Ž . Molecular Brain Research 64 1999 69–79 Research report Expression of calbindin-D in C6 glial cells stabilizes intracellular 28k calcium levels and protects against apoptosis induced by calcium ionophore and amyloid b-peptide Roman P. Wernyj a , Mark P. Mattson b , Sylvia Christakos a, ) a Departments of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical and Graduate School of Biomedical Sciences, 185 South Orange AÕenue, Newark, NJ 07103-2714, USA b Sanders–Brown Research Center on Aging and Department of Anatomy and Neurobiology, UniÕersity of Kentucky, Lexington, KY 40536, USA Accepted 3 November 1998 Abstract The calcium binding protein, calbindin-D is normally present in neurons. Recently we reported that brain injury and tumor necrosis 28k Ž . factors TNFs induce calbindin-D in astrocytes. TNF-treated calbindin expressing astrocytes were resistant to acidosis and calcium 28k Ž ionophore toxicity, suggesting that calbindin may have a cytoprotective role in astrocytes in the injured brain M.P. Mattson, B. Cheng, S.A. Baldwin, V.L. Smith-Swintosky, J. Keller, J. Geddes, Scheff, J.W., Christakos, S., Brain injury and tumor necrosis factors induce Ž . . calbindin-D in astrocytes: evidence for a cytoprotective response, J. Neurosci. Res., 42 1995 257 . In order to obtain direct evidence 28k for a role of calbindin, using the eukaryotic expression vector pREP4, rat calbindin-D was stably expressed in C6 rat astocytoma glial 28k Ž cells. Cytotoxicity in response to calcium ionophore or amyloid b-peptide which accumulates in the brain in Alzheimer’s disease and has . been reported to be neurotoxic was measured by MTT reduction in vector transfected cells and in calbindin transfected clones. Stably Ž . Ž expressed calbindin resulted in increased cell survival in the presence of calcium ionophore 1–10 mM or amyloid b-peptide 10–100 . mM . In addition, the calcium ionophore or amyloid b-peptide mediated rise in intracellular calcium in vector transfected cells was significantly attenuated in calbindin expressing cells. Apoptotic cell death was detected by the Hoechst method in vector transfected C6 Ž . glial cells treated with calcium ionophore or b-amyloid 34–36% apoptotic cellsrculture . The number of apoptotic nuclei was Ž . significantly attenuated in similarly treated calbindin-D transfected clones 10–13% apoptotic cellsrculture; p -0.01 . Our results 28k support the involvement of calcium fluxes in apoptosis and suggest that calbindin-D , by buffering calcium, can suppress death in 28k apoptosis susceptible cells in the central nervous system. q 1999 Elsevier Science B.V. All rights reserved. Keywords: Calbindin-D ; Apoptosis; Calcium; Beta amyloid 28k 1. Introduction Calbindin-D is a major calcium binding protein in 28k brain which is present predominantly in the cytosol and constitutes between 0.1–1.5% of the total soluble proteins w x in brain 13 . Calbindin-D is expressed by subpopula- 28k w x tions of neurons in most brain regions 8,20 . Previous studies provided correlative evidence between decreases in w x neuronal calbindin and neurodegeneration 28,30,32 and a direct relationship between calbindin-D immunoreactiv- 28k ity in hippocampal neurons and resistance to excitotoxic ) Corresponding author. Fax: q1-973-972-5594; E-mail: christak@umdnj.edu w x and ischemic injury has been observed 23,49,60,62 . In addition calbindin positive neurons exhibit reduced free intracellular calcium levels in response to excitatory amino w x acids and calcium ionophore 49 , suggesting a role for calbindin in reducing intracellular calcium levels and thereby preventing calcium mediated neuronal death. Di- rect evidence for a neuroprotective role of calbindin has been obtained from studies of primary neuronal cultures or cell lines overexpressing calbindin. Overexpression of cal- bindin has been reported to result in resistance to degener- ation induced by a variety of insults which involve calcium dependent events including exposure to calcium ionophore w x w x 17 , dexamethasone 17 , IgG from amyotrophic lateral Ž . w x w x sclerosis ALS patients 29 , hypoglycemia 52 , mutant w x w x presenilin 26 and amyloid b peptide 26 . Studies using 0169-328Xr99r$ - see front matter q 1999 Elsevier Science B.V. All rights reserved. Ž . PII: S0169-328X 98 00307-6