Tetrahydrobiopterin causes mitochondrial dysfunction in dopaminergic cells: Implications for Parkinson's disease

Abstract

Parkinson's disease (PD) is a neurodegenerative disorder associated with a selective loss of dopaminergic neurons in the substantia nigra. While the underlying cause of PD is not clearly understood, oxidative stress and mitochondrial dysfunction are thought to play a role. We have previously suggested tetrahydrobiopterin (BH4), an obligatory cofactor for the dopamine synthesis enzyme tyrosine hydroxylase and present selectively in monoaminergic neurons in the brain, as an endogenous molecule that contributes to the dopaminergic neurodegeneration. In the present study, we show that BH4 leads to inhibition of activities of complexes I and IV of the electron transport chain (ETC) and reduction of mitochondrial membrane potential. BH4 appears to be different from rotenone and MPP⁺, the synthetic compounds used to generate Parkinson models, in its effect on complex IV. BH4 also induces the release of mitochondrial cytochrome c. Pretreatment with the sulfhydryl antioxidant N-acetylcysteine or the quinone reductase inducer dimethyl fumarate prevents the ETC inhibition and cytochrome c release following BH4 exposure, suggesting the involvement of quinone products. Together with our previous observation that BH4 leads to generation of oxidative stress and selective dopaminergic neurodegeneration both in vitro and in vivo via inducing apoptosis, the mitochondrial involvement in BH4 toxicity further suggests possible relevance of this endogenous molecule to pathogenesis of PD.

Introduction

Parkinson's disease (PD) is a neurodegenerative disorder affecting 1% of the population above the age of 65 (Zhang et al., 2000) and is characterized by a selective loss of dopaminergic neurons in the substantia nigra pars compacta. Attempts have been made to understand why the dopaminergic cells are particularly vulnerable, i.e. whether factors intrinsic to these cells contribute to the vulnerability. The presence of dopamine, tyrosine hydroxylase (TH), monoamine oxidase, iron, and/or neuromelanin has been suggested to play a role.

Another molecule endogenously present in dopaminergic neurons that can generate pro-oxidants and induce cell death is tetrahydrobiopterin (BH4). BH4 is an obligatory cofactor for TH in dopamine synthesis (Kaufman, 1993) and is produced selectively in monoaminergic neurons in the brain including the nigral dopaminergic neurons (Hwang et al., 1998, Nagatsu et al., 1995). Interestingly, BH4 exerts toxicity on dopamine-producing cell lines (Anastasiadis et al., 2001, Choi et al., 2000, Enzinger et al., 2002) and primary cultured TH-positive mesencephalic neurons (Lee et al., submitted for publication) but not on non-dopaminergic cells (Choi et al., 2000, Choi et al., 2003a). In vivo, administration of BH4 into animals produces nigrostriatal degeneration, dopamine loss, apoptotic cell death, and motor deficit (Kim et al., 2003). The role of BH4 in dopaminergic cell death is also demonstrated by the report that inhibition of BH4 synthesis can prevent kainate-induced (Foster et al., 2003) and stress-induced (Kim et al., 2005) deaths of nigral dopaminergic neurons. Based on these data, BH4 has been suggested as a candidate endogenous molecule involved in the pathogenesis of PD.

Although the underlying cause of dopaminergic cell death or the mechanism by which these cells degenerate in PD is still not clearly understood, oxidative stress (Beal, 2003, Zhang et al., 2000), mitochondrial dysfunction (Greenamyre et al., 2001, Orth and Schapira, 2002), apoptosis (Anglade et al., 1997, Kingsbury et al., 1998, Mochizuki et al., 1996, Tompkins et al., 1997) and protein misfolding (Dawson and Dawson, 2003, McNaught and Olanow, 2003) are thought to play important roles.

Involvement of mitochondrial dysfunction in PD is based on the findings that a significant decrease in the activity of complex I (NADH:ubiquinone oxidoreductase; EC 1.6.5.3) of the electron transport chain (ETC) is observed in the substantia nigra of postmortem brain (Schapira et al., 1989, Schapira et al., 1990) as well as platelet (Haas et al., 1995, Parker et al., 1989) and skeletal muscle (Mizuno et al., 1998) of PD patients. Reduction in complex IV (cytochrome c oxidase; EC 1.9.3.1) activity has also been observed in PD (Benecke et al., 1993, Schapira, 1994). Reduced activity of ETC can lead to dissipation of mitochondrial membrane potential (ΔΨ_m) (Ly et al., 2003), which has also been observed in PD (Schapira, 1999). The loss of ΔΨ_m is related to the release of molecules including cytochrome c and activation of the proapoptotic proteins present close to the outer mitochondrial membrane (Kroemer et al., 1997, Reed, 1997).

Mitochondrial ETC activity is inhibited by reactive oxygen species (ROS) (Brown and Yamamoto, 2003), and extremely sensitive to inhibition by sulfhydryl modifying agents (Gutman et al., 1970a, Gutman et al., 1970b). Exposure of isolated intact mitochondria to dopamine has been demonstrated to lead to impaired oxidative phosphorylation (Berman and Hastings, 1999, Cohen et al., 1997, Khan et al., 2005, Kim et al., 1999), suggesting that the mitochondria can be a target of the species generated by dopamine oxidation. Recent reports suggest the crucial role of quinone products in dopamine-mediated inhibition of mitochondrial function (Khan et al., 2005). Interestingly, our previous study has shown that BH4 facilitates dopamine oxidation leading to formation of reactive quinone products (Choi et al., 2003a), which is important in rendering dopaminergic cells vulnerable.

Based on the findings that (1) the nigral dopaminergic cell death in PD involves mitochondrial dysfunction, oxidative stress and apoptosis; (2) BH4 facilitates generation of oxidative stress and quinone products in dopaminergic cells; and (3) the mitochondria is a target of oxidative damage, it was possible that the BH4-induced dopaminergic cell death involves mitochondrial dysfunction. In the present study we therefore tested whether events related to mitochondrial dysfunction including changes in ETC activity, ΔΨ_m and cytochrome c release might take place in the BH4-exposed dopaminergic cells.