Quantitative Analysis of α-Synuclein Solubility in Living Cells Using Split GFP Complementation

Ahmed Kothawala,Jose Andres Novoa,Laura Segatori,Kiri Kilpatrick,Salvador Ventura

doi:10.1371/journal.pone.0043505

Abstract

Presently incurable, Parkinson's disease (PD) is the most common neurodegenerative movement disorder and affects 1% of the population over 60 years of age. The hallmarks of PD pathogenesis are the loss of dopaminergic neurons in the substantia nigra pars compacta, and the occurrence of proteinaceous cytoplasmic inclusions (Lewy bodies) in surviving neurons. Lewy bodies are mainly composed of the pre-synaptic protein alpha-synuclein (αsyn), an intrinsically unstructured, misfolding-prone protein with high propensity to aggregate. Quantifying the pool of soluble αsyn and monitoring αsyn aggregation in living cells is fundamental to study the molecular mechanisms of αsyn-induced cytotoxicity and develop therapeutic strategies to prevent αsyn aggregation. In this study, we report the use of a split GFP complementation assay to quantify αsyn solubility. Particularly, we investigated a series of naturally occurring and rationally designed αsyn variants and showed that this method can be used to study how αsyn sequence specificity affects its solubility. Furthermore, we demonstrated the utility of this assay to explore the influence of the cellular folding network on αsyn solubility. The results presented underscore the utility of the split GFP assay to quantify αsyn solubility in living cells.

Highlights

Parkinson’s disease (PD) is the most prevalent neurodegenerative movement disorder, affecting 1% of the world’s population over the age of 60 years [1]
The hallmarks of PD pathogenesis are the loss of dopaminergic neurons in the substantia nigra pars compacta and the occurrence of cytoplasmic inclusions called Lewy bodies (LB) in surviving dopaminergic neurons [2]
Assay To study asyn solubility in living cells we adapted a previously reported assay based on split GFP complementation [36]

Summary

Introduction

Parkinson’s disease (PD) is the most prevalent neurodegenerative movement disorder, affecting 1% of the world’s population over the age of 60 years [1]. Post mortem analyses revealed that the main component of LB is the pre-synaptic protein alpha-synuclein (asyn) and of trace amounts of ubiquitin and molecular chaperones [3], suggesting that they result from the aberrant accumulation and aggregation of misfolded, undegraded asyn. Overexpression of asyn results in the formation of inclusion bodies, cytotoxicity and cell death in animal models and cell cultures [11,12,13]. Aberrant accumulation of misfolded asyn plays a key role in development of PD pathogenesis.

Methods

Results

Conclusion