Fig 2: MC1R activation alleviates aSyn oligomerization and neurotoxicity by activating Nrf2 in vitro. HEK293T cells were co-transfected with aSyn 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 aSyn 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 aSyn, 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 aSyn 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 aSyn AAV and lentiviral scRNA or shNrf2 RNA. Two-tail Student’s t test. N LDH release in primary neurons transduced with aSyn 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 aSyn 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

Fig 3: aSyn-induced dopaminergic neurotoxicity is exacerbated by MC1R disruption and reversed by human MC1R transgene. MC1Re/e and WT mice were injected unilaterally with human WT aSyn AAV into the SN. A Contralateral and ipsilateral turns induced by amphetamine 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. B Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. C TH staining and D stereological quantification of TH-positive and -negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 12 mice/group. Scale bar, 100 µm. MC1Re/eTg and MC1Re/e mice were injected unilaterally with human WT aSyn AAV into the SN. E Striatal dopamine content 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. F TH staining and G stereological quantification of TH-positive and negative cells in the SN 16 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 6–7 mice/group. Scale bar, 100 µm. H p-aSyn staining and I quantification of p-aSyn aggregates in the ipsilateral SNpc 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1Re/e mice and multiplying by 100. Two-tail Student’s t test. n = 4 mice/group. Scale bar, 50 µm. J Iba1 staining and morphological classification and K quantification of iba1-positive cells in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 30 µm. L Nrf2 and TH double-labeling and (M) quantification of nuclear and cytoplasmic Nrf2 in the SNpc 12 weeks post-AAV injection. Two-way ANOVA followed by Tukey’s post hoc test. n = 4 mice/group. Scale bar, 20 µm. N mRNA levels of Nrf2 target genes HO-1, NQO1, GCLC, and GCLM in the ipsilateral ventral midbrain 12 weeks post-AAV injection. Measurements were normalized by dividing values by the mean of the MC1Re/e mice. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 4: MC1R disruption amplifies microglia activation and alters Nrf2 response to aSyn overexpression in the nigrostriatal pathway. MC1Re/e and WT mice were injected unilaterally with human WT aSyn AAV into the SN and sacrificed 8 weeks later: A Iba1 staining and B morphological classification and quantification of iba1-positive cells in the SN. n = 4 mice/group. Two-way ANOVA followed by Tukey’s post hoc test. Scale bar, 30 µm. C IL-1a, IL-6, TNFa, and ICAM1 mRNA levels in ventral midbrain. Measurements were normalized by dividing values by the mean of the WT contralateral side. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. D Representative oxyblots for protein carbonyls and the corresponding Ponceau S staining in the ipsilateral ventral midbrain and E quantification of band density. Measurements were normalized by dividing values by the mean of WT control and multiplying by 100. Two-tail Student’s t-test. n = 3 mice/group. F Immunoblot for Nrf2 using ventral midbrain tissue and G quantification of Nrf2 band density in original values or H normalized to contralateral side by dividing values by the mean of the contralateral side and multiplying by 100. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group.I SN sections double-stained for Nrf2 and TH and J quantification of nuclear and cytoplasmic Nrf2. One-way ANOVA followed by Tukey’s post hoc test. n = 3 mice/group. Scale bar, 20 µm. K mRNA levels of Nrf2 target genes HO-1, NQO1, GCLM, and GCLC in the ipsilateral ventral midbrain. Measurements were normalized by dividing values by the mean of WT control. One-way ANOVA followed by Tukey’s post hoc test. n = 5 mice/group. *P < 0.05, **P < 0.01, ***P < 0.001

Fig 5: MC1R disruption exacerbates synucleinopathies in the nigrostriatal pathway in an aSyn AAV mouse model. MC1Re/e and WT mice were injected unilaterally with human WT aSyn AAV into the SN and sacrificed 8 weeks later: A Immunoblot of human aSyn species in Triton X-100-soluble and -insoluble SDS-soluble fractions in ipsilateral ventral midbrain and striatum and B quantification of aSyn monomers and oligomers. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. C p-aSyn staining and D quantification of p-aSyn aggregates in the ipsilateral SN. n = 3 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t-test. Scale bar, 50 µm. MC1Re/e and WT mice were injected unilaterally with BiFC human WT aSyn AAV into the SN and sacrificed 8 weeks later: E VenusYFP fluorescence before and after proteinase K treatment and F quantification of venusYFP fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. One-way ANOVA followed by Tukey's post hoc test. Scale bar, 50 µm. G Thioflavin-S staining and H quantification of thioflavin-S fluorescence density in the ipsilateral SN. n = 4 mice/group. Measurements were normalized by dividing values by the mean of WT and multiplying by 100. Two-tail Student’s t-test. Scale bar, 50 µm. *P < 0.05, **P < 0.01