Misfolded α-synuclein causes hyperactive respiration without functional deficit in live neuroblastoma cells.

C L Ugalde,K Mroczek,S Gordon,M A Perugini,P R Fisher,V A Lawson,S J Annesley,A F Hill,D I Finkelstein

doi:10.1242/dmm.040899

Abstract

ABSTRACTThe misfolding and aggregation of the largely disordered protein, α-synuclein, is a central pathogenic event that occurs in the synucleinopathies, a group of neurodegenerative disorders that includes Parkinson's disease. While there is a clear link between protein misfolding and neuronal vulnerability, the precise pathogenic mechanisms employed by disease-associated α-synuclein are unresolved. Here, we studied the pathogenicity of misfolded α-synuclein produced using the protein misfolding cyclic amplification (PMCA) assay. To do this, previous published methods were adapted to allow PMCA-induced protein fibrillization to occur under non-toxic conditions. Insight into potential intracellular targets of misfolded α-synuclein was obtained using an unbiased lipid screen of 15 biologically relevant lipids that identified cardiolipin (CA) as a potential binding partner for PMCA-generated misfolded α-synuclein. To investigate whether such an interaction can impact the properties of α-synuclein misfolding, protein fibrillization was carried out in the presence of the lipid. We show that CA both accelerates the rate of α-synuclein fibrillization and produces species that harbour enhanced resistance to proteolysis. Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we then assessed the ability of these misfolded species to alter mitochondrial respiration in live non-transgenic SH-SY5Y neuroblastoma cells. Extensive analysis revealed that misfolded α-synuclein causes hyperactive mitochondrial respiration without causing any functional deficit. These data give strong support for the mitochondrion as a target for misfolded α-synuclein and reveal persistent, hyperactive respiration as a potential upstream pathogenic event associated with the synucleinopathies.This article has an associated First Person interview with the first author of the paper.

Highlights

Parkinson’s disease (PD) is a slowly progressing movement disorder clinically defined by the appearance of bradykinesia, rigidity and/or tremor
Because CA is virtually exclusively expressed in the inner mitochondrial membrane, we assessed the ability of these misfolded species to alter mitochondrial respiration in live non-transgenic SH-SY5Y neuroblastoma cells
Western immunoblot was employed to identify small conformations of misfolded α-synuclein that may be resolved by SDSPAGE and showed samples subjected to Protein Misfolding Cyclic Amplification (PMCA) contained a range of various sized species, with monomeric protein observed at 14 kDa and dimer, trimer and tetramer oligomers identified by their increase in molecular weight by intervals of the weight of the monomer (Figure 1B)

Summary

Introduction

Parkinson’s disease (PD) is a slowly progressing movement disorder clinically defined by the appearance of bradykinesia, rigidity and/or tremor. An unstructured protein that is localized to the synapse under normal physiological conditions, the misfolding of α-synuclein is strongly linked to the development of disease. This is evidenced by the finding that various point mutations in the gene encoding α-synuclein, SNCA (such as A53T), causes early onset familial disease (Appel-Cresswell et al, 2013; Kruger et al, 1998; Lesage et al, 2013; Polymeropoulos et al, 1997; Proukakis et al, 2013; Zarranz et al, 2004) as do gene duplications (Chartier-Harlin et al, 2004) and triplications (Singleton et al, 2003). There is a large body of data showing the ability of α-synuclein to adopt pathogenic properties upon misfolding (reviewed in: (Ugalde et al, 2016))

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