Direct Observation of α-Synuclein Amyloid Aggregates in Endocytic Vesicles of Neuroblastoma Cells.
Aggregation of α-synuclein has been linked to both familial and sporadic Parkinson's disease. Recent studies suggest that α-synuclein aggregates may spread from cell to cell and raise questions about the propagation of neurodegeneration. While continuous progress has been made characterizing α-synuclein aggregates in vitro, there is a lack of information regarding the structure of these species inside the cells. Here, we use confocal fluorescence microscopy in combination with direct stochastic optical reconstruction microscopy, dSTORM, to investigate α-synuclein uptake when added exogenously to SH-SY5Y neuroblastoma cells, and to probe in situ morphological features of α-synuclein aggregates with near nanometer resolution. We demonstrate that using dSTORM, it is possible to follow noninvasively the uptake of extracellularly added α-synuclein aggregates by the cells. Once the aggregates are internalized, they move through the endosomal pathway and accumulate in lysosomes to be degraded. Our dSTORM data show that α-synuclein aggregates remain assembled after internalization and they are shortened as they move through the endosomal pathway. No further aggregation was observed inside the lysosomes as speculated in the literature, nor in the cytoplasm of the cells. Our study thus highlights the super-resolution capability of dSTORM to follow directly the endocytotic uptake of extracellularly added amyloid aggregates and to probe the morphology of in situ protein aggregates even when they accumulate in small vesicular compartments.
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
Mihaela M
MM
Apetri
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
Rolf
R
Harkes
FOM Institute AMOLF, Amsterdam, The Netherlands.
Vinod
V
Subramaniam
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
Gerard W
GW
Canters
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
Thomas
T
Schmidt
Huygens-Kamerlingh Onnes Laboratory, Leiden University, Leiden, The Netherlands.
Thijs J
TJ
Aartsma
D000682:Amyloid / Q000378:metabolism*; D045744:Cell Line, Tumor; D004705:Endocytosis; D006801:Humans; D018625:Microscopy, Atomic Force; D009447:Neuroblastoma / Q000473:pathology*; D051844:alpha-Synuclein / Q000378:metabolism*
D000682:Amyloid; D051844:alpha-Synuclein