University of Groningen Inhibition of Calpain Prevents N-Methyl-D-aspartate-Induced Degeneration of the Nucleus Basalis and Associated Behavioral Dysfunction

N-Methyl-D-aspartate (NMDA) receptor-mediated excitotoxicity is thought to underlie a variety of neurological disorders, and inhibition of either the NMDA receptor itself, or molecules of the intracellular cascade, may attenuate neurodegeneration in these diseases. Calpain, a calcium-dependent cysteine protease, has been identified as part of such an NMDA receptorinduced excitotoxic signaling pathway. The present study addressed the question of whether inhibition of calpain can prevent neuronal cell death and associated behavioral deficits in a disease-relevant animal model, which is based on excitotoxic lesions of the cholinergic nucleus basalis magnocellularis of Meynert. Excitotoxic lesions of the nucleus basalis with NMDA induced a markedly impaired performance in the novel object recognition test. Treatment with the calpain inhibitor, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1-yl]benzamide (A-705253), dose-dependently prevented the behavioral deficit. Subsequent analysis of choline acetyltransferase in the cortical mantle of the lesioned animals revealed that application of A-705253 dose-dependently and significantly attenuated cholinergic neurodegeneration. Calpain inhibition also significantly diminished the accompanying gliosis, as determined by immunohistochemical analysis of microglia activation. Finally, inhibition of calpain by A-705253 and the peptidic calpain inhibitor N-acetyl-Leu-LeuNle-CHO did not impair long-term potentiation in hippocampal slices, indicating that calpain inhibition interrupts NMDA excitotoxicity pathways without interfering with NMDA receptormediated signaling involved in cognition. We conclude that inhibition of calpains may represent a valuable strategy for the prevention of excitotoxicity-induced neuronal decline without interfering with the physiological neuronal functions associated with learning and memory processes. Thus, calpain inhibition may be a promising and novel approach for the treatment of various neurodegenerative disorders. Calpains in the central nervous system are Ca -dependent cysteine proteases, which can modulate the conformation, localization, and activity of various proteins. Calpaininduced modifications have a profound affect on biological processes as different as cell cycle progression and vesicular trafficking. They are an integrative part of several signaling cascades, but they can also contribute to apoptotic and necrotic cell death in pathological conditions. For this reason, calpains have been discussed extensively as target for therapeutic interventions in a number of neurodegenerative disease that are associated with neuronal loss (for review, see Huang and Wang, 2001; Goll et al., 2003; Zatz and Starling, 2005). In that respect, it has recently been shown that inhibition of calpains prevents excitotoxic neuronal cell death in vitro (Caba et al., 2002; Ray et al., 2006) and in vivo (Chiu et al., 2005; Takano et al., 2005). Excitotoxicity is caused by an overstimulation of N-methyl-D-aspartate (NMDA) receptors, and there is now growing evidence that calpain cleaves sevArticle, publication date, and citation information can be found at http://jpet.aspetjournals.org. doi:10.1124/jpet.108.142679. □S The online version of this article (available at http://jpet.aspetjournals.org) contains supplemental material. ABBREVIATIONS: NMDA, N-methyl-D-aspartate; A-705253, N-(1-benzyl-2-carbamoyl-2-oxoethyl)-2-[E-2-(4-diethlyaminomethylphenyl) ethen-1yl]benzamide; NBM, nucleus basalis magnocellularis; LTP, long-term potentiation; PBS, phosphate-buffered saline; MK-801, 5-methyl-10,11dihydro-5H-dibenzo[a,d]cyclohepten-5,10imine; ALLN, N-acetyl-Leu-Leu-Nle-CHO; ChAT, choline acetyltransferase; fEPSP, field excitatory postsynaptic potential; SBDP, spectrin breakdown product; APV, (2R)-amino-5-phosphonovaleric acid. 0022-3565/08/3272-343–352$20.00 THE JOURNAL OF PHARMACOLOGY AND EXPERIMENTAL THERAPEUTICS Vol. 327, No. 2 Copyright © 2008 by The American Society for Pharmacology and Experimental Therapeutics 142679/3397325 JPET 327:343–352, 2008 Printed in U.S.A. 343 at U nirsity of G roingen on S etem er 5, 2009 jpet.asjournals.org D ow nladed fom http://jpet.aspetjournals.org/cgi/content/full/jpet.108.142679/DC1 Supplemental Material can be found at: