Fig 1: TNF-α uncovers genotype-specific α-syn accumulation and synaptic dysfunction in SNCA 3x ENLs(A) Ligand-Receptor pair analysis of the scRNAseq data prior to subclustering. This plot depicts pairs that show both a large mean change as well as a large variance in the population (purple). Cells that don’t make the variance cut-off of 0.3 are shown in orange. Cells that don’t make the mean cut-off of 0.3 are shown in grey. (B) Experimental paradigm showing the stimulation of iPSC-ENLs with different cytokines. Generated using BioRender. (C) ELISA of total α-syn (Abcam, cat:ab260052) measured intracellularly in iPSC-ENLs, normalized by total protein. n=3 biological replicates per group, mean ± SEM, *p<0.05 by two-way ANOVA with Sidak post-hoc. Basal refers to cells treated with vehicle used to dilute the cytokines (DPBS+0.1% BSA). (D) Flow cytometry analysis of the percentage of live cells using Live/Dead staining. n=3 biological replicates per group, mean ± SEM. Basal refers to cells treated with vehicle used to dilute the TNF-α (DPBS+0.1% BSA). (E) ELISA of aggregated α-syn (Biolegend, cat:448807) quantified intracellularly in iPSC-ENLs, normalized by total protein. n=3 biological replicates per group, mean ± SEM, *p<0.05 by two-way ANOVA with Sidak post-hoc. Basal refers to cells treated with vehicle used to dilute the TNF-α (DPBS+0.1% BSA). (F) Human cytokine array quantification of the supernatants of iPSC-ENLs previously stimulated for 24h with 100 ng/ml TNF-α. Data represent the mean of n=3 biological replicates per group. (G) Experimental paradigm for the MEA experiments. Generated using BioRender. (H and I) Quantification of the number of spikes (H) and number of active electrodes (I) generated from the MEA data, n=wells of a CytoView MEA 48-well plate, representative of 3 biological replicates per group, mean ± SEM, *p<0.05, **p<0.01 by two-way ANOVA with Sidak post-hoc.