Calpain activation and a-spectrin cleavage in rat brain by ethanol Yadavalli Rajgopal, Mohan C. Vemuri * School of Life Sciences, University of Hyderabad, Hyderabad 500 046, India Received 29 October 2001; received in revised form 8 January 2002; accepted 10 January 2002 Abstract Calpain, a calcium-activated cysteine protease, has been implicated in neuronal degeneration and death. In this study, we have characterized calpain activation in adult rat cerebral cortex and cerebellum, using an experimental paradigm of in vivo chronic ethanol exposure. Ethanol treatment increased the calpain activity in cortex and cerebellum, but to a higher extent in the cortex. Western blot analysis revealed a significant decrease in m-calpain levels while calpastatin levels were unaltered. Calpain activation was further monitored by the proteolysis of a-spectrin (fodrin) and protein kinase C-alpha (PKC-a). Protease specific spectrin breakdown products revealed calpain generated 150- and 145-kDa fragments. In addition, we also observed a 120-kDa fragment characteristic of caspase-3 activation in the cerebellum. PKC-a levels were decreased in the cortex and cerebellum by ethanol. Calpain activation, cleavage of a-spectrin into calpain specific signature fragments and decreased PKC-a protein levels after ethanol treatment provide the evidence of calpain involvement besides caspase-3-mediated cell death in the cortex and cerebellum. Given the role of calpains in cell death, increased calpain activity followed by a-spectrin cleavage in this study suggests that calpains are important effectors in ethanol-mediated cell injury and alcoholic neurodegeneration. q 2002 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Ethanol; m-Calpain; Calpastatin; a-Spectrin; Cerebral cortex; Cerebellum Calpain or calcium-dependent cysteine protease (EC 3.4.22.17) is ubiquitously expressed in mammalian cells [15]. Calpain participates in various calcium-mediated signaling pathways [16] and exists as two isoenzymes, Calpain I or m-calpain, and Calpain II or m-calpain, that are distinguished by their in vitro calcium requirements. Each isoform is composed of a 28-kDa regulatory subunit that is identical for both isoforms, and a 80-kDa catalytic subunit unique to each isoform [15]. Calpastatin, an endo- genous protein inhibitor, is known to regulate calpain activ- ity, and a co-existence of calpain and calpastatin in cells suggests tightly controlled calpain–calpastatin interactions [3]. The widespread distribution of calpain as a cytosolic protease and the property of activation by calcium suggest a role in physiological as well as pathological events. The physiological functions of calpains remain poorly under- stood except for their possible role in long-term potentia- tion, memory formation, synaptic neurotransmission and exocytosis [7]. However, neuronal calpains appear to be activated uncontrollably by sustained elevation of cytosolic calcium levels under pathological conditions such as ische- mia and spinal cord injury [2], as well as in neurodegenera- tive diseases like Alzheimer’s disease [18], Parkinson’s disease [9] and amyotrophic lateral sclerosis [19]. Ethanol is a neuroactive addictive drug, and chronic alco- hol consumption produces a wide spectrum of physiological and pathological changes, including cognitive impairment and seizures. Several studies suggest loss of neurons in specific regions of brain in chronic alcoholics, although the mechanisms underlying such reduced neuronal numbers remain elusive [8]. While the involvement of caspase-3, another cysteine protease, has been attributed in ethanol- induced cell death, the contribution of calpains in ethanol- mediated neuronal cell death and central nervous system injury remains elusive. To our knowledge, the expression and activity of calpain has not been investigated in an experimental alcoholic paradigm. Therefore, in the present study, expression of m-calpain, calpastatin protein and total calpain activity is reported. In addition, proteolytic degrada- tion of a-spectrin (fodrin) and protein kinase C-alpha (PKC- a) was studied as an evidence to assess calpain activation following chronic ethanol treatment. The delineation of Neuroscience Letters 321 (2002) 187–191 0304-3940/02/$ - see front matter q 2002 Elsevier Science Ireland Ltd. All rights reserved. PII: S0304-3940(02)00063-0 www.elsevier.com/locate/neulet * Corresponding author. Department of Pathology, Anatomy and Cell Biology, Building JAH Room # 511, Thomas Jefferson University, 1020 Locust Street, Philadelphia, PA 19107, USA. Tel.: 11-215-503-7837; fax: 11-215-923-3808. E-mail address: mohan.c.vemuri@lycos.com (M.C. Vemuri).