Neuroprotection of active principles from the Cudrania tricuspidata in in vitro models of Parkinsons disease: Effect on the ubiquitin-proteasome system and Nrf2-ARE pathway

학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2018. 2. 마응천.Abstract Neuroprotection of active principles from the Cudrania tricuspidata in in vitro models of Parkinsons disease: Effect on the ubiquitin-proteasome system and Nrf2- ARE pathway Dong-Woo Kim Natural Products Science Major College of Pharmacy Doctor Course in the Graduate School Seoul National University Parkinsons disease (PD) is characterized by severe motor deficits, cogwheel rigidity, bradykinesia, and the loss of dopaminergic neurons. The aetiology of PD has not been clearly identifiedhowever, oxidative stress is thought to be a common factor that leads to cellular dysfunction and neurodegeneration. In particular, the pathological events that occur in PD have been suggested to be linked to protein oxidation caused by oxidative stress, and excessive intracellular ROS induce apoptosis that is characterized by the cleavage of caspase-3, caspase-9 and poly ADP-ribose polymerase (PARP). The neurotoxin 6-hydroxydopamine (6-OHDA), Carbonyl cyanide 3-chlorophenylhydrazone (CCCP) destroys dopaminergic and noradrenergic neurons in the brain by inducing excessive ROS such as superoxide radicals, which leads to protein oxidation and neuronal cell death. Also, ubiquitin-proteasome system play a key role in the etiology of PD. The proteasome selective degrades oxidized proteins via ubiquitin-mediated processes, and its role is essential for cellular protein maintenance. However, dysfunctions in the ubiquitination machinery or in the proteolytic activities of the proteasome induce the accumulation of polyubiquitinated misfolded proteins and oxidized proteins. Subsequently, this induces protein aggregation, further inhibits proteasome activity, generates additional cellular stress, and ultimately leads to neuronal cell death. Additionally, mitophagy play a major role in the etiology of PD. Mitophagy, a specialized autophagy pathway that mediates the clearance of damaged mitochondria by lysosomes, is important for mitochondrial quality control. Defective mitochondria, if left uncleared, can be a source of oxidative stress and compromise the health of the entire mitochondrial network. The several lines of evidence propose that mitochondrial dysfunction is central to the disease. PD-associated mutations in PINK1 or parkin impair parkin recruitment, mitochondrial ubiquitination, and/or mitophagy. In the context of the inherently high mitochondrial oxidative stress in substantia nigra dopamine neurons, loss of parkin-mediated mitophagy could explain the greater susceptibility of substantia nigra neurons to neurodegeneration. Thus, promoting mitophagy and enhancing mitochondrial quality control could benefit dopaminergic neurons. The current therapeutic drugs are based on prohibiting the progress of PD through treatment of dopamine agonist or dopamine precursor. New therapies in development are aimed at protecting dopaminergic neurons. In this study, the effects of natural products on 6-OHDA, CCCP-mediated signaling in SH-SY5Y neuroblastoma cell were investigated to discover new lead compounds for the treatment of PD. Cudrania tricuspidata (Moraceae) is a subtropical tree that is widely distributed in Korea, China, and Japan. The fruits of C. tricuspidata are used in jams, juices, and a fermented alcoholic beverage with sugar, and they are commercially produced as food in Korea. Also, the cortex and root bark of C. tricuspidata have been used as a traditional medicine for inflammation and tumors. A recent study demonstrated that the extracts of C. tricuspidata protect neurons against oxidative stress-induced cytotoxicity and inhibitory effects on nitric oxide synthase (NOS). The compounds isolated from C. tricuspidata are primarily xanthones and flavones in addition to some alkaloids, lignins, coumarins, polysaccharides, and chromones. The isoflavones and chromones from C. tricuspidata have been reported to exert protective effects against 6-OHDA-induced neurotoxicity and to have inhibitory effects against IgE-mediated allergic and inflammatory responses. In first chapter, it was investigated that 5,7-dihydroxychromone (DHC) isolated from the roots of C. tricuspidata for its neuronal cell protection and inhibition of the generation of ROS in 6-OHDA-induced SH-SY5Y cells. Flow cytometric analysis revealed that DHC protected against the 6-OHDA-induced generation of ROS and protected against neuronal cell death. Additionally, DHC increased the nuclear translocation of Nrf2 and the binding of Nrf2 to ARE, which subsequently resulted in the up-regulation of the expression of Nrf2-dependent antioxidant genes, including heme oxygenase 1 (HO-1), NAD(P)H quinone oxidoreductase 1 (NQO1) and glutamate-cysteine ligase, catalytic subunit (GCLc). DHC inhibited the expression of cleaved caspase-3 and caspase-9 and PARP in 6-OHDA-induced SH-SY5Y cells. The addition of Nrf2 siRNA abolished the neuroprotective effect of DHC against 6-OHDA-induced cell death and the expression of Nrf2-mediated antioxidant genes. These findings suggest that the neuroprotective effect of DHC against 6-OHDA-induced toxicity is partly due to the induction of Nrf2-mediated antioxidant gene expression via the activation of the Nrf2-ARE signaling pathway in SH-SY5Y cells. In the second chapter, the effects of ethanol extract from the fruits of C. tricuspidata (CTE) and it active compounds were studied. Among the nine isolates from a 50% ethanol extract from C. tricuspidata fruits (CTE50), orobol (OB), 6-prenylorobol (POB), and 6,8-diprenylorobol (DPOB) showed neuroprotective effects in 6-OHDA-induced SH-SY5Y cell death. In addition, CTE50 and the three orobol derivatives (OB, POB, and DPOB) attenuated the cleavage of caspase-3, caspase-9, and PARP and inhibited the excessive generation of ROS. Furthermore, it enhanced the 6-OHDA-induced dysfunction of proteasome activity and reduced the accumulation of ubiquitin conjugated-proteins and the polyubiquitination of α-synuclein and synphilin-1. The proteasome inhibitor MG132 blocked the neuroprotective effects and the enhanced proteasome activity produced by CTE50 and the three orobol derivatives. These results demonstrate that CTE50 and three orobol derivatives protect against 6-OHDA-induced neurotoxicity by enhancing the ubiquitin/proteasome-dependent degradation of α-synuclein and synphilin-1, suggesting that they might be possible candidates for the treatment of neurodegenerative diseases. In the third chapter, the effects of active compounds from the C. tricuspidata extracts on deubiquitinating enzymes were studied. TH3-125-4 (TH20) isolated from the root barks of the C. tricuspidata protected against CCCP-induced neuronal cell death in Parkin K.D. SH-SY5Y cells. Also, TH20 significantly inhibited USP30 enzyme activity and disassembly of polyubiquitin chain in in vitro assay. Additionally, TH20 decreased protein expression of USP30. Based on the results, it was suggested that TH20 might be promising candidates for the therapy of familiar PD via restoring Parkin-mediated mitophagy.Chapter1 Neuroprotection against 6-OHDA-induced oxidative stress and apoptosis in SH-SY5Y cells by 5,7 Dihydroxychromone: Activation of the Nrf2/ARE pathway 1 1. Introduction 2 2. Material and methods 10 2.1. Chemicals and reagents 10 2.2. Preparation of 5,7-dihydroxychromone (DHC) 11 2.3 Cell cultures 11 2.4 Measurement of cell viability 12 2.5 Measurement of cell necrosis by propidium iodide staining 12 2.6 Measurement of intracellular ROS by Flow cytometry 13 2.7 Nuclear and cytosolic lysate preparations 13 2.8 Electrophoretic mobility shift assay (EMSA) 14 2.9 Nrf2 knockout via the transfection of small interfering RNA (siRNA) 14 2.10 Measurement of mRNA expression 14 2.11 Measurement of protein expression 16 2.12 Nuclear translocation of Nrf2 using fluorescence microscope 16 2.13 Statistical analysis 17 3 . Results 18 3.1 Protective effect of DHC against 6-OHDA-induced neuronal cell death 18 3.2 Inhibitory effect of DHC against 6-OHDA-induced intracellular ROS generation 27 3.3 Effects of DHC on induction of the nuclear Nrf2 and binding affinity of Nrf2/ARE in SH-SY5Y cells 29 3.4 Effects of DHC on HO-1, NQO1 and GCLc protein expression in SH-SY5Y cells 34 3.5 Effects of DHC on HO-1, NQO1 and GCLc mRNA expression in SH-SY5Y cells 37 3.6 The inhibitory effects of DHC on the 6-OHDA-induced apoptotic signal 41 4. Discussion 43 5. Conclusion 47 Chapter2 Orobol derivatives and extracts from Cudrania tricuspidata fruits protect against 6-hydroxydopamine-induced neuronal cell death by enhancing proteasome activity and the ubiquitin/proteasome-dependent degradation of α-synuclein and synphilin- 1 49 1. Introduction 50 2. Material and methods 57 2.1. Chemicals and reagents 57 2.2. Preparation of ethanol extracts from the fruits of C. tricuspidata (CTE) 57 2.3 Ultra performance liquid chromatography (UPLC) analysis of CTE50 58 2.4 Isolation and identification of compounds from CTE50 59 2.5 Cell cultures 59 2.6 Measurement of cell viability 60 2.7 Measurement of intracellular ROS by flow cytometry 60 2.8 Measurement of proteasome activity 61 2.9 Measurement of mRNA expression 62 2.10 Measurement of protein expression 63 2.11 Immunoprecipitation assay 64 2.12 Statistical analysis 65 3. Results 66 3.1 Protective effects against 6-OHDA-induced neuronal cell death in SH-SY5Y cell 66 3.2 Inhibition of 6-OHDA-induced intracellular ROS generation 73 3.3 Neuroprotective effects against 6-OHDA-induced apoptosis 76 3.4 Protective effects against 6-OHDA-induced dysfunction of proteasome activity 78 3.5 Effects of CTE50 and three orobol derivatives on proteasome subunit mRNA expression 82 3.6 Inhibition of 6-OHDA-induced ubiquitin-conjugated proteins 84 3.7 Inhibition of 6-OHDA-induced poly-ubiquitination of α-synuclein, and synphilin-1 86 3.8 A proteasome inhibitor (MG-132) diminished the protective effects of CTE50 and the three orobol derivatives against 6-OHDA-induced neuronal cell death and proteasome dysfunction 89 4. Discussion 96 5. Conclusion 103 Chapter3 Protective effects of TH3-125-4 (TH20) from the root barks of Cudrania tricuspidata on CCCP-induced neuronal cell death via the inhibition of USP30 deubiquitinating enzyme in Parkin knock down SH-SY5Y cells 105 1. Introduction 106 2. Material and methods 109 2.1 Chemicals and reagents 109 2.2 Preparation of TH3-125-4 (TH20) 109 2.3 Cell cultures 110 2.4 Measurement of cell viability 110 2.5 Measurement of mitochondrial membrane potential by JC-1 staining 111 2.6 Measurement of intracellular ROS by Flow cytometry 111 2.7 Mitochondrial and cytosolic fraction preparations 112 2.8 Parkin knock down via the transfection of small interfering RNA (siRNA) 112 2.9 Measurement of protein expression 113 2.10 Immunoprecipitation assay 113 2.11 Measurement of mitophagy by fluorescence microscope 114 2.12 Measurement of deubiquitinating enzyme activity 115 2.13 Measurement of ubiquitin chain disassembly 115 2.14 Statistical analysis 115 3. Results 117 3.1 Inhibitory effect of TH3-125-4 (TH20) on deubiquitinating enzymes, USP15, USP30 117 3.2 Effect of TH3-125-4 (TH20) on ubiquitin chain disassembly 123 3.3 The protective effects of TH3-125-4 (TH20) against CCCP-induced neuronal cell death in parkin knock down SH-SY5Y cells 125 3.4 Inhibitory effect of TH3-125-4 (TH20) on USP30 protein expression in SH-SY5Y cells 129 3.5 Effect of TH3-125-4 (TH20) on CCCP-induced disruption of mitochondrial membrane potential 132 4. Discussion 134 5. Conclusion 137 References 138 Abstract (in Korean) 161Docto