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Am J Physiol Cell Physiol 291: C76-C82, 2006. First published February 8, 2006; doi:10.1152/ajpcell.00508.2005
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RECEPTORS AND SIGNAL TRANSDUCTION

Effect of {gamma}-secretase inhibitors on muscarinic receptor-mediated calcium signaling in human salivary epithelial cells

Young S. Oh and R. James Turner

Membrane Biology Section, Gene Therapy and Therapeutics Branch, National Institute of Dental and Craniofacial Resarch, Bethesda, Maryland

Submitted 12 October 2005 ; accepted in final form 2 February 2006


   ABSTRACT
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 ABSTRACT
MATERIALS AND METHODS
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Altered intracellular Ca2+ signaling has been observed in cells derived from Alzheimer’s disease patients, and a possible link between {gamma}-secretase activity and the content of intracellular Ca2+ stores has been suggested. To test this hypothesis we studied the effects of several {gamma}-secretase inhibitors on muscarinic receptor-mediated intracellular calcium release in the human salivary gland cell line HSG. Although several inhibitors in the peptide aldehyde class partially blocked carbachol-induced Ca2+ transients, these effects did not appear to be due to {gamma}-secretase inhibition, and overall we found no evidence that inhibition of {gamma}-secretase activity had any significant effect on agonist-induced intracellular calcium release in HSG cells. In complementary experiments with presenilin-null cells we found that the reconstitution of {gamma}-secretase activity by transfection with wild-type presenilin 1 likewise had no significant effect on thapsigargin-induced Ca2+ release. In a test of the specific hypothesis that the level of APP intracellular domain (AICD), the intracellular fragment of the beta-amyloid precursor protein (APP) resulting from {gamma}-secretase cleavage, can modulate the Ca2+ content of the endoplasmic reticulum, we were unable to demonstrate any effect of APP small interfering RNA on the magnitude of carbachol-induced intracellular calcium release in HSG cells. Together our data cast considerable doubt on the hypothesis that there is a direct link between {gamma}-secretase activity and the content of intracellular Ca2+ stores.

presenilin; carbachol; inositol 1,4,5-trisphosphate; Alzheimer’s disease

MOST CASES OF EARLY-ONSET, familial Alzheimer’s disease are associated with mutations in the highly homologous proteins presenilin 1 (PS1) and presenilin 2 (PS2) (2, 30, 37). The presenilins are components of an apparently ubiquitous intramembranous protease activity known as {gamma}-secretase (18, 37), whose substrates are a number of type I transmembrane proteins including the beta-amyloid precursor protein (APP). Mutations in the presenilins have been shown to lead to altered processing of APP resulting in increased production of longer neurotoxic forms of beta-amyloid (2, 11, 30), the major component of the senile plaques characteristic of Alzheimer’s disease.

In addition to their well-studied effects on {gamma}-secretase activity, mutations in the presenilins have also been shown to result in alterations of intracellular calcium signaling (19, 34). A number of familial Alzheimer’s disease mutations in the presenilins have been found to be associated with increased levels of calcium in the endoplasmic reticulum, potentiation of inositol 1,4,5-trisphosphate (IP3)-mediated calcium release, and attenuation of capacitative calcium entry. These effects have been documented in both neural and nonneural cells from model animals (3, 16, 20, 28, 35), in transfected mammalian cells and Xenopus oocytes (4, 14, 21, 23, 24, 27, 39), and significantly in (nonneural) cells from familial Alzheimer’s disease patients (9, 17). Because aberrant intracellular calcium signaling has been implicated in the pathogenesis of Alzheimer’s disease, a possible involvement of the presenilins via this pathway has also been hypothesized (19, 34).

Because familial Alzheimer’s disease mutations in the presenilins are associated with modifications in {gamma}-secretase activity, it has been suggested that there may be a link between this activity and the content of intracellular calcium stores. In cells APP is cleaved in its ectodomain to produce a large soluble extracellular fragment and a smaller (~10 kDa) membrane-bound COOH-terminal fragment that is the substrate for {gamma}-secretase (2, 30, 37). Cleavage of this COOH-terminal fragment by {gamma}-secretase produces a ~7-kDa intracellular peptide, referred to as the APP intracellular domain (AICD). In the past, a number of authors have speculated that AICD may have a signaling role analogous to that of the intracellular fragment of Notch, which also results from {gamma}-secretase cleavage (10). In a recent publication Leissring et al. (22) proposed that AICD is involved in regulating the content of intracellular calcium stores. These authors showed that various maneuvers expected to increase or decrease the level of AICD in cultured cells resulted in corresponding changes in the content of intracellular calcium stores and in the magnitude of agonist-induced intracellular calcium release. In particular, they showed that when N2a neuroblastoma cells were treated with various {gamma}-secretase inhibitors (presumably reducing the production of AICD), agonist-induced calcium release from intracellular stores was markedly reduced at concentrations of these compounds that correlated well with their inhibitory effects on {gamma}-secretase activity. In addition, they showed that fibroblasts from APP–/– knockout mice and from PS1–/–/PS2–/– double-knockout mice, neither of which should be able to produce AICD, had similar reduced agonist responses as well as reduced content of intracellular calcium stores, and that in APP–/– cells this defect could be reversed by transfection with AICD.

Although the above results and the results of others provide intriguing evidence for an involvement of the presenilins and {gamma}-secretase activity in intracellular calcium signaling and possible relationships to the pathology of Alzheimer’s disease, other observations suggest that the links among these processes may not be simple. For example, two familial Alzheimer’s disease-linked mutations in PS2 have recently been identified (12, 40) that result in reduced, rather than increased, calcium release from intracellular stores. Thus the association of mutation-related changes in {gamma}-secretase activity and potentiation of intracellular calcium responses is not absolute. There is also evidence that the effects of some presenilin mutants on intracellular calcium release may involve the modulation of phospholipase C by {gamma}-secretase (4, 27). How these observations relate to some of the previous results described above is presently unclear. More generally the role of the presenilins, {gamma}-secretase activity, and intracellular calcium signaling in the pathology of nonfamilial Alzheimer’s disease is still very poorly understood.

In the present study, we have reexamined the relationships among intracellular calcium signaling, {gamma}-secretase activity, and APP expression. In most of our experiments we have used HSG cells, a salivary epithelial cell line derived from a human submandibular gland (32). Salivary glands and salivary cell lines have been extensively used as model tissues to study agonist-induced signaling processes. In salivary glands, agonist (mainly muscarinic) stimulation causes IP3-mediated intracellular calcium mobilization that in turn leads to salivary fluid secretion. Interestingly, salivary flow from the submandibular gland has been found to be significantly impaired in patients with Alzheimer’s disease (31); however, the significance of this observation remains unexplored. Contrary to previous results from other cell types, we do not find any evidence for the involvement of {gamma}-secretase activity in intracellular calcium signaling in HSG cells or in the other cell lines we have examined.


   MATERIALS AND METHODS
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 MATERIALS AND METHODS
RESULTS AND DISCUSSION
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Cell culture and transfection. HSG and HSY cells were cultured in DMEM-F-12 (1:1) medium supplemented with 2 mM glutamine, 100 µg/ml each of penicillin and streptomycin (all from Biofluids), and 10% fetal bovine serum (GIBCO-BRL) in a humidified incubator at 37°C and 5% CO2. Two to three days before use, cells were plated in 60-mm culture dishes at 50–70% confluence. HEK-293 cells were cultured in the same way but in Eagle’s modified essential medium (EMEM) rather than DMEM-F-12. The PS1/PS2 double-knockout cell line BD1 (6), a generous gift from Dr. Alan Bernstein (Mt. Sinai Hospital, Toronto, ON, Canada), was cultured as described previously (41).

BD1 cells were transfected with human PS1, using FuGENE (Roche) according to the manufacturer’s instructions. To obtain stably transfected cells hygromycin B (0.1 mg/ml) was added to the medium 2 days after transfection and cells were subcultured as necessary. Confluent cultures of hygromycin-resistant cells were obtained 2–3 wk later. The human PS1 cDNA clone was generously provided by Dr. Todd E. Golde (Mayo Clinic, Jacksonville, FL).

Treatment with {gamma}-secretase inhibitors. Cells in 60-mm dishes were washed three times with Dulbecco’s PBS (GIBCO-BRL) and then incubated in serum-free culture medium (DMEM-F-12 or EMEM, as appropriate) at 37°C and 5% CO2 for various times with the concentrations of {gamma}-secretase inhibitors (all from Calbiochem) indicated. Control cells were treated in the same way but with vehicle instead of inhibitors.

Intracellular calcium measurements. Intracellular calcium levels were monitored with the Ca2+-sensitive fluorescent indicator fura-2 (Molecular Probes) as follows. Cells in 60-mm dishes were washed three times with Dulbecco’s PBS and then incubated in 2 ml of physiological saline solution (PSS; in mM: 135 NaCl, 5.8 KCl, 1.8 CaCl2, 0.8 MgSO4, 0.73 NaH2PO4, 11 glucose, and 20 HEPES, pH 7.4 with NaOH) containing 1% BSA and 2 µM fura-2 AM (from a 1 mM stock solution in DMSO) for 30 min at room temperature with gentle agitation. After fura-2 loading, cells were washed three times with Ca2+,Mg2+-free PBS and then incubated for 2 min with 2 ml Ca2+,Mg2+-free PBS containing 3 mM EGTA to dissociate them from the culture dish. These dissociated cells were spun down, resuspended in 50–70 µl of PSS, and rested for >10 min at room temperature. For the intracellular calcium measurements a 15-µl aliquot of cells was added to 1.5 ml of Ca2+-free PSS containing 0.1 mM EGTA in a fluorescence cuvette.

Fura-2 fluorescence was monitored in ratio mode with a PTI DeltaRAM V fluorimeter (Photon Technology International) at excitation wavelengths of 340 and 360 nm and an emission wavelength of 510 nm. In some experiments the intracellular calcium concentration was calculated by the method of Grynkiewicz et al. (13).

Small interfering RNA treatment. Small interfering RNA (siRNA) duplexes against human APP and control (nontargeting) siRNA were obtained from Dharmacon Research (catalog nos. M-003731 and D-001206-13, respectively). siRNAs (100 nM) were transfected into HSG cells in 60-mm dishes, using RNAiFect (Qiagen) according to the manufacturer’s instructions, and cells were harvested 2 days later.

Western blot analysis. Particulate fractions were prepared from cultured cells and probed via Western blot analysis, as previously described (5). Primary antibodies against the COOH-terminus of APP (Zymed), the NH2-terminal fragment of PS1 (Santa Cruz Biotechnology), and Na+-K+-ATPase (Santa Cruz Biotechnology) were used at dilutions of 1:1,000, 1:2,000, and 1:1,000, respectively. Secondary antibodies (Pierce) conjugated to horseradish peroxidase were used at a dilution of 1:10,000. Detection was carried out with an ECL kit (Amersham) and X-Omat AR film (Kodak).

Measurement of IP3. HSG cells in 60-mm dishes were treated with or without {gamma}-secretase inhibitors and then suspended in 250 µl of PSS by the method described above for fura-2 studies (but without fura-2 loading). After carbachol stimulation, 0.2 volumes of 20% perchloric acid was added and the samples were left on ice for 20 min. Cellular debris was spun down (2000 g for 15 min), and the supernate was neutralized to pH 7.5 with 1.5 M potassium hydroxide-containing 60 mM HEPES and then spun again as before. IP3 in 100-µl aliquots of the resulting supernates was measured with a commercial kit (Amersham Biosciences; TRK 1000), following the manufacturer’s protocol. Protein was assayed by the bicinchoninic acid method (Pierce).

Data presentation and statistics. Quantitative results are presented as means ± SE of three or more determinations. P values <0.05 calculated from a two-tailed t-test are taken to represent statistically significant differences.


   RESULTS AND DISCUSSION
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 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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Carbachol-stimulated intracellular Ca2+ release in HSG cells. Figure 1 illustrates the responses of HSG cell intracellular Ca2+ stores to the muscarinic agonist carbachol and the sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA) pump inhibitor thapsigargin as monitored via fura-2 fluorescence. In Fig. 1A we show that the application of 100 µM carbachol in Ca2+-free PSS elicited a rapid rise in cytosolic Ca2+ concentration due to release from intracellular stores followed by a return to baseline levels. A subsequent application of 1,000 µM carbachol resulted in only a small effect, indicating that 100 µM carbachol produces a near-maximal response. Adding the SERCA inhibitor thapsigargin after 1,000 µM carbachol produced a further rise in cytosolic Ca2+ concentration, indicating the presence of significant additional thapsigargin-sensitive, carbachol-insensitive stores. However, when thapsigargin was added before carbachol (Fig. 1B), both 100 µM and 1,000 µM carbachol were without effect, demonstrating that all carbachol-sensitive stores in these cells are thapsigargin sensitive.


Figure 1
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  		Fig. 1. Intracellular calcium release induced by carbachol and thapsigargin treatment of HSG cells. A and B: HSG cells in Ca2+-free physiological saline solution (PSS) were exposed to 100 µM carbachol (a), 1,000 µM carbachol (b), and 1 µM thapsigargin (TG) as indicated. The traces represent intracellular fura-2 fluorescence monitored in ratio mode as described in MATERIALS AND METHODS.

 
Effect of {gamma}-secretase inhibitors on carbachol-mediated intracellular Ca2+ release. As indicated above, Leissring et al. (22) recently reported that treatment of N2a neuroblastoma cells with various {gamma}-secretase inhibitors, including the peptide aldehydes IL, YIL, and CIL, markedly reduced agonist-induced calcium release from intracellular stores at concentrations that correlated well with their inhibitory effects on {gamma}-secretase. In Fig. 2, A and B, we demonstrate that 10 µM YIL also blunts carbachol-induced calcium release from intracellular stores in HSG cells. The effect of YIL was time (Fig. 2C)- and dose (Fig. 2D)-dependent but typically only blocked ~65% of the carbachol response even at longer times and/or high YIL concentrations (Fig. 2, C and D). In control studies we found that YIL treatment had no significant effect on resting intracellular calcium levels under the experimental conditions of Fig. 2 (e.g., resting intracellular Ca2+ concentration was 18.2 ± 0.7 nM in untreated HSG cells and 19.5 ± 0.5 nM in cells treated with 10 µM YIL for 4 h; n = 5 in both cases).


Figure 2
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  		Fig. 2. Inhibition of carbachol-induced intracellular calcium release by the {gamma}-secretase inhibitor YIL. A: typical intracellular fura-2 fluorescence trace from HSG cells stimulated with 100 µM carbachol (arrow). B: typical fura-2 trace from HSG cells stimulated with 100 µM carbachol (arrow) after pretreatment with 10 µM YIL for 4 h before fura-2 loading as described in MATERIALS AND METHODS. C: time course of the effect of YIL on carbachol-induced intracellular calcium release. HSG cells were treated with 10 µM YIL for the times indicated before fura-2 loading and stimulation with 100 µM carbachol. The change in the fura-2 ratio above resting levels measured at the peak of carbachol response was used as a measure of the magnitude of the intracellular calcium response. The results are expressed as % of the response observed without YIL treatment. All points are significantly less than control (P < 0.03; n ≥ 6 in all cases). D: effects of 4-h treatment with YIL concentrations of 1 (n = 26), 10 (n = 32), and 50 (n = 3) µM on the response of HSG cells to 100 µM carbachol. All points are significantly less than control (P < 0.005). E: effects of 4-h treatment with 10 µM YIL on the responses of HSY (n = 4) and HEK-293 (n = 13) cells to 100 µM carbachol. Both experimental points are significantly less than control (P < 0.004).

 
In Fig. 2E, we demonstrate that 10 µM YIL also blunted carbachol-induced calcium release in another salivary epithelial cell line, HSY, derived from a human parotid gland (15), as well as in the human embryonic cell line HEK-293. However, the effect of YIL in HEK-293 cells was significantly smaller than that observed in HSG or HSY cells.

In Fig. 3A, we examine the effects of a variety of other {gamma}-secretase inhibitors on carbachol-induced intracellular calcium release in HSG cells. The inhibitors studied include a variety of structurally unrelated compounds (Table 1). What is striking, however, is that aside from the peptide aldehydes there is no relationship between the marginal or negligible effects of these compounds on calcium signaling and their reported IC50 values for inhibition of {gamma}-secretase activity in intact cells (Table 1). Moreover, Compound E and N,[N-(3,5-difluorophenacetyl)-L-alanyl]-S-phenylglycine t-butyl ester (DAPT), the most potent {gamma}-secretase inhibitors tested, have no significant effect on calcium signaling even at concentrations several orders of magnitude above their IC50 values. In additional experiments we confirmed that DAPT treatment has no effect on the dose response of HSG cells to carbachol (the half-maximal responses to carbachol occurred at 35.6 ± 5.6 µM in untreated cells and at 30.6 ± 3.7 µM in cells treated overnight with 10 µM DAPT; n = 3 in both cases). We also tested the effects of YIL and DAPT treatment on intracellular calcium release in HSG cells induced by bradykinin. Consistent with the effects observed for carbachol stimulation, the response to bradykinin was blunted by YIL but not by DAPT (Fig. 3B).


Figure 3
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  		Fig. 3. Effects of various {gamma}-secretase inhibitors on carbachol-induced calcium release in HSG cells. A and B: HSG cells were treated with the concentrations (in µM) of the compounds indicated for 4 h, except in the case of DAPT, where treatment was for 18–20 h. The response to 100 µM carbachol (A) or 100 nM bradykinin (B) was then measured, and results were analyzed as in Fig. 2. All experimental procedures were as described in MATERIALS AND METHODS. In A, DAPT, WPE-31, and Compound E do not have statistically significant effects (n ≥ 5 in all cases; the effect of WPE-31 is marginal with P = 0.08); the effects of all other treatments are statistically significant (P < 0.003; n ≥ 4 in all cases). In B the response of bradykinin is significantly different from control with YIL treatment (P < 0.007) but not with DAPT treatment (n = 4 in both cases). C: particulate fractions from HSG cells incubated overnight in the presence (+) or absence (–) of 10 µM DAPT were analyzed via Western blotting using an antibody directed against the COOH terminus fragment (CTF) of beta-amyloid precursor protein (APP).

 

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  		Table 1. Reported IC50 values for inhibition of {gamma}-secretase activity in cultured cells by compounds tested in Fig. 3D

 
As an additional control we assayed the level of the APP COOH-terminal fragment, the substrate of {gamma}-secretase (see introduction), in untreated HSG cells and in cells treated with DAPT under the conditions used in Fig. 3A. In these experiments we found a dramatic increase in the level of intact APP COOH-terminal fragment in DAPT-treated cells (Fig. 3C), consistent with the presence of {gamma}-secretase activity in HSG cells and with the expected inhibitory effect of DAPT.

In contrast to previous observations with N2a neuroblastoma cells (22), the above results, particularly those with DAPT and Compound E, strongly suggest that inhibition of {gamma}-secretase activity does not blunt agonist-induced calcium release in HSG cells.

Inhibitory mechanism of YIL on intracellular calcium release in HSG cells. Because our results with other classes of {gamma}-secretase inhibitors were inconsistent with the hypothesis that the effects of YIL in Fig. 2 were due to inhibition of {gamma}-secretase, we set out to determine how else YIL might be acting to interfere with the calcium release process. When we monitored thapsigargin-induced calcium release from HSG cells treated with 10 µM YIL for 4 h we observed a small but consistent decrease (~13%) in this response relative to untreated controls (determined by measuring the peak 340-to-360 fura-2 fluorescence ratio after the application of 1 µM thapsigargin; data not shown). Because variations in intracellular IP3 levels can influence both the content of intracellular calcium stores and the magnitude of agonist-induced calcium release, we measured intracellular IP3 directly in YIL-treated HSG cells and in untreated controls before and after 100 µM carbachol stimulation (Fig. 4). In these experiments we found that basal IP3 levels in YIL-treated cells were approximately twofold higher than in controls. Stimulation with carbachol in both control and YIL-treated cells resulted in increased intracellular IP3 levels (Fig. 4), indicating that the phosphoinositide signaling cascade following muscarinic receptor stimulation was intact in both cases.


Figure 4
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  		Fig. 4. Intracellular inositol 1,4,5-trisphosphate (IP3) generation in control and YIL-treated HSG cells. Intracellular IP3 levels were measured as described in MATERIALS AND METHODS before and after 30-s stimulation with 100 µM carbachol in HSG cells treated for 3–4 h with vehicle or 10 µM YIL. Cell treatment and preparation was identical to that for intracellular calcium measurements (see MATERIALS AND METHODS) but without fura-2 loading. IP3 levels after YIL treatment are significantly different from those in vehicle-treated controls in cells before carbachol application (P < 0.04) as well as in cells after carbachol treatment (P < 0.02); n = 6 in all cases.

 
Taken together, the results of Figs. 3 and 4 suggest that the inhibitory effects of YIL on carbachol-induced calcium transients are not due to {gamma}-secretase inhibition but rather may be secondary to a YIL-induced increase in basal intracellular IP3 levels. We have not pursued the explanation of this phenomenon further; however, it was reported previously that elevated cellular IP3 levels can lead to downregulation of IP3 receptors and/or reduced sensitivity of intracellular calcium stores to IP3 (26, 33, 38). These effects could certainly account for the effects of YIL on calcium signaling that we observe.

Reduced APP protein expression has no significant effect on intracellular Ca2+ release in HSG cells. As an alternate test of the proposal that intracellular AICD levels modulate the content of intracellular calcium stores, we used siRNA to reduce APP expression directly. The results of these experiments are shown in Fig. 5. In Fig. 5A we demonstrate the effectiveness of our siRNA treatment. Two days after treatment with siRNA directed against APP (lane 2) we found that APP expression was 24 ± 4% of that observed in cells treated with control siRNA (lane 1), whereas the expression of Na+-K+-ATPase remained the same. However, despite this reduction in APP expression, we observed no significant change in carbachol-dependent calcium release in APP siRNA-treated cells (Fig. 5B).


Figure 5
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  		Fig. 5. Effect of small interfering RNA (siRNA)-mediated knockdown of APP expression on carbachol-induced intracellular calcium release in HSG cells. A: particulate fractions from HSG cells treated with 100 nM control (lane 1) or APP (lane 2) siRNA for 2 days were analyzed via Western blotting using an antibody directed against the COOH terminus of APP and an antibody directed against the {alpha}-subunit of Na+-K+-ATPase as indicated. B: HSG cells were treated with control or APP siRNA, respectively, for 2 days, and their responses to 100 µM carbachol treatment were measured and analyzed as in Fig. 2. The responses to carbachol with and without APP siRNA treatment are not statistically significantly different.

 
Calcium transients in fibroblasts from PS1–/–/PS2–/– mice. As a final test of the involvement of the presenilins and/or {gamma}-secretase activity in modulating intracellular calcium signaling we examined the magnitude of the calcium transients induced by thapsigargin in the immortalized PS1–/–/PS2–/– cell line BD1 derived from PS1–/–/PS2–/– mouse embryos by Donoviel and collaborators (6) and in BD1 cells stably transfected with wild-type PS1 (Fig. 6, A and B). These two cell lines had similar resting intracellular calcium levels [62.8 ± 3.6 nM (n = 5) and 65.7 ± 7.6 nM (n = 3), respectively], allowing for direct comparison of intracellular calcium responses. In these experiments we found that the thapsigargin-induced calcium transients of the two cell lines were also very similar (Fig. 6, A and B); the response of the PS1-transfected BD1 cells was 100 ± 5.7% (n = 9) of the untransfected BD1 cells, indicating that PS1 expression had no significant effect on the content of endoplasmic reticulum calcium stores. In Fig. 6C we show the results of experiments in which we confirmed the expression and lack of expression of PS1 in transfected (lane 2) and untransfected (lane 1) BD1 cells, respectively. We also show that the level of the APP COOH-terminal fragment is markedly lower in the PS1-transfected cells, consistent with the presence of {gamma}-secretase activity.


Figure 6
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  		Fig. 6. Effect of presenilin (PS)1 expression on the content of intracellular calcium stores in PS1–/–/PS2–/– fibroblasts. Typical traces of intracellular fura-2 fluorescence from untransfected BD1 cells (A) and BD1 cells stably transfected with human PS1 (B) after the application of 1 µM thapsigargin (arrow) are shown. No significant difference was detected between the magnitudes of these responses from transfected and untransfected cells (see text). By contrast, Leissring et al. (22) observed that responses to thapsigargin in their independently derived fibroblasts from PS1–/–/PS2–/– mice were ~50% lower than those observed in wild-type controls. C: particulate fractions from untransfected BD1 cells (lane 1) and BD1 cells transfected with human PS1 (lane 2) were analyzed via Western blotting using antibodies directed against the NH2-terminal fragment (NTF) of PS1 and the CTF of APP.

 
Concluding remarks. As already discussed, there is convincing evidence that cells expressing a number of mutant presenilins exhibit abnormalities in intracellular calcium signaling, specifically in the content of their intracellular calcium stores. These effects have been documented in a variety of cell types (3, 4, 14, 16, 20, 21, 23, 24, 27, 28, 35, 39), including cells obtained from familial Alzheimer’s disease patients (9, 12, 17, 40). Because of the well-documented effects of presenilin mutations on {gamma}-secretase activity, it has been suggested that this activity may play a significant role at some step in the calcium signaling cascade and thereby account for these effects. Taken together, however, the data presented here cast considerable doubt on this hypothesis, at least in the cell types we have examined. Setting aside our results with the peptide aldehydes such as YIL, which as we argue above are almost certainly not related to {gamma}-secretase inhibition, we find no evidence that inhibition of {gamma}-secretase activity has any significant effect on agonist-induced intracellular calcium release in HSG cells (Fig. 3). In complementary experiments with PS1–/–/PS2–/– knockout cells we find no evidence that the reconstitution of {gamma}-secretase activity has any effect on the content of thapsigargin-sensitive calcium stores (Fig. 6). Finally as a test of the specific hypothesis that the level of AICD, the intracellular fragment of APP resulting from {gamma}-secretase cleavage, is involved in this process, we were unable to demonstrate any significant effect of APP knockdown on the magnitude of agonist-induced intracellular calcium release in HSG cells (Fig. 5).

As discussed in the introduction, Leissring et al. (22) reached quite different conclusions from their experiments with N2a neuroblastoma cells and their independently derived PS1–/–/PS2–/– fibroblasts. At the moment, we can only speculate about the reasons for these differences between our results and theirs. One possibility is certainly that some of these effects are cell type specific. It is also important to recall that the presenilins have been shown to be involved in a number of cellular processes including cell adhesion, neurite outgrowth, and apoptosis (36), and that at least some of their effects appear to be independent of {gamma}-secretase activity (1, 25). Whether the effects of presenilin mutations on calcium signaling occur via one of these processes remains to be determined.


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This research was supported by the Intramural Research Program of the National Institutes of Health, National Institute of Dental and Craniofacial Research.


   ACKNOWLEDGMENTS
 
We thank Dr. Bruce J. Baum for many useful discussions during the course of this work.


   FOOTNOTES
 

Address for reprint requests and other correspondence: R. J. Turner, National Institute of Dental and Craniofacial Research, Bldg 10, Rm. 1A01, GTTB, Bethesda, MD 20892 (e-mail: rjturner{at}mail.nih.gov)

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.


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 ABSTRACT
 MATERIALS AND METHODS
 RESULTS AND DISCUSSION
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1. Akbari Y, Hitt BD, Murphy MP, Dagher NN, Tseng BP, Green KN, Golde TE, and LaFerla FM. Presenilin regulates capacitative calcium entry dependently and independently of {gamma}-secretase activity. Biochem Biophys Res Commun 322: 1145–1152, 2004.[CrossRef][ISI][Medline]

2. Annaert W and De Strooper B. A cell biological perspective on Alzheimer’s disease. Annu Rev Cell Dev Biol 18: 25–51, 2002.[CrossRef][ISI][Medline]

3. Begley JG, Duan W, Chan S, Duff K, and Mattson MP. Altered calcium homeostasis and mitochondrial dysfunction in cortical synaptic compartments of presenilin-1 mutant mice. J Neurochem 72: 1030–1039, 1999.[CrossRef][ISI][Medline]

4. Cedazo-Minguez A, Popescu BO, Ankarcrona M, Nishimura T, and Cowburn RF. The presenilin 1 {Delta}E9 mutation gives enhanced basal phospholipase C activity and a resultant increase in intracellular calcium concentrations. J Biol Chem 277: 36646–36655, 2002.[Abstract/Free Full Text]

5. Dohke Y and Turner RJ. Evidence that the transmembrane biogenesis of aquaporin 1 is cotranslational in intact mammalian cells. J Biol Chem 277: 15215–15219, 2002.[Abstract/Free Full Text]

6. Donoviel DB, Hadjantonakis AK, Ikeda M, Zheng H, Hyslop PS, and Bernstein A. Mice lacking both presenilin genes exhibit early embryonic patterning defects. Genes Dev 13: 2801–2810, 1999.[Abstract/Free Full Text]

7. Dovey HF, John V, Anderson JP, Chen LZ, de Saint AP, Fang LY, Freedman SB, Folmer B, Goldbach E, Holsztynska EJ, Hu KL, Johnson-Wood KL, Kennedy SL, Kholodenko D, Knops JE, Latimer LH, Lee M, Liao Z, Lieberburg IM, Motter RN, Mutter LC, Nietz J, Quinn KP, Sacchi KL, Seubert PA, Shopp GM, Thorsett ED, Tung JS, Wu J, Yang S, Yin CT, Schenk DB, May PC, Altstiel LD, Bender MH, Boggs LN, Britton TC, Clemens JC, Czilli DL, Dieckman-McGinty DK, Droste JJ, Fuson KS, Gitter BD, Hyslop PA, Johnstone EM, Li WY, Little SP, Mabry TE, Miller FD, and Audia JE. Functional gamma-secretase inhibitors reduce beta-amyloid peptide levels in brain. J Neurochem 76: 173–181, 2001.[CrossRef][ISI][Medline]

8. Esler WP, Kimberly WT, Ostaszewski BL, Ye W, Diehl TS, Selkoe DJ, and Wolfe MS. Activity-dependent isolation of the presenilin-{gamma}-secretase complex reveals nicastrin and a {gamma} substrate. Proc Natl Acad Sci USA 99: 2720–2725, 2002.[Abstract/Free Full Text]

9. Etcheberrigaray R, Hirashima N, Nee L, Prince J, Govoni S, Racchi M, Tanzi RE, and Alkon DL. Calcium responses in fibroblasts from asymptomatic members of Alzheimer’s disease families. Neurobiol Dis 5: 37–45, 1998.[CrossRef][ISI][Medline]

10. Fortini ME. {gamma}-Secretase-mediated proteolysis in cell-surface-receptor signalling. Nat Rev Mol Cell Biol 3: 673–684, 2002.[CrossRef][ISI][Medline]

11. Fraser PE, Yang DS, Yu G, Levesque L, Nishimura M, Arawaka S, Serpell LC, Rogaeva E, and George-Hyslop P. Presenilin structure, function and role in Alzheimer’s disease. Biochim Biophys Acta 1502: 1–15, 2000.[Medline]

12. Giacomello M, Barbiero L, Zatti G, Squitti R, Binetti G, Pozzan T, Fasolato C, Ghidoni R, and Pizzo P. Reduction of Ca2+ stores and capacitative Ca2+ entry is associated with the familial Alzheimer’s disease presenilin-2 T122R mutation and anticipates the onset of dementia. Neurobiol Dis 18: 638–648, 2005.[CrossRef][ISI][Medline]

13. Grynkiewicz G, Poenie M, and Tsien RY. A new generation of Ca2+ indicators with greatly improved fluorescence properties. J Biol Chem 260: 3440–3450, 1985.[Abstract/Free Full Text]

14. Guo Q, Furukawa K, Sopher BL, Pham DG, Xie J, Robinson N, Martin GM, and Mattson MP. Alzheimer’s PS-1 mutation perturbs calcium homeostasis and sensitizes PC12 cells to death induced by amyloid beta-peptide. Neuroreport 8: 379–383, 1996.[ISI][Medline]

15. Hayashi Y, Yanagawa T, Yoshida H, Azuma M, Nishida T, Yura Y, and Sato M. Expression of vasoactive intestinal polypeptide and amylase in a human parotid gland adenocarcinoma cell line in culture. J Natl Cancer Inst 79: 1025–1037, 1987.[ISI][Medline]

16. Herms J, Schneider I, Dewachter I, Caluwaerts N, Kretzschmar H, and Van Leuven F. Capacitive calcium entry is directly attenuated by mutant presenilin-1, independent of the expression of the amyloid precursor protein. J Biol Chem 278: 2484–2489, 2003.[Abstract/Free Full Text]

17. Ito E, Oka K, Etcheberrigaray R, Nelson TJ, McPhie DL, Tofel-Grehl B, Gibson GE, and Alkon DL. Internal Ca2+ mobilization is altered in fibroblasts from patients with Alzheimer’s disease. Proc Natl Acad Sci USA 91: 534–538, 1994.[Abstract/Free Full Text]

18. Kopan R and Ilagan MX. {gamma}-Secretase: proteasome of the membrane? Nat Rev Mol Cell Biol 5: 499–504, 2004.[CrossRef][ISI][Medline]

19. LaFerla FM. Calcium dyshomeostasis and intracellular signalling in Alzheimer’s disease. Nat Rev Neurosci 3: 862–872, 2002.[ISI][Medline]

20. Leissring MA, Akbari Y, Fanger CM, Cahalan MD, Mattson MP, and LaFerla FM. Capacitative calcium entry deficits and elevated luminal calcium content in mutant presenilin-1 knockin mice. J Cell Biol 149: 793–798, 2000.[Abstract/Free Full Text]

21. Leissring MA, LaFerla FM, Callamaras N, and Parker I. Subcellular mechanisms of presenilin-mediated enhancement of calcium signaling. Neurobiol Dis 8: 469–478, 2001.[CrossRef][ISI][Medline]

22. Leissring MA, Murphy MP, Mead TR, Akbari Y, Sugarman MC, Jannatipour M, Anliker B, Muller U, Saftig P, De SB, Wolfe MS, Golde TE, and LaFerla FM. A physiologic signaling role for the {gamma}-secretase-derived intracellular fragment of APP. Proc Natl Acad Sci USA 99: 4697–4702, 2002.[Abstract/Free Full Text]

23. Leissring MA, Parker I, and LaFerla FM. Presenilin-2 mutations modulate amplitude and kinetics of inositol 1,4,5-trisphosphate-mediated calcium signals. J Biol Chem 274: 32535–32538, 1999.[Abstract/Free Full Text]

24. Leissring MA, Paul BA, Parker I, Cotman CW, and LaFerla FM. Alzheimer’s presenilin-1 mutation potentiates inositol 1,4,5-trisphosphate-mediated calcium signaling in Xenopus oocytes. J Neurochem 72: 1061–1068, 1999.[CrossRef][ISI][Medline]

25. Meredith JE Jr, Wang Q, Mitchell TJ, Olson RE, Zaczek R, Stern AM, and Seiffert D. {gamma}-Secretase activity is not involved in presenilin-mediated regulation of beta-catenin. Biochem Biophys Res Commun 299: 744–750, 2002.[CrossRef][ISI][Medline]

26. Oberdorf J, Webster JM, Zhu CC, Luo SG, and Wojcikiewicz RJ. Down-regulation of types I, II and III inositol 1,4,5-trisphosphate receptors is mediated by the ubiquitin/proteasome pathway. Biochem J 339: 453–461, 1999.

27. Popescu BO, Cedazo-Minguez A, Benedikz E, Nishimura T, Winblad B, Ankarcrona M, and Cowburn RF. {gamma}-Secretase activity of presenilin 1 regulates acetylcholine muscarinic receptor-mediated signal transduction. J Biol Chem 279: 6455–6464, 2004.[Abstract/Free Full Text]

28. Schneider I, Reverse D, Dewachter I, Ris L, Caluwaerts N, Kuiperi C, Gilis M, Geerts H, Kretzschmar H, Godaux E, Moechars D, Van LF, and Herms J. Mutant presenilins disturb neuronal calcium homeostasis in the brain of transgenic mice, decreasing the threshold for excitotoxicity and facilitating long-term potentiation. J Biol Chem 276: 11539–11544, 2001.[Abstract/Free Full Text]

29. Seiffert D, Bradley JD, Rominger CM, Rominger DH, Yang F, Meredith JE Jr, Wang Q, Roach AH, Thompson LA, Spitz SM, Higaki JN, Prakash SR, Combs AP, Copeland RA, Arneric SP, Hartig PR, Robertson DW, Cordell B, Stern AM, Olson RE, and Zaczek R. Presenilin-1 and -2 are molecular targets for {gamma}-secretase inhibitors. J Biol Chem 275: 34086–34091, 2000.[Abstract/Free Full Text]

30. Selkoe DJ. Alzheimer’s disease: genes, proteins, and therapy. Physiol Rev 81: 741–766, 2001.[Abstract/Free Full Text]

31. Ship JA, DeCarli C, Friedland RP, and Baum BJ. Diminished submandibular salivary flow in dementia of the Alzheimer’s type. J Gerontol 45: M61–M66, 1990.[ISI][Medline]

32. Shirasuna K, Sato M, and Miyazaki T. A neoplastic epithelial duct cell line established from an irradiated human salivary gland. Cancer 48: 745–752, 1981.[CrossRef][ISI][Medline]

33. Sipma H, Deelman L, Smedt HD, Missiaen L, Parys JB, Vanlingen S, Henning RH, and Casteels R. Agonist-induced down-regulation of type 1 and type 3 inositol 1,4,5-trisphosphate receptors in A7r5 and DDT1 MF-2 smooth muscle cells. Cell Calcium 23: 11–21, 1998.[CrossRef][ISI][Medline]

34. Stutzmann GE. Calcium dysregulation, IP3 signaling, and Alzheimer’s disease. Neuroscientist 11: 110–115, 2005.[Abstract]

35. Stutzmann GE, Caccamo A, LaFerla FM, and Parker I. Dysregulated IP3 signaling in cortical neurons of knock-in mice expressing an Alzheimer’s-linked mutation in presenilin1 results in exaggerated Ca2+ signals and altered membrane excitability. J Neurosci 24: 508–513, 2004.[Abstract/Free Full Text]

36. Thinakaran G and Parent AT. Identification of the role of presenilins beyond Alzheimer’s disease. Pharmacol Res 50: 411–418, 2004.[CrossRef][ISI][Medline]

37. Van Gassen G and Annaert W. Amyloid, presenilins, and Alzheimer’s disease. Neuroscientist 9: 117–126, 2003.[Abstract]

38. Wojcikiewicz RJ, Furuichi T, Nakade S, Mikoshiba K, and Nahorski SR. Muscarinic receptor activation down-regulates the type I inositol 1,4,5-trisphosphate receptor by accelerating its degradation. J Biol Chem 269: 7963–7969, 1994.[Abstract/Free Full Text]

39. Yoo AS, Cheng I, Chung S, Grenfell TZ, Lee H, Pack-Chung E, Handler M, Shen J, Xia W, Tesco G, Saunders AJ, Ding K, Frosch MP, Tanzi RE, and Kim TW. Presenilin-mediated modulation of capacitative calcium entry. Neuron 27: 561–572, 2000.[CrossRef][ISI][Medline]

40. Zatti G, Ghidoni R, Barbiero L, Binetti G, Pozzan T, Fasolato C, and Pizzo P. The presenilin 2 M239I mutation associated with familial Alzheimer’s disease reduces Ca2+ release from intracellular stores. Neurobiol Dis 15: 269–278, 2004.[CrossRef][ISI][Medline]

41. Zhang Z, Nadeau P, Song W, Donoviel D, Yuan M, Bernstein A, and Yankner BA. Presenilins are required for {gamma}-secretase cleavage of beta-APP and transmembrane cleavage of Notch-1. Nat Cell Biol 2: 463–465, 2000.[CrossRef][ISI][Medline]





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