Abstract
BackgroundMultiple 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 α-synuclein both between different patients and between different brain regions.MethodsThe reliable detection of α-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.ResultsUsing our novel buffer conditions, we show that the physicochemical factors that govern the in vitro amplification of α-synuclein can be tailored to generate strain-specific reaction buffers that can be used to reliably study the seeding capacity from MSA-derived α-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 α-synuclein seeding in MSA, we conducted a comprehensive multi-regional evaluation of α-synuclein seeding in 13 different regions from 2 high seeders, 2 intermediate seeders and 2 low seeders.ConclusionsWe have identified unexpected differences in seed-competent α-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 α-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 α-synuclein present. Finally, our methods provide an experimental framework for the development of vitally needed, rapid and sensitive diagnostic assays for MSA.
Highlights
Multiple system atrophy (MSA) is a progressive neurodegenerative disorder with clinical presentation of various combinations of parkinsonism, cerebellar and autonomic dysfunction [1]
Screening 168 RT‐QuIC conditions to detect α‐synuclein seeding in MSA We hypothesized that changing the physicochemical factors that govern the in vitro amplification of amyloidogenic proteins would favor α-synuclein seeding in MSA
Based on the published success of using different ionic environments to enhance the sensitivity of different proteopathic seeding amplification assays [19], we conducted a systematic evaluation of 168 different reaction buffers, using an array of pH and salts, seeded with brain homogenates from one MSA and one Parkinson’s disease (PD) patient (Fig. 1)
Summary
Multiple system atrophy (MSA) is a progressive neurodegenerative disorder with clinical presentation of various combinations of parkinsonism, cerebellar and autonomic dysfunction [1]. The presence of the 140-amino-acid protein, α-synuclein, as a major component of these inclusions linked MSA with Lewy body disorders such as Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) [4]. The presence of different strains is hypothesized to dictate the cell-to-cell spreading of pathology and the cellular impact of the pathological α-synuclein in every individual [5, 6] Consistent with this notion, many experimental findings have indicated that α-synuclein forming GCIs has greater seeding activity compared to Lewy body-associated α-synuclein [7, 8]. We hypothesized that the clinical hetero‐ geneity in MSA is a consequence of variability in the seeding activity of α-synuclein both between different patients and between different brain regions
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