Neurobiology of Aging 21 (2000) 31–38

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Alterations in muscarinic receptor-coupled phosphoinositide hydrolysis
and AP-1 activation in Alzheimer’s disease cybrid cells૾
Patrizia De Sarnoa, Gautam N. Bijura, Rong Lua, Robert E. Davisb, Richard S. Jopea,‫ء‬
a

Department of Psychiatry and Behavioral Neurobiology, University of Alabama at Birmingham, Sparks Center 1057,
Birmingham, AL 35294-0017, USA
b
MitoKor, San Diego, CA 92121, USA
Received 14 September 1999; received in revised form 17 November 1999; accepted 13 January 2000

Abstract
Alzheimer’s disease cybrid cells produced by replacing endogenous mitochondria in human neuroblastoma SH-SY5Y cells with platelet
mitochondria from subjects with Alzheimer’s disease have higher levels of reactive oxygen species than do cybrid cells with mitochondria
from control subjects. These cells were used to test if this chronic mild increase in reactive oxygen species affects muscarinic receptorcoupled signaling activities. Basal and carbachol-stimulated phosphoinositide hydrolysis were higher, and there was less inhibition by
glutathione depletion, in Alzheimer’s disease than control cybrid cells. Elevated phosphoinositide hydrolysis in Alzheimer’s disease cybrid
cells also was evident upon direct activation of G-proteins (Gq/11) linked to phosphoinositide signaling or of phospholipase C, but
immunoblot analyses revealed equivalent levels of Gq/11 and phospholipase C in both cell lines. These results indicate that there is
up-regulation of phosphoinositide signaling in Alzheimer’s disease cybrid cells in association with chronic mild oxidative stress, although
treatment of cells with H2O2 to induce greater acute oxidative stress caused decreases in carbachol-stimulated phosphoinositide hydrolysis
that were similar in Alzheimer’s disease and control cybrid cells. In contrast to phosphoinositide hydrolysis, carbachol-stimulated AP-1
DNA binding activity was lower in Alzheimer’s disease than control cybrid cells, and this deﬁcit was associated with deﬁcient protein kinase
C-mediated activation of AP-1. Overall, these results demonstrate that chronically elevated reactive oxygen species in Alzheimer’s disease
cybrid cells are associated with a more robust phosphoinositide signaling system, but lower signaling to activation of AP-1. These alterations
may represent adaptations to exposure to oxidants, which precede more widespread deﬁcits in signaling associated with more severe
oxidative stress. © 2000 Elsevier Science Inc. All rights reserved.
Keywords: Alzheimer’s disease; Cholinergic signaling; Phosphoinositide; AP-1

1. Introduction
Reactive oxygen species are important intermediates in
the normal physiological functioning of neurons and other
cells, whereas their excess causes oxidative stress, a condition associated with many neuropathological disorders [11,
20]. Although highly susceptible to detrimental effects of
large acute increases in oxidative stress, neurons have
adopted a variety of mechanisms to minimize the deleterious impact of such exposure, including regulating the activities of signaling systems and antioxidant mechanisms
૾
This research was supported by grants from the NIH (AG06569 and
NS37768) and an NRSA award from the NIH to GNB (NS10795).
* Corresponding author. Tel.: ϩ1-205-934-7023; fax: ϩ1-205-9343709.
E-mail address: jope@uab.edu (R. Jope).

[7]. Nonetheless, oxidative stress is thought to be a critical
factor in the neurodegeneration associated with neurological
disorders such as Alzheimer’s disease (AD) and Parkinson’s
disease [14]. Thus, neurons activate measures to counteract
oxidative stress (which in themselves may inﬂuence other
cellular functions), and an intermediate stage of survival
during chronic exposure to elevated levels of reactive oxygen species may be attained until toxic thresholds of oxidative stress are reached that contribute to neurodegenerative diseases.
The association of oxidative stress with Alzheimer’s
disease has been established by multiple experimental strategies [1]. One of these is the identiﬁcation of abnormal
mitochondrial function that causes an excess production of
reactive oxygen species [1,6]. The recently developed AD
and control cybrid cells provide a new experimental approach to study how chronically increased levels of reactive

0197-4580/00/$ – see front matter © 2000 Elsevier Science Inc. All rights reserved.
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P. De Sarno et al. / Neurobiology of Aging 21 (2000) 31–38

oxygen species affect cellular functions, such as signaling
cascades. These cell lines were established by replacing
endogenous mitochondria in human neuroblastoma SHSY5Y cells with mitochondria isolated from the platelets of
subjects with sporadic AD (AD cybrid cells) or agematched control subjects (control cybrid cells) [17,18]. The
AD cybrid cells maintain elevated levels of reactive oxygen
species compared with control cybrid cells and normal SHSY5Y cells, and therefore provide a model system to study
how this condition affects cellular functions.
The present study was undertaken to investigate if chronically elevated reactive oxygen species evident in AD cybrid cells affects cholinergic muscarinic receptor-coupled
signaling activities. In AD brain there is substantial evidence of impaired muscarinic receptor-linked activation of
signaling systems [8]. Additionally, studies in SH-SY5Y
cells have shown that oxidative stress impairs muscarinic
receptor-coupled activation of the phosphoinositide second
messenger system and downstream transcription factor activation [9,10,12,13]. Therefore, cybrids prepared from parental SH-SY5Y cells provide a unique model system to use
to address these goals. The results show that unlike with
acute, severe oxidative stress which severely impairs phosphoinositide signaling, chronic exposure to mildly elevated
reactive oxygen species in AD cybrid cells causes an upregulation of the phosphoinositide signaling system, but
there is impaired coupling to activation of the AP-1 transcription factor.

2. Method
2.1. Materials
Commercial reagents were obtained as follows: DMEM,
penicillin/streptomycin, qualiﬁed fetal bovine serum, antibodies to subtypes of protein kinase C, Klenow enzyme, and
10ϫ REACT buffer from Gibco BRL (Grand Island, NY,
USA), (␣-32P)dCTP from Amersham (Arlington Heights,
IL, USA), horseradish peroxidase-conjugated goat anti-rat
IgG from Calbiochem (San Diego, CA, USA), 2Ј,7Ј-dichlorodihydroﬂuorescein diacetate (DCFH-DA) from Molecular
Probes (Eugene, OR, USA), BCA kit from Pierce (Rockford, IL, USA), (3H)myo-inositol from American Radiolabeled Chemicals (St. Louis, MO, USA), buthionine sulfoximine (BSO) from Sigma (St. Louis, MO, USA).
2.2. Cell culture
Control and AD cybrid cells from Mitokor (San Diego,
CA, USA) were grown in DMEM containing 10% qualiﬁed
fetal bovine serum, 100 U/ml penicillin and 100 ␮g/ml
streptomycin. Cells were plated at a density of 106 cells/100
mm dish and were harvested 48 h later, after the treatments
described below. Glutathione concentrations were measured

by the method of Tietze [22]. Protein concentrations were
determined by the method of Bradford [2].
2.3. Measurement of reactive oxygen species
Cybrid cells were cultured in 24 well tissue culture plates
at a density of 5 ϫ 104 cells per well. Eight wells were
plated with control cybrids and eight wells were plated with
AD cybrids. After 48 h the cells were washed with warm
serum-free DMEM and then incubated for 1 h at 37°C with
serum-free DMEM containing 10 ␮m dichloroﬂuorescin
diacetate, which becomes a ﬂuorescent product, 2Ј, 7Ј-dichloroﬂuorescein (DCF), in the presence of reactive oxygen
species. Cells were washed twice with PBS and DCF ﬂuorescence was measured by a Bio–Tek ﬂuorescence plate
reader (Bio–Tek Instruments Inc., Winooski, VT, USA),
with excitation set at 435 nm and emission at 530 nm. Cells
were then lysed with lysis buffer (10 mM Tris-HCl, pH 7.4,
0.5% NP-40) and total protein was measured by using the
BCA protein assay. Reactive oxygen species production
was quantitated as the ratio of DCF ﬂuorescence to total
protein and the mean reactive oxygen species production
was determined by averaging eight wells for control and
eight wells for AD cybrid cells.
2.4. Phosphoinositide hydrolysis
Cells were prelabeled with 7.5 ␮Ci/ml myo-(2-3H)inositol for 48 h, harvested, resuspended in buffer (30 mM
HEPES, pH 7.4, 122 mM NaCl, 3.6 mM NaHCO3, 1.2 mM
KH2PO4, 1.2 mM MgCl2, 5 mM KCl, 1.3 mM CaCl2, 10
mM LiCl, 11 mM glucose), and washed 2ϫ, as described
previously [12]. Suspended cells were preincubated for 10
min with or without the indicated concentrations of H2O2
followed by incubation with 1 mM carbachol, 20 mM NaF
(with 10 ␮M AlCl3), or 50 ␮M ionomycin (with 2.2 mM
CaCl2) at 37°C for the indicated times. Radioactivity was
measured in samples after fractionation of lipids, inositol
monophosphate, and inositol as described previously [12].
Measurements were made in triplicate for each treatment in
at least three separate experiments. Statistical signiﬁcance
was determined using analysis of variance with a posthoc
Dunnett multiple comparison test, or Student’s t-test was
used for comparing matched treatments.
2.5. Preparation of nuclear and cytosolic extracts
To prepare nuclear extracts, cells were washed two times
with PBS and 500 ␮l of lysis buffer (10 mM Tris-HCl, pH
7.4, 3 mM MgCl2, 10 mM NaCl, 0.5% NP-40, 10 ␮g/ml
leupeptin, 10 ␮g/ml aprotinin, 1 mM sodium vanadate, 5
␮g/ml pepstatin, 1 nM okadaic acid, and 1 mM phenylmethylsulfonyl ﬂuoride) was added. Lysed cells were centrifuged at 4000 ϫ g for 5 min at 4°C. The supernatant
containing the cytosolic proteins was transferred to microfuge tubes. The pellet was resuspended in 50 ␮l of buffer

P. De Sarno et al. / Neurobiology of Aging 21 (2000) 31–38

33

containing 20 mM HEPES, pH 7.9, 20% glycerol, 0.3 M
NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM dithiothreitol
(DTT), 0.1 mM ␤-glycerophosphate, 0.05 mM vanadate, 1
mM phenylmethylsulfonyl ﬂuoride and 1 ␮g/ml each of
pepstatin A, leupeptin and aprotinin. After extraction on ice
for 30 min, the samples were centrifuged at 16 000 ϫ g for
15 min at 4°C. The supernatant containing nuclear proteins
was transferred to a microfuge tube. Protein concentrations
were determined by the BCA method.
2.6. Electrophoretic mobility shift assay (EMSA)
EMSAs were performed using a double-stranded 18 base
pair oligonucleotide (5Ј-CTAGTGATGAGTCAGCCG-3Ј)
containing the AP-1 consensus sequence as described previously [12]. The oligonucleotide (200 pmol) was labeled at
37°C for 1 h in 20 ␮l containing 10 ϫ REACT buffer, 10
␮M each of dATP, dGTP, and dTTP, 100 ␮Ci (␣32
P)dCTP, and 40 U Klenow. After 1 h, the sample was
diluted to 100 ␮l with TE buffer (10 mM Tris-HCl, pH 8.0,
1 mM EDTA) and free probe was removed by centrifugation at 4000 ϫ g for 45 s on a Sephadex G-50 column. For
the binding reaction, 2 ␮g of nuclear protein extract was
incubated in a total volume of 20 ␮l in binding buffer
containing 20 mM HEPES, pH 7.9, 4% glycerol, 1 mM
MgCl2, 50 mM KCl, 0.5 mM DTT, 1 ␮g poly(dI-dC) and
10 000 cpm radiolabeled DNA for 30 min at 4°C. DNAprotein complexes were resolved on a pre-electrophoresed
6% non-denaturing polyacrylamide gel, in 0.25 ϫ TBE
(22.3 mM Tris, 22.3 mM boric acid, and 0.5 mM EDTA) at
4°C for 1.5 h at 150 V. The gel was dried under vacuum and
the amount of DNA-protein complex present was analyzed
using a Phosphor–Imager (Molecular Dynamics, Sunnyvale, CA, USA). Data from treated cells were compared
with untreated control cells using analysis of variance with
a post hoc Bonferroni test or Student’s t-test.
2.7. Immunoblotting
Cells were washed 2ϫ with phosphate-buffered saline
(PBS) and were lysed with buffer A (20 mM tris, pH 8.0,
137 mM NaCl, 2 mM EDTA, 10% glycerol, 1 mM phenylmethanesulfonyl ﬂuoride, 10 ␮g/ml aprotinin, 10 ␮g/ml
leupeptin, 1 mM sodium vanadate, 1% Triton-X-100). After
a 30 min incubation on ice, the lysates were centrifuged at
10 000 ϫ g for 10 min to remove detergent-insoluble material. Cell extracts were mixed with Laemmli sample buffer
(2% SDS) and placed in a boiling water bath for 5 min.
Proteins were resolved in 10% SDS-polyacrylamide gels,
transferred to nitrocellulose and incubated with appropriate
antibodies. Immunoblots were developed using horseradish
peroxidase-conjugated secondary antibodies followed by
enhanced chemiluminescence.

Fig. 1. Reactive oxygen species in control and AD cybrid cells. Reactive
oxygen species were quantitated by DCF ﬂuorescence and values were
calculated as the ratio of ﬂuorescence units to total protein. The values are
given as the percent of reactive oxygen species in control cybrid cells.
Means Ϯ SEM, n ϭ 4, * p Ͻ 0.05.

3. Results
3.1. Reactive oxygen species
Reactive oxygen species were determined in control and
AD cybrid cells by measuring DCF ﬂuorescence. AD cybrid
cells produced approximately 40% greater reactive oxygen
species than control cybrid cells (Fig. 1). This result conﬁrms previous reports [17,21] that the AD cybrid cells
maintain elevated levels of reactive oxygen species.
3.2. Carbachol-stimulated phosphoinositide hydrolysis
and inhibition by H2O2
Basal and carbachol-stimulated phosphoinositide hydrolysis were measured in control and AD cybrid cells.
These experiments were conducted with and without
prior depletion of cellular glutathione because glutathione depletion previously was found to exacerbate the
inhibitory effects of H2O2 on phosphoinositide signaling
[13]. The basal rate of phosphoinositide hydrolysis was
two-times higher (224 Ϯ 47%) in AD than control cybrid
cells (Fig. 2A). To test if glutathione depletion inﬂuenced
basal phosphoinositide hydrolysis, cells were pretreated
with 200 ␮M BSO, an inhibitor of ␥-glutamylcysteine
synthase [16], to deplete glutathione. The glutathione
concentrations were equivalent in AD and control cybrid
cells (7.3 Ϯ 0.8 and 7.0 Ϯ 0.5 ng/␮g protein, respectively, n ϭ 3), and this treatment regimen with BSO
depleted glutathione by at least 99% in both cell lines.
Depletion of glutathione did not alter basal phosphoinositide hydrolysis in either control or AD cybrid cells, with
the AD cybrid cells retaining a 2-fold higher basal rate of

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P. De Sarno et al. / Neurobiology of Aging 21 (2000) 31–38

Fig. 2. Basal (A) and carbachol-stimulated (B) phosphoinositide hydrolysis in
control and AD cybrid cells. Cells were prelabeled with (3H)inositol for 48 h
in the absence or presence of 200 ␮M BSO (which did not alter (3H)inositol
uptake or incorporation into lipids, as reported previously [12]). Phosphoinositide (PI) hydrolysis was measured as described in Section 2 after a 30-min
incubation in the absence (basal) or presence of 1 mM carbachol. Values are
given in (A) as the percent of phosphoinositide hydrolysis measured in untreated control cybrid cells, and in (B) as the percent of carbachol-stimulated
phosphoinositide hydrolysis in control cybrid cells not pretreated with BSO.
Basal values (shown in A) were subtracted to obtain the carbachol-stimulated
values shown in (B). Mean Ϯ SEM (n ϭ 3). *p Ͻ 0.05 compared with values
in control cybrid cells not treated with BSO.

phosphoinositide hydrolysis even after depletion of glutathione (Fig. 2A). Because of this difference, basal activities were subtracted from all further calculations of
stimulated phosphoinositide hydrolysis to allow for com-

parisons of responses to stimulants in control and AD
cybrid cells.
Incubation with 1 mM carbachol induced an approximately 13-fold stimulation of phosphoinositide hydrolysis
in control cybrid cells, and this response to carbachol was
33% greater in AD cybrid cells (calculated after subtracting
the basal activities) (Fig. 2B). The inﬂuence of glutathione
depletion on carbachol-stimulated phosphoinositide hydrolysis was examined by measuring the response to carbachol
in cells pretreated with 200 ␮M BSO for 48 h to deplete
glutathione. In control cybrid cells, carbachol-stimulated
phosphoinositide hydrolysis was reduced by 39% (p Ͻ
0.05) after depletion of glutathione, whereas in AD cybrid
cells carbachol-stimulated phosphoinositide hydrolysis was
only reduced by 11% after glutathione depletion, which was
not a statistically signiﬁcant change from untreated AD
cybrid cells. Thus, both basal and carbachol-stimulated
phosphoinositide hydrolysis were greater in AD cybrids
than control cybrid cells, and the response to carbachol was
more sensitive to inhibition by slight oxidative stress associated with glutathione depletion in control than in AD
cybrid cells. All of these differences indicate that the phosphoinositide signaling system was more robust in AD than
control cybrid cells.
To test the relative sensitivities of phosphoinositide hydrolysis in control and AD cybrid cells to greater oxidative
stress, the inhibitory effect of H2O2 was measured by incubating the cells with H2O2 for 10 min before the addition of
carbachol. H2O2 pretreatment caused a concentration-dependent inhibition of carbachol-stimulated phosphoinositide
hydrolysis in control cybrid cells, with signiﬁcant inhibition
caused by 30 ␮M H2O2, and apparently maximal inhibition
obtained with 300 ␮M H2O2 (Fig. 3). The concentrationdependent inhibition by H2O2 of carbachol-stimulated phosphoinositide hydrolysis was similar in AD cybrid cells,
except inhibition by the lowest effective concentration of
H2O2 (30 ␮M) was only 22%, whereas it was 37% in
control cybrid cells.
To test if glutathione depletion inﬂuenced the inhibitory
effect of H2O2 on carbachol-stimulated phosphoinositide
hydrolysis, cells were pretreated with 200 ␮M BSO, to
deplete glutathione. In control cybrid cells, depletion of
glutathione by the BSO treatment signiﬁcantly reduced carbachol-stimulated phosphoinositide hydrolysis and potentiated the inhibitory effect of H2O2 on carbachol-stimulated
phosphoinositide hydrolysis throughout the 3 to 300 ␮M
H2O2 concentration range that was examined. In AD cybrid
cells, glutathione depletion also potentiated the H2O2 concentration-dependent inhibitory effects in a similar manner
to that observed in control cybrid cells. Thus, in the absence
of H2O2, the phosphoinositide signaling system was more
robust in AD than control cybrid cells, but each showed
similar susceptibilities to the impairment induced by H2O2
without or with prior glutathione depletion.

P. De Sarno et al. / Neurobiology of Aging 21 (2000) 31–38

35

Fig. 4. Activities and levels of G-proteins and phospholipase C associated
with phosphoinositide hydrolysis. (A) Phosphoinositide hydrolysis was
measured after stimulation of G-proteins with 20 mM NaF plus 10 ␮M
AlCl3 or stimulation of calcium-activated phospholipase C with 50 ␮M
ionomycin plus 2.2 mM CaCl2 for 60 min. Basal values were subtracted to
obtain the stimulated values. Mean Ϯ SEM (n ϭ 4). *p Ͻ 0.05 compared
with values in control cybrid cells. (B) Cell extracts were immunoblotted
with antibodies speciﬁc for Gq/11 ␣-subunit or phospholipase C-␤. In AD
cybrid cells, Gq/11 was 101 Ϯ 5% and phospholipase C-␤ was 101 Ϯ 2%
of levels in control cybrid cells. Means Ϯ SEM; n ϭ 4.

Fig. 3. Inhibition by H2O2 of carbachol-stimulated phosphoinositide hydrolysis and potentiation of inhibition by glutathione depletion in (A)
control and (B) AD cybrid cells. Cells were prelabeled with (3H)inositol for
48 h in the absence or presence of 200 ␮M BSO. Prelabeled cells were
suspended in assay buffer, incubated for 10 min with 0, 3, 10, 30, 100, or
300 ␮M H2O2, and then incubated for 30 min with 1-mM carbachol.
Values are given as the percent of phosphoinositide (PI) hydrolysis obtained with carbachol in the absence of treatment with BSO or H2O2.
Mean Ϯ SEM (n ϭ 3) *p Ͻ 0.05 compared with matched cells not treated
with H2O2.

cybrid cells could be due to differences in the function
and/or levels of the G-proteins or phospholipase C that
mediate phosphoinositide hydrolysis. To test function,
phosphoinositide hydrolysis was measured after direct stimulation of G-proteins with 20 mM NaF, or after direct
activation of phospholipase C by calcium attained by treating cells with the calcium ionophore ionomycin (50 ␮M).
These experiments revealed that phosphoinositide hydrolysis stimulated by NaF or by ionomycin was greater in AD
than control cybrid cells (Fig. 4A). Thus, basal phosphoinositide hydrolysis and that activated by each stimulant
was higher in AD than control cybrid cells.
To test if these differences in phosphoinositide hydrolysis activities were due to differences in protein levels, Gq/11
and phospholipase C-␤ levels were measured by quantitative immunoblot analyses. Fig. 4B shows that the levels of
Gq/11 and phospholipase C-␤ were equivalent in AD and
control cybrid cells.

3.3. G-protein and phospholipase C function and levels

3.4. AP-1 DNA binding activity

The higher basal and carbachol-stimulated phosphoinositide hydrolysis activities in AD compared with control

A downstream consequence of activation of the phosphoinositide signal transduction system is stimulation of

36

P. De Sarno et al. / Neurobiology of Aging 21 (2000) 31–38

Fig. 5. Carbachol-stimulated AP-1 DNA binding activity. Cells were
treated with 0, 3, 10, 30, or 100 ␮M carbachol for 1 h, nuclear extracts were
prepared, and AP-1 DNA binding activity was measured by EMSA as
described in Section 2. (A) shows EMSA results from a representative
experiment (only the top portion of the gel is shown), and (B) shows
quantitative values from three independent experiments given as a percent
of AP-1 binding in untreated control cybrid cells. Mean Ϯ SEM (n ϭ 3).
*p Ͻ 0.05 for AD compared with control cybrid cells.

AP-1 DNA binding activity, and carbachol-stimulated activation of AP-1 has been reported to be inhibited by oxidative stress [12]. Therefore, we tested if the greater carbachol-stimulated phosphoinositide hydrolysis activity
observed in AD relative to control cybrid cells resulted in a
corresponding increased activation of AP-1. Contrary to
this, carbachol-stimulated activation of AP-1 was signiﬁcantly lower in AD than control cybrid cells. Fig. 5 shows
that carbachol concentration-dependently increased AP-1
DNA binding activity in both cell lines, but there was an
approximately 40% lower maximal activation in the AD
cybrid cells. Thus, although muscarinic receptor-coupled
phosphoinositide hydrolysis was elevated in AD relative to
control cybrid cells, there was impaired translation in the
intermediate signaling cascade coupling this to activation of
AP-1 DNA binding activity.
3.5. Protein kinase C
To test the activities of signaling intermediates coupling
phosphoinositide hydrolysis to AP-1 activation, AD and

Fig. 6. Protein kinase C-mediated AP-1 DNA binding activity and subtype
levels. (A) Cells were treated with 0.5 ␮M PMA for 1 h, nuclear extracts
were prepared, and AP-1 DNA binding activity was measured by EMSA as
described in Section 2. The ﬁgure contains results representative of four
experiments which showed that PMA-stimulated AP-1 DNA binding activity was 40% lower in AD than in control cybrid cells. (B) Cell extracts
were immunoblotted with antibodies speciﬁc for ␣, ⑀, and ␨ subtypes of
protein kinase C. In AD cybrid cells, protein kinase C-␣, -⑀, and –␨ were
98 Ϯ 4%, 98 Ϯ 6%, and 99 Ϯ 4%, respectively, of levels in control cybrid
cells. Mean Ϯ SEM; n ϭ 4.

control cybrid cells were compared in their responses to
protein kinase C-mediated activation of AP-1. An activator
of protein kinase C, PMA (1 ␮M), increased AP-1 DNA
binding activity in both AD and control cybrid cells (Fig. 6).
However the stimulation of AP-1 DNA binding activity in
AD cybrid cells was 40% lower than the stimulation in
control cybrid cells. This observation indicates that in AD
cybrid cells there is impaired coupling of the increased
carbachol-stimulated phosphoinositide hydrolysis to protein
kinase C-mediated activation of AP-1. Immunoblot analyses of protein kinase C subtypes, including a predominant
representative (␣, ⑀, and ␨) of each of the three classes of
protein kinase C expressed in these cells, did not reveal
differences in their levels between AD and control cybrid
cells (Fig. 6B).

4. Discussion
Substantial evidence has implicated oxidative stress as
an important contributory factor in the neuronal dysfunction
associated with AD [1,14]. An important component of AD
is the loss of cholinergic muscarinic receptor-coupled signaling activity mediated by the phosphoinositide signal
transduction system [8], a deﬁcit that likely aggravates

P. De Sarno et al. / Neurobiology of Aging 21 (2000) 31–38

impaired cholinergic activity caused by diminished acetylcholine production and limits the therapeutic efﬁcacy of
inhibitors of acetylcholinesterase. Because the activity of
this signaling cascade is susceptible to inhibition by oxidative stress [12], oxidative stress may contribute directly to
impaired phosphoinositide signaling activity in AD. The
present investigation was undertaken to determine if studies
using AD cybrid cells might provide further information
about the relationship between oxidative stress and phosphoinositide signaling. Two properties of AD cybrid cells
make them a unique model system with which to study the
effects of long-term oxidative stress on activities of signaling cascades coupled to muscarinic receptors. First, AD
cybrid cells are constantly exposed to increased endogenous
production of reactive oxygen species, a condition that may
resemble in vivo conditions more closely than the widely
used protocol of acute exposure of cells to high levels of
oxidants. Second, these cybrid cells from AD and control
subjects were developed using a parental cell line, human
neuroblastoma SH-SY5Y cells, that endogenously express
muscarinic m3 receptors that are coupled to the phosphoinositide second messenger system [5]. Thus, AD and control cybrid cells provide a model system to assess muscarinic receptor-coupled signaling activities during chronic
exposure to elevated levels of reactive oxygen species. In
conﬁrmation of earlier reports, the AD cybrid cells had a
40% greater level of reactive oxygen species than control
cybrid cells [18,21]. This elevated level of reactive oxygen
species was associated with two unexpected alterations in
signaling activities associated with activation of muscarinic
receptors. First, in AD cybrid cells phosphoinositide hydrolysis was up-regulated, rather than impaired as would be
expected from previous studies of the effects of acute oxidative stress. Second, although phosphoinositide signaling
was increased in AD cybrid cells, there was a reduction in
the downstream activation of AP-1 DNA binding activity,
indicative of impaired coupling between the activity of the
phosphoinositide second messenger system and the associated activation of AP-1.
Three results suggest that the phosphoinositide signal
transduction system is more robust in AD than control
cybrid cells. These include the observations that in AD,
compared with control, cybrid cells 1) the basal phosphoinositide hydrolysis activity was twice as high, 2) the stimulation by carbachol was 33% higher, and 3) there was less
of an inhibitory effect of glutathione depletion (in the absence of added H2O2) on carbachol-stimulated phosphoinositide hydrolysis. Taken together, these ﬁndings indicate
that the activity of the phosphoinositide signal transduction
system is up-regulated in AD cybrid cells, a change that
may reﬂect an adaptation process in AD cybrid cells that
was activated in response to chronically elevated levels of
reactive oxygen species. This is not the ﬁrst adaptive upregulation found in cybrid cells, as both AD and Parkinson’s
disease cybrid cells have been reported to compensate for
their increased burdens of reactive oxygen species by up-

37

regulating several antioxidant enzymes [3,21], although
these remain inadequate for completely neutralizing reactive oxygen species that remain elevated in both types of
cybrid cells. Thus, contrary to expectations, chronic mild
oxidative stress did not impair phosphoinositide signaling,
but actually increased this activity. The increased basal and
carbachol-stimulated phosphoinositide hydrolysis activities
are entirely consistent with previous ﬁndings that basal
calcium levels and the increase in intracellular calcium
stimulated by carbachol are elevated in AD relative to
control cybrid cells [18] because one result of phosphoinositide hydrolysis is an increase in intracellular calcium.
Considering that phosphoinositide hydrolysis stimulated by
activation of muscarinic receptors, G-proteins, or phospholipase C were all elevated in AD cybrid cells relative to
control cybrid cells, it appears that increased coupling to,
and activity of, phospholipase C accounts for the increased
signaling activity.
Although phosphoinositide signaling was robust in AD
cybrid cells, this was not adequate to overcome the effects
of more severe oxidative stress. Thus, exposure to exogenous H2O2 severely impaired phosphoinositide signaling in
both AD and control cybrid cells. Taken together, these
results raise the possibility that the AD cybrid cells represent a model of cellular conditions at an early stage of mild
oxidative stress to which the cells can at least partially
compensate and adapt, before reaching a toxic threshold of
oxidative stress (as modeled by exposure to H2O2), at which
point the cell’s defense and signaling systems cannot adequately compensate, thus becoming severely compromised.
In stark contrast to the increased activity of the phosphoinositide signaling system in AD cybrid cells, there was not
complete compensation for the detrimental effects of reactive oxygen species, as there was a reduction of the downstream muscarinic receptor-coupled signaling leading to activation AP-1 DNA binding activity. This indicates that
signaling intermediates have differential susceptibilities to
detrimental effects of oxidative stress, a proposal consistent
with growing evidence that oxidative stress causes not
global impairments in cellular functions but strikes selective
targets [9,11]. This dysfunctional stimulation of AP-1 DNA
binding activity was identiﬁed as being due to an impairment in protein kinase C-dependent activation of AP-1.
Interestingly, several studies have reported deﬁcits in protein kinase C in AD brain [4,15,19]. Measurements of several subtypes of protein kinase C, including a representative
from each of the three classes of protein kinase C subtypes
that is predominant in SH-SY5Y cells, did not reveal any
differences between AD and control cybrid cells. This indicates that in AD cybrid cells there is an impairment on the
activation of protein kinase C or of signaling components
mediating the downstream activation of AP-1.
In conclusion, in AD cybrid cells activation of the phosphoinositide signal transduction is increased, but downstream activation of AP-1 is decreased. Although these
alterations are most likely attributable to the chronic, low

38

P. De Sarno et al. / Neurobiology of Aging 21 (2000) 31–38

level of oxidative stress present in AD cybrid cells, we
cannot discount potential contributions of other mitochondrial-derived pathophysiology present in these cells. These
results demonstrate the selective effects that oxidative stress
associated with mitochondrial dysfunction can cause on
cellular signaling systems, with some, such as phosphoinositide signaling, able to compensate more fully than others,
such as activation of AP-1. Thus, AD cybrid cells may
model an early stage of neuronal responses to chronic oxidative stress.

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