Tetrahydrobiopterin precursor sepiapterin provides protection against neurotoxicity of 1-methyl-4-phenylpyridinium in nigral slice cultures Jakob Torp Madsen,* Pernille Jansen,* Christian Hesslinger, Morten Meyer,* Jens Zimmer* and Jan Bert Gramsbergen* *Anatomy and Neurobiology, Institute of Medical Biology, University of Southern Denmark, Odense, Denmark  Institute of Pharmacology and Toxicology, Johann Wolfgang Goethe-Universita ¨t, Frankurt am Main, Germany Abstract Complex-I inhibition and oxidative processes have been implicated in the loss of nigral dopamine neurones in Parkin- son’s disease and the toxicity of MPTP and its metabolite MPP + . Tetrahydrobiopterin, an essential cofactor for tyrosine hydroxylase, may act as an antioxidant in dopaminergic neurones and protects against the toxic consequences of glutathione depletion. Here we studied the effects of mani- pulating tetrahydrobiopterin levels on MPP + toxicity in organ- otypic, rat ventral mesencephalic slice cultures. In cultures exposed to 30 lM MPP + for 2 days, followed by 8 days ‘recovery’ in control medium, we measured dopamine and its metabolites in the tissue and culture medium by HPLC, lactate dehydrogenase release to the culture medium, cellular uptake of propidium iodide and counted the tyrosine hydroxylase- immunoreactive neurones. Inhibition of tetrahydrobiopterin synthesis by 2,4-diamino-6-hydroxypyrimidine had no signifi- cant synergistic effect on MPP + toxicity. In contrast, the tetrahydrobiopterin precursor L-sepiapterin attenuated the MPP + -induced dopamine depletion and loss of tyrosine hydroxylase-positive cells in a dose-dependent manner with 40 lML-sepiapterin providing maximal protection. Accordingly, increasing intracellular tetrahydrobiopterin levels may protect against oxidative stress by complex-I inhibition. Keywords: dopamine, MPTP, organotypic culture, oxidative stress, Parkinson’s disease. J. Neurochem. (2003) 85, 214–223. Mitochondrial dysfunction and oxidative stress, including complex-I deficits and glutathione depletion, have been implicated in the loss of nigral dopamine (DA) neurones in Parkinson’s disease (PD) and the selective toxicity of MPTP, its metabolite MPP + (Jenner 1998) and rotenone (Betarbet et al. 2000) against these neurones. Dopaminergic neurones grown as primary dissociated cell cultures or as organotypic mesencephalic slice cultures are, however, preferentially resistant to the oxidative stress induced by glutathione depletion, possibly due to high intrinsic levels of tetrahydro- biopterin (BH 4 ) in DA neurones (Nakamura et al. 2000a,b; Gramsbergen et al.2002).Tetrahydrobiopterinisanessential cofactor for aromatic amino acid hydroxylases, including tyrosine hydroxylase (TH; Kaufman 1993), and all three forms of nitric oxide synthase (NOS; Kwon et al. 1989) and may act as an intrinsic antioxidant, in particular as a scavenger of superoxide (Nakamura et al. 2001). Toxic effects of BH 4 have, however, been reported in a variety of cell lines, including catecholaminergic cells (Choi et al. 2000). Because of its role as a cofactor for TH, BH 4 was considered for use as an antiparkinsonian drug more than a decade ago. A limited number of PD patients have indeed been treated with BH 4 , but with conflicting results (Dissing et al. 1989 and references therein). However, the possible role of BH 4 as an antioxidant or neuroprotective agent in Received April 11, 2002; revised manuscript received November 12, 2002; accepted December 19, 2002. Address correspondence and reprint requests to Dr Jan Bert Gramsbergen, Anatomy and Neurobiology, Institute of Medical Biology (IMB), SDU-Odense University, Winsloewparken 21, DK-5000 Odense C, Denmark. E-mail: jbgramsbergen@health.sdu.dk Abbreviations used:BH 4 , tetrahydrobiopterin; DA, dopamine; DAHP, 2,4-diamino-6-hydroxypyrimidine; DOPAC, 3,4-dihydroxyphenylacetic acid; FBS, fetal bovine serum; GDNF, glial-derived neurotrophic factor; HVA,homovanillicacid;LDH,lactatedehydrogenase;NOS,nitricoxide synthase; PCA, perchloric acid; PD, Parkinson’s disease; PI, propidium iodide;TBS,Tris-bufferedsaline;TH,tyrosinehydroxylase;VM,ventral mesencephalic. Journal of Neurochemistry , 2003, 85, 214–223 doi:10.1046/j.1471-4159.2003.01666.x 214 Ó 2003 International Society for Neurochemistry, J. Neurochem. (2003) 85, 214–223