Journal of Neurochemistry Lippincott—Raven Publishers, Philadelphia © 1997 International Society for Neurochemistry The Amyloid ,8-Protein of Alzheimer’ s Disease Increases Acetylcholinesterase Expression by Increasing Intracellular Calcium in Embryonal Carcinoma P19 Cells Gian Sberna, Javier Sáez-Valero, *Konrad Beyreuther, Cohn L. Masters, and David H. Small Department of Pathology, University of Melbourne, Parkville, Victoria, Australia; and *Centre of Molecular Biology (ZMBH), University of Heidelberg, Heidelberg, Germany Abstract: One of the characteristic changes that occurs in Alzheimer’s disease is the loss of acetylcholinesterase (AChE) from both cholinergic and noncholinergic neurons of the brain. However, AChE activity is increased around amyloid plaques. This increase in AChE may be of sig- nificance for therapeutic strategies using AChE inhibitors. The aim of this study was to examine the effect of amyloid /3-protein (A/3), the major component of amyloid plaques, on AChE expression. A/3 peptides spanning residues 1 — 40 or 25—35 increased AChE activity in P19 embryonal carcinoma cells. A peptide containing a scrambled A/3 2535 sequence did not stimulate AChE expression. To examine the possibility that the increase in AChE expression was mediated by an influx of calcium through voltage-depen- dent calcium channels (VDCCs), drugs acting on VDCCs were tested for their effects. Inhibitors of L-type VDCCs (diltiazem, nifedipine, and verapamil), but not N- or P- or Q-type VDCCs, resulted in a decrease in AChE expres- sion. Agonists of L-type VDCCs (maitotoxin and S(—)- Bay K 8644) increased AChE expression. As L-type VDCCs are known to be modulated by cyclic AMP-de- pendent protein kinase, the effect of the adenylate cy- clase activator forskolin was also examined. Forskolin stimulated AChE expression, an action that was blocked by the L-type VDCC antagonist nifedipine. The A/32535- induced increase in AChE expression was mediated by an L-type VDCC, as the effect was also blocked by nifedi- pine. The results suggest that the increase in AChE ex- pression around amyloid plaques could be due to a dis- turbance in calcium homeostasis involving the opening of L-type VDCCs. Key Words: Acetylcholinesterase— Amyloid /3-protein—Voltage-dependent calcium chan- nel—Alzheimer’s disease—Cyclic AMP. J. Neurochem. 69, 1177—1184 (1997). Accumulation of the amyloid /3-protein (A/3) in the brain is a defining feature of Alzheimer’s disease (AD). The heterogenous processing of the amyloid protein precursor (APP) can result in the production of A/3 peptides, which range from 39 to 43 amino acids in length (Masters et a!., 1985; Kang et al., 1987; Prelli et al., 1988). Increasing evidence indicates that overproduction of A/3, particularly those forms that aggregate into amyloid fibrils, is an early event in the pathogenesis of the disease (Evin et al., 1994). A/3 has been found to be toxic to cells in culture, because it disrupts calcium homeostasis in human cortical neu- rons (Mattson et al., 1992) and other cell types (Joseph and Han, 1992; Davidson et al., 1994; Takenouchi and Munekata, 1994; Brorson et al., 1995; Zhou et al., 1996). The disruption of calcium homeostasis may be mediated in part through the opening of L-type volt- age-dependent calcium channels (VDCCs) (Davidson et al., 1994; Weiss et al., 1994; Ueda et al., 1997). Two of the characteristic neurochemical changes that occur in the AD brain are the loss of acetylcholin- esterase (AChE; EC 3.1.1.7) and choline acetyltrans- ferase activity (Davies and Maloney, 1976; Atack et al., 1983; Fishman et al., 1986). Despite the overall loss of AChE in the brain, AChE activity is increased in plaques and tangles very early in the process of amyloid deposition (Ulrich et al., 1990; Moran et al., 1993). It has been hypothesized that AChE may pro- mote aggregation of A/3 into amyloid (Inestrosa et al., 1996), although other proteins are likely to contribute to this process (Snow et al., 1988; Kalaria et al., 1991; Received January 31, 1997; revised manuscript received April 14, 1997; accepted April 21, 1997. Address correspondence and reprint requests to Dr. D. H. Small at Laboratory of Molecular Neurobiology, Department of Pathology, University of Melbourne, Parkville, Victoria 3052, Australia. Abbreviations used: A/3, amyloid /3-protein; AChE, acetylcholin- esterase; AD, Alzheimer’s disease; APP, amyloid protein precursor; AraC, cytosine arabinoside; S(—)-Bay K 8644, S(—)-l,4-dihydro- 2,6-dimethyl-5-nitro-4- [ 2-(trifluoromethyl)phenyl ] -3-pyridinecar- boxylic acid methyl ester; [Ca 2 ]~, intracellular free calcium con- centration; cAMP, cyclic AMP; DMSO, dimethyl sulfoxide; EC, embryonal carcinoma; fluo-3/AM, fluo-3 acetoxymethyl ester; NS buffer, normal salt buffer; PKA, cyclic AMP-dependent protein kinase; RA, retinoic acid; Rp-cAMPS, Rp-adenosine 3 ‘,5 ‘-cy- clic monophosphothioate triethylamine; scram. A/3 25_35, scrambled A/32535 peptide; VDCC, voltage-dependent calcium channel. 1177