶K消化后表现出不同的神经毒性和免疫印迹模式 在大脑内，msa(多系统萎缩)脑匀浆显著促进α-SYN病理。 与PD匀浆相比，表明MSA和PD具有不同的α-SYN株系。应变- 在神经系统疾病体征、发病时间、病理形态等方面有明显差异。 脑α-SYN沉积及其诱导聚集体的构象特性。此外，不同的菌株 脑内有针对性的不同细胞群和细胞类型，概括了选择性靶向 在α-突触核病症中观察到。为了研究α-SYN菌株在pdd进展中的作用，我们收集了 临床特征良好的患者的脑脊液(CSF)样本进行了纵向追踪。在一个 采用双盲法，以患者脑脊液(含α-SYN种子)为模板扩增α-SYN聚集体， 使用一种成熟的菌株扩增技术--PMCA(蛋白质错误折叠循环扩增)。我们 表征这些α-SYN聚集体的特征，这些聚集体来自具有明确的神经毒性、生化和 生物物理化验。然而，鉴于错误折叠的α-SYN聚集体表现出不同的应变特性 和动态转换，特别是在与环境、遗传、老龄化因素复杂相互作用的情况下，它是 有必要建立一种指纹分析方法来确定这些来自狼疮患者的α-SYN菌株。幸运的是，很小 分子微阵列(SMM)筛选提供了一个结合不同面板的亲和力图谱的平台 数万个小分子与特定的蛋白质靶标。通过比较和分析来分析这一“大数据” 将这些亲和探针作为指纹可以帮助我们鉴定和区分α-SYN菌株。我们有 用我们建立的大环化合物“Rapafucin”文库生成了一个三维SMM，并进行了筛选 SMM抗体外分离的α-SYN毒株。我们发现这些菌株表现出不同的 3D-SMM中的绑定模式(指纹签名)。我们建议将3D-SMM作为一种化学物质 用于鉴定和区分患者α-SYN菌株并开发相关治疗方法的指纹平台 策略。

PROJECT SUMMARY Dementia has significant social and economic implications in terms of direct medical and social care costs. Lewy bodies dementia (LBD) is one of the most common causes of dementia, including Parkinson's disease with dementia (PDD) and dementia with Lewy bodies (DLB). Approximately 30% of Alzheimer' disease (AD) patients also suffer from LBD resulting in a more rapid and severe cognition decline than AD alone. LBD is associated with abnormal deposits of a protein called α-synuclein (α-syn) in the brain. Substantial postmortem studies by Braak et al. show that α-syn pathology spreads in a stereotypical fashion in PDD, and the onset of motor symptoms occurs with loss of dopaminergic neurons in the substantia nigra (SN), and ~80% of patients finally progress to PDD with α-syn pathology in the cortex. In brief, the spread of pathogenic α-syn acts as a major driver of cognitive impairment (CI) in LBD. Recent studies support the notion that pathogenic α-syn may behave in a manner similar to strain-specific prions exhibiting distinct biochemical and pathologic phenotypes. Even recombinant α-syn aggregates (one strain) can convert to another strain after cyclic aggregation, and these two strains exhibited different neurotoxicity and immunoblot patterns after digestion with proteinase K. When injected intracerebrally, MSA (multiple system atrophy) brain homogenates remarkably promote α-syn pathology spreading compared to PD homogenates, suggesting that MSA and PD have different strains of α-syn. Strain- specific difference were observed in the signs of neurological illness, time to disease onset, morphology of cerebral α-syn deposits and the conformation properties of the induced aggregates. Moreover, different strains targeted distinct cellular populations and cell types within the brain, recapitulating the selective targeting observed among α-synucleinopathies. To investigate the role of α-syn strains in PDD progression, we collected cerebrospinal fluid (CSF) samples from the clinically well-characterized patients followed longitudinally. In a double-blinded manner, we amplified α-syn aggregates templated by CSF (containing α-syn seeds) of patients, using a well-established strain amplification technique--PMCA (protein misfolding cyclic amplification). We characterized these α-syn aggregates from patients with well-established neurotoxicity, biochemical, and biophysical assays. However, given that misfolded α-syn aggregates exhibit heterogeneous strain properties and dynamic conversion, particularly in the complicated interplay with environmental, genetic, aging factors, it is necessary to build a fingerprinting method to define these α-syn strains from LBD patients. Fortunately, small molecule microarray (SMM) screening has provided a platform to combine the affinity profile of a diverse panel of tens of thousands of small molecules to certain protein targets. Analysis of this “big data” by comparing and contracting these affinity probes as fingerprints can help us identify and differentiate α-syn strains. We have generated a 3-dimensional SMM using our established macrocyclic compound “rapafucin” library, and screened the SMM against in-vitro-derived distinct α‐syn strains. We have found that these strains exhibited different binding patterns in the 3D-SMM (fingerprinting signatures). We propose to apply the 3D-SMM as a chemical fingerprinting platform to identify and differentiate α‐syn strains from patients and develop related therapeutic strategy.