Seeding variability of different alpha synuclein strains in synucleinopathies

Abstract

Objectives: Currently, the exact reasons why different α-synucleinopathies exhibit variable pathologies and phenotypes are still unknown. A potential explanation may be the existence of distinctive α-synuclein conformers or strains. Here, we intend to analyze the seeding activity of dementia with Lewy bodies (DLB) and Parkinson's disease (PD) brain-derived α-synuclein seeds by real-time quaking-induced conversion (RT-QuIC) and to investigate the structure and morphology of the α-synuclein aggregates generated by RT-QuIC. Methods: A misfolded α-synuclein-enriched brain fraction from frontal cortex and substantia nigra pars compacta tissue, isolated by several filtration and centrifugation steps, was subjected to α-synuclein/RT-QuIC analysis. Our study included neuropathologically well-characterized cases with DLB, PD, and controls (Ctrl). Biochemical and morphological analyses of RT-QuIC products were conducted by western blot, dot blot analysis, Raman spectroscopy, atomic force microscopy, and transmission electron microscopy. Results: Independently from the brain region, we observed different seeding kinetics of α-synuclein in the RT-QuIC in patients with DLB compared to PD and Ctrl. Biochemical characterization of the RT-QuIC product indicated the generation of a proteinase K-resistant and fibrillary α-synuclein species in DLB-seeded reactions, whereas PD and control seeds failed in the conversion of wild-type α-synuclein substrate. Interpretation: Structural variances of α-synuclein seeding kinetics and products in DLB and PD indicated, for the first time, the existence of different α-synuclein strains in these groups. Therefore, our study contributes to a better understanding of the clinical heterogeneity among α-synucleinopathies, offers an opportunity for a specific diagnosis, and opens new avenues for the future development of strain-specific therapies. ANN NEUROL 2019;85:691-703 S ynucleinopathies, such as Parkinson's disease (PD) and dementia with Lewy bodies (DLB), are a class of neurodegenerative diseases characterized by the presence of insoluble intraneural deposits consisting of fibrillary α-synuclein, 1,2 referred to as Lewy bodies. A PD with dementia (PDD) diagnosis is appropriate when the cognitive symptoms View this article online at wileyonlinelibrary.com.

Effect of the micro-environment on α-synuclein conversion and implication in seeded conversion assays

“…Biochemical analyzes confirmed the generation of a fibrillary, proteinase K (PK)-resistant seeding competent αsynuclein species only in DLB-brain seeded RT-QuIC reactions, pointing toward the existence of different αsynuclein strains in DLB and PD cases. Importantly, the RT-QuIC results were neither influenced by the brain region analyzed nor by the co-pathology or other epidemiological factors, such as age, sex, severity of the disease, and age of onset [108]. Controversial findings regarding a lack of α-synuclein seeding in PD patients might be explained by the addition of beads and the preanalytical treatment (fractionation protocol) [104,106].…”

“…Candelise and co-workers recently published a study in which positive RT-QuIC signals could be obtained specifically from DLB-brain seeded reactions [108]. In order to avoid possible contamination from macromolecules that may skew the aggregation process, a new preanalytical fractionation protocol for brain homogenates was established.…”

Modeling Parkinson’s Disease With the Alpha-Synuclein Protein

“…Each a-Syn conformation displays distinct properties in terms of neurotoxicity, stability and seeding and propagation ability. It has been proposed that the existence of structurally distinct a-Syn assemblies or a-Syn "strains" may contribute to explain the clinical-pathological heterogeneity among synucleinopathies and help to develop strain-specific medications (Melki, 2015;Peelaerts et al, 2015;Candelise et al, 2019).…”

Cytosolic Trapping of a Mitochondrial Heat Shock Protein Is an Early Pathological Event in Synucleinopathies

“…Next, we assessed the conversion of monomeric aSyn into fibrils in vitro, using the real-time quaking-induced conversion (RT-QuIC) assay (Candelise et al, 2019;van Rumund et al, 2019). Briefly, WTaSyn or A30PaSyn monomers were incubated in the presence or absence of HSP10, and the aggregation was monitored by measuring thioflavin T fluorescence.…”