α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells
Christian Hansen, … , Jia-Yi Li, Patrik Brundin
Christian Hansen, … , Jia-Yi Li, Patrik Brundin
Published January 18, 2011
Citation Information: J Clin Invest. 2011;121(2):715-725. https://doi.org/10.1172/JCI43366.
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Research Article Neuroscience

α-Synuclein propagates from mouse brain to grafted dopaminergic neurons and seeds aggregation in cultured human cells

Abstract

Post-mortem analyses of brains from patients with Parkinson disease who received fetal mesencephalic transplants show that α-synuclein–containing (α-syn–containing) Lewy bodies gradually appear in grafted neurons. Here, we explored whether intercellular transfer of α-syn from host to graft, followed by seeding of α-syn aggregation in recipient neurons, can contribute to this phenomenon. We assessed α-syn cell-to-cell transfer using microscopy, flow cytometry, and high-content screening in several coculture model systems. Coculturing cells engineered to express either GFP– or DsRed-tagged α-syn resulted in a gradual increase in double-labeled cells. Importantly, α-syn–GFP derived from 1 neuroblastoma cell line localized to red fluorescent aggregates in other cells expressing DsRed–α-syn, suggesting a seeding effect of transmitted α-syn. Extracellular α-syn was taken up by cells through endocytosis and interacted with intracellular α-syn. Next, following intracortical injection of recombinant α-syn in rats, we found neuronal uptake was attenuated by coinjection of an endocytosis inhibitor. Finally, we demonstrated in vivo transfer of α-syn between host cells and grafted dopaminergic neurons in mice overexpressing human α-syn. In summary, intercellularly transferred α-syn interacts with cytoplasmic α-syn and can propagate α-syn pathology. These results suggest that α-syn propagation is a key element in the progression of Parkinson disease pathology.

Authors

Christian Hansen, Elodie Angot, Ann-Louise Bergström, Jennifer A. Steiner, Laura Pieri, Gesine Paul, Tiago F. Outeiro, Ronald Melki, Pekka Kallunki, Karina Fog, Jia-Yi Li, Patrik Brundin

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Figure 7

Inhibition of endocytosis decreases α-syn cellular uptake in vitro and in vivo.

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Inhibition of endocytosis decreases α-syn cellular uptake in vitro and i...
(A) Conditioned medium from GFP–α-syn–expressing HEK cells was concentrated 10 times and incubated with native SH-SY5Y cells at 4°C or 37°C. After 6 hours, GFP α-syn uptake was analyzed by fluorescence microscopy. Original magnification, ×40. (B) HEK cells stably transfected with either DsRed- or GFP–tagged α-syn were cocultured for 3 days in the absence (control) or presence of the endocytosis inhibitors monodansylcadaverine (MDC, 1 μM) or dynasore (1 μM), and intercellular transfer was evaluated as percentage of double-labeled cells with epifluorescence microscopy (n = 3). (C) Alexa Fluor 488 signal detected in cortical sections of the same rat injected with Alexa Fluor 488–labeled α-syn monomers on the left side (control) and Alexa Fluor 488–labeled α-syn monomers together with dynasore (80 μM) on the right side (dynasore). The inset in the control picture shows higher magnification of Alexa Fluor 488–positive cells (green), identified all around the injection site of Alexa Fluor 488–labeled α-syn alone. The neuronal nature of these cells is confirmed by colocalization with MAP2 (red). Scale bars: 50 μm. Original magnification, ×60 (top left panels). (D) The mean Alexa Fluor 488 fluorescence per area unit (AU) measured in the injection site, reflecting cellular uptake as a large majority of extracellular Alexa Fluor 488 α-syn monomers, is likely to have been degraded after 6 hours and is decreased by 40% in the dynasore-injected cortex compared with the control side, confirming the inhibition of cellular uptake of Alexa Fluor 488–labeled α-syn monomers in the presence of dynasore. Error bars represent SD. *P < 0.05; **P < 0.01.