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. Experimental 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 transfected 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 activation 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 transduction mechanism remain unaffected and constitute a redundant, compensatory mechanism within the APP secretory pathway.
We investigated the differential role of protein kinase C (PKC) isoforms in the regulated proteolytic release of soluble amyloid precursor protein (sAPPa) in SH-SY5Y neuroblastoma cells. We used cells stably transfected with cDNAs encoding either PKCa or PKCe in the antisense orientation, producing a reduction of the expression of PKCa and PKCe, respectively. Reduced expression of PKCa and/or PKCe did not modify the response of the kinase to phorbol ester stimulation, demonstrating translocation of the respective isoforms from the cytosolic fraction to specific intracellular compartments with an interesting differential localization of PKCa to the plasma membrane and PKCe to Golgi-like structures. Reduced expression of PKCa significantly impaired the secretion of sAPPa induced by treatment with phorbol esters. Treatment of PKCadeficient cells with carbachol induced a significant release of sAPPa. These results suggest that the involvement of PKCa in carbachol-induced sAPPa release is negligible. The response to carbachol is instead completely blocked in PKCe-deficient cells suggesting the importance of PKCe in coupling cholinergic receptors with APP metabolism.Keywords: Alzheimer's disease; cholinergic receptors; neuroblastoma; phorbol esters; signal transduction.Alzheimer's disease (AD), the most common type of dementia, is characterized by deposition in the brain of fibrillar aggregates of a peptide named beta-amyloid (Ab), derived from proteolytic processing of a larger precursor called amyloid precursor protein (APP) [1]. APP is metabolized by several alternative pathways: in the secretory pathway, it is cleaved extracellularly within the Ab domain by a-secretase to generate a soluble nonamyloidogenic fragment of APP (sAPPa) that is secreted in the conditioned medium of cell cultures, human plasma and in the cerebrospinal fluid. Other enzymes, b-and c-secretase, cleave APP at the N and C termini of Ab, respectively, releasing the amyloidogenic peptide [2,3].APP processing by a-secretase occurs via a constitutive pathway and by receptor-mediated activation of multiple signal trasduction pathways among which protein kinase C (PKC) is a major player.PKC is a family of at least 12 isoenzymes of serine/ threonine protein kinases, central to many signal transduction pathways [4]. Although these isoenzymes share a similar structural domain organization, differences in their substrate specificity, cofactor requirements, tissue and cellular distribution, and subcellular localization suggest that each of the different PKC isoenzymes plays a specific and distinct regulatory role in cellular signal transduction [4][5][6][7][8].The role of individual PKC isoforms in the regulation of APP proteolytic processing is not yet understood. Recently we demonstrated that PKCa was specifically involved in phorbol ester-induced sAPPa release [9], further supporting a series of reports that pointed to a specific role for PKCa in APP processing in vitro (for review see [2]), and most recently also in vivo [10,11] where constitutive o...
We have investigated the effect of ganstigmine (CHF2819), a novel geneserine derived acetylcholinesterase (AChE) inhibitor, on the expression and metabolism of the amyloid precursor protein (APP) in neuroblastoma cell line SH-SY5Y. The rationale was based on the suggestion that cholinergic activity may also be involved in the regulation of APP metabolism. We studied the acute effect on APP metabolism following the secretion of sAPPalpha in the conditioned medium of cells. Following short term treatment (2h), ganstigmine promoted a slight increase in the release of sAPPalpha, the maximal effect approaching on average 1.5 fold baseline value. The data obtained in the long term experiments demonstrate that continuous inhibition of AchE obtained with 100 nM ganstigmine following an exposure of 24 hours did not influence APP isoforms expression. However, the compound appeared to increase the constitutive release of sAPPalpha, with a mechanism that is derived from an indirect cholinergic stimulation.
The triggering events leading to the selective neurodegeneration observed in Alzheimer brains are not yet completely understood. They thus create a great challenge for the definition of a resolutive treatment for the causes and symptoms of Alzheimer's Disease (AD). Since the current therapeutic option for AD patients is the use of acetylcholinesterase inhibitors (AChEIs), several authors have examined whether these drugs can also affect the expression and metabolism of the amyloid precursor protein (AbetaPP). The rationale behind these studies was based on the fact that the literature suggests that cholinergic activities are also involved in the regulation of AbetaPP metabolism. Therefore, the characterization of these aspects of AD pharmacology may allow cholinergic drugs to be tested for their ability to intervene at different levels of the pathogenetic chain, other than providing a replacement therapy for lost neurotransmitters. This paper reviews the evidence that many of these drugs, although with different qualitative effects, are able to modulate the metabolism and expression of AbetaPP. This effect is often sustained by an indirect cholinergic mechanism and does not affect the mRNA expression of the precursor, although some other authors have demonstrated an effect on post-transcriptional regulation of AbetaPP expression. In addition to the effect on AbetaPP processing, we recently explored the possibility that these molecules affect a gene expression program beyond the classical pharmacological effects, for insights on possibly unexplored pathways of intervention in AD.
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