Fig 2: Generation of mDA neurons from hiPSC lines from patients with SNCA mutations display early a-synuclein aggregation and calcium dysregulation.a Representative ICC images of control, A53T, and SNCA x3 mDA neurons showing high TH expression. Scale bar = 50 µm. b Quantification of % MAP2 and TH-positive cells after day 41 of differentiation (ns P > 0.05, two-way ANOVA). c Super-resolved images from control, A53T, SNCA x3 day 27 mDA neurons. Left panel shows phalloidin and aptamer. Middle panel shows only super-resolved aptamer binding events. Last panel shows a magnified version of aggregates detected by DBSCAN. Left panel scale bar = 2 µm. Middle panel scale bar = 2 µm. Right panel = 1 µm. d Quantification showing the number of aggregates per cell in control, A53T and SNCA x3 mDA neurons (ns P > 0.05, *P < 0.05, **P < 0.005, one-way ANOVA). e Quantification showing the length of all aggregates in control, A53T and SNCA x3 mDA neurons represented in a violin plot (****P < 0.0001, one-way ANOVA). f Representative time series Ca2+ images in response to KCl (scale bar = 50 µm). g Quantification of the number of cells with KCl-induced calcium signal at day 34 of differentiation (ns P > 0.05, one-way ANOVA). h Representative images of neuronal marker expression. i Representative single-cell trace in patient mDA neurons. j Quantification of the normalised rate of recovery of Fluo-4 after stimulation with KCl (***P = 0.0002, one-way ANOVA). k Representative traces showing the Fura-2 ratio in response to 50 mM KCl in day 41 control neurons, A53T neurons, and SNCA x3 neurons. l Quantification of the basal calcium Fura-2 ratio ([Ca2+]c) before KCl stimulation (***P < 0.0005, ****P < 0.0001, one-way ANOVA). m Quantification of the rate of calcium ([Ca2+]c) recovery in response to KCl (****P < 0.0001, one-way ANOVA). All values are plotted as ±s.e.m. All N numbers for each experiment can be found in Supplementary Table 5.

Fig 3: Cellular dysfunction and cell death arise later in SNCA PD mDA neurons.a Representative live-cell imaging of mitochondrial fluorescence using the lipophilic cationic dye TMRM at day 48 of differentiation. Scale bar = 10 µm. b Quantification of the normalised fluorescence intensity of TMRM (**P < 0.005, one-way ANOVA). c Trace showing the ratiometric measurement of superoxide generation using dihydroethidium (HEt) at day 48 of differentiation. d Quantification of the rate of superoxide generation based on HEt ratiometric fluorescence (ns P > 0.05, ***P < 0.0005, one-way ANOVA). e Representative live-cell imaging of endogenous glutathione using the fluorescent reporter MCB at day 48 of differentiation. Scale bar = 20 µm. f Quantification of the endogenous level of glutathione based on MCB fluorescence (*P < 0.05, ***P = 0.0009, one-way ANOVA). g Representative live-cell imaging of lysosomes and nuclear marker Hoechst 33342 at day 48 of differentiation (scale bar = 5 µm). h Quantification of the normalised average lysosomal area/size (***P = 0.0002, ****P < 0.0001, one-way ANOVA). i Histogram plot showing the percentage of total lysosomes in each set area bin (0–10 µm2). j Representative time series snapshots of TMRM (red) and Fluo-4 (green) in day 62 old neurons showing the response to KCl and FCCP. The arrow in the control cell highlights polarised mitochondria and calcium response to KCl and FCCP. Arrowhead in the SNCA x3 cell highlights KCL-induced TMRM intensity decrease. Scale bar = 10 µm. k Representative single-cell trace showing TMRM intensity in response to KCl and FCCP in control and SNCA x3 day 62 old neurons. l Quantification showing the decrease in TMRM intensity after KCl stimulation in control, A53T and SNCA x3 neurons (*P < 0.05, one-way ANOVA). m Instant Structured illumination microscopy (iSIM) images of control, A53T, and SNCA x3 day 55 neurons probed for mitochondrial marker Tomm20, and the autophagosome marker LC3B. Scale bar = 5 µm). n Quantification of the number of Tomm20-LC3B colocalizations per cell (*P < 0.05, Welch’s t test). o Quantification of the number of LC3B puncta per cell (*P < 0.05, Welch’s t test). p Live-cell images depicting dead cells in mDA neurons at >day 48 of differentiation using the fluorescent dye SYTOX green (scale bar = 20 µm). q Quantification of the percentage of dead cells (**P < 0.005, one-way ANOVA). All values are plotted as ±s.e.m. All N numbers for each experiment can be found in Supplementary Table 5.

Fig 4: Functional consequences of SNCA mutations in human mDA neurons.a The normalised basal mitochondrial footprint measurement plotted out in control, A53T, and SNCA x3 >day 62 old neurons (*P < 0.05, **P < 0.005, one-way ANOVA). b The normalised basal number of lysosomes per cell in controls, A53T and SNCA x3 >day 62 old neurons (*P < 0.05, **P < 0.005, one-way ANOVA). c The normalised percentage of cell death in control, A53T, and SNCA x3 >day 62 old neurons (**P < 0.005, ****P < 0.0001 one-way ANOVA). d Representative bright-field images showing >day 70 neurons patched for electrophysiological recordings. Scale bar = 20 µm. e Quantification of electrophysiological recordings showing the resting membrane potential in control and A53T neurons, and control and SNCA x3 neurons (**P = 0.0068, ***P = 0.0008, Welch’s t test). f Quantification of electrophysiological recordings showing the input resistance in control and A53T neurons, and control and SNCA x3 neurons (*P < 0.05, Welch’s t test). g Quantification of the threshold for AP generation in control and A53T neurons, and control and SNCA x3 neurons (*P < 0.05, ****P < 0.0001, Welch’s t test). h Quantification of the AP amplitude in control and A53T neurons, and control and SNCA x3 neurons (**P < 0.005, ****P < 0.0001, Welch’s t test). i Quantification of the AP repolarisation rate in control and A53T neurons, and control and SNCA x3 neurons (*P < 0.05, **P < 0.005, Welch’s t test). All values are plotted as ±s.e.m. All N numbers for each experiment can be found in Supplementary Table 5.