| Objective: To study the effect and underlying mechanism of dopamine D2 like receptor agonists on autophagy and subsequent degradation of aggregate-prone proteins like α-synuclein in dopaminergic cells.Methods: In vitro experiments involve a variety of dopaminergic cell lines including PC12, MES23.5, SH-SY5 Y and primary rat midbrain neurons, with PC12 cells as the main cellular model of this study. Dopamine D2-like receptor agonists(pramipexole and quinpirole) and antagonists were used as pharmacologic tool drugs. Western Blotting analysis was extensively used to detect the changes in protein expression or phosphorylation of various proteins. SH-SY5 Y cells were differentiated by retinoic acid(RA) in combination with phorbol 12-myristate 13-acetate(TPA). Reverse-transcription PCR and Quantitative PCR were applied to determine the dopamine D2-like receptors and BECN1 m RNA changes. CCK-8 was used to evaluate the cell viability and the cleaved caspase-3 as an indicator of apoptosis. The formation of EGFP-LC3, GFP-RFP-LC3 dots and the distribution of dopamine D2/D3 receptor subtype were observed under the confocal microscope; Ultrastructure and autophagosome changes in pramipexole and quinpirole treated-cells were observed by Transmission Electron Microscope(TEM). Calcium imaging was used to monitor the dynamic change of intracellular calcium levels. Beclin 1 and c-Fos plasmid or si RNA were introduced by transient transfection. Co-immunoprecipitation(Co-IP) was used to observe the protein-protein interaction of Beclin 1 and Ptd Ins3K; Chromatin immunoprecipitation(Ch IP) was used to study the binding of c-Fos protein to BECN1 promoter; Luciferase double-standard test was used to detect the activity of BECN1 promoter. Lentiviral vector driving the recombinant eukaryotic expression vector of p Lenti VENUS-YFP-SNCA that contains wild type synonymous mutation or its A53 T and A30 P pathogenic mutation were overexpressed in PC12 cells; Adenovirus vector driving the first 552 amino acids of wild type(18Q) and mutant(100Q) Htt were also used to infect PC12 cells. In vivo study, 15-month-old wild-type and A53T-SNCA transgenic mice were intraperitoneal injected with pramipexole at 0.5 mg/kg twice daily for 3 consecutive weeks. An equal volume of saline was given as a negative control.Results:1. The expression patterns of dopamine D2-like receptors in different dopaminergic cell lines: PC12 cells mainly expressed dopamine D2 receptor, while MES23.5 cells expressed both dopamine D2 and D3 subtype receptors. Undifferentiated SH-SY5 Y cells expressed few dopamine D2 and D3 receptors, while RA/TPA differentiated SH-SY5 Y cells exhibited higher expressions of D2 and D3 receptors, especially the D3 subtype.2. The effects of D2-like receptor agonists on autophagy activity and cell viability: 1) The protein levels of LC3-II and Beclin 1, two autophagy-related markers, increased in dose-and time-dependent manners following pramipexole(dopamine D2-like receptor agonist) treatment in both PC12, MES23.5 and RA/TPA differentiated SH-SY5 Y cells, while P62 protein level decreased. It did not produce similar effects in undifferentiated SH-SY5 Y cells, Similar observations were obtained in quinpirole(another dopamine D2-like receptor agonist) –treated PC12 cells. By contrast, dexpramipexole, which is the R(+) enantiomer of pramipexole and virtually devoid of any of the DA agonist effects, failed to affect LC3-II, Beclin 1 or P62 protein levels. Pramipexole and quinpirole still increased the LC3-II level in the presence of bafilomycin A1 and chloroquine. 2) Pramipexole also induced massive LC3 puncta formation in PC12 cells transfected with EGFP-LC3. When PC12 cells were transfected with RFP-GFP-LC3, pramipexole increased both GFP and RFP puncta, and the RFP puncta increased more obviously. 3) Pramipexole induced massive formation of LC3 puncta in rat primary midbrain neurons. 4) Pramipexole and quinpirole induced massive autophagosome, which was observed by Transmission Electron Microscope(TEM). 5) Pretreatment with D2-like receptor Inhibitors inhibited the pramipexole-induced up-regulation of LC3-II and Beclin 1. 6) Neither pramipexole nor quinpirole treatment for 24 h, within the tested concentration range, affected the cell viability in PC12 cells. These results fully demonstrated dopamine D2-like receptor agonists(as a representative with pramipexole) can induce autophagy in dopamine cells without any significant effect on cell viability within the tested concentration range.3. Mechanisms results: 1) c-Fos levels increased time-dependently following pramipexole treatment in PC12 cells. 2) Pramipexole induced a time-dependent increase in p-Ca MKIV(Thr196) and p-CREB(S133) phosphorylation. 3) The phosphorylation of JNK(Thr183/Tyr185) and c-Jun(S63) was not changed. 4) Pramipexole and quinpirole failed to reduce the phosphorylation of p-m TOR(S2448), p-p70S6K(Thr389), p-ULK1(S757), and increase the phosphorylation of p-ULK1(S555) compared with the canonical starvation-induced autophagy. In contrast, pramipexole significantly upregulated p-m TOR(S2248), p-p70S6K(Thr389) and p-ULK1(S757) phosphorylation levels at 30 minutes to 1 hour after treatment; 5) BECN1 1 m RNA increased dose-dependently following pramipexole treatment; The Beclin 1 and Ptd Ins3 K protein-protein interaction was significantly enhanced following treatment with pramipexole or quinpirole. 6) Pramipexole triggered an elevation of intracellular Ca2+([Ca2+]i). [Ca2+]i chelator BAPTA/AM reversed the increases of c-Fos and autophagic markers LC3-II and Beclin 1 caused by pramipexole treatment. 7) After BECN1 gene silencing, pramipexole could not induce any significant increase in LC3-II; Beclin 1 overexpression significantly increased LC3-II; 8) After c-Fos gene silencing, pramipexole could not induce any increase in both Beclin 1 and LC3-II; c-Fos overexpression caused significant increases in Beclin 1 and LC3-II; 9) The chromatin immunoprecipitation(Ch IP) assay revealed that c-Fos bound to the putative AP-1 site(5’-TGCCTCA-3), and pramipexole treatment signi?cantly enhanced its binding to rat BECN1 promoter region; 10) The BECN1 luciferase reporter analysis displayed that pramipexole remarkably enhanced the BECN1 promoter activity, and this was abolished in cells transfected with mutant BECN1 reporter in which the putative AP-1 binding site was deleted.4. The effect of pramipexole to aggregate-prone proteins: 1) Pramipexole reduced the α-synuclein aggregation in rotenone-treated normal and WT, A53 T, A30P-SNCA overexpressing PC12 cells; 2) Intraperitoneal injection with pramipexole increased the level of autophagy and decreased α-synuclein aggregation in the substantia nigra and the striatum of normal and A53T-SNCA mice; 3) Pramipexole also reduced Htt-552-18 and Htt-552-100 Q protein aggregation.Conclusion: Dopamine D2-like receptor agonist promotes the degradation of α-synuclein and huntingtin protein via enhancing autophagy activity. The underlying mechanism involves the elevation of intracellular calcium and activation c-Fos / Beclin1 signaling pathway. |
