Cui-Zan Cai1, He-Feng Zhou1, Ning-Ning Yuan1, Ming-Yue Wu1, Simon Ming-Yuen Lee1, Jiao-Yan Ren2, Huan-Xing Su1, Jin-Jian Lu1, Xiu-Ping Chen1, Min Li3, Jie-Qiong Tan4, Jia-Hong Lu5. 1. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China. 2. School of Food Science and Engineering, South China University of Technology, Wushan RD., Tianhe District, Guangzhou, China. 3. Mr. and Mrs. Ko Chi Ming Centre for Parkinson's Disease Research, School of Chinese Medicine, Hong Kong Baptist University, Hong Kong SAR, China. 4. Center for Medical Genetics, School of Life Sciences, Central South University, Changsha, Hunan, China. Electronic address: tanjieqiong@sklmg.edu.cn. 5. State Key Laboratory of Quality Research in Chinese Medicine, Institute of Chinese Medical Sciences, University of Macau, Macao SAR, China. Electronic address: jiahonglu@umac.mo.
Abstract
BACKGROUND: Parkinson's disease (PD) is an age-dependent progressive movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Accumulation of <alpha>-synuclein (<alpha>-syn) positive protein aggregates in the substantia nigra is a pathological hallmark of PD, indicating that protein turnover defect is implicated in PD pathogenesis. PURPOSE: This study aims to identify neuroprotective compounds which can alleviate the accumulation of <alpha>-syn in neuronal cells and dissect the underlying mechanisms. METHODS: High throughput screening was performed by dot blot assay. The degradation of different forms of <alpha>-syn by candidate compounds were assessed by western blot. The autophagy lysosome pathway and ubiquitin-proteasome system were examined to dissect the degradation pathway. The UPS activity was assessed by cellular UPS substrates degradation assay and biochemical proteasome activity assay. Q-PCR was performed to test the mRNA level of different proteasome subunits. Furthermore, Neuroprotective effect of candidate compound was tested by LDH assay and PI staining. RESULTS: Through the high throughput screening, harmine was identified as a potent <alpha>-syn lowering compound. The time-dependent and dose-dependent effects of harmine on the degradation of different forms of <alpha>-syn were further confirmed. Harmine could dramatically promote the degradation of UPS substrates GFP-CL1, Ub-R-GFP and Ub-G76V-GFP, and activate cellular proteasome activity. Mechanistically, harmine dramatically enhanced PKA phosphorylation to enhance proteasome subunit PSMD1 expression. PKA inhibitor blocked the effects of harmine in activating UPS, up regulating PSMD1 and promoting <alpha>-syn degradation, indicating that harmine enhances UPS function via PKA activation. Moreover, harmine efficiently rescued cell death induced by over-expression of <alpha>-syn, via UPS-dependent manner. CONCLUSION: Harmine, as a new proteasome enhancer, may have potential to be developed into therapeutic agent against neurodegenerative diseases associated with UPS dysfunction and aberrant proteins accumulation.
BACKGROUND:Parkinson's disease (PD) is an age-dependent progressive movement disorder characterized by a profound and selective loss of nigrostriatal dopaminergic neurons. Accumulation of <alpha>-synuclein (<alpha>-syn) positive protein aggregates in the substantia nigra is a pathological hallmark of PD, indicating that protein turnover defect is implicated in PD pathogenesis. PURPOSE: This study aims to identify neuroprotective compounds which can alleviate the accumulation of <alpha>-syn in neuronal cells and dissect the underlying mechanisms. METHODS: High throughput screening was performed by dot blot assay. The degradation of different forms of <alpha>-syn by candidate compounds were assessed by western blot. The autophagy lysosome pathway and ubiquitin-proteasome system were examined to dissect the degradation pathway. The UPS activity was assessed by cellular UPS substrates degradation assay and biochemical proteasome activity assay. Q-PCR was performed to test the mRNA level of different proteasome subunits. Furthermore, Neuroprotective effect of candidate compound was tested by LDH assay and PI staining. RESULTS: Through the high throughput screening, harmine was identified as a potent <alpha>-syn lowering compound. The time-dependent and dose-dependent effects of harmine on the degradation of different forms of <alpha>-syn were further confirmed. Harmine could dramatically promote the degradation of UPS substrates GFP-CL1, Ub-R-GFP and Ub-G76V-GFP, and activate cellular proteasome activity. Mechanistically, harmine dramatically enhanced PKA phosphorylation to enhance proteasome subunit PSMD1 expression. PKA inhibitor blocked the effects of harmine in activating UPS, up regulating PSMD1 and promoting <alpha>-syn degradation, indicating that harmine enhances UPS function via PKA activation. Moreover, harmine efficiently rescued cell death induced by over-expression of <alpha>-syn, via UPS-dependent manner. CONCLUSION:Harmine, as a new proteasome enhancer, may have potential to be developed into therapeutic agent against neurodegenerative diseases associated with UPS dysfunction and aberrant proteins accumulation.