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 α-synuclein both between different patients and between different brain regions. Methods The 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. Results Using 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. Conclusions We 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.

Background and purpose Progressive supranuclear palsy (PSP) encompasses a broader range of disease courses than previously appreciated. The most frequent clinical presentations of PSP are Richardson syndrome (RS) and PSP with a predominant Parkinsonism phenotype (PSP‐P). Time to reach gait dependence and cognitive impairment have been proposed as prognostic disease milestones. Genetic polymorphisms in TRIM11 and SLC2A13 genes have been associated with longer disease duration (DD). Methods Methods used include retrospective chart review, genetic single nucleotide polymorphism analyses (in three cases), and neuropathology. Results We identified four cases with long (>10–15 years) or very long (>15 years) DD. Stage 1 PSP tau pathology was present in two cases (one PSP‐P and one undifferentiated phenotype), whereas pallidonigroluysian atrophy (PSP‐RS) and Stage 4/6 (PSP‐P) PSP pathology were found in the other two cases. Three cases were homozygous for the rs564309‐C allele of the TRIM11 gene and the H1 MAPT haplotype. Two were heterozygous for rs2242367 (G/A) in SLC2A13, whereas the third was homozygous for the G‐allele. Conclusions We propose a protracted course subtype of PSP (PC‐PSP) based on clinical or neuropathological criteria in two cases with anatomically restricted PSP pathology, and very long DD and slower clinical progression in the other two cases. The presence of the rs564309‐C allele may influence the protracted disease course. Crystallizing the concept of PC‐PSP is important to further understand the pathobiology of tauopathies in line with current hypotheses of protein misfolding, seeding activity, and propagation.

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