Fig 2: Effects of PACAP or VIP treatment on BV2 microglia inflammatory profile after exposure to rotenone. Real-time qPCR and Western blot analyses of microglial activation markers, pro- and anti-inflammatory cytokines in cells exposed to rotenone alone (0.1 µM), PACAP alone (0.001 µM), VIP alone (0.001 µM) or in combination for 24 h. Gene expression changes of (a) IL-6, (b) NOS2, (c) CD11b, (d) MMP-9, (e) IL-10 and (f) Arg1 in response to treatments are shown. Relative changes in mRNA levels were determined using the ?CT method and normalised to the ribosomal protein subunit S18, used as the housekeeping gene. (g–i) Western blot analyses and densitometry of (g) Iba1 and (i) Arg1 protein expression. Quantifications were performed using the ImageJ software, and band densities were normalised to GAPDH, the loading control. Data represent the mean of 3–6 biological replicates for each group. Results are expressed as mean ± SEM. *P < 0.05, **P < 0.01 or ****P < 0.0001 compared to untreated controls. ##P < 0.01 or ####P < 0.0001 compared to rotenone treated cells as determined by one-way ANOVA followed by Sidak’s post hoc test. PACAP, pituitary adenylate cyclase-activating polypeptide; VIP, vasoactive intestinal peptide; s18, ribosomal protein s18; GAPDH, Glyceraldehyde 3-phosphate dehydrogenase; kDa, kilodalton; n.s., not significant; Ctl, untreated controls. IL-6, interleukin-6; NOS2, nitric oxide synthase 2; MMP-9, matrix metallopeptidase 9; IL-10, interleukin-10; Arg1, arginase-1; Iba1, ionised calcium binding adaptor molecule 1

Fig 3: Buspirone prevents PACAP and VIP neuropeptide dysregulations in the brain of rotenone-treated mice. Real-time qPCR analyses of PACAP and VIP mRNA expression in the midbrain (A,B), striatum (F,G), prefrontal cortex (K,L), amygdala (P,Q), and hippocampus (U,V) following the administration of rotenone (10 mg/kg) and/or buspirone (1, 3, or 10 mg) daily for 21 days. Fold-changes were calculated using the ??Ct method after normalization to s18 (ribosomal protein s18 gene), the housekeeping gene. Each data point represents the mean value from n = 5–8 mice per each group. Representative Western blots and densitometry of PACAP and VIP protein expression in the midbrain (C–E), striatum (H–J), prefrontal cortex (M–O), amygdala (R–T), and hippocampus (W–Y). Protein expression was normalized to GAPDH, the loading control. Western blots are cropped and removed lanes depicting drug-treatment controls are included in Supplementary Figure S6. Densitometric results are expressed as means ± S.E.M from n = 5–8 mice per each group. * p < 0.05, ** p < 0.01, *** p < 0.001, or **** p < 0.0001, compared to vehicle-treated controls; or # p < 0.05, ## p < 0.01, ### p < 0.001, or #### p < 0.0001, compared to rotenone (10 mg/kg)-treated mice, as determined by an ANOVA followed by Sidak’s post-hoc test. PACAP, pituitary adenylate cyclase-activating peptide; VIP, vasoactive intestinal peptide; s18, ribosomal protein s18 gene; GAPDH, glyceraldehyde 3-phosphate dehydrogenase; kDa, Kilodalton.

Fig 4: Dose–response effects of PACAP on rotenone-treated microglia. PACAP titration experiments showing dose-dependent changes in morphology, cell viability and nitric oxide release. (a) BV2 cells were either treated with rotenone alone (0.1 µM) or co-treated with increasing concentrations of PACAP (0.0001–1 µM) for 24 h and representative photomicrographs (using the embossing filter settings) were taken (scale bar = 60 µm). (b) Morphological assessment was performed by assigning cells into three gross categories (normal, activated or apoptotic) and by calculating the percentage of cells in each category. For these analyses, at least eight representative micrographs per condition were appraised (n = 8). (c) Cell viability and (d) NO release were assessed under the same experimental conditions. Data reported as mean ± SEM from at least three independent experiments run using eight biological replicates per group (n = 24). ****P < 0.0001 vs untreated controls or ####P < 0.0001 vs rotenone-treated cells, as determined by one-way ANOVA followed by Sidak’s post hoc test. PACAP, pituitary adenylate cyclase-activating polypeptide; NO, nitric oxide

Fig 5: Expression of PACAP, VIP and related receptors, PAC1, VPAC1 and VPAC2 in the hippocampus of 2- and 7-month-old WT and NZBWF1 mice. Depicted are real-time qPCR results of (A) PACAP, (B) VIP, (C) PAC1, (D) VPAC1 and (E) VPAC2 gene expression. Relative changes in mRNA levels were determined using the ?CT method and were normalised to the ribosomal subunit S18 which was used as a housekeeping gene. (F,I) Representative Western blots and related densitometry of (G) PACAP, (H) VIP, (J) PAC1, (K) VPAC1 and (L) VPAC2 protein expression. Densitometric analyses were performed using the ImageJ software and were normalised to GAPDH bands, here used as loading control. Data represent the mean of (n = 5) 2 mo WT, (n = 7) 2 mo NZBWF1, (n = 4) 7 mo WT and (n = 9) 7 mo NZBWF1 mice. Results are expressed as mean ± S.D. * p = 0.05 or ** p = 0.01 vs. WT at the corresponding age, as determined by two-way ANOVA followed by Tukey post hoc test. PACAP = pituitary adenylate cyclase-activating peptide; VIP = vasoactive intestinal peptide; PAC1 = PACAP receptor 1; VPAC1 = VIP receptor 1; VPAC2 = VIP receptor 2.