Brain Research 975 (2003) 214–221 www.elsevier.com / locate / brainres Research report Rapid reduction of ATP synthesis and lack of free radical formation 1 by MPP in rat brain synaptosomes and mitochondria 1 * Carlos Fonck , Michel Baudry Neuroscience Program, HNB124, USC, Los Angeles, CA 90089-2520, USA Accepted 21 March 2003 Abstract MPTP is a neurotoxin thought to damage dopaminergic neurons through free radical formation. MPTP is metabolized in the brain to 1 MPP , which is taken up into dopaminergic neurons via the dopamine transporter and assumed to impair mitochondrial function. We used 1 striatal synaptosomes and telencephalic mitochondria to further investigate MPP mechanism of action. For comparison, the respiratory toxins FCCP, a cyanide analog that uncouples mitochondrial ATP production, and rotenone, a NADH dehydrogenase inhibitor, were also 1 3 tested. FCCP, MPP and rotenone caused a rapid but stable decrease in [ H]dopamine (DA) uptake by striatal synaptosomes. Two free 1 radical scavengers, the salen-manganese complex EUK-134, and the spin trap s-PBN, did not prevent MPP -induced decrease in DA 1 3 uptake. However, addition of ATP during synaptosome preparation resulted in partial recovery of MPP -induced [ H]DA uptake 1 decrease. Generation of oxygen free radicals by treatment of telencephalic mitochondria with MPP , FCCP, or rotenone, was evaluated by 1 measuring DCF fluorescence, while light emission by the luciferin-luciferase complex was used to determine ATP levels. MPP , unlike rotenone, did not produce oxygen free radicals, but rather blocked ATP production in mitochondria, as did FCCP and rotenone. Taken 1 together, these results suggest that MPP toxicity, at least during its initial stages, is primarily due to a decrease in ATP synthesis by mitochondria and not to free radical formation.  2003 Elsevier Science B.V. All rights reserved. Theme: Disorders of the nervous system Topic: Neurotoxicity 1 Keywords: MPTP; MPP ; Rotenone; FCCP; Dopaminergic; Free radical; Parkinson’s disease; Synaptosome 1. Introduction toxic metabolite 1-methyl-4-phenylpyridinium ion 1 (MPP ). Dopaminergic neurons are the primary target of 1 1-Methyl,1-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP- MPP because this metabolite is a substrate for the )-induced degeneration of dopaminergic neurons has been dopamine transporter present in dopaminergic neurons 1 proposed as an experimental animal model to study [26]. MPP accumulates inside mitochondria where it Parkinson’s disease (recently reviewed in Ref. [14] ). In interrupts the respiratory chain by inhibiting NADH dehy- humans [13,27], monkeys [5], and rodents [22,24], MPTP drogenase, one of the complex I enzymes of the electron 1 selectively damages dopaminergic neurons in the sub- transport chain [34]. Binding of MPP to NADH dehydro- stantia nigra, thus causing Parkinson’s disease-like symp- genase is thought to occur near the binding site for the toms. Inside the brain, the dopamine-degrading enzyme, respiratory toxin rotenone [39]. Disruption of the electron monoamine oxidase B [7,31] transforms MPTP into the transport chain may lead not only to ATP depletion [6], but also to superoxide radical formation, as NADH would no longer transfer reducing equivalents to oxidized ubiquin- 1 *Corresponding author. Tel.: 11-213-740-9188; fax: 11-213-740- one [23]. It has been suggested that MPP -induced 5687. formation of reactive oxygen species (ROS) could func- E-mail address: baudry@neuro.usc.edu (M. Baudry). 1 tionally and structurally damage mitochondria as well as Present address: Division of Biology, California Institute of Technology, Pasadena, CA 91125, USA. other cellular components and ultimately cause dopa- 0006-8993 / 03 / $ – see front matter  2003 Elsevier Science B.V. All rights reserved. doi:10.1016 / S0006-8993(03)02675-1