Fig. 5

MC1R activation alleviates αSyn oligomerization and neurotoxicity by activating Nrf2 in vitro. HEK293T cells were co-transfected with αSyn and MC1R or vector (GPRC5A-Tango). Non-transfected cells served as controls (NC): A Immunoblot and quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. B Nrf2 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. C Immunoblot and quantification of pCREB and CBP. One-way ANOVA followed by Tukey's post hoc test. D ChIP-qPCR analysis of pCREB binding in the Nrf2 promoter. Chromatins were immunoprecipitated using pCREB or IgG as negative control (Neg). Values were calculated by subtracting the cycle threshold (Ct) values for immunoprecipitated DNA from adjusted Ct of input DNA to get delta Ct, followed by raising 2 to the power of the delta Ct and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. E Nrf2 staining and quantification of nuclear and cytoplasmic Nrf2. Nuclei were stained with DAPI. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 10 µm. F Nrf2 target gene HO-1 mRNA levels. One-way ANOVA followed by Tukey's post hoc test. G and H Immunoblot and quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. HEK293T cells were transfected with αSyn, MC1R or vector control, and shNrf2 RNA or scRNA control: G Immunoblot and I and J quantification of MC1R and Nrf2. One-way ANOVA followed by Tukey's post hoc test. G Immunoblot and K quantification of αSyn species. Measurements were normalized by dividing values by the mean of vector control and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Primary cortical neurons were prepared from embryonic day 16–17 WT mice: L MAP2 and MC1R staining at DIV5. Scale bar, 10 µm. (M) Nrf2 mRNA levels in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-tail Student’s t test. N LDH release in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA and treated with PBS or NDP-MSH. Two-way ANOVA followed by Tukey’s post hoc test. O MAP2 staining and N quantification of MAP2-postive cells in primary neurons transduced with αSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 10 µm. Visual field area = 0.13508 mm². *P < 0.05, **P < 0.01, ***P < 0.001, ns = not statistically significant. n = 3 replicates. Experiments were repeated ≥ 3 times