Neuroprotective effect of zVAD against the neurotoxin 3-nitropropionic acid involves inhibition of calpain Nicolas Bizat a , Marie-Christine Galas b,c , Carine Jacquard a , Fre´de´ric Boyer a , Jean-Michel Hermel a , Serge N. Schiffmann b , Philippe Hantraye a , David Blum b,d , Emmanuel Brouillet a, * a URA CEA-CNRS 2210, Service Hospitalier Fre ´de´ric Joliot and Program ImaGene, DRM, DSV, CEA, 4 place du Ge ´ne ´ral Leclerc, 91401 Orsay, France b Laboratoire de Neurophysiologie, ULB-Erasme, CP601, 808 route de Lennik, 1070 Brussels, Belgium c INSERM U422, 1 place de Verdun, 59045 Lille cedex, France d Laboratoire de Neurochirurgie Expe ´rimentale and IRIBHM, ULB-Erasme, CP602, 808 route de Lennik, 1070 Brussels, Belgium Received 11 January 2005; received in revised form 25 April 2005; accepted 28 April 2005 Abstract The contribution of calpains and caspases to cell death has been widely studied using pharmacological inhibitors. Among them, the caspase inhibitor N-benzyloxycarbonyl-valyl-alanyl-aspartyl-fluoromethylketone (zVAD) has been used as a specific caspase inhibitor in nearly 1000 published studies. However, several studies showed that zVAD also behaves as a calpain inhibitor in peripheral cells. The effects of zVAD as a calpain inhibitor have never been assessed in neurodegeneration models. We examined here whether zVAD could reduce neurodegeneration in Huntington’s disease models using the mitochondrial inhibitor 3- nitropropionic acid (3NP). In these models, 3NP toxicity has been shown to require calpain activation. In rats, intra-cerebro- ventricular infusion of zVAD significantly reduced 3NP-induced striatal degeneration, and decreased the 3NP-induced activation of calpain and calpain-dependent cleavage of fodrin. zVAD (100 mM) also blocked 3NP-induced death of cultured striatal neurons. In vitro, zVAD inhibited purified m-calpain with high affinity (IC 50 Z 10 nM). The present data demonstrate that zVAD protects neurons against 3NP through calpain inhibition. This suggests that, in certain models of neuronal death where zVAD showed protective effects, caspases but also calpains may be involved. Ó 2005 Elsevier Ltd. All rights reserved. Keywords: Neurodegeneration; Succinate dehydrogenase; Mitochondrial complex II inhibitor; Calpain; Caspase; Calpain inhibitor; Neuroprotection 1. Introduction There is compelling evidence that the cysteine protease caspases play a major role in apoptosis of neurons during brain development (Thornberry and Lazebnik, 1998). Activation of upstream caspases such as caspase-9 produces downstream activation of effector caspases, leading to neuronal death. Abnormal activation of caspases may also play a role in acute and chronic pathological situations affecting the central nervous system, such as brain ischemia and neurodegenerative diseases, respectively. In addition, the Ca 2C -activated neutral protease calpains can also play a major role in pathological neuronal death (Wang, 2000). Brain cal- pains mainly include m-calpain and m-calpain, the * Corresponding author. URA CEA-CNRS 2210, Neuronal Death Group, Department of Medical Research, Service Hospitalier Fre´de´ric Joliot and Program ImaGene, DRM, DSV, CEA, 4 place du Ge´ne´ral Leclerc, 91401 Orsay, France. Tel.: C33 1 69 86 78 15; fax: C33 1 69 86 77 45. E-mail address: brouille@shfj.cea.fr (E. Brouillet). 0028-3908/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.neuropharm.2005.04.030 Neuropharmacology 49 (2005) 695e702 www.elsevier.com/locate/neuropharm