the presenilin 1 ∆e9 mutation gives enhanced basal … · 4 hydrolysis of phosphoinositides (pi)...

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The presenilin 1 ∆E9 mutation gives enhanced basal phospholipase C

activity and a resultant increase in intracellular calcium concentrations.

Angel Cedazo-Mínguez*1, Bogdan O. Popescu*1, Maria Ankarcrona1,2, Takeshi

Nishimura2 and Richard F. Cowburn1,2.

1. Karolinska Institutet. Neurotec, Section of Experimental Geriatrics,

NOVUM, KFC, 141 86 Huddinge, Sweden.

2. Karolinska Institutet. Neurotec, Section of Experimental Geriatrics, KASPAC,

NOVUM, 141 57 Huddinge, Sweden.

*These authors contributed equally to the work.

Address correspondence to:

Angel Cedazo-Mínguez

Karolinska Institutet, Neurotec,

Section of Experimental Geriatrics

Novum, KFC, plan 4

S-141 86 Huddinge, Sweden

Phone: +46 8 585 83751

FAX: +46 8 585 83880

e-mail: [email protected]

Running title: Presenilin 1 ∆E9 mutation enhances PLC activity.

Copyright 2002 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on July 16, 2002 as Manuscript M112117200 by guest on Septem

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Summary

We studied effects of the familial Alzheimer's disease (FAD) presenilin 1 (PS1) exon

9 deletion (PS1-∆E9) mutation on basal and carbachol-stimulated phosphoinositide (PI)

hydrolysis and intracellular Ca2+ concentrations ([Ca2+]i) in human SH-SY5Y

neuroblastoma cells. We demonstrate that PS1-∆E9 cells have an enhanced basal PI

hydrolysis and [Ca2+]i as compared with both wild type PS1 (PS1-WT) and non-

transfected (NT) cells. Both were reversed by the phospholipase C (PLC) inhibitor

neomycin. The PS-1∆E9 related high basal [Ca2+]i was also reversed by xestospongin C

confirming that this effect was inositol trisphosphate receptor (IP3R)-mediated. Carbachol

gave a greater stimulation of [Ca2+]i in PS1-∆E9 cells that took longer to return to basal as

compared to responses seen in NT and PS1-WT cells. This long tail-off effect seen in

PS1-∆E9 cells after carbachol stimulation was reversed by xestospongin C and dantrolene

suggesting that it was mediated by IP3R and ryanodine receptor amplification of Ca2+.

Ruthenium red only reduced carbachol peak elevations of [Ca2+]i in NT and PS1-WT

cells and not in PS1-∆E9 cells. No significant between cell type differences were seen for

basal and carbachol stimulated [Ca2+]i with either ryanodine or the endoplasmic reticulum

Ca2+ ATP-ase inhibitor cyclopiazonic acid. Immunostaining experiments revealed that for

all the cell types PS1 is present at the plasma membrane and co-localises with N-

cadherin, a component of the cell-cell adhesion complex. Immunoblotting of cell extracts

for PLC-β1 showed that compared to NT and PS1-WT cells, the PS1-∆E9 transfectants

gave a relative increase in levels of the calpain generated N-terminal fragment (100 kDa)

over full length (150 kDa) PLC-β1. Our results suggest that the PS1-∆E9 mutation causes

upstream changes in PI signalling with enhanced basal PLC activity as a primary effect

that leads to a higher [Ca2+]i. This may provide a novel mechanism by which the PS1-∆E9

mutation sensitises cells to apoptotic stimuli and enhanced Aβ generation.

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1. Introduction

Mutations in the presenilin 1 (PS1) gene on chromosome 14 account for a large

proportion of the early onset familial cases of Alzheimer’s disease (FAD) (1). Two

pathogenic mechanisms have been proposed by which PS1 mutations cause FAD. One

mechanism involves altered proteolytic processing of the amyloid precursor protein

(APP). Fibroblasts from PS1 mutation bearing individuals, brains from mice

overexpressing mutant PS1, as well as cells transfected with FAD-linked PS1 variants, all

produce a greater proportion of longer, more fibrillogenic forms of β-amyloid (Aβ) (2, 3,

4). Several recent studies have shown that PS1 plays an important role in regulating the γ-

secretase cleavage of APP (5, 6, 7) and it has been hypothesised that PS could in fact be

the γ-secretase enzyme (8).

A second mechanism proposes that PS1 mutations make cells more vulnerable to

undergo death by apoptosis. Guo et al, showed that the PS1 L286V mutation increases the

vulnerability of PC12 cells to apoptosis induced by Aβ (9) and nerve growth factor

withdrawal (10). The pro-apoptotic effects of PS1 have been shown to involve increases

in intracellular Ca2+ concentration ([Ca2+]i) and peroxide levels under conditions of

oxidative and excitotoxic stress (9, 10). Hippocampal neurons from L286V PS1 mutant

knockin mice are more vulnerable to excitotoxicity associated with increased [Ca2+]i

levels (10). These data are in accordance with older reports showing that bradykinin

stimulated fibroblasts from FAD patients have higher [Ca2+]i than fibroblasts from age-

matched controls (11, 12). Dysregulation of [Ca2+]i may be of importance in the

pathogenesis of AD, since it has also been shown to contribute to enhanced Aβ

generation (13) and tau protein hyperphosphorylation (14).

A large proportion of Ca2+ mobilisation and regulation in neurons can be attributable

to cholinergic neurotransmission (15). Activation of phospholipase C (PLC) results in


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hydrolysis of phosphoinositides (PI) to give formation of the second messengers inositol

1,4,5 trisphosphate (IP3) and diacylglycerol (DAG). Whilst IP3 releases Ca2+ from

intracellular stores, DAG activates protein kinase C (PKC) (for review see 16).

Cholinergic stimulation of PI hydrolysis has been shown to be severely impaired in brain

tissue from AD subjects (for review, see 17). In addition, two molecules that play an

essential role in AD, namely Aβ and apolipoprotein E4, have both been shown to disrupt

acetylcholine muscarinic receptor stimulated PI hydrolysis in vitro (18, 19).

Recent studies from Leissring and colleagues have provided some insight into the

mechanisms by which PS1 mutations affect Ca2+ signalling. Using Xenopus oocytes

loaded with caged IP3, they demonstrated that PS1 modulates the IP3 mediated Ca2+

release from internal stores and that this was enhanced in cells expressing the PS1

M146V mutation (20, 21). PS1 effects on Ca2+ signalling were suggested to be owing to

an abnormal elevation of Ca2+ within the endoplasmic reticulum (ER) rather than to

perturbations in either the number or activity of IP3-activated Ca2+ release channels (22).

At present, the effects of PS1 mutations on cholinergic signalling processes upstream of

the IP3 receptor have not been determined.

A previous report by Moerman and Barger (23) showed that the PS1-L286V

mutation dampened glutamate-induced Ca2+ responses mediated by α-amino-3-hydroxy-

5-methyl-4-isoxazolepropionic acid receptors in human NT2 neuroblastoma cells. This

study suggested that PS1 mutations may alter upstream neurotransmission processes with

resultant consequences for survival at the cellular level and for cognition at the

physiological level.

To test the hypothesis that FAD-linked PS1 mutations influence muscarinic receptor-

stimulated signalling, we used SH-SY5Y neuroblastoma cells stably transfected with

either wild type PS1 (PS1-WT) or the FAD causing exon 9 deletion mutation of PS1


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(PS1-∆E9) to compare basal and carbachol stimulated PI hydrolysis and [Ca2+]i. SH-

SY5Y neuroblastoma cells have been extensively used as a neuronal model to study PI

hydrolysis (for review see 24). We also examined downstream effects of the PS1-∆E9

mutation on Ca2+ release from the endoplasmic reticulum (ER) through IP3 receptors

(IP3R), ryanodine receptors (RyR) and Ca2+-transporting ATP-ases under basal conditions

and following acetylcholine muscarinic receptor stimulation.


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2. Materials and methods

Materials

Chemicals and isotopes were purchased from the following companies: myo [2-3H]

inositol (10 Ci / mmol) from NEN, Du Pont, Europe. Carbamylcholine chloride

(carbachol), DOWEX 1X8-200 (chloride form), ruthenium red, ryanodine, dantrolene,

cyclopiazonic acid, and probenecid were from Sigma-Aldrich, Sweden. Neomycin

(neomycin sulfate) and xestospongin C, were from Calbiochem-Novabiochem

(Darmstadt, Germany). Fluo-3 acetoxymethyl (AM) ester and Pluronic F-127 were

purchased from Molecular Probes (Europe BV, The Netherlands).

All other chemicals were standard laboratory reagents.

Cell Culture

Human SH-SY5Y neuroblastoma cells stably transfected with either PS1-WT or the

PS1-∆E9 mutation were established in our lab as described previously (25).

SH-SY5Y neuroblastoma cells were cultured at 37°C, 5% CO2, in Minimum

essential medium (MEM) with Earle´s salts containing 10% Foetal Calf Serum (FCS), 2

mM L-Glutamine, 100 units/ml penicillin, 100 µg/ml streptomycin. Transfected cells

were additionally supplemented with 200 µg/ml geneticin. All cell culture supplies were

purchased from GIBCO-BRL, Life Technologies (European Division).

Immunoblot analysis

The expression of PS1 in non-transfected (NT) and transfected cell lines was checked

by Western blotting as previously described (25). To obtain cell extracts, cells were


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washed twice with ice-cold Ca2+- and Mg2+- free phosphate buffered saline (PBS),

harvested with a cell-scraper and collected by 3000 rpm centrifugation for 3 min. Pellets

were resuspended in 100 µl of lysis buffer (20 mM Tris-HCl, 137 mM NaCl, 2 mM

EDTA, 2% Nonidet P-40, 2% Triton-X100 and 400 µg/ml protease inhibitors-Complete

TM mini, Boehringer Mannheim, Germany) and centrifuged at 11000 rpm for 10 min at

4°C. Samples were stored at -20°C until use. Protein amounts in cell extracts were

quantified by the Protein assay kit (Pierce, Rockford, IL, USA). Equivalent amounts of

protein were separated by SDS-PAGE using 10% or 7% acrylamide gels for the PS-1 or

PLC expression analyses respectively. Proteins were transferred onto Hybond ECL

nitrocellulose membranes (Amersham, UK) by trans-blot Electrophoretic Transfer for 4 h

at a constant current of 200 mA. After blocking for 1 h using 5% (w/v) dried milk in Tris-

buffered solution containing 0.1% Tween-20 (TBS-t) the membranes were incubated

overnight with the first antibody at the following concentrations; 1:2000 (PS1 antibody),

1:1000 (PLC-β1 and PLC-γ antibodies; Transduction Laboratories, Lexington, KY,

USA). The membranes were washed in TBS-t for 15 min and then three times for 5 min.

The secondary antibody (anti-rabbit or anti-mouse horseradish peroxidase linked)

(Amershan Life Science, UK) diluted in blocking solution (1:2000) was added to the

membranes for 1 h at room temperature. The membranes were then washed as described

above, plus a further wash of 1 h. Bound antibody was detected by the ECL method

(Amersham, UK) after exposure to Hyper film MP (Amersham, UK). The relative density

of immunoreactive bands on Western blots was calculated from the optical density

multiplied by the area of the selected band, following acquisition of the blot image

through Image Master (Pharmacia Biotech, Sweden).


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PI hydrolysis assay

Cells were cultured until 75-80 % confluence in 75 cm2 flasks. One day prior to the

experiment, cells were changed to serum free media containing 5 µCi/ml myo [2-3H]

inositol and incubated for 24 h. PI hydrolysis was measured as described previously (19).

Cells were harvested by scraping with a rubber policeman in 4 ml PBS. Contents were

centrifuged at 1500 rpm for 15 min. Pellets were washed twice with 37°C PBS and re-

suspended in 3 ml 37°C Krebs-Henseleit bicarbonate buffer containing 10 mM LiCl

(KHB/Li), gassed with 5% CO2 / 95% O2 and centrifuged again (15 000 rpm, 15 min).

Cell pellets were re-suspended in 210 µl KHB/Li, regassed and 50 µl added to glass

centrifuge tubes containing 250 µl KHB/Li buffer with or without 100 µM carbachol. The

tubes were incubated at 37°C under an atmosphere of 5% CO2 / 95% O2 with gentle

agitation for 25 min. Incubations were stopped by adding 940 µl of chloroform: methanol

(1:2). Tubes were incubated on ice for 30 min and phases separated by adding 310 µl

chloroform and 310 µl water followed by vortexing and centrifugation. 750 µl of the

aqueous phase were removed and labelled inositol phosphates (IPs) separated from [3H]

myoinositol by DOWEX chromatography, as described by Berridge (26). The chloroform

phase was extracted with 75 µl HCl and tubes were vortexed again for 20 s, followed by 5

min centrifugation. The chloroform phase was removed, placed into scintillation vials and

allowed to evaporate before determination of “lipid dpm” by scintillation spectroscopy.

Results were expressed as dpm IPs / (dpm IPs + dpm lipid). This unit is independent of

the number of cells aliquoted in each tube and upon the degree of labelling of inositol

phospholipids (27).

For the treatments with neomycin, one day prior to the experiment cells were

changed to serum free media containing 5 µCi/ml myo [2-3H] inositol, and incubated for


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21 h. The medium was then replaced with that containing neomycin (500 µM) plus 5

µCi/ml myo [2-3H] inositol for 3 hr prior to PI hydrolysis measures.

Intracellular calcium measurements

Intracellular Ca2+ concentrations were essentially determined as previously described

(28). In brief, cells were loaded with medium containing 5 µM Fluo-3 AM ester, 0.5%

(V/V) Pluronic F-127 and 1 mM of the organic anion-transport inhibitor probenecid (90

min in the dark, at room temperature). After loading, cells were incubated for 120 min in

MEM without phenol red with 1 mM probenecid in the dark, at room temperature, to

allow intracellular esterases to decompose the Fluo-3 AM ester. For the measurements of

basal [Ca2+]i, the medium was removed and Ca2+ and Mg2+ free PBS added. Basal [Ca2+]i

was measured repeatedly during 10 min at 37 ºC to certify that a steady state of [Ca2+]i

was obtained. Preliminary experiments showed that for all three cell types, basal [Ca2+]i

in the absence of treatments was steady during the entire incubation time courses used

(data not shown). Neomycin, ruthenium red, ryanodine, and cyclopiazonic acid were

added in PBS at indicated concentrations at 37 ºC for the 10 min when basal [Ca2+]i was

measured. Xestospongin C and dantrolene were added at indicated concentrations in

MEM without phenol red during the 120 min of incubation time and in PBS for the 10

minutes when basal [Ca2+]i was measured. The PBS was then removed and 100 µM

carbachol in PBS solution at 37 ºC added (100 µl to each well). Carbachol was present for

all subsequent [Ca2+]i measures. Fluorescence was measured by a CytoFluor Series 4000

multi-well plate reader (PerSeptive Biosystems GmbH, Wiesbaden, Germany), the

excitation and emission wavelengths being 485 and 530 nm, respectively. Slit widths

were 20 nm for both excitation and emission. Apparent [Ca2+]i corresponding to

fluorescent value F was calculated with the formula: [Ca2+]i = Kd (F-Fmin)/(Fmax-F), where


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Kd for Fluo-3-Ca2+ was taken as 390 nm, as indicated by the manufacturer’s protocol

(Molecular Probes, Europe BV, The Netherlands). Fmin was determined by measuring the

signal of the unloaded cells and Fmax was determined by addition to the cells of a 100 mM

CaCl2 buffer containing 1% (V/V) Triton-X 100.

Immunocytochemistry and confocal microscopy

Cells grown on cover-slips were fixed at 4°C in 2% paraformaldehyde + 0.2%

glutaraldehyde for 20 min and subsequently rinsed in PBS (3 x 10 min). Cells were

permeabilised with 0.2 % Triton X-100 in PBS for 10 min and then 10 % goat-serum

was added for 30 min to block unspecific binding of antibodies. Cells were incubated

over-night at 4°C with primary antibodies diluted in 10 % serum. Polyclonal anti PS1 N-

terminal antibody was diluted 1:500 and monoclonal anti α-N-cadherin antibody was

diluted 1:500. Next day cells were rinsed in PBS (3 x 10 min) before incubation with

anti-mouse antibody conjugated to Alexa green (1:400 dilution) and anti-rabbit antibody

conjugated to cy3 (1:1000 dilution) in PBS + 0.3% Triton X-100 for 1h in the dark.

Finally cells were rinsed in PBS (3 x 10 min) and mounted in glycerol:PBS 1:1 on glass-

slides.

Fluorescence images were excited using the 488 nm line of a krypton laser and the

543 nm line of a helium-neon laser on a Bio-Rad Radiance Plus confocal microscope.

Cy3 fluorescence was collected using a HQ 590/70 emission filter (red) and Alexa green

was collected with a HQ 515/30 emission filter (green).

Statistical analyses.

Analyses of differences were carried out by ANOVA followed by Fisher’s PLSD

post-hoc test. A value of p<0.05 was considered statistically significant.


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3. Results

PS1 expression in transfected cell lines

SH-SY5Y neuroblastoma cells stably transfected to express moderate levels of PS1-

WT and PS1-∆E9 were established previously in our lab (25). The expression of PS1 in

these cell lines was confirmed by sequencing and checked by immunoblotting using a

PS1 N-terminal antiserum. The pattern of PS1 expression was similar as reported before

(25). NT cells were found to express very low levels of PS1 holoprotein, most of PS1

being identified as approximately 28 kDa band corresponding to endogenous PS1 N-

terminal fragment (NTF). PS1-WT cells (clones 1-1 and 1-14) showed accumulation of

full length PS1 (48 kDa) as well as of NTF. PS1-∆E9 cells (clones 1-2 and 2-4) lack the

cleavage site where the physiological proteolytic cleavage occurs (site between residues

291 and 299), and showed accumulation of a truncated full-length protein and a reduced

endogenous NTF consistent with the deletion of the cleavage region (Figure 1). Both WT

and mutant clones showed similar PS1 mRNA levels following transfection (25). The 1-2

clone of PS1-∆E9 cells shows the highest levels of truncated holoprotein and lower levels

of endogenous NTF indicative of higher expression, as compared to the 2-4 clone.

Effects of the PS1-∆E9 mutation on basal and carbachol stimulated PI hydrolysis.

Figure 2A shows that basal PI hydrolysis was significantly higher in both clones of

PS1-∆E9 cells (1-2 and 2-4) as compared to NT and to both clones of PS1-WT cells (1-1

and 1-14). Carbachol (100 µM) stimulated PI hydrolysis was significantly higher in both

clones of PS1-∆E9 cells as compared to both clones of PS1-WT cells (Figure 2A). The

clone 2-4 of PS1-∆E9 cells also showed a significantly higher carbachol stimulated PI

hydrolysis as compared to NT cells.


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However, analysis of the carbachol stimulated PI hydrolysis as the difference from

basal did not show significant differences between any of the three cell types (Figure 2B).

Effects of the PS1-∆E9 mutation on basal and carbachol stimulated intracellular Ca2+

concentrations.

Basal [Ca2+]i (values given for time 0 before addition of carbachol) was similar in NT

(36.2 ± 1.3 nM) and in PS1-WT cells (36.1 ± 0.9 nM; and 38.6 ± 1.2 nM, respectively for

clones 1-1 and 1-14). The PS1-∆E9 transfected cells showed significantly higher basal

[Ca2+]i (58.6 ± 3.2 nM and 65.1 ± 3.2 nM, respectively for clones 1-2 and 2-4) as

compared with both NT and both clones of PS1-WT cells (p<0.001; ANOVA, Fisher's

post-hoc test) (Figure 3).

Treatment of cells with 100 µM carbachol gave a rapid peak elevation of [Ca2+]i.

Peak elevations were found at approximately 30 sec after addition of carbachol. No

significant differences were found for peak [Ca2+]i elevations between PS1-WT (64.0 ±

3.6 nM and 73.0 ± 2.33 nM, respectively for clones 1-1 and 1-14) and NT cells (65.1 ±

3.3 nM). The PS1-∆E9 clones showed a carbachol induced peak [Ca2+]i elevation of 105.4

± 3.9 nM (clone 1-2) and 133.6 ± 12.4 nM (clone 2-4). These values were statistically

significantly higher than those seen for both NT and both clones of PS1-WT cells

(p<0.001; ANOVA, Fisher's post-hoc test). [Ca2+]i returned to basal after approximately

120 sec in NT and PS1-WT cells. In contrast, the PS1-∆E9 cells showed a long tail-off

effect where [Ca2+]i was still significantly higher than basal even at 5 min after addition

of carbachol (p<0.01; ANOVA, Fisher's post-hoc test) (Figure 3).

For subsequent experiments of PI hydrolysis and [Ca2+]i measurements we selected

the 1-1 clone of PS1-WT and the 1-2 clone of PS1-∆E9. The 1-2 clone of PS1-∆E9 gave


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lower basal and carbachol stimulated PI and [Ca2+] responses as compared to the 2-4

clone and was chosen so as to avoid potential ceiling effects.

Neomycin reversal of basal PI hydrolysis and [Ca2+]i in PS1-∆E9 cells to levels seen in

PS1-WT cells.

An enhanced high basal PI hydrolysis could lead to a rise in [Ca2+]i. Alternatively,

high [Ca2+]i could activate various PLC isoforms thereby resulting in an enhanced PI

hydrolysis. To investigate the relationship between PLC activity and [Ca2+]i, we treated

cells with neomycin, an inhibitor of PLC (29), and then determined both basal and

carbachol stimulated PI hydrolysis and [Ca2+]i.

Pre-treatment for 3 h with neomycin (500 µM) gave drastic reductions of both basal

and carbachol stimulated PI hydrolysis in all cell lines. Neomycin (500 µM) decreased

the high basal and carbachol stimulated PI hydrolysis seen in PS1-∆E9 cells to the same

level as for PS1-WT and NT cells (Figure 4A).

Neomycin (100 and 500 µM), treatment for 10 min before carbachol stimulation did

not affect basal [Ca2+]i in either NT or PS1-WT cells. In contrast, both neomycin

concentrations significantly reduced basal [Ca2+]i in PS1-∆E9 cells (to 43.9 ± 1.0 nM and

39.8 ± 1.3 nM, for 100 µM and 500 µM, neomycin respectively) (Figure 4B). Neomycin

(500 µM) completely reversed the significantly higher basal [Ca2+]i in PS1-∆E9 cells to

levels comparable to those seen in NT and PS1-WT cells (Figure 4B).

Neomycin treatment also significantly reduced the carbachol elevation of [Ca2+]i in

all cell lines (to 55.4 ± 4.9 nM, 52.0 ± 5.9 nM and 84.7 ± 6.2 nM in NT, PS1-WT and

PS1-∆E9 cells, respectively for 100 µM neomycin and to 52.7 ± 4.1 nM, 46.9 ± 5.0 nM

and 71.1 ± 4.2 nM in NT, PS1-WT and PS1-∆E9 cells, respectively for 500 µM

neomycin). The neomycin (both 100 µM and 500 µM) reduction of carbachol stimulated


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[Ca2+]i seen in PS1-∆E9 cells did not come down to the same level as seen for either NT

(p<0.05; ANOVA, Fisher's post-hoc test) or PS1-WT cells (p<0.01; ANOVA, Fisher's

post-hoc test) (Figure 4B).

Regulation of intracellular Ca2+ pools in NT, PS1-WT and PS1-∆E9 cells.

Xestospongin C, a specific antagonist of IP3R (30), did not induce significant

changes of basal [Ca2+]i in either NT or PS1-WT cells. In contrast, for PS1-∆E9 cells

xestospongin C gave a significant reduction of basal [Ca2+]i to the levels seen in NT and

PS1-WT cells (to 49.0 ± 1.5 nM and to 44.2 ± 1.2 nM for 1 µM and 10 µM, respectively)

(Figure 5). Xestospongin C (10 µM) significantly reduced the carbachol elevation of

[Ca2+]i in NT and PS1-WT cells to levels approaching basal [Ca2+]i (to 44.1 ± 3.5 nM in

NT and to 50.5 ± 3.0 nM in PS1-WT cells). In PS1-∆E9 cells, xestospongin C gave a

dose-dependent reduction of the carbachol elevation of [Ca2+]i (to 67.1 ± 5.3 nM and to

53.3 ± 3.7 nM respectively for 1 and 10 µM xestospongin C). These reduced levels were

similar to those seen in NT and PS1-WT cells (Figure 5). Both 1 and 10 µM xestospongin

C treatments also abolished the long tail-off effect on [Ca2+]i seen in PS1-∆E9 cells after

carbachol stimulation (Figure 5).

Ruthenium red, a ryanodine receptor and mitochondrial uniporter competitive

antagonist gave a reduction of basal [Ca2+]i in all cell lines in a dose-dependent manner

(to 28.2 ± 2.2 nM, 28.3 ± 1.7 nM and 50.5 ± 3.9 nM in NT, PS1-WT and PS1-∆E9 cells,

respectively, for 10 µM ruthenium red and to 22.9 ± 3.8 nM, 25.1 ± 2.8 nM and 37.3 ±

1.8 nM in NT, PS1-WT and PS1-∆E9 cells, respectively for 30 µM ruthenium red)

(Figure 6). Basal [Ca2+]i in PS1-∆E9 cells remained significantly higher as compared with

both NT and PS1-WT cells (p<0.01; ANOVA, Fisher's post-hoc test) after treatment with

both concentrations of ruthenium red (Figure 6).


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Both ruthenium red concentrations significantly reduced carbachol peak elevations of

[Ca2+]i in NT and PS1-WT cells, but not in PS1-∆E9 cells (Figure 6).

Treatment of the cells with ryanodine (either 1 µM and 10 µM) for 10 min did not

induce significant changes in either basal or carbachol stimulated [Ca2+]i levels in any of

the cell lines (Figure 7).

Combined treatment with 10 µM ryanodine and 30 µM ruthenium red decreased

basal [Ca2+]i in all cell lines to similar levels seen with 30 µM ruthenium red alone (to

27.9 ± 6.4 nM, 26.8 ± 2.7 nM, 44.2 ± 3.1 nM in NT, PS1-WT and PS1-∆E9 cells,

respectively) (Figures 6 and 7). None of the cell lines showed changes in carbachol peak

elevations of [Ca2+]i after 10 µM ryanodine + 30 µM ruthenium red (Figure 7).

Dantrolene, a specific blocker of RyR (31), used at concentrations of 100 nM, 1 µM

and 10 µM did not induce any significant changes of basal [Ca2+]i in NT and PS1-WT

cells (Figure 8). In contrast, in PS1-∆E9 cells both 1 µM and 10 µM dantrolene

significantly reduced basal [Ca2+]i (to 52.7 ± 2.5 nM and 54. 8 ± 2.0 nM respectively).

Basal [Ca2+]i in PS1-∆E9 cells was still significantly higher than that for either NT or

PS1-WT (p<0.01; ANOVA, Fisher's post-hoc test) after treatment with both dantrolene

concentrations (Figure 8). Ten µM dantrolene significantly reduced the carbachol-

induced peak of [Ca2+]i in NT and PS1-WT cells, but not in PS1-∆E9 cells. Dantrolene

treatment of PS1-∆E9 cells abolished the long tail-off effect on [Ca2+]i seen after

carbachol stimulation (Figure 8).

Cyclopiazonic acid (CPA, 10 µM and 30 µM), an ER Ca2+ ATP-ase inhibitor, did not

significantly change basal [Ca2+]i in any of the cell lines, even though a tendency towards

an increase was seen in PS1-∆E9 cells at early time points (Figure 9). CPA treatment did

not affect carbachol elevation of [Ca2+]i in any of the cell lines (Figure 9).


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PLC levels in NT, PS1-WT and PS1-∆E9 cells.

To determine whether the enhanced basal PI hydrolysis seen in PS1-∆E9 cells was

due to increased PLC levels, we investigated the expression of PLC-β1 and PLC-γ

isoenzymes in NT, PS1-WT and PS1-∆E9 cells. PLC-β1 is the β-isoform most widely

expressed in the brain and primary neurons and which is at highest concentrations in brain

areas affected in AD. PLC-γ is also expressed in brain where it is concentrated in

astroglia. In addition to its function in PI hydrolysis, PLC-γ is involved in regulating PI-3

kinase activity and therefore contributes to the regulation of anti-apoptotic pathways (for

review, see 16).

The monoclonal N-terminal PLC-β1 antibody recognised two groups of

inmunoreactive bands at approximately 150 and 100 kDa (Figure 10A). The 150 kDa

molecular weight band represents full length PLC-β1, whereas the 100 kDa band

represents the N-terminal fragment of PLC-β1 after proteolytic cleavage by calpain (32).

Densitometric analysis of the blots showed that 150 kDa full length PLC-β1 was

decreased in PS1-∆E9 cells, as compared with both NT and PS1-WT cells (Figure 10B).

In contrast, both the PS1-WT and PS1-∆E9 cells showed a significantly higher level of

the 100 kDa fragment as compared to NT cells.

The monoclonal PLC-γ antibody recognised one band at approximately 148 kDa. No

differences in PLC-γ expression were seen between NT, PS1-WT and PS1-∆E9 cells

(Figure 10A).

Localization of PS1 in NT, PS1-WT and PS1-∆E9 cells.

PS1 is known to be principally localised in the Golgi apparatus and the endoplasmic

reticulum. Recent reports have shown that PS1 is also localised at the plasma membrane

where it has been shown to form complexes with components of the cadherin-catenin cell


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to cell adhesion system (33, 34, 35). To determine whether PS1 was also found at the

plasma membrane in the cells used in our study, we performed double immunostaining

experiments with anti N-terminal PS1 and anti N-cadherin antibodies and examined cells

with laser scanning confocal microscopy. In agreement with previous reports (34, 35)

confluent cells from both NT as well as PS1-WT and PS1-∆E9 transfected cell lines

showed a granular PS1 immunofluorescence pattern that was strong at sites of cell to cell

contact (Figure 11, first column). As expected, immunoreactivity of N-cadherin, a

component of the cell adhesion system, was found concentrated at the plasma membrane

at sites of cell-cell contact (Figure 11, second column). Two-color immunofluorescence

of PS1 and N-cadherin also showed co-localisation of these proteins at sites of cell to cell

contact (Figure 11, yellow).


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4. Discussion

Mutations in PS1 account for the majority of FAD cases. Either an altered APP

processing leading to a more abundant production of more amyloidogenic forms of Aβ (2,

3, 4) or an increased susceptibility to apoptosis (9, 10) have been described as the putative

mechanisms responsible for the association of PS1 mutations and FAD. The pro-apoptotic

effects of PS1 have been demonstrated to involve disturbances in [Ca2+]i (9, 10).

Cholinergic signalling is one of the major sources of Ca2+ regulation and

redistribution in neuronal cells (for review, see 15) and it is well known to be impaired in

AD (17). In the present paper we investigated the possibility that PS1 mutations could

influence acetylcholine muscarinic receptor-mediated PI hydrolysis and Ca2 +

homeostasis. To this end, we chose to study the PS1-∆E9 mutation, this having been

previously shown by us to give an increased basal [Ca2+]i when transfected into SH-

SY5Y cells (28).

We demonstrated that both basal PI hydrolysis and basal [Ca2+]i were greatly

increased in PS1-∆E9 as compared with both PS1-WT and NT cells. Neomycin, a PLC

inhibitor, was able to reduce both high basal PI hydrolysis and high basal [Ca2+]i in PS1-

∆E9 cells. The high basal [Ca2+]i in PS1-∆E9 cells came down to a level comparable to

those seen in NT and PS1-WT cells following neomycin treatment. This suggests that

high basal [Ca2+]i in PS1-∆E9 cells was due to a high PLC activity and not the opposite

way round. The specific IP3R blocker xestospongin C also reduced basal [Ca2+]i in PS1-

∆E9 cells to the levels of NT and PS1-WT cells. Together this data is consistent with a

mechanism whereby the PS1-∆E9 mutation increases basal PLC activity to raise the

levels of IP3 available to gate Ca2+ release via IP3Rs.

The specific RyR antagonist dantrolene also decreased basal [Ca2+]i only in PS1-∆E9

cells, but not down to a level similar to that for NT and PS1-WT cells. This result


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suggests that a component of the increased basal [Ca2+]i in PS1-∆E9 cells due to

enhanced PLC activity is mediated via RyRs.

Previous studies on Ca2+ regulation in cells containing PS1 mutations have focused

on Ca2+ release from the endoplasmic reticulum. Guo et al (9, 36) showed that PC12 cells

overexpressing the PS1-L286V mutation have greater Ca2+ responses to bradykinin,

carbachol (9) and thapsigargin (36). Also, Chan et al showed enhanced Ca2+ responses to

caffeine both in PC12 cells over expressing the PS1-L286V mutation and in primary

cortical neurons from PS1-M146V knockin mice (37). Neither the L286V or M146V PS1

point mutations were shown to affect basal [Ca2+]i (9, 36, 37). More recently, Leissring

and colleagues showed that the PS1-M146V mutation enhances IP3 mediated Ca2+

signalling in Xenopus oocytes (20, 21, 22).

The mechanism by which the PS1-∆E9 mutation increases basal PI hydrolysis is

currently unclear. PS1 is enriched in the Golgi apparatus and endoplasmic reticulum (38).

We show here that PS1 is also present at the plasma membrane, where it colocalised with

N-cadherin, a component of the cell-cell adhesion system. This is in agreement with

previous reports (33, 34, 35), where cell surface PS1 has been suggested to regulate cell-

cell adhesion and synaptic contacts (35). Others have shown that PS1 can stabilise

cadherin-catenin complexes to stimulate Ca2+ dependent cell to cell adhesion (39).

However, PS1-∆E9 mutants failed to stabilise cadherin-catenin complexes and stimulate

cell-cell adhesion (39). Interestingly, the γ isoform of PLC can be activated by integrins

(40, 41), another component of the cell to cell adhesion complex, and integrin functions

in cell adhesion and signalling have been shown to be regulated by the Ca2+ dependent

enzyme calpain (42, 43). We showed that, compared to NT and PS1-WT cells, the PS1-

∆E9 transfectants gave a relative increase in levels of the calpain generated N-terminal

fragment (100 kDa) over full length (150 kDa) PLC-β1. Both full length PLC-β1 and N-


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terminal fragment have been shown to possess catalytic activity in purified enzyme

preparations (32). However, it is currently unknown as to which of these contributes most

to cellular PI hydrolysis. The C-terminal region of PLC-β1 is responsible for the

membrane association of the enzyme (44) with the 150 kDa full length protein being

present in the particulate fraction. In contrast, the 100 kDa protein is mainly cytosolic

(45) and is more active but also less stable than full length PLC-β1 (32). From such data

it is tempting to speculate that the enhanced basal PI hydrolysis seen in PS1-∆E9 cells

occurs due to a mutation related disruption of the cell adhesion complex resulting in

dysregulated PLC.

The PS1-∆E9 cells treated with carbachol gave [Ca2+]i responses that were relatively

increased as compared to NT and PS1-WT cells. Carbachol stimulated [Ca2+]i responses

in PS1-∆E9 cells also had a long tail-off effect, even after treatment with the PLC

inhibitor neomycin. This would suggest that in addition to the effect on PLC activity the

PS1-∆E9 mutation also affects some other downstream process of Ca2+ signal

amplification. One mechanism could be that the ability of Ca2+ itself to induce Ca2+

release through IP3R or RyR is higher in PS1-∆E9 cells. Both IP3R and RyR have been

described in SH-SY5Y neuroblastoma cells (46). A recent report has shown that PC12

cells transfected with the L286V or M146V PS1 mutations and cortical neurons from

PS1-M146V knock-in mouse have increased levels of type 3 RyR (37). We showed that

the specific IP3R blocker xestospongin C decreased the carbachol stimulated [Ca2+]i in all

cell types to similar levels and also abolished the long tail-off effect seen in PS1-∆E9

cells. The carbachol induced long tail-off effect on [Ca2+]i found in PS1-∆E9 cells was

also reduced by dantrolene treatments. Therefore, the long tail-off effect seen following

carbachol stimulation of [Ca2+]i in PS1-∆E9 cells likely occurred due to Ca2+ signal

amplification via IP3R and RyR. This effect was not seen with the less specific RyR and


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mitochondrial uniporter competitive antagonist ruthenium red. Interestingly, both

dantrolene (10 µM) and ruthenium red (10 and 30 µM) only reduced carbachol peak

elevations of [Ca2+]i in NT and PS1-WT cells but not in PS1-∆E9 cells. Since no

consistent changes of basal [Ca2+]i were seen with the CPA treatment, it is suggested that

the ER Ca2+ ATP-ase is not involved in the mechanism of action of the PS1-∆E9

mutation.

We previously reported that PS1-∆E9 cells show higher [Ca2+]i than PS1-WT cells

not only under basal conditions but also during and after exposure to the Ca2+ ionophore

A23187, suggesting that PS1-∆E9 also alters Ca2+ buffering when facing high [Ca2+]i

(28). The PS1-∆E9 mutation has also been shown to suppress capacitative Ca2+ entry

(CCE) in SH-SY5Y cells following depletion of acetylcholine muscarinic receptor

coupled intracellular Ca2+ stores (47). A suppression of CCE has also been reported in

SH-SY5Y and CHO cells transfected with the PS1-M146L mutation where it was also

shown that this mutation increased IP3 mediated Ca2+ release from the ER (48).

Leissring and co-workers (20, 21, 22) reported that Xenopus oocytes carrying the

PS1-M146V mutation show a greater IP3 mediated Ca2+ release than cells carrying PS1-

WT. In addition, they showed that the Ca2+ binding protein calsenilin decreased IP3

mediated Ca2+ release in both PS1-WT and PS1-M146V transfected cells to the same

level (49). The fact that calsenilin has been demonstrated to bind the C-terminus of PS

(50) suggests that this region may mediate PS1 effects on [Ca2+]i. In our model, we used

the PS1-∆E9 mutation that lacks the cleavage site for physiological proteolytic processing

of PS1 (51). Therefore, the effects that we observed on [Ca2+]i are likely mediated by

mechanisms not involving the C-terminal fragment of PS1. The idea that the PS1-∆E9

mutation has cellular effects distinct from those of other FAD PS1 mutations is supported

indirectly by data showing that the brains of Alzheimer’s disease cases due to the PS1-


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∆E9 mutation show unique amyloid plaque pathology with few neuritic changes and little

glial cell activation (52).

In summary, the present study examined a variety of potential cellular mechanisms

by which the FAD linked PS1-∆E9 mutation could alter Ca2+ signalling under basal

conditions and following acetylcholine muscarinic receptor stimulation. We demonstrated

that PS1-∆E9 cells have an enhanced basal PLC activity that leads to increased basal

[Ca2+]i. Enhanced basal PLC activity leading to a higher [Ca2+]i may be a mechanism by

which the PS1-∆E9 mutation sensitises cells to apoptotic stimuli (24, 28) and enhanced

Aβ generation (53). In addition and in agreement with previous reports, our results also

suggest that IP3R- and RyR-mediated Ca2+ amplification is disturbed by PS1 mutations.


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Acknowledgements

This research was supported by grants from Alzheimerfonden, Åke Wiberg and

Gamla Tjänarinnor foundations, the World Bank and Romanian Government (CNCSIS-

C253/2000).


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FIGURE LEGENDS.

FIGURE 1.

Presenilin 1 expression in control and transfected cell lines.

Immunoblotting of PS1 expression in non-transfected (NT), PS1 wild type (PS1-WT

clones 1-1 and 1-14) , and PS1 exon 9 deletion mutation (PS1-∆E9 clones 1-2 and 2-4)

stably transfected human SH-SY5Y neuroblastoma cells.


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FIGURE 2

Effects of the PS1-∆E9 mutation on basal and carbachol-stimulated PI hydrolysis.

Histograms show means ± SEM of at least 8 independent experiments. Data is given

for two PS1-WT clones (1-1 and 1-14) and two PS1-∆E9 clones (1-2 and 2-4). Figure 2A

shows basal and carbachol (100 µM)-stimulated PI hydrolysis. Figure 2B shows the

differences (Carbachol-stimulated minus Basal). Statistical analysis of the results was

carried out using ANOVA followed by Fisher’s post-hoc test. ***, p<0.001 as compared

with values for NT cells. #, p<0.05; ###, p<0.001 as compared with PS1-WT cells.


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FIGURE 3

Effects of the PS1-∆E9 mutation on basal and carbachol-stimulated intracellular

Ca2+ concentrations.

Basal [Ca2+]i was measured repeatedly during 15 minutes to certify that a steady state

of Fluo-3 loading was obtained. The graph shows [Ca2+]i measurements 4 min prior to

addition of carbachol (100 µM) (time 0). Basal [Ca2+]i levels were significantly higher in

both clones of PS1-∆E9 cells (1-2 and 2-4) as compared to both NT (***:p<0,001;

ANOVA, Fisher's post hoc) and both clones of PS1-WT cells (1-1 and 1-14) (###:p<0,001;

ANOVA, Fisher's post hoc). After treatment with 100 µM carbachol, peak elevations of

[Ca2+]i were found after approximately 30 sec. Peak [Ca2+]i was significantly higher in

PS1-∆E9 cells as compared with NT (***:p<0,001; ANOVA, Fisher's post hoc) and PS1-

WT (###:p<0,001; ANOVA, Fisher's post hoc) cells. In NT and PS1-WT cells [Ca2+]i

returned to basal levels after approximately 120 seconds following addition of carbachol.

In contrast, a long tail-off effect was found in PS1-∆E9 cells. Data are expressed as

means of 14 independent experiments. The value from each experiment is the mean of

measurements from 6 cell wells.


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FIGURE 4

Neomycin reverses basal PI hydrolysis and intracellular Ca2+ levels in PS1-∆E9 cells

to those of NT and PS1-WT cells.

Figure 4A: Pre-treatment (3 h) with 500 µM neomycin drastically reduced both basal

and carbachol (100 µM)-stimulated PI hydrolysis in NT, PS1-WT (clone 1-1) and PS1-

∆E9 cells (clone 1-2). Histograms show means ± SEM of 3 independent experiments.

Statistical analysis of the results was carried out using ANOVA followed by Fisher’s

post-hoc test. *, p<0.05; ***, p<0.001 as compared with NT cells. #, p<0.05; ###, p<0.001

compared with PS1-WT cells.

Figure 4B: Pretreatment for 10 minutes with neomycin (100 and 500 µM) did not

affect basal [Ca2+]i levels in either NT or PS1-WT cells. In contrast, in PS1-∆E9 cells,

both neomycin concentrations significantly reduced basal [Ca2+]i to levels comparable to

those seen in NT and PS1-WT cells. Neomycin treatment also significantly reduced the

carbachol elevation of [Ca2+]i in all cell types (*: p<0.05; ANOVA, Fisher's post-hoc

test), but the neomycin (both 100 µM and 500 µM) reduction of carbachol stimulated

[Ca2+]i seen in PS1-∆E9 cells did not come down to the same level as for either NT.

(p<0.05; ANOVA, Fisher's post-hoc test) or PS1-WT cells (p<0.01; ANOVA, Fisher's

post-hoc test for comparison to untreated cells). Data are expressed as means of at least 4

independent experiments. The value from each experiment is the mean of measurements

from 6 cell wells.


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3 3

FIGURE 5

Xestospongin C reduces PS1-∆E9 mutation-related increase in basal and carbachol

intracellular Ca2+ to similar levels seen in NT, PS1-WT cells.

Xestospongin C (1 µM and 10 µM) reduced basal and carbachol stimulated [Ca2+]i in

PS1-∆E9 cells (clone 1-2) to similar levels at those seen in both NT and PS1-WT cells

(clone 1-1). Xestospongin C (10 µM) also significantly reduced carbachol peak elevations

of [Ca2+]i in NT and PS1-WT cells (clone 1-1). Significances are shown for comparisons

with untreated cells (*: p<0.05; ***: p<0.001; ANOVA, Fisher's post-hoc test). Data are

expressed as means of at least 3 independent experiments. The value from each

experiment is the mean of measurements from 6 cell wells.


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3 4

FIGURE 6

Effects of rutheniun red on basal and carbachol increased intracellular Ca2+ levels in

NT, PS1-WT and PS1-∆E9 cells.

Ruthenium red (10 µM and 30 µM) reduced basal [Ca2+]i levels in all cell lines (*:

p<0.05; ***: p<0.001; ANOVA, Fisher's post-hoc test for comparisons with untreated

cells). Basal [Ca2+]i in PS1-∆E9 cells (clone 1-2) was significantly higher as compared

with both NT and PS1-WT (clone 1-1) (p<0.01; ANOVA, Fisher's post-hoc test) even

after treatment with both concentrations of ruthenium red. Both ruthenium red

concentrations significantly reduced carbachol peak elevations of [Ca2+]i in NT and PS1-

WT cells (clone 1-1) (*: p<0.05; ANOVA, Fisher's post-hoc test) but not in PS1-∆E9

cells (clone 1-2). Data are expressed as means of at least 4 independent experiments. The

value from each experiment is the mean of measurements from 6 cell wells.


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3 5

FIGURE 7

Effects of ryanodine and ryanodine plus ruthenium red on basal and carbachol

increased intracellular Ca2+ levels in NT, PS1-WT and PS1-∆E9 cells.

Ryanodine (1 µM and 10 µM) treatment for 10 minutes did not induce significant

changes in either basal or carbachol stimulated [Ca2+]i in either NT, PS1-WT (clone 1-1)

or PS1-∆E9 cells (clone 1-2). Combined treatment with 10 µM ryanodine and 30 µM

ruthenium red decreased basal [Ca2+]i levels in all cell lines to similar levels seen with 30

µM ruthenium red alone. None of the cell lines showed changes in carbachol peak

elevations of [Ca2+]i after 10 µM ryanodine + 30 µM ruthenium red. Data are expressed as

means of at least 4 independent experiments. The value from each experiment is the mean

of measurements from 6 cell wells. *, p<0.05; **: p<0.01; ANOVA, Fisher's post-hoc

test for comparisons with untreated cells.


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3 6

FIGURE 8

Effects of dantrolene on basal and carbachol increased intracellular Ca2+ levels

in NT, PS1-WT and PS1-∆E9 cells.

Dantrolene (1 µM and 10 µM) reduced basal [Ca2+]i in PS1-∆E9 cells (*: p<0.05,

ANOVA, Fisher's post-hoc test for comparisons with untreated cells). Dantrolene (100

nM, 1 µM and 10 µM) abolished the carbachol induced long tail off [Ca2+]i seen in PS1-

∆E9 cells (clone 1-2) (*: p<0.05, **:P<0.01; ANOVA, Fisher's post-hoc test). Dantrolene

10 µM significantly reduced carbachol peak elevations of [Ca2+]i in NT and PS1-WT cells

(clone 1-1) (*: p<0.05; ANOVA, Fisher's post-hoc test) but not in PS1-∆E9 cells. Data

are expressed as means of at least 4 independent experiments. The value from each



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3 7

FIGURE 9

Lack of effects of CPA on basal and carbachol increased intracellular Ca2+ levels in

NT, PS1-WT and PS1-∆E9 cells.

Cyclopiazonic acid (CPA, 10 µM and 30 µM), did not induce any significant change

of either basal [Ca2+]i or carbachol elevation of [Ca2+]i in any of the cell lines. Data are

expressed as means of at least 4 independent experiments. The value from each



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3 8

FIGURE 10

PLC levels in NT, PS1-WT and PS1-∆E9 cells

Figure 10A shows an immunoblot for PLC-β1 and PLC γ expression in NT, PS1-WT

(clone 1-1), and PS1-∆E9 cells (clone 1-2). The monoclonal N-terminal PLC-β1 antibody

recognised two inmunoreactive bands at approximately 150 and 100 kDa. Densitometric

analyses of the bands obtained for 3 separate blots of PLC-β1 (means ± SEM) are shown

in Figure 10B. Statistical analysis of the results was carried out using ANOVA followed

by Fisher’s post-hoc test. *, p<0.05 and ***, p<0.001 as compared with values for NT

cells. ###, p<0.001 as compared to PS1-WT cells.


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3 9

FIGURE 11

Localisation of PS1 in the plasma membrane in complex with N-cadherin.

Confocal micrographs of NT, PS1-WT (clone 1-1) and PS1-∆E9 cells (clone 1-2)

double stained with anti N-terminal PS1 and anti-N-cadherin antibodies. PS1

immunofluorescence showed a cytosolic granular pattern that was also strong at sites of

cell to cell contact (red). N-cadherin, immunofluorescence was concentrated at the plasma

membrane (green). Superimposed images showed co-localization of both PS1 and N-

cadherin at the level of the plasma membrane (yellow). A: NT (bar = 11.3 µm); B: PS1-

WT (bar = 11.3 µm); C: PS1-∆E9 (bar = 11.3 µm); D: PS1-∆E9 (bar = 43.6 µm)


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50 kDa

30 kDa

PS1-WT PS1- E9 (1-1) (1-14) (1-2) (2-4)

Figure 1

NT ∆


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0

0.025

0.05

0.075

0.1

NT PS1-WT (1-1)

PS1-WT (1-14)

PS1- (1-2)

E9

Carbachol

Basal

D E9

#

***###

0

0.005

0.01

0.015

0.02

0.025

NT PS1-WT (1-1)

PS1-WT (1-14)

E9 (1-2)

E9 (2-4)

Carbachol - basal

PS1-D

IPs

/ (IP

s+I

lipid

)

D

PS1-D

***###

*#

IPs

/ (IP

s+I

lipid

)

Figure 2

A

B

PS1- (2-4)

∆∆

∆ ∆


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0

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0 60 120 180 240 300 360 420 480 540 600

time (sec)

[Ca2+

] i (n

M)

PS1-∆E9 (2-4)

NTPS1-WT (1-1)

***### ***

###***###

***###

***### ***

###***###

***###

PS1-∆E9 (1-2)

PS1-WT (1-14)


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Figure 3

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0

0.01

0.02

0.03

0.04

0.05

0.06

NT NT +

Neomycin

PS1-WT PS1-WT+

Neomycin

PS1-D E9 PS1-D E9+

Neomycin

Carbachol

Basal

***###

*#

IPs

/ (IP

s+I

lipid

)

∆∆

3 5


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Figure 4A

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by guest on September 22, 2018http://www.jbc.org/Downloaded from

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0

10

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0 60 120 180 240 300 360 420 480 540 600

time (sec)

[Ca2

+]i

(nM

)

*

0

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0 60 120 180 240 300 360 420 480 540 600

time (sec)

[Ca2

+]i

(nM

)

**

* ****

*********

0

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0 60 120 180 240 300 360 420 480 540 600

time (sec)

[Ca2

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*

PS1-WTNT PS1-∆E9


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Figure 5

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0

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0 60 120 180 240 300 360 420 480 540 600

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[Ca2

+]i

(nM

)

**

**

0

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0 60 120 180 240 300 360 420 480 540 600

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[Ca2

+]i

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)

**

**

0

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0 60 120 180 240 300 360 420 480 540 600

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[Ca2

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)

**

**

NT PS1-WT PS1-∆E9


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Figure 6

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0

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*

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+ ]i (

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)

**

0

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0 60 120 180 240 300 360 420 480 540 600

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[Ca2

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)

*

PS1-WTNT PS1-∆E9


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Figure 7

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0

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)

*

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)

*

* *

*

*

* * *

0

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)

*

PS1-WTNT PS1-∆E9

*****


Alce

Figure 8

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0

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NT PS1-WT PS1-∆E9


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NT NT PS1- PS1- PS1- PS1- WT D E9 WT D E9

150

100

PLC- 1 PLC-

0

1

2

3

4

5

O.D

NT PS1-WT PS1 -DE9

100 kDa

150 kDa

***###

* ***

A

B

Figure 10

b g


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Figure 11

A

B

C

D

PS-1 N-Cadherin Merged


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and Richard F. CowburnAngel Cedazo-Minguez, Bogdan O. Popescu, Maria Ankarcrona, Takeshi Nishimura

activity and a resultant increase in intracellular calcium concentrationsThe presenilin 1 {capital delta}E9 mutation gives enhanced basal phospholipase C

published online July 16, 2002J. Biol. Chem.

10.1074/jbc.M112117200Access the most updated version of this article at doi:

Alerts:

When a correction for this article is posted•

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