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The Journal of Neuroscience, May 15, 2002, 22(10):3963-3968
 -Amyloid-Induced Synthesis of the Ganglioside Gd3 Is a
Requisite for Cell Cycle Reactivation and Apoptosis in Neurons
Agata
Copani1,
Daniela
Melchiorri3,
Andrea
Caricasole3,
Francesca
Martini4,
Patrizio
Sale4,
Roberto
Carnevale4,
Roberto
Gradini4,
Maria Angela
Sortino2,
Luisa
Lenti4,
Ruggero
De
Maria5, and
Ferdinando
Nicoletti3, 6
Departments of 1 Pharmaceutical Sciences and
2 Experimental and Clinical Pharmacology, University of
Catania, 95125 Catania, Italy, Departments of
3 Human Physiology and Pharmacology and
4 Experimental Medicine and Pathology, University of Rome
"La Sapienza," 00185 Rome, Italy, 5 Laboratory
of Hematology and Oncology, Istituto Superiore di Sanità, 00185 Rome, Italy, and 6 I.N.M. Neuromed, 86077 Pozzilli, Italy
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ABSTRACT |
We have shown that cortical neurons challenged with toxic
concentrations of  -amyloid peptide (AP) enter the S phase of the cell cycle before apoptotic death. Searching for a signaling molecule that lies at the border between cell proliferation and apoptotic death,
we focused on the disialoganglioside GD3. Exposure of rat cultured
cortical neurons to 25 µM AP(25-35) induced a
substantial increase in the intracellular levels of GD3 after 4 hr, a
time that precedes neuronal entry into S phase. GD3 levels decreased but still remained higher than in the control cultures after 16 hr of
exposure to AP(25-35). Confocal microscopy analysis showed that the
GD3 synthesized in response to AP colocalized with nuclear chromatin. The increase in GD3 was associated with a reduction of
sphingomyelin (the main source of the ganglioside precursor ceramide) and with the induction of  -2,8-sialyltransferase
(GD3 synthase), the enzyme that forms GD3 from the monosialoganglioside GM3. A causal relationship between GD3, cell-cycle activation, and
apoptosis was demonstrated by treating the cultures with antisense oligonucleotides directed against GD3 synthase. This treatment, which
reduced AP(25-35)-stimulated GD3 formation by ~50%, abolished the neuronal entry into the S phase and was protective against AP(25-35)-induced apoptosis.
Key words:
Alzheimer's disease; -amyloid; cell cycle; ganglioside GD3; apoptosis; neurodegeneration
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INTRODUCTION |
It is currently believed that
neuronal degeneration in Alzheimer's disease (AD) is caused by
extracellular -amyloid peptide (AP) (for review, see Selkoe,
2001 ). Cultured neurons exposed to AP predominantly show an
apoptotic phenotype (Forloni et al., 1993; Loo et al., 1993), although
neuronal apoptosis by -amyloid is not the only factor that
contributes to the pathophysiology of AD (Behl, 2000; Mattson, 2000;
Joseph et al., 2001; Roth, 2001; Small et al., 2001). Molecular
determinants of AP-induced neuronal death have been investigated
extensively, but they are still poorly defined. In vivo and
in vitro studies have shown that an untimely activation of a
cell cycle in terminally differentiated neurons may be a requisite
antecedent to neuronal apoptosis in AD (Herrup and Busser, 1995;
Vincent et al., 1996; Arendt et al., 1998; Busser et al., 1998; Nagy et
al., 1998; Copani et al., 1999; Giovanni et al., 1999, 2000; McShea et
al., 1999; Yang et al., 2001). We have shown that full-length AP
(fragment 1-42) and its active fragments AP(1-40) and
AP(25-35) promote the activation of a cell cycle in differentiated
cultured cortical neurons. In particular, AP-treated cortical
neurons express the repertoire of proteins necessary to exit quiescence
and eventually enter S phase. These neurons undergo apoptosis before
entering the G2/M phase (Copani et al.,
1999).
Because AP-induced activation of this "neuronal cycle" seems to
be critical for the development of apoptosis, it becomes important to
disclose the signaling pathway(s) leading to reactivation of the cell
cycle in neurons.
Gangliosides, sialic acid-containing glycosphingolipids, constitute a
signaling system involved in the modulation of processes of neuronal
proliferation and differentiation. GD3 is highly expressed in the
embryonic nervous system, particularly in neuroprogenitor cells. GD3
levels are low in the adult brain (Percy et al., 1991; Svennerholm et
al., 1991; Goldman and Reynolds, 1996; Kawai et al., 1998), although
they increase in the brains of patients with AD (Kalanj et al., 1991)
and Creutzfeldt-Jakob disease (Ando et al., 1984). Interestingly,
endogenous GD3 neosynthesis is associated with the appearance of a
tumor phenotype in melanocytes (Birkle et al., 2000), and
overexpression of GD3 synthase (the -2,8-sialyltransferase that
generates GD3 from GM3) enhances the proliferation rate of both rat C6
glioma (Sottocornola et al., 1998) and PC12 pheochromocytoma cell lines
(Fukumoto et al., 2000). Thus, it appears that GD3 is able to modify
the cell proliferation status under physiological and pathological conditions.
In the present study we show that mature rat cortical neurons in
culture, which respond to AP by re-entering the cell cycle, show an
early increase in the intracellular levels of GD3. GD3 synthesis in
AP-treated neurons is required for their entrance into the S phase
and contributes to the development of apoptosis.
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MATERIALS AND METHODS |
Pure neuronal culture. Cultures of pure cortical
neurons were obtained from rats at embryonic day 15 as described
previously (Copani et al., 1999). Briefly, dissociated cortical cells
were plated in a medium consisting of DMEM/Ham's F12 (1:1)
supplemented with the following components: 10 mg/ml bovine serum
albumin, 10 µg/ml insulin, 100 µg/ml transferrin, 100 µM putrescine, 20 nM progesterone, 30 nM selenium, 2 mM glutamine, 6 mg/ml glucose, 50 U/ml penicillin, and 50 µg/ml streptomycin. Cortical cells were
plated at a density of 2 × 106
cells/dish on 35 mm Nunc (Roskilde, Denmark) dishes precoated with 0.1 mg/ml poly-D-lysine.
Cytosine--D-arabinofuranoside (10 µM) was
added to the cultures 18 hr after plating to avoid the proliferation of
non-neuronal elements and was kept for 3 d before medium
replacement. This method yields >99% pure neuronal cultures, as
judged by immunocytochemistry for glial fibrillary acidic protein and
neuron-specific microtubule-associated protein 2 (Copani et al.,
1999). AP has always been applied to mature cultures at 8 d
in vitro (DIV).
Handling of -amyloid peptide. AP(25-35) and the
reverse peptide AP(35-25) were purchased from Bachem AG (Bubendorf,
Switzerland). Different lots of peptides were used. AP(25-35) and
AP(35-25) were solubilized in sterile, doubly distilled water at an
initial concentration of 2.5 mM and stored frozen
at  20°C. They were used at a final concentration of 25 µM in the presence of the glutamate receptor
antagonists MK-801 (10 µM) and DNQX (30 µM) to prevent the excitotoxicity mediated by
endogenous glutamate (Copani et al., 1999).
Addition of antisense oligonucleotides to the cultures.
Cultures were also treated with the following "end-capped"
phosphorothioate antisense oligonucleotides directed against the enzyme
-2,8-sialyltransferase (GD3 synthase): 5'-CAGTACAGCCATGGCCCCTCT-3'.
A scrambled oligonucleotide was used as a control:
5'-CGACCTACCTATGCGCT-ACCG-3'. Oligonucleotides (3 µM) were applied to the cultures 16 hr before
the addition of AP(25-35).
Fluorescence-activated cell sorter analysis.
Fluorescence-activated cell sorter analysis was performed as described
previously (Copani et al., 1999). Cells were harvested by incubation
with 0.25% trypsin for 3 min, and the suspension was centrifuged at low speed after addition of 50% fetal calf serum. Each pellet was
washed with PBS and finally fixed in 70% ethanol. Before
staining with propidium iodide (50 µg/ml in the dark for 30 min),
suspended cells were treated for 1 hr at 37°C with RNase (100 µg/ml). The DNA content and ploidy were assessed using a Coulter
Elite flow cytometer (Beckman, Fullerton, CA). The Multicycle AV
software program (Phoenix Flow Systems, San Diego, CA) was used to
analyze cell-cycle distribution profiles.
Evaluation of sphingomyelin hydrolysis. Cortical neurons
were incubated in the presence of
[3H]serine (specific activity, 26 Ci mmol 1; Amersham Biosciences,
Milan, Italy) for 72 hr before exposure to AP(25-35) for 4 hr. The
reaction was stopped by adding methanol:chloroform:HCl (100:100:1,
v/v/v) and a balanced salt solution containing 10 mM EDTA; the aqueous and lipid phases were
separated by centrifugation. Glycerophospholipids present in the lipid
phase were saponified in methanolic KOH (0.1 M
for 1 hr at 37°C) before resolution of sphingomyelin by sequential
one-dimensional TLC, using chloroform:benzene:ethanol (80:40:75, v/v/v)
followed by chloroform:methanol:28% ammonia (65:25:5, v/v/v) as
solvents. Plates were analyzed using a digital autoradiographer
(Berthold, Bad Wildbad, Germany).
Assessment of intracellular GD3 levels. Cultures were washed
twice with ice-cold PBS, pH 7.4, and cells were scraped from the dishes
and homogenized. Gangliosides were extracted according to the method of
Svennerholm and Fredman (1980) as described previously (Dotta et al.,
1998) and analyzed by high-performance TLC (HPTLC) using analytical
precoated Silica gel 60 HPTLC plates (Merck, Darmstadt, Germany). All
plates were first activated by heating to 100°C for 30 min. Samples
were spotted onto plates with a Hamilton syringe in
chloroform:methanol:0.25% KCl (5:4:1, v/v/v). Authentic GD3 (provided
by Fidia S.p.A., Abano Terme, Italy) was used as standard. GD3
was immunodetected by using the R24 anti-GD3 monoclonal antibody
(1:100). The plates were incubated for 1 hr at room temperature with
the primary antibody, washed twice with PBS-Tween 20, and then
incubated for 45 min at room temperature with a horseradish peroxidase-conjugated rabbit anti-mouse antibody (1:200; Sigma, St.
Louis, MO). Detection was performed by ECL (Amersham Biosciences). The
bands were quantified by scanning densitometric analysis.
Immunofluorescence analysis of GD3. Cultures were fixed with
2% paraformaldehyde. After incubation with 3% nonimmunized mouse serum in PBS, the R24 anti-GD3 monoclonal antibody (1:100) was applied
at 4°C for 72 hr. Cells were washed three times and FITC-conjugated anti-mouse Ig (1:200; Cappel, ICN Biomedicals, Milan, Italy) was applied for 1 hr at room temperature to visualize the labeled sites.
Nuclei were stained with propidium iodide (50 µg/ml) in PBS.
Fluorescence was detected by a Zeiss (Oberkochen, Germany) LSM510 laser
scanner microscope.
Reverse transcriptase-PCR analysis of GD3 synthase. Total
RNA was extracted from cultures of primary cortical neurons essentially as described previously (Auffray and Rougeon, 1980), except that cells
were washed twice with ice-cold PBS and then scraped in 2 ml of cold 3 M LiCl/6 M urea and the
procedure was scaled down appropriately. Finally, total RNA was
subjected to DNase I treatment (Boehringer Mannheim, Indianapolis, IN)
according to the manufacturer's instructions. Two micrograms of total
RNA were then used for cDNA synthesis, using Superscript II
(Invitrogen, San Diego, CA) and an oligo(dT) primer according to
the manufacturer's instructions. The reverse transcriptase (RT)
product was diluted to 100 µl with sterile, distilled water, and 1 µl of cDNA was used in each subsequent PCR amplification.
Amplification of GD3 synthase cDNA was performed using the following
primers: forward (5'-CCAGCATAATTCGCCAGAGA-3') and reverse
(5'-TTGCATGTTCACGGAGAAGG-3'). For -actin cDNA amplification, the
primers were those described by Roelen et al. (1994), which span an
intron and yield products of different sizes depending on whether cDNA
or genomic DNA is used as a template (400 bp for a cDNA-derived product
and 600 bp for a genomic DNA-derived amplification). Reaction
conditions included an initial denaturation step (94°C for 3 min)
followed by 45 cycles of 94°C for 30 sec, 55°C for 30 sec, and
72°C for 30 sec. A final extension step (72°C for 10 min) concluded
the reaction. PCR products (one-third of the reaction) were analyzed
electrophoretically on 2% agarose gels poured and run in 1× Tris
acetate-EDTA.
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RESULTS |
We have shown previously that AP(1-42) or its active fragments
AP(1-40) and AP(25-35) reactivate the cell cycle and induce apoptotic death in pure cultures of cortical neurons (Copani et al.,
1999). Because identical effects were seen with the three peptides, we
used AP(25-35) in the present study. AP(25-35) (25 µM) was applied to mature cultures (8-9 DIV) in the
presence of a mixture of ionotropic glutamate receptor antagonists (10 µM MK-801 plus 10 µM
DNQX) to avoid any endogenous excitotoxic component (Copani et al.,
1999). As expected, ~8-10% of cultured neurons were found in S
phase 16 hr after the addition of AP(25-35), whereas no S phase was
seen at earlier times (4 or 8 hr). The number of apoptotic neurons
increased progressively after 16 hr, reaching >50% of the cell
population at 20 hr (see also Copani et al., 1999).
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Figure 2.
Immunofluorescence analysis of GD3 in control
cultures (A) and in cultures exposed to reverse
AP(35-25) (B) or to AP(25-35)
(C) for 16 hr. GD3 immunofluorescence is in
green. Fluorescence staining of DNA with propidium
iodide is in red. Colocalization between GD3 and DNA is
in yellow. The count of GD3-positive nuclei is shown in
D. Values were calculated by a blinded observer from three random
fields per culture dish for a total of four to six culture dishes per
condition in two to three independent experiments. Each single dish has
been considered as an individual value in the statistical analysis
(i.e., n = 4-6). Values are means ± SEM. *p < 0.05 (one-way ANOVA plus Fisher's least
significant difference) versus controls or reverse
AP(35-25). Reverse AP(35-25) was not toxic in these
experiments.
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TLC analysis combined with immunodetection showed a large increase in
GD3 content 4 hr after the addition of AP(25-35) (i.e., at a time
that precedes both neuronal entry into S phase and apoptotic death)
(Fig. 1). GD3 levels decreased, but were
still higher than in control cultures, 16 hr after the addition of
AP(25-35) (Fig. 1A,B).
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Figure 1.
Intracellular GD3 levels in cultured cortical
neurons exposed to AP(25-35) for 4 or 16 hr. A, A
representative TLC stained with anti-GD3 antibodies. B,
A densitometric analysis of three independent experiments. Four culture
dishes (2 × 106 neurons per dish) were pooled
for each condition in any experiment. Values are expressed as
percentages of controls and represent means ± SEM.
*p < 0.05 (one-way ANOVA plus Fisher's least
significant difference) versus controls.
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In Figure 2A,B,D,
double-fluorescence analysis of GD3 (green) and
nuclear chromatin (red) showed few neurons expressing GD3 in
control cultures or in cultures exposed to the reverse peptide AP(35-25). Nearly all neurons became immunopositive for GD3 in neurons exposed to AP(25-35). Immunoreactivity was mostly detected in cell nuclei (yellow) after the addition of
AP(25-35), although it was also found outside the nuclear region in
late degenerating neurons (Fig. 2C,D).
The early increase in GD3 expression paralleled a reduction of the
sphingomyelin content in neurons exposed to AP(25-35) for 4 hr
(Fig. 3A), suggesting that GD3
is synthesized after AP(25-35)-induced sphingomyelin hydrolysis.
RT-PCR analysis showed that AP(25-35) induced the expression of GD3
synthase mRNA after 2, 8, and 16 hr (Fig. 3B).
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Figure 3.
A, Exposure of cultured cortical
neurons to AP(25-35) for 4 hr reduces sphingomyelin
(SM) levels. Values are means ± SEM of five
individual determinations. *p < 0.01 (Student's
t test) compared with control cultures.
B, Expression of GD3 synthase in primary cultures of rat
cortical neurons exposed to AP(25-35) for the indicated times. The
results of two representative experiments are shown.
CTRL, Control cultures. Amplification of -actin cDNA
was performed to confirm the integrity of the cDNA preparations and to
control for genomic DNA contamination. The 600 bp of -actin was not
detected, thus excluding any genomic contamination.
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To examine whether the increase in GD3 levels was causally related to
the reactivation of the cell cycle and apoptotic death induced by
AP, we treated the cultures with a 3 µM concentration of end-capped antisense oligonucleotides directed against GD3 synthase
for 16 hr before the addition of AP(25-35). GD3 synthase antisense
oligonucleotides substantially reduced the rise of neuronal GD3 content
induced by AP(25-35) at 4 hr, whereas a scrambled oligonucleotide
had a smaller effect (Fig.
4A).
Cytofluorometric analysis showed that GD3 synthase antisense
oligonucleotides abolished the neuronal re-entry into the S phase of
the cell cycle in response to AP(25-35) (Fig. 4B)
and substantially protected against AP(25-35)-induced apoptosis
(Fig. 4C). Scrambled oligonucleotides could also reduce AP(25-35)-induced S phase and apoptosis, but their effect was much
smaller and was significantly different from that produced by GD3
synthase antisense oligonucleotides (Fig. 4B,C).
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Figure 4.
Intracellular GD3 levels
(A), percentage of neurons in the S phase of the
cell cycle (B), and percentage of apoptotic
neurons (C) in cultured cortical neurons
pretreated for 16 hr with GD3 synthase antisense oligonucleotides
(GD3S-As, 3 µM) or a scrambled
oligonucleotide (GD3S-Scr, 3 µM), and then
exposed to AP(25-25) for 4 hr (A) or 20 hr
(B, C). Densitometric analysis of GD3 levels from three
independent experiments is shown in A. Four culture
dishes (2 × 106 neurons per dish) were pooled
for each condition in any experiment. Values in B and
C were calculated from eight individual culture dishes
from three independent experiments. Each single dish has been
considered as an individual value in the statistical analysis (i.e.,
n = 8). p < 0.05 (one-way
ANOVA plus Fisher's least significant difference) if compared
with cultures treated with AP(25-35) alone (*) or with AP plus
GD3S-As (#).
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Together, these results indicate that AP(25-35)-induced GD3
synthesis is antecedent and causally related to neuronal cell-cycle reactivation and apoptosis.
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DISCUSSION |
After the induction of apoptosis, GD3 accumulates in non-neuronal
cells, where it contributes to the death pathway by a dual mechanism
that involves the opening of mitochondrial permeability transition
pores and the suppression of the nuclear factor- B-dependent survival
pathway (De Maria et al., 1997; Kristal and Brown, 1999; Malisan and
Testi, 1999; Scorrano et al., 1999; Rippo et al., 2000; Colell et al.,
2001). In neurons, a number of studies have been performed with
exogenously added gangliosides (Favaron et al., 1988; Manev et al.,
1990; Saito et al., 1998, 1999; Ryu et al., 1999), but the functional
role of endogenously generated gangliosides in neurodegenerative
processes has never been investigated.
In this study, we show that GD3 accumulates in rat cortical neurons
that have been exposed to AP(25-35). We have demonstrated previously that mature neurons must re-enter the cell cycle and cross
the G1/S transition before undergoing
AP-induced apoptosis (Copani et al., 1999). AP-treated cortical
neurons express a battery of proteins that are typically expressed by
proliferating cells during G1/S phases, such as
cyclin D1, phosphoretinoblastoma, cyclin E, and cyclin A. Neurons
eventually enter the S phase, as shown by quantitative cytofluorometric
analysis, and then die by apoptosis before crossing the border between
the S and G2 phases (Copani et al., 1999). The
use of a dexamethasone-inducible cyclin D1 mRNA antisense, a negative
dominant mutant of cyclin-dependent kinase type 2 (CDK2) or chemical
CDK inhibitors has shown that the unscheduled cell cycle is causally
related to apoptotic death in neurons exposed to AP (Copani et al.,
1999, 2001). These in vitro studies have their in
vivo counterpart in the AD brain, in which Yang et al. (2001)
provided evidence for chromosomal replication in "at-risk" neurons.
The present data indicate that GD3 is an essential component of the
signaling pathway(s) leading to the reactivation of the cell cycle in
AP-treated neurons (Fig. 5). This
evidence is consistent with the regulatory functions of GD3 in cellular
proliferation and differentiation processes. PC12 cells overexpressing
the GD3 synthase gene showed an enhanced rate of proliferation
attributable to a sustained activation of the Ras/mitogen-activated
protein-extracellular signal-regulated kinase
kinase/extracellular signal-regulated kinase pathway and failed to differentiate in response to NGF (Fukumoto et al., 2000). In
malignant cells, GD3 induces the suppression of differentiation phenotypes and promotes proliferation (Sottocornola et al., 1998; Birkle et al., 2000).
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Figure 5.
Hypothetical model of the role of GD3 in
AP-induced cell-cycle activation and apoptosis. SM,
Sphingomyelin.
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In neurons that degenerate in response to AP, neosynthesized GD3
accumulated inside the nuclear region and colocalized with nuclear
chromatin. In other cell types, in which apoptosis is not associated
with a reactivation of the cell cycle, GD3 is instead consistently
found outside the nucleus (Malisan and Testi, 1999). To date, only
gangliosides of the "a" series have been described in the cell
nucleus. GM1 is found in the nuclear membrane, where its expression is
upregulated during axonogenesis (Wu et al., 1995, 2001;
Kozireski-Chuback et al., 1999). Interestingly, GM1 inhibits DNA
synthesis and the activity of DNA polymerase in isolated nuclei of
HeLa cells (Ohsawa et al., 1988). An attractive hypothesis is that GD3
acts as a functional counterpart of GM1 by increasing cell
proliferation via a nuclear mechanism. Accordingly, in neurons exposed
to AP, GD3 might provide a nuclear signal for the aberrant DNA
replication. The evidence that the antisense-induced decrease in GD3
levels was highly effective in preventing the unscheduled S phase, and
the ensuing apoptotic phenotype strengthens the hypothesis of a causal
relationship among GD3 formation, cell-cycle activation, and neuronal
death. However, we cannot exclude the possibility that GD3 contributes
to AP-induced apoptosis through additional mechanisms (Fig. 5), for
example by targeting the mitochondrial pathway of cell death.
Ceramide released from sphingomyelin hydrolysis is a likely source
for GD3 synthesis (De Maria et al., 1998). Sphingomyelin hydrolysis
might follow the interaction of AP aggregates with a membrane
receptor, the identity of which is unknown. The p75 low-affinity
neurotrophin receptor is a possible candidate (Yaar et al., 1997),
because this receptor has been shown to transduce the extracellular
signal via the activation of acidic sphingomyelinase (Brann et al.,
1999; Bilderback et al., 2001). The finding of an early induction of
GD3 synthase in response to AP is particularly interesting because
it provides the first evidence that this enzyme is upregulated in
response to a death signal. This suggests a novel strategy against
AP-induced neurotoxicity based on the pharmacological regulation of
GD3 synthase expression.
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FOOTNOTES |
Received Jan. 9, 2002; revised Feb. 22, 2002; accepted Feb. 26, 2002.
This work was supported by Ministero dell' Istruzione, dell'
Universitá e della Ricerca cofin 2000 (M.A.S.), by Ricerca
Finalizzata 2000 (F.N.), and by Alzheimer's Association Grant
IIRG-01-2824 (A.C.).
Correspondence should be addressed to Dr. Agata Copani, Department of
Pharmaceutical Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. E-mail: acopani{at}katamail.com.
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