Calpain activity contributes to the control of SNAP-25 levels in neurons Carlotta Grumelli a , Paul Berghuis b , Davide Pozzi a , Matteo Caleo c , Flavia Antonucci c , Giambattista Bonanno d , Giorgio Carmignoto e , Marton Benedek Dobszay b , Tibor Harkany b,f , Michela Matteoli a,g , Claudia Verderio a, ⁎ a Istituto CNR di Neuroscienze, Dipartimento di Farmacologia Medica, Universita' di Milano, Italy b Division of Molecular Neurobiology, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, Stockholm, Sweden c Istituto CNR di Neuroscienze, Pisa, Italy d Department of Experimental Medicine, Center of Excellence for Biomedical Research, University of Genova, Italy e Istituto CNR di Neuroscienze, Dipartimento di Scienze Biomediche Sperimentali, Università di Padova, Italy f Institute of Medical Sciences, College of Life Sciences and Medicine, Foresterhill, University of Aberdeen, Scotland, United Kingdom g IRCCS Fondazione Don Gnocchi, Milano, Italy abstract article info Article history: Received 25 February 2008 Revised 4 July 2008 Accepted 8 July 2008 Available online 29 July 2008 Calpains are a family of calcium-dependent proteases with abundant expression in the CNS, and potent in cleaving some synaptic components. Assessment of calpain activity by its fluorescent substrate, Boc-Leu- Met-CMAC, revealed that cultured neurons display a significant level of constitutive enzyme activity. Notably, calpain activity differs in distinct neuronal populations, with a significantly higher level of activity in GABAergic cells. Using selectively-enriched cultures of fast-spiking GABAergic interneurons, we show that calpain activity partially contributes to the post-translational down regulation of SNAP-25, a calpain substrate, in differentiated GABA cells. In addition, we demonstrate that SNAP-25 is cleaved by calpain in response to acute seizures induced by intraperitoneal kainate injection in vivo. These data indicate that calpains in neurons are active even at physiological calcium concentrations and that different levels of calpain activation in selected neuron subtypes may contribute to the pattern of synaptic protein expression. © 2008 Elsevier Inc. All rights reserved. Introduction Calpains are a family of calcium-dependent neutral proteases which cleave several cytosolic, membrane or cytoskeleton-associated proteins. Proteolysis by calpain is likely to change the integrity, localization, and/or activity of endogenous proteins, and results in either the activation or the inhibition of substrate functions. Despite the fact that more than 100 proteins have been identified as calpain substrates, the sequential/structural determinants of calpain recogni- tion are not completely understood. Various enzymatic determinants, such as amino acid preference, secondary structure, and PEST segments, have been shown to correlate with the cleavage site selected by calpain (Tompa et al., 2004). At least three ubiquitous calpains, calpain-1 (μ-calpain), calpain-2 (m-calpain), and calpain-10 are highly expressed in the central nervous system along with the preferential localization of several calpain substrates, including voltage-gated calcium channels (Hell et al., 1996), NMDA receptor subunits (Guttmann et al., 2001), postsynaptic density proteins (Lu et al., 2000), kinases, and phosphatases, to synaptic compartments in neurons (Wu and Lynch, 2006). Recent data suggest that some components of the fusion machinery, in particular the SNARE proteins SNAP-25 and SNAP-23 are substrates of various members of the calpain family in neurons (Ando et al., 2005), as previously described in alveolar epithelial cells (Zimmerman et al., 1999), platelets (Lai and Flaumenhaft, 2003; Rutledge and Whiteheart, 2002) and pancreatic islets (Marshall et al., 2005). Whether the cleavage of these substrates occurs only under pathological (Xu et al., 2007) or physiological conditions is still ambiguous. SNAP-25 and SNAP-23 are plasma membrane proteins which together with their partner SNAREs, the membrane protein syntaxin and the synaptic vesicle protein synaptobrevin/VAMP, form the fusion complex mediating regulated exocytosis. The calpain members proteolitically cleaving SNARE proteins include calpain-1 (Rutledge and Whiteheart, 2002) and calpain-10 (Marshall et al., 2005), an atypical calpain, which lacks the calcium/calmodulin domain (Ma et al., 2001). Analysis of SNAP-23 cleavage products suggests that the calpain cleavage site(s) is situated in the C-terminal third of the molecule, potentially between the cysteine-rich acyl attachment sites and the C-terminal coiled-coil domain (Rutledge and Whiteheart, 2002). The calpain cleavage site in SNAP-25 has been instead localized to the N-terminal third of the molecule in cultured cerebellar granule cells (Ando et al., 2005) and to a region adjacent to the N-terminus in insulinoma INS-1 cells (Marshall et al., 2005). Molecular and Cellular Neuroscience 39 (2008) 314–323 ⁎ Corresponding author. via Vanvitelli 32 20129 Milano, Italy. Fax: +39 027490574. E-mail address: c.verderio@in.cnr.it (C. Verderio). 1044-7431/$ – see front matter © 2008 Elsevier Inc. All rights reserved. doi:10.1016/j.mcn.2008.07.011 Contents lists available at ScienceDirect Molecular and Cellular Neuroscience journal homepage: www.elsevier.com/locate/ymcne