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. 2017 Feb 1;10(2):127-139.
doi: 10.1242/dmm.027185. Epub 2016 Dec 22.

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

Gabor Maasz  1   2 , Zita Zrinyi  1 , Dora Reglodi  3 , Dora Petrovics  2 , Adam Rivnyak  3 , Tibor Kiss  4 , Adel Jungling  3 , Andrea Tamas  3 , Zsolt Pirger  5
Affiliations

Pituitary adenylate cyclase-activating polypeptide (PACAP) has a neuroprotective function in dopamine-based neurodegeneration in rat and snail parkinsonian models

Gabor Maasz et al. Dis Model Mech. .

Abstract

Pituitary adenylate cyclase-activating polypeptide (PACAP) rescues dopaminergic neurons from neurodegeneration and improves motor changes induced by 6-hydroxy-dopamine (6-OHDA) in rat parkinsonian models. Recently, we investigated the molecular background of the neuroprotective effect of PACAP in dopamine (DA)-based neurodegeneration using rotenone-induced snail and 6-OHDA-induced rat models of Parkinson's disease. Behavioural activity, monoamine (DA and serotonin), metabolic enzyme (S-COMT, MB-COMT and MAO-B) and PARK7 protein concentrations were measured before and after PACAP treatment in both models. Locomotion and feeding activity were decreased in rotenone-treated snails, which corresponded well to findings obtained in 6-OHDA-induced rat experiments. PACAP was able to prevent the behavioural malfunctions caused by the toxins. Monoamine levels decreased in both models and the decreased DA level induced by toxins was attenuated by ∼50% in the PACAP-treated animals. In contrast, PACAP had no effect on the decreased serotonin (5HT) levels. S-COMT metabolic enzyme was also reduced but a protective effect of PACAP was not observed in either of the models. Following toxin treatment, a significant increase in MB-COMT was observed in both models and was restored to normal levels by PACAP. A decrease in PARK7 was also observed in both toxin-induced models; however, PACAP had a beneficial effect only on 6-OHDA-treated animals. The neuroprotective effect of PACAP in different animal models of Parkinson's disease is thus well correlated with neurotransmitter, enzyme and protein levels. The models successfully mimic several, but not all etiological properties of the disease, allowing us to study the mechanisms of neurodegeneration as well as testing new drugs. The rotenone and 6-OHDA rat and snail in vivo parkinsonian models offer an alternative method for investigation of the molecular mechanisms of neuroprotective agents, including PACAP.

Keywords: 6-OHDA; DJ-1; Dopamine; PACAP; PARK7; PD models; Rotenone.

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Conflict of interest statement

Competing interests

The authors declare no competing or financial interests.

Figures

Fig. 1.
Fig. 1.
HPLC-MS analysis of DA in SN of the rat. (A) Extracted ion chromatogram of DA at 1.95 min retention time in single ion monitoring (SIM) mode. (B) MS spectrum of DA identified at 154.09 m/z parent ion protonated form [M+H]+. (C) Extracted ion chromatogram of DA fragment at 1.94 min retention time in MS/MS mode. (D) MS/MS spectrum of DA identified at 137.06 m/z fragment ion protonated form [F+H]+ (219 fmol DA was injected). The calculated DA content was 3.87 µg/g SN tissue.
Fig. 2.
Fig. 2.
HPLC-MS analysis of 5HT in SN of the rat. (A) Extracted ion chromatogram of 5HT at 3.89 min retention time in single ion monitoring (SIM) mode. (B) MS spectrum of 5HT identified at 177.10 m/z parent ion protonated form [M+H]+. (C) Extracted ion chromatogram of 5HT fragment at 3.88 min retention time in MS/MS mode. (D) MS/MS spectrum of 5HT identified at 160.08 m/z fragment ion protonated form [F+H]+ (143 fmol DA was injected). The calculated 5HT content was 3.35 µg/g SN tissue.
Fig. 3.
Fig. 3.
Survival, locomotion and feeding of snails after treatment with PACAP, rotenone or rotenone+PACAP. (A) Survival in controls and after treatment with PACAP, rotenone or rotenone+PACAP for 12 days. The total number of animals was n=40 from 4 independent experiments in each group. (B) Locomotor activity of snails of control (n=8), rotenone (n=8) and rotenone+PACAP (n=8) groups. Mean distances covered during the 3 min test period by snails are shown. One-way ANOVA: F(2)=13.99, P<0.001; post hoc test between control and rotenone groups (P<0.001), control and rotenone+PACAP (P<0.01); rotenone and rotenone+PACAP (P<0.05). (C) Feeding activity in snails of control (n=8), rotenone (n=8) and rotenone+PACAP (n=8) groups. Mean number of bites counted during 2 min time window are shown. One-way ANOVA: F(2)=32.66, P<0.001; post hoc test between control and rotenone (P<0.001), control and rotenone+PACAP (P<0.01); rotenone and rotenone+PACAP groups (P<0.001) (C). *P<0.05, **P<0.01, ***P<0.001 between control and treated groups; #P<0.05, ##P<0.01, ###P<0.001 between rotenone and rotenone+PACAP groups.
Fig. 4.
Fig. 4.
Monoamine concentrations in snails treated with rotenone and rotenone+PACAP. (A) Monoamine concentration in snail control group in µg/g tissue. (B) DA content as a percentage of control (n=30) in rotenone (n=28) and rotenone+PACAP (n=30) groups. One-way ANOVA, F(2,86)=39.03, P<0.001; post hoc test between control and rotenone groups (P<0.001), control and rotenone+PACAP groups (P<0.001); rotenone and rotenone+PACAP groups (P<0.05). (C) 5HT content as a percentage of control (n=30) in rotenone (n=28) and rotenone+PACAP (n=30) groups. One-way ANOVA, F(2,86)=18.41, P<0.001; post hoc test between control and rotenone (P<0.001), control and rotenone+PACAP (P<0.001). Total number of animals per group represents 3 independent experiments. ***P<0.001 between control and treated groups; #P<0.05 between rotenone and rotenone+PACAP groups.
Fig. 5.
Fig. 5.
Analysis of DA metabolic enzymes in snail brain homogenates. (A) Western blot with anti-COMT. The bottom panel shows detection of anti-β-actin as a loading control. The top panel shows S- and MB-COMT localization at ∼23 and 26 kDa. A non-linear adjustment (+0.5% level adjustment) was applied to the MB-COMT band in rotenone group. Densitometry evaluations of S-COMT (B) and MB-COMT (C) (n=3, calculated from independent WB experiments). S-COMT: F(2)=135.80, P<0.001; post hoc test between control and rotenone groups (P<0.001), control and rotenone+PACAP groups (P<0.001). MB-COMT: F(2)=50.68, P<0.001; post hoc test between control and rotenone (P<0.001), rotenone and rotenone+PACAP groups (P<0.001). ***P<0.001 between control and treated groups; ###P<0.001 between rotenone and rotenone+PACAP groups.
Fig. 6.
Fig. 6.
Monoamine concentrations in rat SN after treatment with 6-OHDA. (A) DA content of the SN 1, 3, 7, 9, 12, 14 and 16 days after operation (n=14). The y-axis represents the ratio of DA levels in the hemispheres. Data were calculated using the following formula: 100−(ct/cc×100), where ct=DA concentration of treated SN (6-OHDA-injected, left hemisphere); cc=DA concentration of the control SN (right hemisphere). (B) Monoamine content (DA and 5HT) of control group presented in µg/g tissue. (C,D) DA and 5HT content as a percentage of control (n=4) in 6-OHDA (n=6) and 6-OHDA+PACAP (n=7) groups. One-way ANOVA, DA: F(2,18)=15.49, P<0.001; post hoc test between control and 6-OHDA groups (P<0.001), control and 6-OHDA+PACAP groups (P<0.05); 6-OHDA and 6-OHDA+PACAP groups (P<0.01). 5HT: F(2,12)=12.87, P<0.001; post hoc test between control and 6-OHDA groups (P<0.05), control and 6-OHDA+PACAP groups (P<0.01) (D). *P<0.05, **P<0.01, ***P<0.001 between control and treated groups; ##P<0.01 between rotenone and rotenone+PACAP groups.
Fig. 7.
Fig. 7.
Analysis of DA metabolic enzymes in rat SN after treatment with 6-OHDA. (A) S- and MB-COMT at 23 and 26 kDa in control, 6-OHDA and 6-OHDA+PACAP groups are represented. Middle row shows the intensity of MAO-B. Anti-β-actin was used as an internal control (lower row). MAO-B (middle) and β-actin (bottom) levels were similar in all groups. Changes in concentration of S-COMT (B), MB-COMT (C) and MAO-B (D) detected by densitometry were identified at 23 kDa, 26 kDa and 55 kDa, respectively (n=3, calculated from independent WB experiments). S-COMT: F(2)=108.09; P<0.001; post hoc test between control and 6-OHDA groups (P<0.001), control and 6-OHDA+PACAP groups (P<0.001). MB-COMT: F(2)=134.42; P<0.001; post hoc test between control and 6-OHDA (P<0.001), 6-OHDA and 6-OHDA+PACAP groups (P<0.001). ***P<0.001 between control and treated groups; ###P<0.001 between 6-OHDA and 6-OHDA+PACAP groups.
Fig. 8.
Fig. 8.
Comparison of PARK7 levels in toxin-treated snail CNS and rat SN. Values were measured in control (n=3) and after treatment with the different toxins (rotenone, n=5; 6-OHDA, n=4) and toxin+PACAP-treated groups (n=5). Concentration of PARK7 protein is given in μg/g unit. Snails: F(2)=24.59, P<0.05; post hoc test between control and rotenone groups (P<0.05), control and rotenone+PACAP groups (P<0.05). Rat: F(2)=6.90, P<0.05; post hoc test between control and 6-OHDA (P<0.05), 6-OHDA and 6-OHDA+PACAP groups (P<0.05). *P<0.05 between control and treated groups; #P<0.05 between toxin and toxin+PACAP groups.
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