Oxyphylla A Promotes Degradation of α-Synuclein for Neuroprotection via Activation of Immunoproteasome
Zhou Hefeng1, Li Shengnan1, Li Chuwen1, Yang Xuanjun1,2, Li Haitao1, Zhong Hanbing2, Lu Jia-Hong1,*, Lee Simon Ming-Yuen1,*
1State Key Laboratory of Quality Research in Chinese Medicine and Institute of Chinese Medical Sciences, University of Macau, Macao, China. 2Department of Biology, South University of Science and Technology, Shenzhen, China.
Parkinson’s disease (PD), the second most common neurodegenerative disorder, is neuropathologically characterized by the loss of dopaminergic neurons in the substantia nigra pars compacta (SNc) and the presence of Lewy bodies in surviving neurons. α-synuclein (α-syn) is the major component of Lewy bodies and its deposition in neurons is critical pathological event in the pathogenesis of PD. Herein, we reported that Oxyphylla A, a novel lead compound from the fruit of Alpinia oxyphylla, significantly promoted α-syn degradation in a cellular PD model. When exploring the molecular pathways, we found that Oxyphylla A promoted α-syn degradation in a ubiquitin proteasome system (UPS)-dependent and autophagy-independent manner. We further confirmed that Oxyphylla A enhanced UPS activity by upregulating 20S subunit PSMB8 expression. A mechanism study revealed that Oxyphylla A activated the PKA/Akt/mTOR pathway to trigger PSMB8 expression and enhance UPS activity. Finally, we illustrated that Oxyphylla A alleviated the accumulation of both Triton-soluble and Triton-insoluble forms of α-syn and protected against α-syn-induced neurotoxicity in A53T α-syn transgenic mice. These findings suggest that the activation of UPS, via small molecular UPS enhancers including Oxyphylla A, may be a therapeutic strategy for intervention against PD and related diseases.
Figure 1. Oxyphylla A promotes the degradation of WT and mutant α-syn in cell models. (A) Chemical structure of Oxyphylla A. Inducible PC12/A53T-α-syn cells were treated with different concentrations of Oxyphylla A or 0.1% (v/v) DMSO for 24 h, and then analyzed by MTT assay (B) and LDH assay (C). Inducible PC12/WT-α-syn (D), A30P-α-syn (E) and A53T-α-syn (F) cells were treated with 1 μg/ml Dox for 24 h to induce the expression of WT or mutant α-syn, and then with different concentrations of Oxyphylla A or 0.1% (v/v) DMSO for another 24 h, cells were lysed in RIPA lysis buffer. (G) PC12/A53T-α-syn cells were induced with 1 μg/ml Dox for 24 h, and then treated with Oxyphylla A (100 μM) for different time periods. (H) After treatment with Oxyphylla A (100 μM), cells were lysed in 1% Triton X-100. The Triton-soluble fractions were subjected to Western blotting analysis. (I) Triton-insoluble fractions were also subjected to Western blotting analysis. Representative Western blotting and data analysis of three independent experiments are shown. Data are represented as the mean ± SD. ###P < 0.001 as compared to control group; *P < 0.05, **P < 0.01, ***P < 0.001, as compared to Dox group.
Figure 2. Oxyphylla A promotes α-syn degradation via the UPS. (A) Inducible PC12/A53T-α-syn cells were treated with or without 1 μg/ml Dox for 24 h, and then with 100 μΜ Oxyphylla A, 30 μΜ CQ, 0.1 μΜ MG132 or 0.1% (v/v) DMSO for another 24 h. (B) Inducible PC12/A53T-α-syn cells were treated with 1 μg/ml Dox for 24 h, and then treated with 100 μΜ Oxyphylla A for different time periods. Representative Western blotting and data analysis of three independent experiments are shown. The proteasomal enzyme activities were measured using specific bioluminogenic enzyme substrates for chymotrypsin-like (C), trypsin-like (D), and caspase-like (E) activity. Data from three independent experiments are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3. Oxyphylla A promotes α-syn degradation through upregulating the expression of genes encoding proteasome subunit beta type-8 (PSMB8). (A) PC12/A53T-α-syn cells were treated with 1 μg/ml Dox for 24 h and then with 100 μΜ Oxyphylla A for another 24 h. The levels of PSMB8 mRNA were determined by qRT-PCR. (B) PC12/A53T-α-syn cells were treated with Oxyphylla A for 24 h and the protein level of PSMB8 was detected by Western blotting analysis. (C) PC12/A53T-α-syn cells were transduced by PSMB8-shRNA or control-shRNA, treated with Dox for 24 h and then treated with 100 μΜ Oxyphylla A for another 24 h. (D) PC12/A53T-α-syn cells were transfected with PSMB8 or empty vector for 24 h and treated with 1 μg/ml Dox for another 24 h. Cell lysates were subjected to Western blotting analysis. Data are represented as mean ± SD of three independent experiments. *P < 0.05, **P < 0.01, ***P < 0.001, as compared to control group.
Figure 4. Oxyphylla A promotes PSMB8 expression and enhances proteasomal activity through the PKA/Akt/mTOR pathway. Inducible PC12/A53T-α-syn cells were treated with 1 μg/ml Dox for 24 h and then with Oxyphylla A (100 μM) for the indicated time periods. The expression ratios of phosphorylated PKA/total PKA (A), phosphorylated Akt/total Akt (B), phosphorylated p-mTOR/total mTOR (C) and phosphorylated p-p70s6k/total p70s6k (D) were detected by Western blotting analysis. PC12/A53T-α-syn cells were pretreated with or without PKA inhibitor H-89 (10 μM) for 1 h and then treated with Oxyphylla A (100 μM) for 30 min. The expression ratios of phosphorylated Akt/total Akt (E), phosphorylated p70S6K/total p70S6K (F) were analyzed by Western blotting. PC12/A53T-α-syn cells were pretreated with or without PKA inhibitor H-89 (10 μM) for 1 h and then treated with Oxyphylla A (100 μM) for another 24 h. The protein levels of PSMB8 were determined by Western blotting (G). Cellular chymotrypsin-like activity was determined by specific bioluminogenic enzyme substrates (H). Representative Western blotting and data analysis of three independent experiments are shown. Data are represented as the mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5. Oxyphylla A protects mice against α-syn-induced neurotoxicity. (A) Experimental scheme. Ten-month-old WT or homozygous A53T human α-syn Tg mice were gavaged daily with Oxyphylla A (30 mg/kg) or olive oil (0.2 ml) for 4 weeks. (B) In the rotarod test, the time spent on the rotating drum before falling was recorded and analyzed for each mouse (n = 10). (C) Representative images of tyrosine hydroxylase positive (TH+) neurons in the SNc (n = 5). (D) Quantification of TH+ cells in the SNc (n = 5). Data are represented as means ± SD. ##P < 0.01, compared to the WT group; *P < 0.05, compared to Tg group.
Figure 6. Oxyphylla A promotes the degradation of Triton-soluble and Triton-insoluble α-syn in A53T α-syn Tg mouse brains. Ten-month-old homozygous A53T human α-syn Tg mice were gavaged daily with Oxyphylla A (30 mg/kg) or olive oil (0.2 ml) for 4 weeks. The cortex (A), midbrain (B) and striatum (C) were homogenized and A53T α-syn was analyzed by Western blotting. (D) Whole brain levels of Triton-insoluble α-syn were analyzed by Western blotting and semi-quantified by densitometry. The expression ratios of phosphorylated PKA/total PKA (E), p-Akt/total Akt (F), p-p70S6K/total p70S6K (G) and the levels of PSMB8 (H) in whole brain were detected by Western blotting and semi-quantified by densitometry. Data are represented as means ± SD (n = 5). *P < 0.05, **P < 0.05, ***P < 0.001 compared to control.
Figure 7. A proposed schematic of Oxyphylla A promotes degradation of α-syn via modulation of PKA/AKt/mTOR/PSMB8 pathway.
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