Calpain activation and CaMKIV reduction in spinal cords from hSOD1G93A mouse model Myriam Gou-Fabregas a,1 , Omar Ramírez-Núñez b , Daniel Cacabelos b , Nuria Bahi a , Manuel Portero b , Ana Garcera a,2 , Rosa M. Soler a, ⁎ ,2 a Unitat de Senyalització Neuronal, Dept Ciencies Mediques Basiques, Facultat de Medicina, Universitat de Lleida, IRBLLEIDA, Rovira Roure, 80, 25198 Lleida, Spain b Dept Medicina Experimental, Facultat de Medicina, Universitat de Lleida, IRBLLEIDA, Rovira Roure, 80, 25198 Lleida, Spain abstract article info Article history: Received 24 October 2013 Revised 17 July 2014 Accepted 21 July 2014 Available online 22 July 2014 Keywords: ALS Neurodegeneration Intracellular calcium CaMKIV Calpain hSOD1G93A Amyotrophic Lateral Sclerosis (ALS), a severe neurodegenerative disease, affects the upper and lower motor neu- rons in the brain and spinal cord. In some studies, ALS disease progression has been associated with an increase in calcium-dependent degeneration processes. Motoneurons are specifically vulnerable to sustained membrane de- polarization and excessive elevation of intracellular calcium concentration. The present study analyzed intracel- lular events in embryonic motoneurons and adult spinal cords of the hSOD1G93A ALS mouse model. We observed activation of calpain, a calcium-dependent cysteine protease that degrades a variety of substrates, and a reduction in calcium–calmodulin dependent protein kinase type IV (CaMKIV) levels in protein extracts from spinal cords obtained at several time-points of hSOD1G93A mice disease progression. However, in cultured embryonic motoneurons these differences between controls and hSOD1G93A mutants are not evident. Our re- sults support the hypothesis that age-dependent changes in calcium homeostasis and resulting events, e.g., calpain activation and CaMKIV processing, are involved in ALS pathogenesis. © 2014 Elsevier Inc. All rights reserved. Introduction Amyotrophic Lateral Sclerosis (ALS) is a fatal, adult-onset, neurode- generative disease causing the degeneration of cranial and spinal cord motor neurons (MNs), which leads to muscle atrophy and paralysis. Nu- merous ALS studies have analyzed subgroups of familial cases originat- ing from mutations in the SOD1 gene (Rosen et al., 1993). The precise mechanisms whereby mutant SOD1 is toxic to MNs are not defined. However, studies in mutant hSOD1 transgenic mice have revealed many pathogenic changes in degenerating MNs, including hyper- excitability, disturbed calcium homeostasis, SOD1 aggregation, and acti- vation of cell death signals (Bento-Abreu et al., 2010; Cleveland and Rothstein, 2001; Pasinelli and Brown, 2006). Several of these pathogenic changes are related to calcium deregulation in neurons. Unfortunately, whether these are primary or secondary events or represent compensa- tory mechanisms remains unknown. MNs are specifically vulnerable to membrane depolarization and in- creased intracellular calcium concentration (Arakawa et al., 2002). Levels of intracellular calcium determine neuronal dependence on neu- rotrophic factors and susceptibility to cell death, although how calcium induces MN cell death is not fully understood. Diverse molecular mech- anisms that lead to neuronal degeneration and death in response to ex- cessive calcium influx are being elucidated, among them the activation of specific enzymes such as protein phosphatases, endonucleases, or proteases. One of these enzymes is calpain, the calcium-sensitive prote- ase that mediates cell death when intracellular calcium is increased (Das et al., 2005). Previous results from our group and others have shown that chron- ically depolarizing conditions induces cell death in mouse MNs through increased intracellular calcium and calpain activation (Gou-Fabregas et al., 2009; Kaiser et al., 2006). This protease can become over- activated under extreme conditions — for example, as a consequence of sustained elevation of cytosolic calcium levels, which in turn is asso- ciated with apoptotic or non-apoptotic cell death (Wang, 2000). In the central nervous system, calpain activation is related to neuronal damage in ischemia, stroke, and Alzheimer and Huntington diseases (Cowan et al., 2008; Shields et al., 2000; Yamashima, 2013). One of these calpain substrates is the calcium–calmodulin dependent protein kinase type IV (CaMKIV). This kinase is highly expressed in the nervous system and is Molecular and Cellular Neuroscience 61 (2014) 219–225 Abbreviations: ALS, Amyotrophic Lateral Sclerosis; MN, spinal cord motor neurons; hSOD1G93A, mutant human superoxide dismutase 1; CaMKIV, calcium–calmodulin de- pendent protein kinase IV; NTF, neurotrophic factor; BDNF, brain derived neurotrophic factor; GDNF, glial cell line-derived neurotrophic factor; CNTF, ciliary neurotrophic factor; CT-1, cardiotrophin I; HGF, hepatocyte growth factor; WT, wild type. ⁎ Corresponding author at: Edif. Biomedicina I. Lab 4.1, Universitat de Lleida-IRBLLEIDA, Rovira Roure, 80, 25198-Lleida, Spain. E-mail address: rosa.soler@cmb.udl.cat (R.M. Soler). 1 Present address: Dept of Morphology & Biomedical Research Institute, Hasselt University, Diepenbeek BE3590, Belgium. 2 AG and RMS are senior co-authors. http://dx.doi.org/10.1016/j.mcn.2014.07.002 1044-7431/© 2014 Elsevier Inc. All rights reserved. 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