DEVELOPMENTAL NEUROSCIENCE NEUROREPORT

Guanosine protects SH-SY5Y cells against

b-amyloid-induced apoptosis
Kathleen M. Pettifer,1 Sonya Kleywegt,1,2 Christian J. Bau,1 James D. Ramsbottom,1,2,3 Eva Vertes,1
Renata Ciccarelli,2 Francesco Caciagli,2 Eva S. Werstiuk1,CA and Michel P. Rathbone1

1
Department of Medicine, McMaster University, Health Sciences Centre, 4N711200 Main Street West, Hamilton, ON Canada, L8N 3Z5;
2
Department of Biomedical Sciences, School of Medicine, University of Chieti,Via dei Vestini 31, 66013 Chieti; 3Department of Biomedical Sciences,
Pharmacology, University of Trieste,Via Licio Giorgieri 7, 34127 Trieste, Italy
CA
Corresponding Author: wrstiuke@mcmaster.ca

Received 3 January 2004; accepted 3 February 2004

DOI: 10.1097/01.wnr.0000123386.87650.ab

Apoptosis is implicated in the pathophysiology of Alzheimer’s dis-
ease. Extracellular guanosine inhibits staurosporine-induced apop-
tosis in astrocytes.We examined whether guanosine protects SH-
SY5Y human neuroblastoma cells against b-amyloid (bA)-induced
apoptosis. Addition of bA (fragment 25-35, 5 mM for 24 h) to SH-
SY5Y cells increased the number of apoptotic cells, as evaluated
by oligonucleosome ELISA. Guanosine pre-treatment decreased
bA-induced apoptosis (maximal e¡ect after 24 h, 300 mM,
po0.05). The anti-apoptotic e¡ect of guanosine was reduced by
LY294002 (PI3K inhibitor) or PD98059 (MEK inhibitor) (po0.05).
Guanosine increased phosphorylation of Akt/PKB, and this was
abolished by inhibiting PI3K or MEK, (po0.001, 5 min). Thus, the
protective e¡ect of guanosine against bA-induced apoptosis of
SH-SY5Ycells is mediated via activation of the PI3K/Akt/PKB and
MAPK pathways. NeuroReport 15:833^ 836
c 2004 Lippincott
Williams & Wilkins.

Key words: Apoptosis; Alzheimer’s disease; Beta-amyloid; Cell survival; Guanosine; SH-SY5Y Neuroblastoma cells PI3K/Akt/PKB; MAPK

INTRODUCTION neuroblastoma cells, and characterized the key intracellular

Neurodegeneration in Alzheimer’s disease (AD) is asso-
ciated with abnormal accumulation of neurotoxic b-amyloid
protein (bA) [1], which causes apoptosis of brain cells [2].
Apoptosis, or programmed cell death, has been implicated
in the pathophysiology of AD [1]. Activation of unique
biochemical pathways ultimately leads to the destruction of
cells [3]. Mitochondria and endoplasmic reticulum (ER) are
involved in this process [1,3] by activating a set of cysteine-
dependent proteases known as caspases [3]. These enzymes
cleave multiple cellular targets, including nuclear DNA into
characteristic fragments, considered to be the hallmark of
apoptotic cells [4]. Recently a number of intracellular
pathways have been identified, which protect cells against
apoptosis. These include the phosphatidylinositol 3-kinase
(PI3 K)/Akt/protein kinase B (PKB) pathway (for review
see [5,6]) and the mitogen-activated protein (MAP) kinase
pathway [7]. The downstream targets of Akt/PKB include
activation of transcription factors CREB and NF-kB, and inhi-
bition of the pro-apoptotic proteins caspase-9 and Bad [5].
Guanosine and other nonadenine-based purines have a
number of neurotrophic and neuroprotective roles, such as
stimulation of astrocyte proliferation [8,9], promotion of
neurite outgrowth [10] and increased synthesis and release
of NGF [11]. Recently, we have shown that guanosine also
protects astrocytes against staurosporine-induced apoptosis,
and this is mediated via the PI3K/Akt/PKB pathway [12].
In the present study we examined whether guanosine
protects against bA-induced apoptosis in SH-SY5Y human
pathways, which mediate this effect.
MATERIALS AND METHODS
Cell culture and treatments: SH-SY5Y human neuroblas-
toma cells (ATCC) were cultured using 45% minimum
essential medium (Gibco-BRL, Gaithersburg, MD, USA),
45% F12 Hamilton’s (Gibco-BRL) medium, supplemented
with 10% fetal bovine serum (Gibco-BRL), 100 U/ml
penicillin and 100 mg/ml streptomycin (Gibco-BRL) at pH
7.4 and maintained in a humidified 5% CO2 atmosphere at
371C. Culture medium was changed every 3–4 days.
Neuroblastoma cells were subcultured at a ratio of 1:20
every 7–10 days. Cells were maintained in a medium
containing 1% FBS for 24 h before the start of experiments.
All experiments were performed using 70 to 80% confluent
cultures. bA (protein fragment 25-35) (Sigma-Aldrich) used
to induce apoptosis, was dissolved in 1% acetic acid, and
stored at 201C. In all experiments, guanosine (Sigma-
Aldrich, Canada) was dissolved in sodium hydroxide (1 N
NaOH) and added to the culture medium at a final
concentration of 0.01% sodium hydroxide. Vehicle control
cells (control) were exposed to 0.01% sodium hydroxide. In
experiments, where enzyme inhibitors were tested, SH-
SY5Y cells were pre-treated with various agents for 20 min
prior to the addition of guanosine. These treatments
included: the selective inhibitor of PI3K, [2-(4-mor-
pholinyl)-8-phenyl-1(4 H)-benzopyran-4-1-hydrochloride]

0959- 4965

c Lippincott Williams & Wilkins Vol 15 No 5 9 April 2004 833
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
 NEUROREPORT
(LY294002; Calbiochem, Ontario, Canada) or the selective
inhibitor of MEK, [2-(2-amino-3-methoxyphenyl)-4H-1-ben-
zopyran-4-1] (PD98059; Calbiochem, Ontario, Canada).
LY294002 and PD98059 were dissolved in, and added to
the culture medium at a final concentration of 0.01%
dimethylsulfoxide (DMSO). All compounds were purchased
from Sigma-Aldrich unless otherwise stated. Enzyme
inhibitors, guanosine or bA, once added to cultures were
maintained for the entire duration of the experiment.
Evaluation of apoptosis: DNA fragmentation was evaluated
by two methods: histochemically, using acridine orange-
ethidium bromide staining (Sigma-Aldrich), by fluorescence
microscopy, or by the oligonucleosomal ELISA (Roche Diag-
nostics, IN, USA). For the acridine orange assay, cells were
seeded onto poly-D-lysine-coated glass coverslips (diameter
10 mm; 3  104 cells/coverslip) and exposed to bA. After 24 h,
neuroblastoma cells were fixed in 1% paraformaldehyde for
15 min on ice and then incubated with 70% ethanol for 4 h on
ice. RNase solution A (1 mg/ml) was added to cells and
incubated at 371C for 30 min. After a brief incubation (30–45 s)
with 0.1 M HCl at room temperature, acridine orange staining
solution (6 mg/ml) was added to cells. All these steps were
followed by three washes with PBS. Observations were carried
out using a fluorescence microscope (Nikon, Tokyo, Japan). The
number of fragmented nuclei and condensed chromatin were
determined by counting Z 200 cells. The percentage of apoptotic
cells is defined as: (total number of cells with apoptotic nuclei/
total number of cells)  100. Experiments were performed in
duplicate, and each point represents the mean7s.e.m. of three
independent assays. Oligonucleosome ELISAs were carried out
according to the manufacturer’s instructions. The cytosolic
fraction of the cell lysates was placed into streptavidin-coated
wells, a mixture of biotin-linked anti-histone antibody and
peroxidase-linked anti-DNA antibody were added and incu-
bated for 2 h at room temperature. Plates were washed with the
incubation buffer to remove the unfixed anti-DNA antibody and
the peroxidase activity was determined spectrophotometrically,
with ABTS (2,20 -azino-bis[3-ethylbenzthiazoline-6-sulfonic acid])
as the substrate (absorbance at 405 nm).
Western immunoblot analysis: Phosphorylated Akt/PKB
was detected by Western immunoblot analysis. Following
various drug treatments, cells were washed once with PBS
then harvested at 41C using a lysis buffer (25 mM Tris–HCl pH
7.4, 10 mM NaCl, 10 mM EDTA, 100 ml/10 ml Tween 20,
10 mM sodium pyrophosphate decahydrate, 10 mM sodium
orthovanadate, 5 mg/ml leupeptin, 10 mM glycerophosphate).
Cells were disrupted by sonication and aliquots (25 ml) were
removed for the determination of protein concentrations
using the bicinchoninic acid (BCA) protein assay (Pierce, IL,
USA). Proteins were separated on 12% SDS-polyacrylamide
gels and electrophoretically transferred to nitrocellulose
membranes (PALL, MI, USA). Membranes were incubated
with a specific primary antibody (polyclonal rabbit phospho-
Akt (Ser473), diluted 1:1500, Cell Signaling, Canada) for 1 h at
room temperature then exposed to a secondary antibody for
1 h at room temperature (anti-rabbit IgG horseradish per-
oxidase-linked antibody, diluted 1:200 000, Cell Signaling,
Canada). Immunocomplexes were visualised using a chemi-
luminescence substrate (Sigma-Aldrich). Immunoblots were
quantified by densitometric analyses, using the Northern
Eclipse program (EPIX). Unless otherwise specified, all
products were purchased from Invitrogen, Ontario, Canada.
834 Vol 15 No 5 9 April 2004
Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
K. M. PETTIFER ETAL.
Data analysis: Data are expressed as the mean7s.e.m. of
Z 3 independent experiments. Statistical comparisons were
performed by ANOVA followed by Fisher’s LSD post-hoc
comparison test (when the variance within groups was the
same), or by Kruskal-Wallis test followed by Student-
Newman-Keuls post-hoc test (when variance between
groups was significantly different). p o 0.05 was considered
statistically significant.
RESULTS
b-Amyloid fragment induces apoptosis in SH-SY5Y neuro-
blastoma cells: Exposure of SH-SY5Y neuroblastoma cells
to various concentrations of bA, (1–15 mM) for 24 h increased
the percentage of apoptotic cells, as determined by the
acridine orange-ethidium bromide histochemical assay
(data not shown). These experiments established that the
administration of 5 mM bA to neuroblastoma cells induced
significant apoptosis. These results were confirmed using
the oligonucleosome ELISA (Fig. 1). The proportion of
apoptotic cells in untreated controls was 15.474.0%, and
this increased to 26.571.7% in cells exposed to 5 mM bA
(p o 0.05). In all further experiments apoptosis was induced
by exposure of cells to 5 mM bA for 24 h.
Guanosine protects SH-SY5Y neuroblastoma cells against
b-amyloid induced apoptosis: In a pilot study cultured
SH-SY5Y cells were pre-treated with different concentra-
tions of guanosine (10, 50, 100 and 300 mM) for 1 h prior to
the administration of bA. Guanosine was present for the
entire duration of the experiment. Apoptosis was evaluated
by the acridine orange-ethidium bromide assay. Pre-treat-
35 §
#
(% of positive control)
30
Amount of Apoptosis
*
25
20
‡
15
10
5
0
LY294002 30 µM − − − − + −
PD98059 10 µM − − − − − +
Guanosine 100 µM − + − + + +
β-amyloid 5 µM − − + + + +
Fig. 1. Guanosine protects cultured SH-SY5Yneuroblastoma cells against
b-amyloid-induced apoptosis. Neuroblastoma cells were maintained in cul-
tures containing 1% FBS for 24 h, then cells were treated with b-amyloid
(bA), (fragment 25-35, 5 mM, for 24 h) and the extent of apoptosis was evalu-
ated by the oligonucleosome ELISA. Guanosine was added to some cultures
(100 mM, for1h) prior to the addition of bA.Other cultures were treated with
guanosine alone (100 mM) for 25 h. Some cultures were also pre-treated with
the selective inhibitor of PI3K, LY294002 (30 mM), or MEK, PD98059 (10 mM)
for 20 min prior to the addition of guanosine. Enzyme inhibitors, guanosine
and bA once added, were present for the entire duration of the experiment.
Data are presented as the percentage of apoptotic cells relative to positive
control (n¼3).The percentage of apoptotic cells increased signi¢cantly in the
b-amyloid-treated cells compared to control (*p o 0.05). Exposure of cells to
guanosine prior to bA (guanosine + b-amyloid) reduced the number of
apoptotic cells signi¢cantly, compared to those treated with bA alone (b-
amyloid; wp o 0.05). Addition of LY294002 or PD98059 to cells pre-treated
with guanosine plus b-amyloid increased the percentage of apoptotic cells
compared to those exposed to guanosine plus b-amyloid (}p o 0.05 and
#p o 0.05, respectively).
 GUANOSINE IS ANTI-APOPTOTIC IN SH-SY5Y CELLS NEUROREPORT

1200 1200
**
Amount of phosphorylated

*
Akt/PKB (% of control)

Amount of phosphorylated
1000 1000

Akt/PKB (% of control)
*
800
800
600
600
400
400
200
200
0
0 50 100 150 200 250 300
0
Concentration of guanosine (µM) 0 5 10 15 20
Fig. 2. Concentration-dependence of guanosine-stimulated Akt/PKB Time (min)
phosphorylation in cultured SH-SY5Yneuroblastoma cells. Neuroblasto- Fig. 3. Time course of the guanosine-stimulated Akt/PKB phosphoryla-
ma cells were treated with various concentrations of guanosine alone (10, tion in cultured SH-SY5Yneuroblastoma cells. Neuroblastoma cells were
50, 100 and 300 mM) and the amount of phosphorylated Akt/PKB was de- treated with guanosine alone (300 mM) and the amount of phosphorylated
termined at 5 min in guanosine-treated and control cells, using Western Akt/PKB was determined at various time points (2.5, 5, 10 and 20 min) in
blot analysis. Immunoblots were quantitated using the program North- guanosine-treated and in control cells, using Western blot analysis. Immu-
ern Eclipse (EPIX). Data are presented as the percentage of the amount noblots were quantitated using the program Northern Eclipse (EPIX).
of phosphorylated Akt/PKB relative to control (n¼3). The addition of Data are presented as the percentage of the amount of phosphorylated
100 mM or 300 mM guanosine increased the amount of phosphorylated Akt/PKB relative to control (n¼3). The amount of phosphorylated Akt/
Akt/PKB signi¢cantly (*p o 0.05 and **p o 0.01, respectively). PKB was signi¢cantly increased at 5 min compared to 0 min (*p o 0.05).

ment of cells with all concentrations of guanosine reduced 1200

Amount of phosphorylated
the proportion of apoptotic cells significantly, with 100 mM *

Akt/PKB (% of control)
1000
guanosine being the most effective (data not shown). These
results were confirmed using the oligonucleosome ELISA. 800
The proportion of apoptotic cells was 26.571.7% in the bA-
600
treated cells and this was reduced to 11.572.9% in cells pre-
treated with 100 mM guanosine (p o 0.01; Fig. 1). 400
§ #
200
Effect of guanosine on the cell survival pathways: SH-
SY5Y cells were pre-treated with the PI3K inhibitor LY294002 0
(30 mM) or with the MEK inhibitor PD98059 (10 mM) for
20 min prior to the addition of guanosine. The inhibitors and LY294002 30 µM − − + + − − + +
guanosine were present for the entire duration of the PD98059 10 µM − − − − + + + +
experiment. Inhibition of the PI3K or the MAPK pathway Guanosine 300 µM − + + − + − + −
abolished the protective effect of guanosine against bA-
Fig. 4. The e¡ect of selected inhibitors on the guanosine-stimulated Akt/
induced apoptosis as determined at 24 h (p o 0.05; Fig. 1). PKB phosphorylation in cultured SH-SY5Yneuroblastoma cells. Neuroblas-
toma cells were treated with guanosine alone (300 mM) for 5 min and the
Guanosine increases the phosphorylation of Akt/PKB: We amount of phosphorylated Akt/PKB was determined in guanosine-treated
next evaluated the effects of guanosine treatment alone on and control cells using Western blot analysis. Some cells were pre-treated
Akt/PKB phosphorylation. Various concentrations of gua- with the selective inhibitor of PI3K, LY294002 (30 mM), or with the selective
nosine were added to SH-SY5Y cells (10, 50, 100, or 300 mM) inhibitor of MEK, PD98059 (10 mM) for 20 min prior to treatment with gua-
and Akt/PKB phosphorylation was determined by Western nosine. Inhibitors and guanosine once added to cells were maintained for
the entire duration of the experiment (total time¼25 min). Immunoblots
immunoblot analysis after 5 min guanosine exposure. were quantitated using the program Northern Eclipse (EPIX). Data are
Phosphorylated Akt/PKB increased in a concentration- presented as the percentage of the amount of phosphorylated Akt/PKB re-
dependent manner (Fig. 2). Maximal phosphorylation was lative to control (n¼3).The amount of phosphorylated Akt/PKB stimulated
detected in the presence of 300 mM guanosine (986752%) by guanosine was signi¢cantly di¡erent from control (*p o 0.05). Phos-
compared to controls (p o 0.01). phorylated Akt/PKB in the presence of LY294002 plus guanosine, or
Guanosine-induced Akt/PKB phosphorylation (300 mM) PD98059 plus guanosine was signi¢cantly di¡erent from that in the pre-
sence of guanosine only (}p o 0.001 and #p o 0.001, respectively).
was rapid and transient. Phosphorylated Akt/PKB was
detectable by 2.5 min after the addition of guanosine. It sine-stimulated Akt/PKB phosphorylation in neuroblasto-
reached maximal values at 5 min (986752% compared to ma cells is mediated via the PI3K and the MAPK pathways.
control, p o 0.05), and declined to basal values by 20 min
(Fig. 3). When cells were pre-treated with the PI3K inhibitor
LY294002 (30 mM) or the MEK inhibitor PD98059 (10 mM) DISCUSSION
20 min prior to the addition of guanosine, guanosine- In previous studies we have shown that guanosine protects
stimulated Akt/PKB phosphorylation was reduced to astrocytes against staurosporine-induced apoptosis, and this
values comparable to those of control (p o 0.001; Fig. 4). is mediated by the activation of the PI3K/Akt/PKB and
Inhibition of both, PI3K/Akt/PKB and MAPK pathways MAPK pathways [12]. We therefore examined whether
prior to guanosine administration abolished guanosine- guanosine also protects neuroblastoma cells against apop-
stimulated phosphorylation of Akt/PKB, so this was tosis induced by bA. This peptide is neurotoxic, and has
undetectable (p o 0.001). These data indicate that guano- been shown to promote apoptosis in a number of different

Vol 15 No 5 9 April 2004 835

Copyright © Lippincott Williams & Wilkins. Unauthorized reproduction of this article is prohibited.
 NEUROREPORT
cells, including neurons [13] and cultured SH-SY5Y human
neuroblastoma cells [14]. Several distinct mechanisms have
been implicated in bA-induced apoptosis, including activa-
tion of p75 receptors [15], induction of ER stress [16],
activation and release of caspase 12 from the ER [17], and
the promotion of cytochrome c release from the mitochon-
dria [18]. Although the exact pathways, which mediate bA-
induced apoptosis of neuroblastoma cells have yet to be
identified, these are known to be cell-type dependent [19].
In the present study we found that bA caused significant
apoptosis of neuroblastoma cells. When these cells were
exposed to guanosine prior to the administration of bA, the
number of apoptotic cells was significantly reduced. Pre-
treatment of cells with the inhibitors of the PI3K and MAPK
pathways abolished the protective effect of guanosine against
bA-induced apoptosis. These data therefore confirm, that the
anti-apoptotic effect of guanosine is mediated by activation of
the PI3K/Akt/PKB and MAPK pathways. Thus, guanosine
protects SH-SY5Y cells against bA-induced apoptosis by
activating similar cell-survival pathways in neuroblastoma
cells as in astrocytes [12]. Survival factors suppress apoptosis
by activating the enzyme, PI3K (for review see [6,20]), and this
in turn phosphorylates and activates the serine/threonine
kinase, Akt/PKB (for review see [5]). Martı́n et al. [21] reported
that this pathway is activated transiently in response to bA
fragment (25-35), and protects PC12 cells against the pro-
apoptotic action of this peptide. When SH-SY5Y cells were
exposed to guanosine, there was a rapid and significant
increase in the amount of phosphorylated Akt/PKB. This
guanosine-stimulated phosphorylation of Akt/PKB was
mediated via activation of the PI3K/Akt/PKB and the MAPK
pathways. We observed similar results in astrocytes. Guanosine
administration to astrocytes also increased the amount of
phosphorylated Akt/PKB, by activating the same pathways,
the PI3K/Akt/PKB and the MAPK [12], and this was mediated
through the Gi-protein coupled putative guanosine receptor
[11,22,23]. Thus, in neuroblastoma cells as in astrocytes,
guanosine activates the same intracellular enzyme, Akt/PKB,
via the same survival pathways. The MAPK pathway promotes
cell survival via a dual effect: by inhibiting the pro-apoptotic
protein, Bad, and by inducing pro-survival genes, in a CREB-
dependent manner [7]. The PI3K/Akt/PKB and MAPK
pathways actually converge on these common intracellular
target proteins and inhibit apoptosis [24]. These two pathways
have been implicated in the excitoprotective effects of secreted
amyloid precursor protein in hippocampal neurons [25]. In the
present study we also found that the protective effect of
guanosine is exerted by the activation of both pathways.
CONCLUSION
Guanosine protects SH-SY5Y neuroblastoma cells against
bA-induced apoptosis. This anti-apoptotic effect of guano-
sine is mediated by the activation of the PI3K/Akt/PKB and
MAPK pathways. Our findings suggest that targeting
selected anti-apoptotic pathways may provide an effective
therapeutic strategy for the treatment of AD.
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