Abstract
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.
Highlights
Progressive accumulation and deposition of specific protein aggregates is a characteristic of many neurodegenerative disorders, including Parkinson’s disease (PD)
In this study we used direct stochastic reconstruction microscopy to follow the uptake of α-syn aggregates by the non-transfected SH-SY5Y human neuroblastoma cells. direct stochastic optical reconstruction microscopy (dSTORM) has been used in several studies to probe the morphology of protein aggregates such as Aβ-aggregates in Alzheimer’s disease [20, 21], Huntingtin protein aggregates in Huntingtin disease [17, 26] and α-syn amyloid fibrils in Parkinson’s disease (PD) [18]
We demonstrated that using dSTORM, it is possible to follow noninvasively the endocytotic uptake of extracellularly added amyloid aggregates and get morphological details doi:10.1371/journal.pone.0153020.g006
Summary
Progressive accumulation and deposition of specific protein aggregates is a characteristic of many neurodegenerative disorders, including Parkinson’s disease (PD). In PD, α-synuclein(αsyn), a small presynaptic protein (~15 kDa), is the main fibrillar component of the intraneuronal protein aggregates (Lewy bodies) that represent the pathological feature of this disease [1]. Α-syn is predominantly a cytosolic protein, recent studies suggest the protein exerts a pathogenic effect inside the cells, but an extracellular pathogenic action as well. When applied to cultured cells, α-syn preformed aggregates are internalized via endocytosis and targeted to the lysosomes for degradation [5,6,7,8,9]. Few groups reported that α-syn taken-up from the extracellular space induces the aggregation of the endogenous protein leading to the formation of Lewy body-like inclusions [9,10,11,12,13,14]. Cell to cell transmission of α-syn pathological aggregates most likely through sequential exocytosis and endocytosis, has been demonstrated in neuronal cultured cells as well as in animal models [15, 16]
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