Structure-function relationships in alpha-synuclein strain biology - Cryo-electron microscopy (cryo-EM) studies have shown that protein fibrils isolated from patient samples adopt distinct misfolded conformations, which are associated with specific synucleinopathies, including Lewy body dementia (LBD) and multiple system atrophy (MSA). These data support the prion strain hypothesis, or the idea that misfolded protein structure determines a patient’s clinical phenotype, including both cognitive and motor signs. However, both how protein structure impacts disease presentation and how strains evolve are unknown. This knowledge gap is due to two key obstacles: 1) the widespread use of α-synuclein (α-syn) mutations in transgenic (Tg) mouse models, even though only 2% of Lewy body disease cases are caused by SNCA mutations; and 2) the low yield of protein fibrils from Tg rodent brains impeding successful cryo-EM determination. Recent advances from our groups now enable us to overcome these barriers. We reported that α-syn strains transmit neurological disease following intracranial (i.c.) injection into the WT-expressing TgM20+/- mouse model. Additionally, our preliminary data show our ability to isolate protein fibrils and generate high-resolution cryo-EM maps from Tg rodent models. Drawing on these discoveries, we are well-positioned to investigate the structure-function relationship for α-syn strains, leading to our understanding of how α-syn structure encodes both cognitive and motor impairment in LBD and the ~30% of MSA patients who develop cognitive impairment. The long-term goal of our research is to use our understanding of α-syn disease pathogenesis to successfully develop diagnostics and therapeutics for synucleinopathy patients. Our objective in this application is to establish the first structure-function relationship for a prion-like protein, which will yield fundamental discoveries about the role of strains in driving the clinical presentation of disease. Building on recently published data from the Woerman Lab showing deformed templating of α-syn in Tg mice, we will test the hypothesis that small differences in protein structure cause a change in the biological properties of α-syn strains, both in vivo and in vitro. Our innovative approach will capitalize on a panel of α-syn biosensor cell lines developed by the Woerman Lab to determine the effect of structural changes on α-syn misfolding, which will synergize with Dr. Merz’s expertise in cryo-EM. In Aim 1, we will use i.c. injection of two α-syn strains to investigate the effect of the A53T mutation on α-syn strain maintenance, both structurally and biologically, including the onset of distinct neurological deficits. In Aim 2, we will used a forced evolution approach to investigate the unique neurological signs induced by small structural rearrangements after the same α-syn strain is forced to replicate using mutant monomer. This work is significant because it will establish the first structure-function relationship for any prion-like protein, enabling us to mechanistically define the role of strain biology on both cognitive and motor decline in synucleinopathies, including LBD and MSA.
