Figure 7. DAMP signaling via RAGE drives inflammatory activation in midbrain astrocytes.

(A) Schematic of experimental design for DAMP transfer experiments. Astrocytes were treated with NCM in the presence of FPS-ZM1 or control for 24 hours prior to qRT-PCR profiling. (B) qRT-PCR profiling of indicated genes in astrocytes treated for 24 hours with NCM derived from indicated conditions. (C and D) ELISA analysis of HMGB1 protein levels in supernatants of SH-SY5Y cells treated with MPP⁺ (C) or midbrain homogenates from WT mice 3 days post-MPTP treatment (D). n = 4–8 replicates per time point in C. (E) qRT-PCR profiling of indicated genes in human midbrain astrocytes treated for 24 hours with NCM derived from indicated conditions in the presence of neutralizing antibodies against HMGB1 (1 μg/mL) or an isotype control antibody. (F–H) qRT-PCR analysis of indicated genes in WT murine midbrain astrocytes (F) or midbrain astrocytes derived from indicated genotypes (G and H) 24 hours following treatment with recombinant HMGB1 (F and G) or S100β (H). (I) qRT-PCR analysis of indicated genes in ACSA2⁺ astrocytes sorted via MACS from brains of mice 24 hours following ICV administration of HMGB1 (200 ng). n = 3 cultures/group (B), 8 cultures/group for viability data and 2–4 cultures per group for HMGB1 expression (C), 5–6 mice/group (D), 6 cultures/group (E), 4 cultures/group (F and G), and 4 mice/group (H). Data are represented as mean values with scatterplots depicting individual biological replicate values, except in C, where data are represented as mean values ± SEM. Comparisons via 2-tailed t test (D) or 2-way ANOVA with Holm-Šídák multiple-comparison test (B and E–I). *P < 0.05, **P < 0.01, ***P < 0.001. A was created with Biorender.com.