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  • Quantum-dot-labeled synuclein seed assay identifies drugs modulating the experimental prion-like transmission.

Quantum-dot-labeled synuclein seed assay identifies drugs modulating the experimental prion-like transmission.

Communications biology (2022-06-30)
Yukio Imamura, Ayami Okuzumi, Saki Yoshinaga, Akiko Hiyama, Yoshiaki Furukawa, Tomohiro Miyasaka, Nobutaka Hattori, Nobuyuki Nukina
ABSTRACT

Synucleinopathies are neurodegenerative disorders including Parkinson disease (PD), dementia with Lewy body (DLB), and multiple system atrophy (MSA) that involve deposits of the protein alpha-synuclein (α-syn) in the brain. The inoculation of α-syn aggregates derived from synucleinopathy or preformed fibrils (PFF) formed in vitro induces misfolding and deposition of endogenous α-syn. This is referred to as prion-like transmission, and the mechanism is still unknown. In this study, we label α-syn PFF with quantum dots and visualize their movement directly in acute slices of brain tissue inoculated with α-syn PFF seeds. Using this system, we find that the trafficking of α-syn seeds is dependent on fast axonal transport and the seed spreading is dependent on endocytosis and neuronal activity. We also observe pharmacological effects on α-syn seed spreading; clinically available drugs including riluzole are effective in reducing the spread of α-syn seeds and this effect is also observed in vivo. Our quantum-dot-labeled α-syn seed assay system combined with in vivo transmission experiment reveals an early phase of transmission, in which uptake and spreading of seeds occur depending on neuronal activity, and a later phase, in which seeds induce the propagation of endogenous misfolded α-syn.

MATERIALS
Product Number
Brand
Product Description

Sigma-Aldrich
γ-Aminobutyric acid, ≥99%
Sigma-Aldrich
Anti-DARPP-32 Antibody, from rabbit, purified by affinity chromatography
Sigma-Aldrich
Anti-Tubulin Antibody, beta III isoform, CT, clone TU-20 (Similar to TUJ1), ascites fluid, clone TU-20 (Similar to TUJ1), Chemicon®
Sigma-Aldrich
Cytoplasmic Dynein Inhibitor, Ciliobrevin D, Ciliobrevin D is a cell-permeable, reversible, and specific blocker of AAA+ ATPase motor cytoplasmic dynein. Disrupts spindle pole focusing and kinetochore-microtubule attachment (~10 to 40 µM).