Calcium regulates processing of the Alzheimer amyloid protein precursor in a protein kinase C-independent manner.

Abstract

Various first messengers linked to phospholipase C, including acetylcholine and interleukin 1, regulate the production both of the secreted form of the amyloid protein precursor (APP) and of amyloid a-protein. We have now identified intracellular signals which are responsible for mediating these effects. We show that activation of phospholipase C may affect APP processing by either of two pathways, one involving an increase in protein kinase C and the other an increase in cytoplasmic calcium levels. The effects ofcalcium on APP processing appear to be independent of protein kinase C activation. The observed effects of calcium on APP processing may be of therapeutic utility.Alzheimer disease is characterized by distinct neuropathological lesions, including intracellular neurofibrillary tangles, extracellular parenchymal and cerebrovascular amyloid deposits; and selective cell death that particularly affects cholinergic neurons in the basal forebrain (1). The principal component of parenchymal amyloid plaque cores and cerebrovascular amyloid is the amyloid (3-protein (A(3) (2-4). It has been shown that this -4-kDa protein is produced by various cultured cells (5-7), including transfected cells stably expressing the amyloid protein precursor (APP), from which AB is derived (8)(9)(10)(11)(12)(13)(14).During the past few years, a variety of evidence has emerged indicating that the processing of APP is regulated by signal transduction pathways. Thus, phorbol esters (activators of protein kinase C) and okadaic acid (an inhibitor of protein phosphatases 1 and 2A) increase APP metabolism and secretion (15-18). More recently, it has been shown that first messengers known to activate the phospholipase C/protein kinase C cascade increase the secretion of APP (17,19). It was also shown that the formation of a peptide with properties similar to those of Af was decreased by phorbol esters, by okadaic acid, by direct activators of phospholipase C, and by first messengers that activate phospholipase C (20-22). However, activation of phospholipase C not only activates protein kinase C (through the formation ofdiacylglycerol) but also increases cytoplasmic calcium (through the action of inositol 1,4,5-trisphosphate, IP3). For this reason, we undertook an investigation to determine whether the IP3/calcium limb of this pathway might, like the diacylglycerol/protein kinase C limb, affect APP processing. MATERIALS AND METHODSCell culture conditions and the sources of analytical reagents have been described (17,20 Pulse-chase labeling of cells was carried out on confluent cell monolayers in six-well culture dishes (Corning) with 1 ml of methionine-free Dulbecco's modified Eagle's medium (DMEM) supplemented with 1 mCi (37 MBq) of [35S]methionine/cysteine (EXPRE35S35S; NEN). Metabolic labeling was carried out for 2 hr, followed by a chase period of 2 hr. The chase was initiated by replacing the labeling medium with DMEM containing 0.2 mM unlabeled methionine. Two minutes after the start of the chase, the indicated test compounds ...

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