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

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약학대학 약학과
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서울대학교 대학원
neuroprotection57-DihydroxychromoneNrf2/ARE pathwayorobol derivativesproteasome activityubiquitinationdeubiquitinating enzymePINK1ParkinmitophagyCudrania tricuspidataoxidative stress6-OHDA
학위논문 (박사)-- 서울대학교 대학원 : 약학대학 약학과, 2018. 2. 마응천.

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 identified
however, 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.
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