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

Abstract

The 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 nontransgenic 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.

An intact membrane is essential for small extracellular vesicle‐induced modulation of α‐synuclein fibrillization

“…Complementary to the ThT data, the generation of mature αsyn fibrils using PMCA was further confirmed using TEM. Using TEM, protein exposed to PMCA was found to form rod‐like fibrils (Figure 1b) consistent with the morphology of fibrils produced using PMCA reported by others (Herva et al., 2014; Ugalde et al., 2020). No fibrils were observed in the non‐PMCA, −80°C sample (Figure 1b).…”

“…The ability of PMCA to induce misfolding in αsyn was first confirmed following a previously published method that involved exposing human recombinant protein (90 μM) to PMCA for a total process time of 72 h (Ugalde et al., 2020). Misfolded αsyn protein was characterized using standard techniques routinely used to detect misfolded protein: Thioflavin T (ThT) fluorescence, western immunoblot (protease resistance and soluble/insoluble fractionation) and TEM.…”

“…Recombinant human αsyn was purchased from Monash Protein Production facility, Monash University Clayton VIC and prepared as described elsewhere (Ugalde et al., 2020). Briefly, protein was produced using wild‐type αsyn:pRSET B transformed into BL21 (DE3) Gold cells and amplified in luria broth (10 g/l bacterial tryptone (Merck, Kilsyth, VIC, Australia), 5 g/l yeast extract, 5 g/l NaCl, pH 7.5) containing ampicillin (100 μg/ml, Astral Scientific, Gymea, NSW, Australia).…”

“…We additionally examined the effects of sEVs on PMCA-mediated αsyn fibrillization in vitro using sedimentation velocity analytical centrifugation as described previously (Ugalde et al, 2020). To provide a robust assessment of composition differences between samples, raw absorbance-detected sedimentation profiles ( Figure S1A-C) were analysed using the model-independent enhanced van Holde-Weischet (vHW) method (Demeler & van Holde 2004).…”

“…Here, αsyn misfolding was studied using Protein Misfolding Cyclic Amplification (PMCA); an assay that was originally developed to study the misfolding of the prion protein, PrP C , into its misfolded isoform, PrP Sc (Saborio, Permanne, & Soto, 2001) that is associated with another NDP, the prion disorders. The PMCA is an attractive tool to study αsyn misfolding because it has been shown to be capable of rapidly inducing misfolding in αsyn (Herva et al., 2014; Ugalde et al., 2020) and, given that a range of misfolded conformations present in human synucleinopathy disorders including oligomers and protofibrils (Garcia‐Esparcia et al., 2015; Kramer & Schulz‐Schaeffer 2007), the PMCA has advantages over traditional fibrillization techniques due to its ability to produce a heterogenous mix of various sized species (Herva et al., 2014; Ugalde et al., 2020). Using PMCA, we confirm sEVs alter αsyn misfolding and thoroughly characterise these changes using a range of assays: Thioflavin T (ThT), transmission electron microscopy (TEM), western immunoblot coupled with protease resistance assays and soluble/insoluble fractionation and analytical centrifugation.…”