Inhibition of the cdk5/p25 fragment formation may explain the antiapoptotic effects of melatonin in an experimental model of Parkinson’s disease Introduction Parkinson’s disease (PD) is the second most frequently diagnosed neuro-degenerative disorder and is characterized by rigidity, slowness in voluntary movements, and postural instability [1]. The symptoms of PD result from a loss of dopaminergic neurons in the substantia nigra pars comp- acta [1, 2]. Currently, the primary drug treatment against this disease is the administration of l-dopa and inhibitors of l-dopa decarboxylase, the aim being to increase brain levels of dopamine. Other treatments involve the adminis- tration of dopaminergic agonist and MAO B inhibitors [2]. The mechanism underlying dopaminergic neuronal death in this disease remains unknown. While the exact cause or mechanism of neuronal loss remains unknown, the use of neurotoxins to reproduce this disease in laboratory animals may facilitate our understanding of the mechanisms involved [3]. Thus, the neurotoxin 1-methyl-4-phenyl- 1,2,3,6-tetrahydropyridine (MPTP) and its metabolite 1- methyl-4-phenylpyridinium ion (MPP + ) are useful tools for inducing PD in experimental animal models, as well as in humans [4–9]. Furthermore, the utilization of MPP + clarifies understand the general role of mitochondria, and specifically the inhibition of the mitochondrial complex I in neuronal cell death [10–12]. In this neuronal degenerative process, following the inhibition of complex I, a well- known dramatic increase in oxidative stress occurs which is neurotoxic for neurons [7, 8]. Furthermore, this mitoch- ondrial alteration induces a release of such pro-apoptotic proteins as cytochrome c (Cyt c), apoptosis inducing factor, and Smac/diablo which regulate the apoptotic process in caspase-dependent or -independent pathways [4]. The first approach to achieve a neuronal antiapoptotic action employed antioxidants. Several studies have dem- onstrated that antioxidants such as vitamin E, vitamin C, melatonin, and deferoxamine exert neuro-protective effects in vitro, as well as in animals models of PD. Interestingly, a potential antioxidant drug for the treatment of PD currently in phase II trial is coenzyme Q 10 [7, 8]. These data confirm the importance of oxidative stress in the pathogenesis of this disease. Previous studies have demon- strated that MPP + -induced apoptosis is mediated through the release of Cyt c and by the activation of caspase-3 [13, 14]. However, the role of caspase activation in the neuronal Abstract: In this study, the effects of melatonin on MPP + -treated cerebellar granule neurons (CGNs) in culture were investigated. Results showed that MPP + treatment significantly decreased cell viability and increased the apoptotic cell population at 24 and 48 hr. Calpain and caspase-3 activation was also determined, with results showing a strong increase in calpain (74%) and caspase 3 activity (70%), as measured by a-spectrin cleavage and fluorometric and colorimetric analysis, respectively. There are several studies suggesting that the activation of the cdk5/p35 pathway at its cleavage to cdk5/p25 may play a role in neuronal cell death in neurodegenerative diseases. Moreover, these studies indicate that this cleavage is mediated by calpains, and that MPP + prompted an increase in cdk5 expression, as well as the cleavage of p35– p25, in a time-dependent manner. 1 mm Melatonin not only reduced the neurotoxic effects of MPP + on cell viability, but also prevented apoptosis mediated by this Parkinsonian toxin in CGNs. 1 mm Melatonin reduced cdk5 expression, as well as the cleavage of p35–p25. These data indicate that melatonin possesses some neuro-protective properties against MPP + -induced apoptosis. Moreover, these data suggest that the calpain/cdk5 signaling cascade has a potential role in the MPP + -mediated apoptotic process in CGNs. Daniel Alvira 1 *, Marta Tajes 1 *, Ester Verdaguer 1 , Dario Acun ˜ a- Castroviejo 2 , Jaume Folch 3 , Antoni Camins 1  and Merce ` Pallas 1  1 Unitat de Farmacologia i Farmacogno ` sia, Facultat de Farmacia, Universitat de Barcelona, Nucli Universitari de Pedralbes, Barcelona; 2 Departamento de Fisiologı ´a, Instituto de Biotecnologı ´a, Universidad de Granada, Granada; 3 Unitat de Bioquı ´mica i Biologı ´a Molecular, Facultat de Medicina i Cie ` ncies de la Salut, Universitat Rovira i Virgili, Reus, Spain Key words: apoptosis, calpain, caspase, cdk5, cerebellar granule cells, MPP + Address reprint requests to Merce Palla `s, Unitat de Farmacologia i Farmacogno `sia, Facultat de Farma ` cia, Universitat de Barce- lona, Nucli Universitari de Pedralbes, E-08028 Barcelona, Spain. E-mail: pallas@ub.edu *The first two authors contributed equally to this work.   Senior co-authors. Received September 21, 2005; accepted November 18, 2005. J. Pineal Res. 2006; 40:251–258 Doi:10.1111/j.1600-079X.2005.00308.x Ó 2006 The Authors Journal compilation Ó 2006 Blackwell Munksgaard Journal of Pineal Research 251