Fig. 7: RIPK signaling is required for functional markers of astrocyte activation downstream of α-synuclein PFFs.

a Primary human midbrain astrocytes were treated with RIPK inhibitors and/or PFFs, as indicated. After 24 h, an astrocyte conditioned medium (ACM) was applied (1:1) to differentiated SH-SY5Y cultures for 24 h followed by endpoint analyses. b Viability of SH-SY5Y cells following treatment with ACM derived from astrocyte cultures treated with the indicated inhibitors was measured via ATP-luciferase assay (Cell Titer Glo). c, d Primary human midbrain astrocyte cultures were treated with PFFs or PBS control solution. Cultures were pretreated (30 min) with inhibitors of RIPK3 (GSK872) or NF-κB (JSH-23) signaling prior to the addition of PFFs. Following this, CSFE-labeled neuronal debris was added for 24 h. Uptake was measured via flow cytometric analysis of CSFE signal in astrocytes. Data were represented as histograms (c) and quantified using mean geometric fluorescence intensity (GMFI) values (d). e Secondary analysis of microarray profiling of human postmortem substantia nigra samples from patients with PD or healthy controls (GSE26927). Z-scores and corrected p values were calculated for necroptosis pathway components indicated and displayed via heatmap. ns not significant, *p < 0.05, **p < 0.01, ***p < 0.001. Bars represent group means. n = 6 independent replicates in (b). n = 3 independent replicates in (c, d).