Alpha-synuclein seeding shows a wide heterogeneity in multiple system atrophy

Background: Multiple system atrophy (MSA) is a neurodegenerative condition characterized by variable combinations of parkinsonism, autonomic failure, cerebellar ataxia and pyramidal features. Although the distribution of synucleinopathy correlates with the predominant clinical features, the burden of pathology does not fully explain observed differences in clinical presentation and rate of disease progression. We hypothesized that the clinical heterogeneity in MSA is a consequence of variability in the seeding activity of alpha-synuclein both between different patients and between different brain regions. Methods: The reliable detection of alpha-synuclein seeding activity derived from MSA using cell-free amplification assays remains challenging. Therefore, we conducted a systematic evaluation of 168 different reaction buffers, using an array of pH and salts, seeded with fully characterized brain homogenates from one MSA and one PD patient. We then validated the two conditions that conferred the optimal ability to discriminate between PD- and MSA-derived samples in a larger cohort of 40 neuropathologically confirmed cases, including 15 MSA. Finally, in a subset of brains, we conducted the first multi-region analysis of seeding behaviour in MSA. Results: Using our novel buffer conditions, we show that the physicochemical factors that govern the in vitro amplification of alpha-synuclein can be tailored to generate strain-specific reaction buffers that can be used to reliably study the seeding capacity from MSA-derived alpha-synuclein. Using this novel approach, we were able to sub-categorize the 15 MSA brains into 3 groups: high, intermediate and low seeders. To further demonstrate heterogeneity in alpha-synuclein seeding in MSA, we conducted a comprehensive multi-regional evaluation of alpha-synuclein seeding in 13 different regions from 2 high seeders, 2 intermediate seeders and 2 low seeders. Conclusions: We have identified unexpected differences in seed-competent alpha-synuclein across a cohort of neuropathologically comparable MSA brains. Furthermore, our work has revealed a substantial heterogeneity in seeding activity, driven by the PBS-soluble alpha-synuclein, between different brain regions of a given individual that goes beyond immunohistochemical observations. Our observations pave the way for future subclassification of MSA, which exceeds conventional clinical and neuropathological phenotyping and considers the structural and biochemical heterogeneity of alpha-synuclein present. Finally, our methods provide an experimental framework for the development of vitally needed, rapid and sensitive diagnostic assays for MSA.