Insights into the Structure and Pathology of Alpha-Synuclein Inclusions in Dementia with Lewy Bodies

Millions of people around the world suffer from neurodegenerative diseases. While specific treatments may be able to relieve some of the symptoms of neurodegenerative diseases, in most cases, these complex neurological disorders do not have a cure as we do not completely understand the cause of the disease. The current theory for the mechanism of neurodegenerative diseases is that the causative protein misfolds into a beta-sheet conformation triggering the formation of amyloid fibrils that aggregate into distinctive cellular inclusions. One such protein is alpha-synuclein (aSyn) which accumulates into fibrils in Parkinson's disease (PD), dementia with Lewy bodies (DLB), and multiple system atrophy (MSA). When aSyn amyloid fibrils clump together in diseases such as PD and DLB, they form Lewy bodies. As amyloid fibrils are thought to be the causative agent of the disease, many therapeutic strategies are being developed to inhibit the formation of the fibrils. To facilitate structure-based drug design, several methods have emerged in recent years to generate disease fibrils in vitro and obtain the atomic resolution 3D structure using cryo-electron microscopy. Of the many structures which are emerging from these studies, one major question is if these in vitro-generated structures are the same as what is seen in human disease. In this research, I will use multiple electron microscopy approaches to understand the physiological structure of aSyn fibrils in DLB, which is the second most common form of dementia after Alzheimer's disease. My first aim is to determine the in vitro structure of aSyn fibrils extracted from DLB patients. Cryo-EM is a tool that allows us to get the high-resolution structure of the fibrils in native conditions by plunge freezing in liquid ethane. Several software packages have been developed for electron microscopy structure determination, including RELION (REgularised LIkelihood Optimization); My second aim is to use room-temperature correlative light and electron microscopy (CLEM) method on resin-embedded brain sections to obtain the global morphology of aSyn inclusions in DLB brain donors. The inclusions will be first localized in the brain slices by fluorescence microscopy, and the structure will be analyzed by electron microscopy and electron tomography. I will investigate the inclusions in different brain regions and multiple patients. This analysis will inform us about different inclusion types present in DLB brains and define the targets for high-resolution cryo-CLEM; My final aim is to obtain the high-resolution structure directly within post-mortem human brain samples by cryo-CLEM. This will provide a direct representation of the pathological fibril structure, which can be used for drug discovery and mouse models. Cryo-CLEM and CLEM will be used to study the fibrillar structures in different parts of the post-mortem human brain with DLB. By correlating the information from post-mortem human brains with in vitro fibr