Secretion and Uptake of \u03b1-Synuclein Via Extracellular Vesicles in Cultured Cells

Gabriel Gustafsson,Emma Persson,Shuko Takeda,Bence György,Camilla Lööv,Bradley T Hyman,Martin Ingelsson,Dag Sehlin,Tiago F Outeiro,Joakim Bergström,Diana F Lázaro,Xandra O Breakefield,Anna Erlandsson,Leonora Balaj,Martin Hallbeck

doi:10.1007/s10571-018-0622-5

Abstract

In Parkinson’s disease and other Lewy body disorders, the propagation of pathology has been accredited to the spreading of extracellular α-synuclein (α-syn). Although the pathogenic mechanisms are not fully understood, cell-to-cell transfer of α-syn via exosomes and other extracellular vesicles (EVs) has been reported. Here, we investigated whether altered molecular properties of α-syn can influence the distribution and secretion of α-syn in human neuroblastoma cells. Different α-syn variants, including α-syn:hemi-Venus and disease-causing mutants, were overexpressed and EVs were isolated from the conditioned medium. Of the secreted α-syn, 0.1–2% was associated with vesicles. The major part of EV α-syn was attached to the outer membrane of vesicles, whereas a smaller fraction was found in their lumen. For α-syn expressed with N-terminal hemi-Venus, the relative levels associated with EVs were higher than for WT α-syn. Moreover, such EV-associated α-syn:hemi-Venus species were internalized in recipient cells to a higher degree than the corresponding free-floating forms. Among the disease-causing mutants, A53T α-syn displayed an increased association with EVs. Taken together, our data suggest that α-syn species with presumably lost physiological functions or altered aggregation properties may shift the cellular processing towards vesicular secretion. Our findings thus lend further support to the tenet that EVs can mediate spreading of harmful α-syn species and thereby contribute to the pathology in α-synucleinopathies.

Highlights

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are the two most common disorders with Lewy body and Lewy neurite pathology
The plasmids used were pcDNA3.1+ with the following inserts: wild-type α-syn (WT), hemi-peptides of Venus yellow fluorescent protein (YFP) fused to full-length wild-type α-syn (Venus 1–157 N-terminally fused to α-syn (V1S), Venus 158–238 C-terminally fused to α-syn (SV2) or V1S + SV2 at an equal ratio (BiFC)) (Fig. 1a), α-syn-2A full-length green fluorescent protein (GFP), or α-syn with any of the six diseasecausing point mutations (A30P, E46K, H50Q, G51D, A53E, and A53T)
In order to characterize the EV content in the SH-SY5Y cell-derived extracellular fractions (EV; extracellular vesicles and FFP; free-floating protein), we investigated various markers by Western blot

Summary

Introduction

Parkinson’s disease (PD) and dementia with Lewy bodies (DLB) are the two most common disorders with Lewy body and Lewy neurite pathology. Oligomers of α-syn seem to be prone to spread (Danzer et al 2012) and may have a capacity to sequester monomeric protein in recipient cells, suggesting that they can be responsible for a prion-like propagation of pathology (Danzer et al 2009). Via such mechanisms, α-syn could spread from one brain region to another, which would explain the hierarchical pattern by which pathology occurs in the PD brain (Braak et al 2006b). The discovery of early α-syn pathology in the nerve plexus of the gastrointestinal tract suggests that pathology may even initiate outside the central nervous system (CNS) and transfer to the brain stem via enteric neurons (Braak et al 2006a)

Methods

Results

Conclusion