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.^2,3,4,5 At the cell surface or in a secretory vesicle in its immediate proximity, a protease named ' secretase' cleaves APP in the extracellular domain and releases the ectodomain (sAPP or soluble APP) into the extracellular space. This proteolytic cleavage constitutes the non-amyloidogenic pathway because it occurs within the -amyloid (A) sequence, thereby preventing the formation of amyloidogenic fragments. The A peptide is formed and secreted as a physiological product of cell metabolism as a product of '-' and '-secretase' that cleave at the N and C termini of A respectively (reviewed by Mills and Reiner⁶ and Racchi and Govoni⁷). Both pathways are physiologically present in virtually all cell types and result in constitutive secretion of sAPP and A. In normal cellular processing, APP can be committed either to the non-amyloidogenic secretory pathway or to A generating pathways. The choice between these two pathways appears to be a highly regulated process that can be influenced 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 -secretase-mediated sAPP secretion and reduces cellular secretion of A 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 A 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 fibroblasts, which suggested an alteration in the characteristics of the kinase. Subsequently, the described changes in PKC were correlated to a specific 30% reduction in PKC 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 sAPP by phorbol ester with an EC₅₀ two-fold higher than control fibroblasts. This result indicates a fundamental defect in the mechanisms supporting PKC-mediated sAPP secretion.¹¹ These data suggested that the routing of APP into the -secretase pathway in AD fibroblasts is defective because of a reduced PKC, leading to a possible diversion of APP metabolism towards the amyloidogenic pathway.

Although PKC is suggested as a central mechanism regulating sAPP release, a number of intracellular signalling pathways interact to form a complex network. In fact, it has been demonstrated that in addition to the direct activation of PKC, the metabolic processing of APP can be stimulated by a variety of ligands acting on their receptors. Among these, acetylcholine was the first to be described in a key paper in 1992 by Nitsch et al,¹² which suggested that cholinergic agonists could also affect AD pathogenesis by modulating APP metabolism. Downstream of cholinergic receptors and most other G-protein-coupled receptors, the activation of PKC, the mitogen-activated protein kinase (MAP-K) cascade, and the activation of unspecified Tyr kinases have been suggested^13,14 as either connected or independent events. There are at least three major signal transduction pathways involved in receptor-mediated APP processing, and the role of individual PKC isoforms in such complex interactions has not yet been addressed.

The goal of the present study was to characterize the role of PKC isoforms in the regulated secretion of APP. Because of the data on sporadic AD fibroblasts, we wanted to study the direct activation of PKC in a system where the chronic down-regulation of PKC could be reproduced to determine whether a specific isoform of PKC is directly responsible for the activation of APP secretion. Furthermore, we sought to determine whether the specific PKC isoform is involved in the complex interaction of signalling pathways downstream of cholinergic receptors leading to APP proteolytic processing.

Materials and methods

Materials

All culture media, supplements, and fetal calf serum (FCS) were obtained from Gibco Life Technologies (Paisley, Scotland, UK). Electrophoresis reagents were obtained from Bio-Rad (Hercules, CA, USA). All other reagents were of the highest grade available and were purchased from Sigma Chemical Co. (St Louis, MO, USA), unless otherwise indicated. Phorbol 12-myristate 13-acetate (PMA), GF109203X (Calbiochem, Darmstadt, Germany), PD98059 (Alexis Biochemicals, San Diego, CA, USA), and Gö6976 (Calbiochem, Darmstadt, Germany) were dissolved in dimethyl sulfoxide (DMSO) and stored at -20°C. Stocks were diluted in serum-free medium prior to the experiments. Carbachol and neomycin were dissolved and diluted to working concentration in serum-free minimum essential medium (MEM) at the moment of use.

Cell cultures and experimental treatments

SH-SY5Y neuroblastoma cells were cultured in Eagle's MEM supplemented with 10% FCS, penicillin/streptomycin, non-essential amino acids, and sodium pyruvate (1 mM) at 37°C in 5% CO₂/95% air. The cell line with stable antisense down-regulation of PKC was provided by Dr Thomas B Shea (McLean Hospital, Boston, MA, USA) and was grown in the same medium with the addition of the selecting agent G418 (Gibco Life Technologies, Paisley, Scotland, UK) at 400 g/ml. For the experiments, 410⁶ cells were seeded on 60 mm dishes and cultured for 24 h. Prior to the experiment, confluent monolayers of cells were washed twice with phosphate-buffered saline (PBS) and once with serum-free culture medium. Experimental treatments for the detection of sAPP released into the conditioned medium were performed in serum-free MEM with incubation for 2 h at 37°C. Experiments for the detection of activated mitogen-activated protein kinase (MAP kinase) were performed with incubations of 10 min. In all experiments involving the use of inhibitors such as neomycin, GF109203X, PD98059, or Gö6976, the compounds were preincubated for 10–30 min prior to the addition of PMA or carbachol.

Immunodetection of sAPP and PKC

Conditioned medium was collected after 2 h of incubation and centrifuged at 13 000 g for 5 min to remove detached cells and debris. Proteins in the medium were quantitatively precipitated by the deoxycholate/trichloroacetic acid procedure as previously described.¹¹ Cell monolayers were washed twice with ice-cold PBS and lysed on the tissue culture dish by the addition of ice-cold lysis buffer (50 mM Tris/HCl pH 7.5, 150 mM NaCl, 5 mM EDTA, and 1% TritonX-100). An aliquot of the cell lysate was used for protein analysis with the Bio-Rad Bradford kit for protein quantification. Normalization of protein loading on each blot was obtained by loading a volume of sample of conditioned medium standardized to total cell lysate protein concentration. Proteins were subjected to sodium dodecylsulphate-polyacrylamide gel electrophoresis (SDS-PAGE) (10%) and then transferred onto PVDF membrane (DuPont NEN, Boston, MA, USA). The membrane was blocked for 1 h with 10% non-fat dry milk in Tris-buffered saline (TBS) containing 1% Tween 20 (TBST). Membranes were immunoblotted with the antibodies 22C11 or 6E10 (at least once for each set of experiments) (Chemicon-Prodotti Gianni, Milano, Italy), which gave rise to the same pattern of bands. The detection was carried out by incubation with horseradish peroxidase conjugated goat anti-mouse IgG (Kirkegaard and Perry Laboratories, Gaithersburgh, MD, USA) for 1 h. The blots were then washed extensively and sAPP visualized using enhanced chemiluminescent methods (Pierce, Rockford, IL, USA). For the detection of PKC, cells were homogenized in a buffer constituted of 20 mM Tris/HCl pH 7.5, 2 mM EDTA, 0.2 mM phenylmethylsulfonylfluoride, 20 g/ml leupeptin and 25 g/ml aprotinin, and 0.5% TritonX-100. Proteins were measured as described earlier and subjected to Western blot analysis with the method indicated previously using isoform specific monoclonal antibodies from Transduction Laboratories (Lexington, KY, USA) and from Santa Cruz Biotechnology (Santa Cruz, CA, USA).

Western blot for extracellular signal-regulated protein kinase (ERK) phosphorylation

SH-SY5Y cells were cultured in serum-free medium overnight before stimulation with agonists for 10 min with or without preincubation for 30 min with PD- 98059. After stimulation, the cells were lysed in lysis buffer (62.5 mM Tris/HCl pH 6.8, 2% SDS, 10% glycerol, 50 mM DTT, 0.1% bromphenol blue). Cell lysates were boiled for 5 min and then centrifuged at 13 000 rpm for 5 min. Lysate aliquots of 25 l were loaded on 10% SDS-PAGE gels and the separated proteins were then subjected to electrophoretic transfer to PVDF membranes. Blots were probed with either a rabbit polyclonal antibody specific for ERK (p44/p42 MAP kinase) (New England Biolabs, Beverly, MA, USA) or a monoclonal antibody for phosphorylated ERK (phospho-44/42 MAP kinase) (Upstate Biotec Inc., Lake Placid, NY, USA).

Densitometry and statistics

Analysis of Western blot images was performed by calculating the relative intensity of the immunoreactive bands after acquisition of the blot image through a Nikon CCD video camera module and analysis by means of the Image 1.47. program (Wayne Rasband, NIH, Research Service Branch, Nimh, Bethesda, MD, USA). The relative densities of the bands were expressed as arbitrary units and normalized to data obtained from control samples run under the sample conditions. Controls were processed in parallel with stimulated samples and always included in the same blot. Preliminary experiments with serial dilutions of secreted protein allowed the determination of an optimal linear range for chemiluminescence reaction. Statistical analysis was made by the one-way analysis of variance test followed, when significant, by the two-tailed Student's t-test or multiple comparison test, where appropriate; a P value <0.05 was considered significant.

Results

For our investigation, we chose to use the human neuroblastoma cell line SH-SY5Y as the cellular model. We have obtained a cell line stably expressing the cDNA for PKC in the antisense orientation from Dr TB Shea (McLean Hospital, Boston, MA, USA).¹⁵ We refer to this line as SY4 and compare it to parental SH-SY5Y cells, which we refer to as SYwt. Western blot analysis (Figure 1) confirmed the specific down-regulation of the target isoform and the maintenance of the expression levels for other isoforms including PKC and PKC, in accordance with the pattern shown in previous experiments using these cell lines.^15,16 The cellular model was also chosen because SH-SY5Y cells express muscarinic receptors of the m1 and m3 subtype,^17,18 and this characteristic makes the cell line suitable for the characterization of PKC-dependent and receptor-mediated APP metabolism without further manipulation. These cells secrete sAPP, which can be detected by either the 22C11 or 6E10 monoclonal antibodies, with the same SDS gel band pattern. The sAPP band appears as a doublet with an apparent molecular weight of 100–120 kDa (Figure 2),¹⁹ with a pattern that reflects the expression of the three major isoforms of APP. These isoforms include mRNAs containing exon 7 (encoding the KPI domain) and exon 8, isoforms containing only exon 7, and APP mRNAs where both exons are spliced out.²⁰ The cells respond to phorbol esters and carbachol by releasing sAPP ranging two to three-fold above basal levels (Figure 2). Furthermore, following simultaneous treatment with carbachol and phorbol ester, these cells secrete sAPP to an extent that is less than the net sum of the independent effects of these two compounds. This decreased secretion as a result of combined treatment suggests that the two pathways may be at least partially independent of each other (Figure 2). The effect of PKC down-regulation on phorbol-ester-stimulated APP metabolism was examined by treating SYwt cells and SY4 cells for 2 h with increasing concentrations of PMA. As shown in Figure 3, both cell lines responded to PMA-driven activation of PKC by a concentration-dependent increase in the release of sAPP, but with significantly different patterns. In particular, the SYwt cell line responded with a significant increase of sAPP release at low concentrations of the activator. A 10 nM concentration of PMA is sufficient to elicit an approximately two-fold increase in the basal release of sAPP. The effect of phorbol ester activation reached a maximum at 100 nM with an increase of approximately 2.5-fold of basal release and remained substantially equivalent at higher concentrations (1 M PMA). Cells with down-regulated expression of PKC remained silent at low concentrations of PMA (10 nM) while responding at increasing concentrations to a lesser extent than SYwt. At 1 M PMA the average increase of sAPP released remained lower than that of SYwt cells; however, differences were not significant.

Figure 1.

Expression of PKC isoforms in SYwt and SY4 cells. The expression of PKC isoforms has been evaluated using specific mouse monoclonal anti-PKC antibodies. Samples of rat cerebellum homogenate have been included as positive controls and molecular size identification. These examples of Western blot show that the antisense expression of PKC in SY4 neuroblastoma cells significantly reduces its immunoreactivity (a). Quantitative analysis showed a reduction of PKC expression of an average of 40%. No differences in the expression of PKC (b) and (c) isoforms have been observed between SYwt and SY4 cells.

Full figure and legend (41K)

Figure 2.

Effect of cholinergic and phorbol ester stimulation on sAPP release in SH-SY5Y cells. Cells were exposed to vehicle, PMA (100 nM) or CCh (1 mM) or both simultaneously, for 2 h in serum-free MEM. The Western blot is representative of the maximal effect obtained with the direct activation of PKC by the phorbol ester PMA and by receptor-mediated activation of PKC and other signal transduction pathways. It also shows the sum of effects of both compounds.

Full figure and legend (36K)

Figure 3.

SH-SY5Y cells transfected with PKC antisense cDNA show a reduced sensitivity to PMA stimulation of sAPP release compared with wild-type cells. Cells were incubated for 2 h with serum-free medium alone or increasing concentrations of PMA (10 nM, 100 nM, 1 M). Proteins secreted into the conditioned media were collected and subjected to Western blot analysis for sAPP. The blots were quantified by densitometry as described in the 'Materials and methods' section. Data are expressed as a percentage of basal release and are representative of three to four independent experiments.

Full figure and legend (21K)

Treatment of SYwt cells with increasing concentrations of carbachol results in a concentration-dependent increase in sAPP release with a maximally effective concentration of 1 mM (Figure 4). The same treatment applied to SY4 cells produced similar results. In contrast to the findings with PMA stimulation, the intracellular pathways linking cholinergic receptors to sAPP release were not affected by PKC down-regulation.

Figure 4.

Carbachol promotes the non-amyloidogenic metabolism of APP similarly in wild-type cells and in antisense transfected neuroblastoma cells. Cells were incubated for 2 h with serum-free medium alone or increasing concentrations of CCh (10 M, 100 M, 1 mM). Proteins released into the conditioned media were collected and subjected to Western blot analysis for sAPP. The blots were subsequently quantified by densitometry as described in the 'Materials and methods' section. Data are expressed as a percentage of basal release and are representative of three to four independent experiments.

Full figure and legend (21K)

The pathway downstream of muscarinic receptors involved in the regulation of APP metabolism is complex, and some authors suggest that it involves the activation of ERKs and the MAP-kinase pathway.¹³ Stimulation of SYwt and SY4 with carbachol resulted in equally increased ERK phosphorylation (Figure 5a) in the two cell lines. Since it has been suggested that ERKs are involved in carbachol-induced sAPP release, we blocked ERKs activation by preincubation of the cells with PD98059, a well-known MEK inhibitor. Blockade of ERKs phosphorylation (Figure 5a) resulted in partial inhibition of carbachol-mediated sAPP release in both cell lines (Figure 5b). Finally, the signalling pathway involved in carbachol-mediated sAPP release depends in part on the activation of phospholipase C (PLC) as indicated by the fact that the effect is partially inhibited by pretreatment with the PLC inhibitor neomycin²¹ (Figure 6a). This would include the involvement of either Ca²⁺ or inositol turnover. Pretreatment of the cells (SYwt) with the PKC inhibitor GF109203X, which is not selective for PKC isoforms, reduced the stimulatory effect of 1 mM carbachol by an average of 40% (Figure 6b). However, selective inhibition of PKC using the compound Gö6976, which is selective for Ca²⁺-dependent PKC isoforms, had no significant effect on the stimulation of sAPP release by carbachol. This result suggests that the limited involvement of PKC in cholinergic activation of sAPP release is not dependent on PKC.

Figure 5.

Carbachol-mediated sAPP release is partially dependent on MAP-kinase activation. Cells (SYwt and SY4) were preincubated overnight with serum-free MEM and then treated for 10 min with 1 mM carbachol following 30 min pretreatment with vehicle or 50 M PD98059. For sAPP release (panel (b)), the cells were incubated in the presence or absence of carbachol for 2 h. Cell lysates and conditioned media were collected and treated as indicated in the 'Materials and methods' section. As indicated in panel (a), pretreatment with PD98059 effectively blocks ERKs phosphorylation, while only partially blocking (panel (b)) the effect of carbachol on sAPP release.

Full figure and legend (92K)

Figure 6.

Carbachol-mediated sAPP release in the presence of PLC and PKC inhibitors. SH-SY5Y (SYwt) cells were treated for 2 h with 1 M carbachol following pretreatment with vehicle and/or 100 mM neomycin for 10 min (panel (a)). SH-SY5Y (SYwt) cells were treated for 2 h with 1 M carbachol following pretreatment for 30 min with vehicle with and without 2.5 M GF109203X (a non-selective PKC inhibitor) or 2 M Gö6976 (a selective inhibitor of PKC , and isoforms) (panel (b)). The figure shows a representative immunoblot (out of three independent experiments).

Full figure and legend (51K)

Discussion

We demonstrate here that PKC is specifically involved in phorbol ester-induced APP metabolism and that its contribution is specific for direct PKC-mediated pathways. Whereas down-regulation of PKC can significantly impair phorbol-ester-induced sAPP release from SH-SY5Y cells, the down-regulation of the kinase isoform does not affect carbachol-mediated sAPP release.

There are multiple intracellular second messengers that contribute to the regulation of APP metabolism and they have extremely complex interactions.⁷ PKC was one of the first signal transduction related molecules to be implicated in the regulation of APP metabolism.^6,7 The extensive studies on regulation of APP processing by PKC suggest that the A forming amyloidogenic pathway and the non-amyloidogenic -secretase pathway are balanced, the latter being activated by PKC. Although this simple view of APP metabolism may not fully reflect the complexity of the system,²² PKC-defective pathways have been described in peripheral tissues of sporadic AD patients^10,23 and these defects have been associated with aberrant APP processing.¹¹ The central role of PKC in APP metabolism is therefore also connected to the fact that defective PKC is one of the most consistent findings in AD brain and peripheral tissues.^8,24 In our previous work, we found that the defective APP metabolism described in AD fibroblasts is paralleled by a defective PKC activity and, in particular, PKC was found to be reduced in AD fibroblasts.¹¹ The average reduction in the expression of PKC protein in AD fibroblasts was 30%, similar to the levels of down-regulation of approximately 40% observed in the SY4 neuroblastoma cellular model used in this study.

We show here that the phorbol ester-stimulated secretion of sAPP in SY4 cells is defective. Interestingly, the pattern of response to phorbol ester shown by the SY4 cell line is remarkably similar to that previously observed with AD fibroblasts.¹¹ In particular, the cells seem to be totally unresponsive to low concentrations of phorbol ester (10 nM) while SYwt cells respond to phorbol ester treatment at the same concentration with a two-fold increase of sAPP released above basal secretion. The fibroblast data suggest that these cells may have lost a high-affinity binding site for phorbol esters and that PKC is responsible for the effect. Among the different kinase isoforms, PKC and the other calcium-dependent isoforms have the highest affinity for phorbol esters,²⁵ of the same order of magnitude of the lowest concentration used. Higher concentrations are necessary to activate other kinase isoforms that may be novel PKCs. In our current experiments using SY4 cells as well as in published experiments using AD fibroblasts, a significant secretory response was elicited only at 100 nM phorbol ester. The current data indicate that PKC is specifically involved in the phorbol-ester-mediated APP processing to the extent of physiological levels of direct activators. Higher concentrations would drive the activation of other kinases and possibly overcome the defect of a single isoform.

Many results have been reported in the literature that suggest a role for PKC and other isoforms in the regulated secretion of APP. Initially, it was demonstrated that over-expression of PKC in Swiss 3T3 fibroblasts increased the sensitivity of APP processing to phorbol esters.²⁶ Subsequently, other investigators in various experimental systems involving either pharmacological down-regulation or inhibition of PKC^27,28 or over-expression of PKC²⁹ have indicated that PKC plays a significant role in APP metabolism. We provide here for the first time a demonstration of the role of PKC in a system that has been manipulated to reproduce the long-term down-regulation of PKC described in fibroblasts from AD patients.

Other authors pointed more specifically to the role of another isoform, PKC. Kinouchi et al showed that over-expression of PKC also induces an increase of sAPP .²⁹ Other strategies pursuing the inhibition of PKC provided evidence of a role of that isoform in APP processing. The over-expression of the PKC V1 region, which binds specifically to the receptor for activated C-kinase (RACK), blocked phorbol-ester-induced enhancement of APP secretion.³⁰ However, that result was obtained in B103 neuroblastoma cells over-expressing APP. Those cells reportedly do not express endogenous APP and therefore may not include the completely physiological machinery for APP processing. A more recent indication on the role of PKC was reported by Zhu et al,³¹ who showed that expression of a peptide inhibitor of PKC reduced phorbol-ester-mediated sAPP release. This result ruled out the involvement of PKC because of the ineffectiveness of Gö6976, which is a specific inhibitor of PKC, , and isoforms.

The purpose of our study was to investigate the role of PKC in the regulated secretory processing of APP. We studied the involvement of this kinase isoform within the complex intracellular signalling pathway⁷ involved in APP processing, and we focussed on a receptor-mediated pathway that, according to the literature, partially involves PKC activation. Our experiments with phorbol ester established that PKC is significantly involved in the PKC pathway. The degree of activation of the pathway by phorbol esters depends on the concentration used and can influence the pattern of kinase isoforms involved, so we turned our attention to the cholinergic receptor-mediated APP processing. The first study indicating that APP processing could be modulated by cholinergic receptors was conducted in HEK293 cells over-expressing muscarinic m1 receptors.¹² This result was followed by multiple confirmations.^6,7 The demonstration that cholinesterase inhibitors studied for AD therapy also induced sAPP release by an indirect cholinergic mechanism further substantiated this result.²⁰ Stimulation of G-protein-coupled receptors by neurotransmitters regulates APP processing by PKC-dependent signalling pathways. In particular, the cholinergic receptor stimulation of sAPP release is blocked by staurosporine¹² and GF109203X.^14,20 We show here that both cell lines respond equally to carbachol in spite of the defective PKC in the SY4 cell line. This result suggests that the involvement of PKC in the carbachol-regulated APP processing is not significant or at least that redundant signalling pathways are activated that can bypass the defective kinase. Although PKC activation can undoubtedly stimulate sAPP release, this is not the only mechanism activated downstream of the cholinergic receptor that is linked to the activation of APP processing. This has been clear since it was shown that the inhibition of APP secretion by GF109203X was not complete,¹⁴ and thus it was suggested that other kinase pathways may be involved. In particular, Slack et al suggested the involvement of a Tyr kinase¹⁴ together with the involvement of a MAP-kinase pathway.¹³

The signalling pathways downstream of the m3 muscarinic receptor clearly involve both PKC-dependent and -independent mechanisms coupled to the activation of the MAP-kinase pathway.³² However, for the latter mechanism as it relates to APP metabolism, there are contrasting reports. Desdouit-Magnen et al³³ were not able to demonstrate a block of carbachol-mediated sAPP release in PC12-M1 cells treated with the MEK inhibitor PD98059. On the same cellular model, however, Haring et al¹³ showed a significant inhibition of carbachol-mediated sAPP release by PD98059. In our system using SH-SY5Y, which endogenously expresses both APP and m1/m3 muscarinic receptors, we observed a reduction of carbachol-mediated sAPP release by PD98059, suggesting at least a partial involvement of MAP-kinase pathways. Interestingly, no differences in the activation of the MAP-kinase system were detected between SY4 and SYwt cells, suggesting that the recruitment of ERKs was not affected by the specific down-regulation of PKC. This also suggests that although MAP kinases can be activated by PKC following phorbol ester treatment^33,34 and that such activation may promote sAPP release, PKC is not the kinase isoform involved. This is consistent with data in the literature that suggests that PKC is the only kinase isoform in SK-N-BE2(C) neuroblastoma cells involved in MAP-kinase activation upon m3 muscarinic receptor challenge.³⁵

A further demonstration that PKC is not significantly involved in the pathway downstream of cholinergic receptors comes from the experiments with neomycin and with selective PKC inhibitors. Neomycin is a phosphatidylinositide-specific PLC inhibitor²¹ that partially blocks the effect of carbachol on sAPP release in SYwt cells. The blockade of PLC would reduce the contribution of Ca²⁺ and phosphatidylinositol, thus blocking the contribution of cofactors for the activation of PKC. As described before, the inhibition of carbachol-mediated sAPP release can be partially obtained, with the PKC inhibitor GF109203X,^14,20 which is not selective for PKC isoforms. We show here that Gö6976, the specific inhibitor of Ca²⁺-dependent PKC isoforms , , and , did not block the effect of carbachol, thus conclusively demonstrating that PKC is not involved in receptor-mediated sAPP release. Among the other isoforms present in SH-SY5Y cells and because of data in the literature concerning both APP metabolism^29,30,31 and the pharmacology of muscarinic receptor signalling,³⁵ we favor the specific involvement of PKC and investigations of this hypothesis are under way.

The description of the role of PKC here is relative to the data obtained with cholinergic receptors, and other receptor-mediated effects (glutamate, serotonin) have to be investigated specifically. These data indicate that multiple isoforms of PKC are differentially involved and each can specifically contribute to the complexity of the intracellular pathways regulating APP proteolytic metabolism. The data also suggest that the defects observed in fibroblasts from affected patients may be tissue specific according to the blend of expressed kinases and may be largely compensated by the described redundant receptor-mediated mechanisms controlling APP metabolism. On the other hand, these subtle defects may decrease the functional reserve of the system, leading with time or in the presence of additional damage to the unbalancing of APP metabolism toward - and -secretase pathways.

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Acknowledgements

We are grateful to Dr Thomas B Shea for the gift of SH-SY5Y cells transfected with antisense PKC (SY4) and the parental non-transfected SH-SY5Y cell line (SYwt). We are also grateful to Prof. Mark O Lively of Wake Forest University School of Medicine, Winston Salem, NC, USA for helpful discussions and a careful proofreading of the manuscript. The financial support of TELETHON—Italy, Grant # E.0866 to SG is gratefully acknowledged. This research was also funded by MIUR—Cofin 2000 (SG) and Ministero Sanità—Progetto Alzheimer (MR).