Particular vulnerability of rat mesencephalic dopaminergic neurons to tetrahydrobiopterin: Relevance to Parkinson’s disease So Yeon Lee, Younghye Moon, Dong Hee Choi, Hyun Jin Choi, and Onyou Hwang ⁎ Department of Biochemistry and Molecular Biology, University of Ulsan College of Medicine, 388-1 Pungnap-dong, Songpa-ku, Seoul, 138-736, Korea Received 16 June 2006; revised 21 August 2006; accepted 30 August 2006 Available online 16 October 2006 We determined whether tetrahydrobiopterin(BH4), an endogenous cofactor for dopamine(DA) synthesis, causes preferential damage to DArgic neurons among primary cultured rat mesencephalic neurons and whether the death mechanism has relevance to Parkinson’s disease (PD). DArgic neurons were more vulnerable to BH4 than non-DArgic neurons, exhibiting sensitivity at lower concentrations, evident by morphological and neurotransmitter uptake studies. BH4-exposed DArgic neurons showed (1) increased TUNEL staining and activated caspase-3 immunoreactivity, indicative of apoptotic death; (2) mito- chondrial membrane potential loss and increased cytosolic cytochrome c, suggesting mitochondrial dysfunction; (3) increased level of oxidized proteins and protection by antioxidants, indicative of oxidative stress; and (4) increased ubiquitin immunoreactivity, suggesting alteration of protein degradation pattern. Percent of cells positive for these para- meters were much higher for DArgic neurons, demonstrating prefer- ential vulnerability. Therefore, the DArgic neuronal damage induced by BH4, the molecule synthesized and readily upregulated in DArgic neurons and activated microglia, suggests physiological relevance to the pathogenesis of PD. © 2006 Elsevier Inc. All rights reserved. Keywords: Tetrahydrobiopterin; Parkinson’s disease; Rat primary mesen- cephalic neuronal culture; Dopamine; Apoptosis; Oxidative stress Parkinson’ s disease (PD) is a neurodegenerative disorder characterized by a selective loss of dopaminergic (DArgic) neurons in the substantia nigra (SN) pars compacta. While oxidative stress, apoptosis, mitochondrial dysfunction, and protein aggregation/ misfolding are thought to be involved in the cell death mechanism (reviewed by Moore et al., 2005), as to why the DArgic neurons are particularly vulnerable is not fully understood. The findings that a number of genetic mutations, which would affect all cells in the body, as well as systemic exposure to environmental toxins and proteasome inhibitors lead to preferential damage to the SN DArgic neurons suggest the presence of intrinsic factors that render these cells particularly vulnerable. The presence of DA, tyrosine hydro- xylase (TH), monoamine oxidase, iron, and neuromelanin in these cells has been suggested to play a role (Obata, 2002; He et al., 2003; Hirsch et al., 1988). Tetrahydrobiopterin (BH4) is an obligatory cofactor for TH in DA synthesis (Kaufman, 1993), and among the brain neurons, it is present selectively in monoaminergic cells including the nigral DArgic cells (Hwang et al., 1998; Nagatsu et al., 1995). Production of BH4 in DArgic cells can be upregulated by increased calcium in vitro (Hwang et al., 1999) and by stress in vivo (Kim et al., 2005a,b). In addition, as a cofactor of nitric oxide synthase (Kwon et al., 1989), BH4 synthesis is induced in activated microglia (Cho et al., 1999), the macrophage-like immune cells that are present particularly in high density in the SN region (Kim et al., 2000). BH4 is readily released from and taken up into cells (Anastasiadis et al., 1994; Choi et al., 2003c). Therefore, the nigrostriatal DArgic neurons can be exposed to a relatively high level of BH4 in the brain. That BH4 might be an intrinsic factor which can contribute to the DArgic neuron’ s vulnerability has been suggested. BH4 toxicity has been observed in DA-producing cell lines including CATH.a, SK-N- BE(2)C and PC12 (Choi et al., 2000; Anastasiadis et al., 2001) as well as in the nigrostriatal DArgic system when injected intrastriatally (Kim et al., 2003), intranigrally (Choi et al., 2003b), and intraventicularly (Kim et al., 2004). BH4 is readily autooxidized to generate superoxide and hydrogen peroxide (Kirsch et al., 2003), which in turn facilitates autooxidation of DA to DA quinone (Graham et al., 1978). DA quinone species can cause cytotoxicity by generating quinone proteins (LaVoie and Hastings, 1999; Kuhn et al., 1999), and exposure to BH4 indeed leads to accumulation of quinone proteins in DArgic cells (Choi et al., 2003a, 2005). DA quinone is also a source of oxidative stress, as it can attack low molecular weight sulfhydryls including reduced glutathione or cyclize to become aminochrome, whose redox-cycling generates further reactive oxygen species (ROS). Therefore, the concerted actions between BH4 and DA can elicit cellular damage in DArgic system, and this might explain the selective vulnerability as in PD. The present study was undertaken in order to evaluate whether the BH4-induced damage indeed occurs preferentially in DArgic neurons, using primary rat mesencephalic culture containing both DArgic and non-DArgic neurons, and whether the BH4-induced www.elsevier.com/locate/ynbdi Neurobiology of Disease 25 (2007) 112 – 120 ⁎ Corresponding author. Fax: +822 3010 4248. E-mail address: oyhwang@amc.seoul.kr (O. Hwang). Available online on ScienceDirect (www.sciencedirect.com). 0969-9961/$ - see front matter © 2006 Elsevier Inc. All rights reserved. doi:10.1016/j.nbd.2006.08.024