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 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.

Keywords: Cardiolipin; Mitochondria; PMCA; Parkinson's disease; Synucleinopathy; α-Synuclein.

Fig. 1.

α-synuclein PMCA. (A) Workflow of α-synuclein PMCA: lyophilized recombinant wild-type protein was reconstituted in buffer to 90 µM and 60 µl was aliquoted into PCR tubes. Samples were exposed to PMCA in the presence of beads. Sonications were set to occur for 20 s every 29 min 40 s for a total process time of 72 h. This figure was created with Biorender.com. (B–D) The extent of fibrillization in protein exposed to PMCA and non-PMCA controls (−80°C) was assessed by (B) western immunoblot analysis using α-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118–123), (C) transmission electron microscopy (TEM) (representative images shown) and (D) thioflavin T (ThT) fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean±s.e.m. (n=3) and represent every experimental replicate performed.

Fig. 2.

Production of misfolded α-synuclein species in PBSN buffer produces comparable misfolded content to α-synuclein species generated in PCB. Lyophilized recombinant wild-type protein was reconstituted in PBSN (α-synuclein:PBSN) or PCB (α-synuclein:PCB) and subjected to PMCA for a total process time of 0, 24, 48 or 72 h. (A,B) Extent of fibrillization in samples was assessed using (A) western immunoblot analysis using α-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118–123) and (B) silver staining, where the asterisk indicates saturation of the protein signal. (C) Analytical ultracentrifugation sedimentation velocity analyses of non-PMCA and PMCA-generated α-synuclein. Continuous mass distribution [c(M)] distributions are shown as a function of molar mass. Inset shows zoomed-in image of values between 0 and 200 kDa. Best fits to experimental data yielded a root mean square deviation of 0.0055 and 0.0045, f/f₀ of 2.2 and 1.1, and Runs Test Z of 12 and 0.59 for non-PMCA and PMCA-generated α-synuclein, respectively. (D) Secondary structure of non-PMCA (0 h) or PMCA-generated (24 h or 72 h) α-synuclein:PBSN and α-synuclein:PCB was determined using circular dichroism (CD) spectroscopy (n=1, representative spectra). (E) TEM of fibrils produced in PBSN after 72 h PMCA (representative image). (F) α-Synuclein:PBSN exposed to PMCA for 72 h or non-PMCA (0 h) monomeric controls were further characterized by ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean±s.e.m. (n=3) and represent every experimental replicate performed.

Fig. 3.

PMCA-generated α-synuclein selectively associates with cardiolipin. A hydrophobic membrane strip dotted with 15 different lipids (long-chain >diC16 synthetic analogues) was incubated with PMCA-generated α-synuclein. (A) Binding of α-synuclein to lipids was assessed via western immunoblotting using α-synuclein-specific monoclonal antibody MJFR1 (amino acid specificity: 118–123). (B) Unlike PMCA-generated misfolded α-synuclein, monomeric α-synuclein (−80°C) did not show affinity to any lipid class, n=1. To determine whether cardiolipin (CA) modulates α-synuclein fibrillization, CA was dissolved in MeOH/PBSN (MeOH) and added to wild-type α-synuclein to a final concentration of 5.7 μM and 57 µM CA. MeOH/PBSN alone added to α-synuclein:PBSN served to control for the effect of the solvent. (C) Samples were subjected to PMCA for a total process time of 0, 6, 8, 10, 12, 24 and 72 h. Tubes containing PBSN only (no α-synuclein) with solvent or CA exposed to PMCA for 0 h and 72 h served to detect any inherent fluorescence caused by CA or the solvent. Extent of fibrillization was assessed by measuring ThT fluorescence, where the values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. RFU, relative fluorescent units. Data are presented as mean±s.e.m. (n=4 for all groups except for 0 h and 72 h, for which n=3) and represent every experimental replicate performed. To determine the effect of CA on α-synuclein fibrillization compared to control samples, a mixed-effects ANOVA with Dunnett's multiple comparisons test was employed. *P<0.05, **P<0.01. (D) ThT fluorescence was repeated in the presence of CA, MeOH or cholesterol (C), which showed no binding affinity to α-synuclein in panel A. ThT was measured after 8 h, which represented the earliest detectable rise in ThT by CA observed in panel C. The values obtained for each experimental replicate represented the average fluorescence of triplicate wells after subtraction of a blank well to account for background fluorescence. Data are presented as mean±s.e.m. (n=3) and represent every experimental replicate performed. (E) The biochemical properties of α-synuclein species formed after 72 h PMCA (PMCA) or not (−80°C) was assessed by its resistance to proteolysis following digestion in proteinase K (PK) at a final concentration of 0, 2 or 10 µg/ml. PK-resistant species were detected by immunoblotting using MJFR1. Areas of differential PK resistance between PMCA-generated species are identified by red arrows. n=1. (F) Ultracentrifugation was performed on α-synuclein in the presence of 5.7 µM and 57 µM CA or buffer control to determine the insoluble load. n=1.

Fig. 4.

PMCA-generated misfolded α-synuclein causes mitochondrial respiration to become hyperactive in SH-SY5Y cells. (A–F) SH-SY5Y cells were incubated with PMCA-generated α-synuclein (PMCA), monomeric α-synuclein (−80°C) or buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate and mitochondria respiration was measured in adhered cells using the Seahorse XFe24 Analyzer. This was performed by detecting changes in oxygen (referred to as the oxygen consumption rate; OCR) following the addition of pharmacological agents: oligomycin, carbonyl cyanide m-chlorophenyl hydrazone (CCCP), rotenone and antimycin A. In doing so, the following parameters were measured: (A) basal OCR, (B) ATP synthesis, (C) max OCR, (D) complex I activity, (E) complex II activity and (F) non-mitochondrial respiration. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean±s.e.m. (n=16, 13, 18 for PMCA, −80°C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey's multiple comparisons test with a statistical criterion of 0.01. **P<0.01, ***P<0.001, ****P<0.0001.

Fig. 5.

PMCA-generated misfolded α-synuclein does not cause functional deficits in mitochondria of SH-SY5Y cells. SH-SY5Y cells were incubated with PMCA-generated α-synuclein (PMCA), monomeric α-synuclein (−80°C) or either buffer alone or left untreated, which collectively represented the control group (Control). Medium-containing cells were plated into each of four wells per sample of a Seahorse XFe24 plate pre-coated with Matrigel. The Seahorse XFe24 Analyzer measured mitochondria respiration by detecting changes in oxygen (referred to as the OCR) following the addition of pharmacological agents: oligomycin, CCCP, rotenone and antimycin A. The functioning of mitochondria was determined as parameters expressed as % of max or basal OCR (depending on the functional readout). Spare capacity was measured as the difference between max and basal OCR, and proton leak was measured as the difference between basal and ATP synthesis and non-respiratory. The average of five wells was taken for each sample per experimental replicate. Data are presented as mean±s.e.m. (n=16, n=13, n=18 for groups PMCA, −80°C and Control, respectively) and represent every experimental replicate performed. Statistical significance was examined by ANOVA and Tukey's multiple comparisons test with a statistical criterion of 0.01. No statistical significance was found for all experimental comparisons.