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.