STRUCTURAL BIOLOGY OF ALPHA-SYNUCLEIN IN LEWY BODY DEMENTIA

帕金森病(PD)的病理定义是α-突触核蛋白(ASYN)纤维聚集在神经细胞质和神经元包涵体中，称为路易小体和路易神经突起。在罕见的家族性PD中发现编码Asyn基因的显性突变(SNCA)，支持Asyn在PD发病机制中的作用。痴呆症在帕金森病患者中高发。它有时与运动症状几乎同时开始(通常被称为路易体痴呆或DLB)，或在运动症状开始后长达20年(PD伴痴呆或PDD)。路易体痴呆(LBD)一词涵盖了这一系列临床表现，并与大脑中广泛沉积的Asyn纤维有关，特别是新皮质。针对Asyn堆积的多种治疗方法正在进行中。另一个优先事项是开发一种PET显像剂，以量化Asyn在活体中的沉积，作为靶点参与和疾病进展的生物标记物。了解LBD的Asyn纤维结构可以指导Asyn靶向治疗和显像剂的开发。在这个项目中，我们将使用冷冻电子显微镜(Cryo-EM)来确定LBD中Asyn纤维的原子分辨结构，并结合固态核磁共振(SS核磁共振)来精炼结构。我们将分析和比较从早发和晚发痴呆症尸检病例的多个亚组中分离出的Asyn纤维的结构，以及是否存在共发生的淀粉样蛋白β积聚。我们还将利用冷冻电子断层扫描、SS核磁共振光谱分析和新的单抗将Asyn原纤维结构的分析扩展到更多的尸检病例。为了促进这些结构研究的翻译，我们将利用低温EM来确定PET成像配体的主要候选结合位置，并使用纤维生长分析来确定Asyn蛋白中对纤维生长和稳定性至关重要的特定氨基酸残基。

Parkinson’s disease (PD) is defined pathologically by the accumulation of alpha-synuclein (Asyn) fibrils in neuronal cytoplasmic and neuritic inclusions known as Lewy bodies and Lewy neurites. The role of Asyn in the pathogenesis of PD is supported by the identification of dominant mutations in the gene encoding Asyn (SNCA) in rare familial versions of PD. Dementia occurs frequently in PD. It sometimes begins at approximately the same time as motor symptoms (often referred to as dementia with Lewy bodies or DLB), or up to 20 years after motor symptoms begin (PD with dementia or PDD). The term Lewy body dementia (LBD) encompasses this spectrum of clinical presentations and is associated with widespread deposition of Asyn fibrils throughout the brain, particularly neocortex. Multiple therapeutic approaches targeting Asyn accumulation are being pursued. A further priority is to develop a PET imaging agent to quantify the deposition of Asyn in living individuals, as a biomarker for target engagement and disease progression. Understanding Asyn fibril structure in LBD can guide the development of Asyn-targeted therapies and imaging agents. In this project, we will use cryo-electron microscopy (cryo-EM) to determine atomic resolution structures of Asyn fibrils in LBD, in conjunction with solid-state NMR (SSNMR) for refinement of structures. We will analyze and compare structures of Asyn fibrils isolated from multiple subgroups of LBD autopsy cases defined by early versus late onset of dementia, as well as the presence or absence of co-occurring amyloid β accumulation. We will also utilize cryo-electron tomography, SSNMR spectral analysis and new monoclonal antibodies to extend the analysis of Asyn fibril structure to additional autopsy cases. To promote the translation of these structural studies we will utilize cryo-EM to determine binding sites of leading candidates for PET imaging ligand and use fibril growth assays to identify specific amino acid residues in the Asyn protein that are important for fibril growth and stability.