Article Figures & Data
Figures
- Figure 1.
Deletion of ghrelin negates the protective effect of CR. A, CR significantly elevates plasma acylated ghrelin. B, C, Overall reduction in plasma insulin levels in response to CR in both genotypes. D, Representative images showing MPTP-induced TH cell loss in the SN and microglial (IBA) activation. E, F, Stereological quantification of TH neurons in the SN showing CR has no significant effect in MPTP-treated ghrelin KO mice (F) but is protective in ghrelin WT mice (E). G, H, Stereological quantification of IBA1 microglia in the SN shows elevated cell number following MPTP treatment but no effect of genotype. I, J, Stereological quantification of SN GFAP shows that MPTP administration increased GFAP cell number to a lesser extent in ghrelin WT compared with ghrelin KO mice. K, Representative images showing MPTP-induced astrocyte (GFAP) activation in the SN (TH, green; GFAP, red). Data are represented as mean ± SEM (n = 6–10, two-way ANOVA, p < 0.05). A, Significant compared with saline ad libitum controls. b, Significant compared with MPTP ad libitum controls. Scale bar, 50 μm.
- Figure 2.
CR reduces small volume TH cell loss and enhances dopamine turnover in ghrelin WT but not KO mice. A, Overall cell volume for ghrelin WT mice showed a significant (p < 0.05) effect of MPTP administration but no effect of genotype or diet. B, Ghrelin KO mice showed no overall effect of diet, treatment, or genotype. The red dotted line represents the average cell volume of ghrelin WT MPTP-treated mice. When the cells were separated based on number and volume distribution as shown in C and D, the effect of CR is apparent. C, Ghrelin WT have a significant (p < 0.05) effect between ad libitum and CR cell volume in smaller (1000–2000 μm3) cells. There was no significant difference in the ghrelin KO mice (D). E, H, CR attenuates striatal DA loss in ghrelin WT but not ghrelin KO mice after MPTP administration. F, I, MPTP reduced DOPAC with no effect of genotype. G, J, CR reduced the elevation of the DOPAC/DA ratio in MPTP-treated mice compared with ad libitum, in ghrelin WT but not ghrelin KO mice. a, Significant compared with saline ad libitum controls. b, Significant compared with MPTP ad libitum controls.*p < 0.05, **p < 0.01. Data are represented as mean ± SEM (n = 6–10, two-way ANOVA, p < 0.05).
- Figure 3.
The protective effect of CR is concomitant with striatal dopamine and elevated pAMPK, an effect not observed in ghrelin KO mice. A, B, Representative Western blot images of MPTP-induced reduction in TH levels in the SN and striatum. C, D, Quantification of TH levels in ghrelin WT and KO mice showed that MPTP significantly (p < 0.05) reduced TH expression in the SN. E, F, Quantification of TH levels in the striatum revealed that MPTP significantly (p < 0.05) reduced TH expression, this effect was rescued in CR ghrelin WT mice but not in KO mice. G, H, Representative Western blot images of pAMPK, AMPK, pACC, and ACC levels in the SN and striatum after either ad libitum or CR paradigms followed by MPTP or saline treatment. I, M, Quantification of pAMPK/AMPK and pACC/ACC levels in the SN reveals no effect in ghrelin WT mice, however, in KO mice there was a significant (p < 0.05) reduction between MPTP ad libitum and MPTP CR groups (J, N), showing that CR KO mice could not adapt appropriately to MPTP-induced cell degeneration. K, L, MPTP and CR individually increased striatal pAMPK/AMPK in ghrelin WT mice but not in ghrelin KO mice, as no change from baseline with either MPTP or CR was observed. O, P, MPTP-induced an increase in striatal pACC/ACC in ghrelin WT but not ghrelin KO mice, mimicking the effects seen with pAMPK/AMPK. Q, R, Representative Western blots for LC3 I and LC3 II in the SN and striatum of ghrelin WT and KO mice. S, LC3 II in the SN is significantly reduced in ghrelin WT mice after MPTP treatment; however, this was not observed in ghrelin KO mice (T). U, V, There was no effect of CR on LC3 II in the striatum from ghrelin WT and KO mice. However, there was a significant main effect of MPTP to increase LC3 II in WT but not KO mice. a, Significant compared with saline controls. b, Significant compared with a low dose of ghrelin. *p < 0.05, **p < 0.01. Data are represented as mean ± SEM (n = 5–7, one-way ANOVA, p < 0.05).
- Figure 4.
Exogenous ghrelin elevates TH and AMPK activation. A, Representative Western blot images of cultured dopaminergic neurons shows an increase in pAMPK levels in response to acyl ghrelin, JMV2894 (ghrelin agonist) or oligomycin treatment. Quantification of pAMPK/AMPK levels reveals a significant increase in response to acyl ghrelin (B), JMV2894 (C), and oligomycin (D) treatment. E, F, Representative Western blot images of pAMPK, AMPK, pACC, and ACC levels in the SN and striatum. G, H, Quantification of the pAMPK/AMPK and pACC/ACC in the SN (G) in response to a high dose of ghrelin reveals a significant elevation in response to the high dose of ghrelin. I, J, Quantification of pAMPK/AMPK and pACC/ACC in the striatum reveals no change between saline and ghrelin doses. K, Representative Western blot images of TH levels in the SN and striatum. Quantification of TH levels in the SN (L) and striatum (M) show that intraperitoneal ghrelin significantly increases TH expression in response to a high dose of ghrelin. Representative Western blot images of LC3 II expression in the SN and striatum (N). Quantification of LC3 II revealed high dose caused a significant increase in the SN (O) but not the striatum (P). a, Significant compared with saline/saline controls. b, Significant compared with saline/MPTP controls. Data are represented as mean ± SEM (n = 6–8, two-way ANOVA, p < 0.05).
- Figure 5.
Ghrelin activates AMPK to elicit neuroprotection in an AMPK-dependent manner. A, DAT CRE mice crossed with the tdTomato line shows a >90% colocalization (B) between TH (green) and tdTomato (red) neurons. Scale bar, 100 μm. C, Representative tiled image showing TH (green) and tdTomato (red) where each tile represents a 20× image. D, tdTomato labeled TH neurons were sorted via FACs to show the selective deletion of AMPKβ1 and AMPKβ2 in AMPK WT but not AMPK KO mice. Product size for AMPKβ1 = 386 bp, AMPKβ2 = 395 bp. The ghrelin receptor (GHSR) is unaffected by deletion of AMPKβ1 and AMPKβ2 in SN TH neurons. E, Representative images showing TH neurons from AMPK WT and KO mice after chronic ghrelin treatment. F, G, Stereological quantification of TH neurons from AMPK WT (F) and KO (G) mice shows a protective effect of ghrelin treatment in WT but not KO mice. H, Stereological quantification of IBA1 microglia in the SN shows that ghrelin suppresses IBA1 cells relative to saline controls following MPTP treatment; however, this is not observed in AMPK KO mice (I). J, K, Stereological quantification of GFAP in the SN shows that ghrelin attenuates the MPTP-induced increase in GFAP cell numbers in AMPK WT (J) but not AMPK KO (K) mice. L, Representative images showing MPTP-induced astrocyte (GFAP) activation in the SN (TH, green; GFAP, red). a, Significant compared with saline/saline controls. b, Significant compared with saline/MPTP controls. Data are represented as mean ± SEM (n = 6–8, two-way ANOVA, p < 0.05). Scale bar, 100 μm.
- Figure 6.
Chronic ghrelin injection enhances dopamine turnover and behavioral outcomes in AMPK WT but not KO mice. A, B, Overall cell volume showing no reduction in response to genotype or treatment. When cells were separated based on number and volume distribution as shown in C and D, there was no overall effect of genotype or treatment. HPLC data show that ghrelin significantly attenuates the MPTP-induced decrease in striatal dopamine concentration in AMPK WT but not AMPK KO mice (E, H). MPTP reduced DOPAC with no effect of genotype (F, I). Ghrelin treatment significantly attenuates the MPTP-induced increase in the DOPAC/dopamine ration in AMPK WT but not AMPK KO mice (G, J). K–N, Behavioral analysis showing latency to fall on an accelerating rotarod. K, L, No difference in latency to fall in mice not exposed to MPTP. In mice given MPTP, latency to fall is not effected by genotype in mice pretreated with saline (M). However, in mice pretreated with ghrelin there is a significant protective effect in AMPK WT but not KO as evidence by increased latency to fall (N). a, Significant compared with saline/saline controls. b, Significant compared with saline/MPTP controls. Data are represented as mean ± SEM (n = 6–12, two-way ANOVA, p < 0.05).
- Figure 7.
Chronic ghrelin injections increase nigrostriatal TH expression and AMPK activation in an AMPK-dependent manner. A, B, representative Western blot images of the SN (A) and striatum (B) showing TH levels. In both the SN and the striatum there is a significant protective effect of ghrelin administration on TH levels in AMPK WT mice (C, E) that is absent in AMPK KO mice (D, F). G, H, Representative Western blot images showing pAMPK, AMPK, pACC, and ACC levels in the SN (G) and striatum (H). There was no significant change in the pAMPK/AMPK (I, J) or pACC/ACC (M, N) ratio in the SN of AMPK WT or AMPK KO mice in response to MPTP or ghrelin. MPTP-induced an increase in the pAMPK/AMPK and pACC/ACC ratio in AMPK WT mice (K, O) but not AMPK KO mice (L, P). In the striatum, MPTP induced an increase in the pAMPK/AMPK and pACC/ACC ratio AMPK WT mice (K, O) but not AMPK KO mice (L, P). Q, R, Representative Western blot images of LC3 II expression in the SN (Q) and striatum (R). There was no significant effect of MPTP or ghrelin administration on LC3-II levels in the SN (S, T) or striatum (U, V). a, Significant compared with saline/saline controls. b, Significant compared with saline/MPTP controls. Data are represented as mean ± SEM (n = 6–8, two-way ANOVA, p < 0.05).
