Elsevier

Neuroscience Research

Volume 66, Issue 1, January 2010, Pages 124-130
Neuroscience Research

Endogenous catecholamine enhances the dysfunction of unfolded protein response and α-synuclein oligomerization in PC12 cells overexpressing human α-synuclein

https://doi.org/10.1016/j.neures.2009.10.005Get rights and content

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder characterized by the selective loss of dopaminergic neurons and the presence of Lewy bodies. α-Synuclein is a major component of Lewy bodies. Recently, many studies have focused on the interaction between α-synuclein and catecholamine in the pathogenesis of PD. However, no detailed relationship between cathecholamine and α-synuclein cytotoxicity has been elucidated. Therefore, this study established PC12 cell lines which overexpress human α-synuclein in a tetracycline-inducible manner. The overexpression of human α-synuclein increased the number of apoptotic cells in a long-term culture. Moreover, human α-synuclein expressing PC12 cells demonstrated an increased vulnerability to several stressors in a short culture period. Thapsigargin increased the SDS soluble oligomers of α-synuclein associated with catecholamine-quinone. The unfolded protein response (UPR) study showed that thapsigargin increased eIF2α phosphorylation and nuclear GADD153/CHOP induction under α-synuclein overexpressed conditions. The activities of the ATF6α and IRE1α pathways decreased. These findings suggest that an overexpression of α-synuclein partly inactivates the UPR. α-Methyltyrosine inhibited the dysfunction of the UPR caused by an overexpression of human α-synuclein. Therefore, these findings suggest that the coexistence of human α-synuclein with catecholamine enhances the endoplasmic reticulum stress-related toxicity in PD pathogenesis.

Introduction

Parkinson's disease (PD) is the most common movement disorder and is pathologically characterized by selective dopaminergic neuronal death. Abundant evidence points to a causative role for the presynaptic protein α-synuclein (α-syn) in the pathogenesis of PD (Spillantini et al., 1998, Mizuno et al., 2008). α-Syn is a major component of Lewy Bodies, cellular inclusion bodies that are the hallmark pathological feature of PD (Spillantini et al., 1998). The duplication and triplication of the α-syn gene appears to be the cause of PD in rare cases of familial forms of PD (Singleton et al., 2003, Chartier-Harlin et al., 2004, Ibáñez et al., 2004). An overexpression of α-syn leads to neurodegeneration in mouse, rat, fly, and nematode models of PD (Cooper et al., 2006, Auluck et al., 2002, Lo Bianco et al., 2002, Masliah et al., 2000, Cao et al., 2005). These data show that storage of α-syn may be involved in the pathogenesis of PD. Recent studies have shown that an overexpression of α-syn can induce a mitochondrial deficit (Hsu et al., 2000), enhanced vulnerability to oxidative stress (Hsu et al., 2000, Prasad et al., 2004) and inhibition of endoplasmic reticulum (ER)-Golgi trafficking (Cooper et al., 2006, Sugeno et al., 2008). Moreover, other reports suggest that catecholamine (CA) such as dopamine (DA) and DOPA can stabilize the protofibrillar form of α-syn (Conway et al., 2001) and endogenous DA enhances cell death associated with soluble α-syn protein (Xu et al., 2002). However, the association of endogenous CA with α-syn toxicity is still unclear.

Numerous studies of PD rely on drug models using 1-methyl-4-phenyl-pyrisinium (MPP+), rotenone and 6-hydroxydopamine (6-OHDA). These agents cause a DA-neuronal death and PD-like phenotype in animal models. These agents also induce the unfolded protein response (UPR) in ER (Holtz and O’Malley, 2003, Ryu et al., 2002). Dysfunction of Parkin, a gene product responsible for autosomal recessive juvenile Parkinsonism (AR-JP), is linked to ER stress and the UPR (Imai et al., 2002, Imai et al., 2001). Accumulating genetic and molecular evidence suggests that defects in the ER contribute to the pathogenesis of PD.

Previous studies have demonstrated that UPR plays an important role in the pathogenesis of PD, and α-syn relates to a part of UPR. However, it is unclear whether CA is involved in the α-syn pathogenesis in response to ER stress. A human α-syn overexpressing PC12 cell line that could be controlled in tetracycline dependent manner was established to investigate how ER stress and CA enhance the pathogenesis of human α-syn.

Section snippets

Chemicals and antibodies

Nerve growth factor (NGF) was purchased from Invitrogen (Carlsbad, CA, USA). α-Methyltyrosine (α-MT) was purchased from PFALTZ&BAUER (Waterbury, CT, USA). Thapsigargin and 2-melcaptoethanol were obtained from Wako (Osaka, Japan). Tunicamycin and rotenone were purchased from Sigma (Taufkirchen, Germany) and Calbiochem (Darmstadt, Germany), respectively. Mouse monoclonal antibody against human α-syn and β-syn are purchased from BD Transduction laboratory (clone 42; Lexington, KY, USA). Anti-GRP78

Overexpression of human α-syn causes dopaminergic cell death

The PC12 cells expressed α-syn in a Dox dose and time dependent manner (Fig. 1A and B). The overexpression of human α-syn induced cell death significantly at 7 days (Fig. 1C). α-MT, a specific inhibitor of tyrosine hydroxylase, was used to evaluate the association with CA. This reduced the number of apoptotic cells in response to α-syn overexpression in 7 days (Fig. 1D). Consequently, endogenous CA metabolites were thus suggested to enhance the cytotoxicity of α-syn.

Overexpression of human α-syn enhances cell vulnerability relating to CA

The overexpression of human

Discussion

Recent findings suggest that oligomers, rather than the fibrillar amyloid deposits of α-syn, represent the principal toxic species in PD (Kayed et al., 2004). In vitro studies have also demonstrated that CA stabilizes the protofibrillar form of α-syn, thus forming a CA-α-syn adduct (Conway et al., 2001). Therefore, the oligomerization of α-syn may interact with CA in the pathogenesis of PD. On the other hand, environmental stressors have been used as a PD model both in vitro and in animals.

Acknowledgments

This work was supported in part by Grants-in-Aid for Scientific Research from the Ministry of Education, Culture, Sports, Science and Technology of Japan (Nakaso), the Research Committee on Neurodegenerative Diseases, Ministry of Health, Labor and Welfare, Japan (Nakashima), and the Venture Business Laboratory, Tottori University (Ito).

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