ORIGINAL RESEARCH ARTICLE Role of protein kinase Ca in the regulated secretion of the amyloid precursor protein M Racchi, M Mazzucchelli, A Pascale, M Sironi and S Govoni Department of Experimental and Applied Pharmacology, University of Pavia, Italy Keywords: amyloid precursor protein; signal transduc- tion; protein kinase C; neuroblastoma cells; phorbol ester; cholinergic Protein kinase C (PKC) has a key role in the signal transduction machinery involved in the regulation of amyloid precursor protein (APP) metabolism. Direct and indirect receptor-mediated activation of PKC has been shown to increase the release of soluble APP (sAPPa) and reduce the secretion of b-amyloid peptides. Experi- mental evidence suggests that specific isoforms of PKC, such as PKCa and PKCe, are involved in the regulation of APP metabolism. In this study, we characterized the role of PKCa in the regulated secretion of APP using wild-type SH-SY5Y neuroblastoma cells and cells trans- fected with a plasmid expressing PKCa antisense cDNA. Cells expressing antisense PKCa secrete less sAPPa in response to phorbol esters. In contrast, carbachol increases the secretion of sAPPa to similar levels in wild-type cells and in cells transfected with antisense PKCa by acting on APP metabolism through an indirect pathway partially involving the activation of PKC. These results suggest that the direct PKC-dependent activa- tion of the APP secretory pathway is compromised by reduced PKCa expression and a specific role of this isoform in these mechanisms. On the other hand, indirect pathways that are also partially dependent on the mitogen-activated protein kinase signal transduc- tion mechanism remain unaffected and constitute a redundant, compensatory mechanism within the APP secretory pathway. Molecular Psychiatry (2003) 8, 209–216. doi:10.1038/ sj.mp.4001204 Introduction The metabolic fate of the amyloid precursor protein (APP) is one of the key factors in the pathogenesis of Alzheimer’s disease (AD). 1 The routes of APP metabolism result in different pathways, leading to proteolytic processing of the precursor by at least three proteolytic activities that have been more closely characterized in recent years. 2–5 At the cell surface or in a secretory vesicle in its immediate proximity, a protease named ‘a secretase’ cleaves APP in the extracellular domain and releases the ectodomain (sAPPa or soluble APPa) into the extra- cellular space. This proteolytic cleavage constitutes the non-amyloidogenic pathway because it occurs within the b-amyloid (Ab) sequence, thereby preventing the formation of amyloidogenic frag- ments. The Ab peptide is formed and secreted as a physiological product of cell metabolism as a product of ‘b-’ and ‘g-secretase’ that cleave at the N and C termini of Ab respectively (reviewed by Mills and Reiner 6 and Racchi and Govoni 7 ). Both pathways are physiologically present in virtually all cell types and result in constitutive secretion of sAPPa and Ab. In normal cellular processing, APP can be committed either to the non-amyloidogenic secretory pathway or to Ab generating pathways. The choice between these two pathways appears to be a highly regulated process that can be influ- enced by extracellular signals and intracellular second messengers. 7 Several reports suggest a pivotal role of protein kinase C (PKC) in routing APP between the two different pathways: phorbol ester activation of PKC increases a-secretase-mediated sAPP secretion and reduces cellular secretion of Ab peptide. 6,7 The pivotal involvement of PKC in the regulated secretory processing of APP assumes particular importance in the pathogenesis of AD because several authors have demonstrated that both PKC activity and amount are reduced in AD brain. 8,9 These data led to the hypothesis that the observed impairment of the PKC signal transduction pathway may participate in the dysregulation of APP processing in AD patients, leading to the deposition of Ab peptide. In addition, data from a number of laboratories, including ours, have demonstrated a decreased PKC activity in sporadic AD fibroblasts. 10 We also observed an increase of the Kd (dissociation constant) for phorbol ester binding in the cytosolic fraction of AD fibro- blasts, which suggested an alteration in the character- istics of the kinase. Subsequently, the described changes in PKC were correlated to a specific 30% reduction in PKCa immunoreactivity. 11 In fibroblasts from sporadic AD patients we have demonstrated a reduced basal sAPP release from AD cells, suggesting the presence of a constitutive defect in the secretory mechanisms of the amyloid precursor. In addition, we demonstrated that AD cells have a lower sensitivity to stimulation of release of sAPPa by phorbol ester with an EC 50 two-fold higher than control fibroblasts. This result indicates a fundamental defect in the mechan- isms supporting PKC-mediated sAPPa secretion. 11 These data suggested that the routing of APP into the Molecular Psychiatry (2003) 8, 209–216 & 2003 Nature Publishing Group All rights reserved 1359-4184/03 $25.00 www.nature.com/mp