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Extracellular ATP and nerve growth factor intensify
hypoglycemia-induced cell death in primary neurons: role
of P2 and NGFRp75 receptors
Fabio Cavaliere,* Giuseppe Sancesario,*,� Giorgio Bernardi*,� and Cinzia Volonte*,�
*Fondazione Santa Lucia, Rome, Italy
�University of Rome Tor Vergata, Department of Neuroscience, Rome, Italy
�CNR Institute of Neurobiology and Molecular Medicine, Rome, Italy
Abstract
In this study, we monitored the direct expression of P2
receptors for extracellular ATP in cerebellar granule neurons
undergoing metabolism impairment. Glucose deprivation for
30–60 min inhibited P2Y1 receptor protein, only weakly
modulated P2X1, P2X2 and P2X3, and up-regulated by about
two-fold P2X4, P2X7 and P2Y4. The P2X/Y antagonist basilen
blue, protecting cerebellar neurons from hypoglycemic cell
death, maintained within basal levels only the expression of
P2X7 and P2Y4 proteins, but not P2X4 or P2Y1. Glucose
starvation transiently increased (up to three-fold) the expres-
sion of NGFRp75 receptor protein and strongly stimulated the
extracellular release of nerve growth factor (NGF; about
10-fold). Exogenously added NGF then augmented hypo-
glycemic neuronal death by about 60%, increasing the per-
centage of Hoechst-positive nuclei (from approximately
62 to 95%), reducing lactate dehydrogenase (LDH) release
(from about 50 to 14%) and significantly overstimulating the
hypoglycemia-induced expression of P2X7 and P2Y4. Con-
versely, extracellular ATP augmented hypoglycemic neuronal
death by about 80%, reducing the number of Hoechst-positive
nuclei (from approximately 62% to 14%), augmenting LDH
outflow (by about 30%) and further increasing the hypogly-
cemia-induced expression of NGFRp75. Our results indicate
that P2 and NGFRp75 receptors are modulated during glu-
cose starvation and that extracellular ATP and NGF drive
features of, respectively, necrotic and apoptotic hypoglycemic
cell death, aggravating the consequences of metabolism
impairment in cerebellar primary neurons.
Keywords: apoptosis, necrosis, NGF release, P2X7, P2Y4.
J. Neurochem. (2002) 83, 1129–1138.
Binding to their specific P1 and P2 cellular receptors, the
extracellular purines are among the phylogenetically most
ancient signaling factors playing crucial biological roles
(Chow et al. 1997; von Lubitz 1999; Picano and Abbracchio
2000). Purinergic receptors can in fact drive many physio-
logical functions, including smooth muscle contraction
(Lewis and Evans 2000), immune responses (Di Virgilio
et al. 2001), pain (Chizh and Illes 2001; Burnstock 2002a),
neurotransmission (Pintor et al. 2000; Rathbone et al. 1999),
fertilization and embryonic development (Burnstock 2002b).
Even pathological insults can be functionally correlated to
purinergic mechanisms. For instance, the inhibition of P2
receptors prevents cell death in CNS neurons, during both
excitotoxic-necrotic injuries caused by high glutamate, and
apoptotic insults evoked by serum-potassium deprivation
(Volonte and Merlo 1996; Volonte et al. 1999). Furthermore,
brain ischemia induces massive extracellular release of ATP,
adenosine and other neurotransmitters (Balazs et al. 1992;
Phillis et al. 1993; Juranyi et al. 1999) and selected P2
receptor antagonists counteract hypoglycemic cell death by
inhibiting cellular swelling, LDH release, chromatin and
nuclei fragmentation and caspase 2 activity (Cavaliere et al.
2001a,b).
Received July 5, 2002; revised manuscript received September 4, 2002;
accepted September 6, 2002.
Address correspondence and reprint requests to Cinzia Volonte
Fondazione Santa Lucia. Via Ardeatina, 354, 00179 Rome, Italy.
E-mail: cinzia@in.rm.cnr.it
Abbreviations used: BB, basilen blue; BME, Eagle’s basal medium;
CGN, cerebellar granule neurons; DIV, day(s) in vitro; LDH, lactate
dehydrogenase; NGF, nerve growth factor; PBS, phosphate-buffered
saline; SDS–PAGE, sodium dodecyl sulfate–polyacrylamide gel elec-
trophoresis.
Journal of Neurochemistry, 2002, 83, 1129–1138
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1129–1138 1129
A direct interplay between P2 and additional receptor
systems has been suggested, since ATP is often coreleased
with other neurotransmitters, such as, depending on the
individual neuron’s specific transmitter repertoire, acetylcho-
line (Zhang et al. 2000), noradrenaline (Bobalova and
Mutafova-Yambolieva 2001) and GABA (Jo and Schlichter
1999). In particular, Sokolova et al. (2001) demonstrated
negative cross-talk between GABA and P2X receptors in rat
dorsal root ganglion neurons; Rubio and Soto (2001) showed
that synapses expressing P2X receptors are also glutamater-
gic in the cerebellum and hippocampus; Volonte and Merlo
(1996) and Amadio et al. (2002) considered glutamate and
purine receptors as separate components of a single func-
tional unit acting in synergism to regulate the efficiency of
the synaptic transmission. Recently, an interplay between P2
and growth factor receptors, in particular P2X3 and NGF,
was demonstrated in spinal cord and dorsal root ganglia
(Ramer et al. 2001). An interaction between ATP and NGF
signaling in the neuritic outgrowth and survival of PC12 cells
has also been demonstrated (D’Ambrosi et al. 2000, 2001).
In dissociated neuronal cultures (Friedman 2000) and in vivo
in oligodendrocytes not expressing the high affinity NGF
receptor tyrosine kinase TrkA (Frade et al. 1996), NGF
exerts toxic actions through the low affinity NGFRp75
protein. Although NGF and NGFRp75 are induced in
neurons after brain ischemia or seizures (Roux et al. 1999;
Park et al. 2000), no direct evidence has yet established
whether this induction plays detrimental or beneficial roles.
In this paper, we examine the contribution of P2 and
NGFRp75 receptor proteins to metabolism impairment and
address the issue of a potential interaction between these
receptor systems in the mechanisms underlying neuronal
death in cerebellar granule neurons (CGN).
Materials and methods
Dissociated primary cell cultures
CGN from 8-day-old Wistar rat cerebellum were prepared as
described elsewhere (Levi et al. 1989) and seeded on poly L-lysine-
coated dishes, in Eagle’s basal medium (BME) (Gibco BRL, Milan,
Italy), supplemented with 25 mM KCl, 2 mM glutamine, 0.1 mg/mL
gentamycin, 10% heat inactivated fetal calf serum (Gibco BRL). At
1 DIV [day(s) in vitro], 10 lM cytosine arabinoside was added to thecultures, which were then kept for 7–9 days without replacing the
culture medium.
Glucose starvation
CGN at 8 DIV were maintained for different lengths of time in
Earl’s balanced salt solution (116 mM NaCl, 25 mM KCl, 1.8 mM
CaCl2, 0.8 mM MgSO4, 26 mM NaHCO3, 0.6 mM NaH2PO4,
pH 7.4) with or without the addition of 1 mg/mL glucose and in
the presence or absence of various agents. The cells were then
returned to their previously saved culture media and cell survival
was evaluated at different times thereafter, by direct count of the
intact viable nuclei obtained after solubilization of the cellular
membranes by means of a mild detergent (Volonte et al. 1994;
Stefanis et al. 1998).
ELISA
CGN (7 · 106) at 8 DIV were glucose deprived for 1 h in Earl’s
balanced salt solution. This solution was collected and subjected to
ELISA immunoassay, using the commercial kit Emax (Promega,
Milan, Italy). Positive reactions were evaluated using the ELISA
reader Multiscan EX (Labsystems, Orlando, FL, USA).
LDH assay
Necrotic cell death was evaluated by measuring the extracellular
presence of LDH. CGN (6 · 105) at 8 DIV were glucose starved in
Earl’s balanced salt solution for different lengths of time, in the
presence of various agents. Supernatants were collected and 2X
Reaction buffer [60 mM phosphate buffer, 2 mg bNAD, and100 mM lactate in phosphate-buffered saline (PBS)] was added.
Enzymatic LDH activity was evaluated by kinetic NADH reduction
for 10 min at 37�C, performed with a Lambda bio 20 spectropho-tometer (Perkin Elmer, Milan, Italy) and measured at 340 nm.
Chromatin staining by Hoechst 33258
CGN at 8 DIV were maintained in the absence or presence of glucose
for 3 h, with or without exogenous NGF (50 ng/mL) and with or
without 500 lM ATP. They were fixed for 20 min in 4% parafor-
maldehyde, washed three times with PBS and permeabilized in 0.2%
Triton-X in PBS. The cells were incubated for 2 min with 1 lg/mLHoechst 33258 [excitation (Ex.) at 345, emission (Em.) at 478 nm]
and the chromatin was visualized with a fluorescence microscope.
Total protein extraction
CGN were glucose starved for different lengths of time, in the
presence of different compounds. Total protein from 2.5 · 106 cellswas extracted for 30 min at 0�C with RIPA buffer [PBS, 1% NP-40,
0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS),
0.5 lM phenylmethylsulfonyl fluoride, 10 lg/mL leupeptin] and
then centrifuged for 10 min at 4�C (10 000 g). An equal amount of
total protein from each sample was subjected to SDS–polyacryla-
mide gel electrophoresis (PAGE).
SDS–PAGE and western blotting
Protein concentration was determined by the Bradford method
(Bradford 1976). Separation of proteins was performed on 12%
polyacrylamide gels, as described elsewhere (Laemmli 1970),
loading the same amount of total protein for each experimental
condition. Proteins were transferred to nitrocellulose membranes
(Hybond C; Amersham, Milan, Italy) and blots were probed for 2 h
at room temperature with the specified antisera and horseradish
peroxidase-coupled secondary antibody. The immunoreactions were
analyzed using enhanced chemiluminescence (Santa Cruz, Milan,
Italy). Quantification was performed using a Kodak Image Station
(KDS IS440CF 1.1). Only statistically significant immunoblot band
intensity data are reported. P2X1,2,4,7 receptor antisera (Alomone,
Jerusalem, Israel) were used at 1 : 200 dilution. P2Y1 was used at
1 : 400, P2Y4 at 1 : 300, P2X3 (Santa Cruz Biotechnology, Santa
Cruz, CA, USA) at 1 : 2000 and NGFRp75 (H-137) (Santa Cruz) at
1 : 200 dilution. As specified in the analysis certificate for each P2
1130 F. Cavaliere et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1129–1138
receptor antiserum, all sera were affinity purified and raised against
highly purified peptides (identity confirmed by mass spectroscopy
and amino acid analysis), corresponding to specific epitopes not
present in any other known protein (P2X1 : 382–399; P2X2 : 457–
472; P2X3(A-16); P2X4 : 370–388; P2X7 : 576–595; P2Y1 : 242–
258; P2Y4 : 337–350). Specificity for each P2 receptor signal was
also directly tested by immunoreactions in the presence of
neutralizing peptides (peptide/antiserum ratio of 1 : 1). All secon-
dary antisera were used at 1 : 15 000 dilution.
Immunoprecipitation
CGN at 8 DIV were glucose deprived for different lengths of time.
Total protein from 7 · 106 cells was collected in RIPA buffer and
precleared for 1 h at 4�C with 1 lg of rabbit preimmune serum.Anti-NGFRp75 (H-137) (Santa Cruz) (1 lg) was incubated with thecell lysate for 1 h at 4�C; 30 lL of Protein G Plus-agarose (Santa
Cruz Biotechnology) was then added for 20 h at 4�C. The immunecomplexes were washed three times in RIPA buffer, dissolved in
sample buffer and separated by 10% SDS–PAGE.
Statistical analysis
Statistical differences were evaluated by t-test analysis of the data,
and are reported as follows: *p < 0.05, **p < 0.01, ***p < 0.001.
Results
Glucose deprivation modulates the expression
of P2 receptor proteins
ATP released extracellularly with glutamate and other
excitotoxic amino acids during oxygen and/or glucose
deprivation causes serious neuronal cell loss (Phillis et al.
1993; Juranyi et al. 1999). In order to investigate the
involvement of P2 receptor proteins in hypoglycemic cell
death, we adopted the cellular model system of CGN, since
they constitute an abundant, almost pure neuronal population
(90–98% homogenous after 8–9 DIV) and are vulnerable to
necrosis, apoptosis, oxidative stress and mitochondrial
dysfunction. In particular, in CGN glucose deprivation
causes full commitment to cell death as early as 0.5 h post-
treatment (Cavaliere et al. 2001a,b). Therefore, in these
neurons we monitored the direct expression of ionotropic
P2X and metabotropic P2Y receptor proteins after different
lengths of glucose deprivation. P2X1, P2X2 and P2X3proteins were very weakly affected by hypoglycemia,
eliciting at most 30–40% up-regulation (data not shown).
Glucose deprivation (30–60 min) instead increased two-fold
the expression of P2X4, P2X7 and P2Y4 (Fig. 1). Fully
preventing hypoglycemic cell death in CGN (Cavaliere et al.
2001a,b), the P2X/Y-non-selective antagonist basilen blue
(BB) maintained within basal levels only the expression of
P2X7 and P2Y4 proteins, but not P2X4. Conversely, glucose
deprivation inhibited the cellular content of P2Y1, despite the
presence of BB (Fig. 1). We positively established the
specificity of each signal by performing immunoreactions in
the presence of a neutralizing peptide for each P2 receptor
subtype (Fig. 1, lane c; Amadio et al. 2002). Only in the case
of P2X7, we did observe the presence of an additional,
strong, non-specific signal (Fig. 1, and manufacturer’s ana-
lysis certificate). Furthermore, the molecular masses of all P2
receptor proteins detected by the antisera corresponded with
results obtained using other cell types or with cloning data
(Brake et al. 1994; Valera et al. 1994; Bo et al. 1995;
Surprenant et al. 1996; Collo et al. 1997; Vulchanova et al.
1997; Bogdanov et al. 1998; Chan et al. 1998; Le et al.
1998; Webb et al. 1998).
Glucose deprivation up-regulates NGFRp75 receptor
protein and causes release of NGF
CGN lack the high affinity NGF receptor TrkA and, in its
absence, NGFRp75 is well documented to mediate detri-
mental effects and to sustain apoptotic signaling. This occurs
when NGFRp75 is ligand activated (Barret 2000), auto-
stimulated by multimerization (Wang et al. 2001) or simply
over-expressed in vitro (Rabizadeh et al. 1993; Barret and
Bartlett 1994). Despite these notions, little is still known of
a direct role of NGFRp75 as a death effector during oxygen
and/or glucose deprivation (Park et al. 2000). In this study,
we demonstrated in CGN that NGFRp75 protein is maxi-
mally (three-fold) and transiently up-regulated after glucose
deprivation (as demonstrated by immunoprecipitation and
SDS–PAGE/western blot analysis, Fig. 2a). In addition, the
extracellular presence of NGF remarkably increased (about
10-fold) after 1 h of glucose deprivation (as demonstrated by
ELISA immunoassay, Fig. 2b) and this occurred before any
detectable sign of cell death.
Extracellular ATP and NGF increase hypoglycemic
cell death
We then investigated the contribution of extracellularly added
NGF or ATP to hypoglycemia-induced cell death. Although
NGF, unlike ATP (Amadio et al. 2002), was per se not toxic
(see also Courtney et al. 1997), it augmented by about 60%
the extent of cell death triggered by glucose deprivation in 1 h
(Fig. 3). Consistently, an NGF-blocking antibody (aNGF,which totally impaired the NGF-induced differentiation of
PC12 cells, data not shown) almost completely (70–80%)
abolished hypoglycemic cell death, either in the absence or in
the presence of extracellularly added NGF (Fig. 3). Because
the activation of P2 receptors by exogenous ATP is toxic for
CNS neurons (Amadio et al. 2002), extracellular ATP (used
at 500 lM during glucose deprivation for 1 h) augmented byabout 80% the CGN cell death (Fig. 3). As expected, the
inhibitor of ATP co-release, guanethidine (Poelchen et al.
2001), reduced hypoglycemic cell death to a final 18%, both
in the absence (Fig. 3) or in the presence (data not shown) of
extracellular ATP. NGF and ATP not only increased, but also
interfered with the features of cell death (Table 1). NGF (used
Dying by glucose starvation: P2 and NGFRp75 receptors 1131
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1129–1138
for 3 h during hypoglycemia) increased the percentage of
Hoechst-positive nuclei (from about 62 to 95%) and reduced
LDH release (from about 50 to 14%). Under similar
experimental conditions, extracellular ATP conversely aug-
mented LDH outflow (by about 30%) and diminished the
number of Hoechst-positive nuclei (from approximately 62%
to 14%) [Table 1; note that 100% LDH release (control) was
obtained in the presence of 100 lM glutamate for 30 min,
necrotic paradigm, and 100% Hoechst-positive nuclei were
instead obtained in the absence of serum and in the presence
of 5 mM potassium, apoptotic paradigm]. Two main cellular
morphologies generally distinguish these neurons when are
maintained in the absence of glucose and are committed to
cell death: swollen and shrunken cellular bodies (Fig. 4b),
with blown or condensed nuclei and chromatin, as shown by
Hoechst staining (Fig. 4f). We proved here that hypoglyce-
mic CGN become more pyknotic in shape with compacted
chromatin when cultured simultaneously with NGF (Figs 4c
and g) and, conversely, that they are induced to swell with
dispersed chromatin in the presence of extracellular ATP
(Figs 4d and h).
ATP and NGF reciprocally modulate the expression
of NGFRp75 and P2 receptor proteins
We then assessed whether extracellular NGF or ATP,
exacerbating the hypoglycemic cell death of CGN, might
Fig. 1 Hypoglycemia directly modulates P2
receptor protein expression. CGN at 8 DIV
were maintained for different lengths of time
under hypoglycemic conditions, in the
presence or absence of 100 lM BB. West-
ern blotting and immunoreactions were
performed and quantitative analysis was
obtained with a Kodak Image Station, using
an antiserum against Erk1/2 (Erk1/2) as
internal standard. Lane c represents
immunoreactions performed with neutral-
izing peptides. The quantitative analysis
was performed, respectively, to: the 46 kDa
protein band for P2X4; the 68 kDa protein
band for P2X7; the 110 kDa protein band for
P2Y1; the 90 kDa protein band for P2Y4.
The analysis of additional specific P2X/Y
protein signals provided comparable
results. Only statistically significant immu-
noblot band intensity data are reported
(*p < 0.05, **p < 0.01).
1132 F. Cavaliere et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1129–1138
act directly on the expression levels of NGFRp75 and P2
proteins. We found that NGF failed to significantly increase
the homologous NGFRp75, both in the presence and in the
absence of glucose (Fig. 5), but it strongly stimulated the
expression of P2X7 receptor protein (two-fold or 50%
increase with or without glucose, respectively; Fig. 6).
Extracellular NGF acted similarly with regard to P2Y4protein, inducing a three- to four-fold increase in the
presence of glucose and a two-fold increase without it; the
NGF-blocking antibody significantly prevented these effects
(Fig. 6). P2X7 and P2Y4 proteins were never found to
immunoprecipitate together with NGFRp75 receptor and the
expression of P2X1,2,3,4 and P2Y1 proteins was instead never
modulated by NGF, under all the experimental conditions
tested (data not shown). Conversely, extracellular ATP,
without significantly modifying the P2X7 and P2Y4 protein
content (Fig. 6), strongly augmented the expression of the
heterologous NGFRp75 (about five-fold in the presence and
two-fold in the absence of glucose, respectively) while
guanethidine prevented this effect (Fig. 5).
Discussion
We previously found that several P2 receptor antagonists can
efficiently prevent cell death evoked not only by excitotoxicity
(Volonte and Merlo 1996) and apoptosis (Volonte et al.
1999), but also by metabolism impairment (Cavaliere et al.
2001a,b), in primary neuronal dissociated and organotypic
CNS cultures. The attempt to uncover the P2 receptor
subtypes mostly contributing to these effects thus became
crucial and, to this end, in the present study we began by
investigating the expression of both metabotropic and
ionotropic P2 receptor proteins. We can summarize our
results by saying that CGN die in response to hypoglycemia,
modulating a combination of selected P2X and P2Y receptor
subtypes. For instance, glucose starvation weakly modulated
P2X1, P2X2 and P2X3, up-regulated P2X4, but down-
regulated P2Y1. Nevertheless, not one of these proteins
was affected by the neuroprotective P2 receptor antagonist
(b)
(a)
Fig. 2 Hypoglycemia transiently up-regulates NGFRp75 and causes
NGF release. (a) CGN at 8 DIV were maintained for different lengths
of time under hypoglycemic conditions and NGFRp75 was immuno-
precipitated and then detected by western blot, using anti-NGFRp75.
Quantitative analysis was obtained using immunoglobulin heavy-chain
(Ig-HC) as an internal standard. Only statistically significant immuno-
blot band intensity data are reported. (b) Release of NGF in the culture
medium during 1 h of glucose deprivation was measured by ELISA
assay. Similar results were obtained in three independent experi-
ments. Statistical differences were evaluated by t-test analysis of the
data (**p < 0.01).
Fig. 3 Extracellular NGF and ATP increase hypoglycemic cell death.
CGN at 8 DIV were maintained for 1 h in the presence or absence of
glucose and with or without different compounds. Cell death was
evaluated by direct count of intact nuclei and data represent means ±
SEM (n ¼ 6). Statistical differences were evaluated by t-test analysis
of the data (**versus control condition; #,##versus hypoglycemia;
§§versus hypoglycemia + NGF).
Table 1 Extracellular HGF and ATP: features of cell death
Cell death (%) LDH (%) Apoptosis (%)
CTRL 8 ± 2 10 ± 4 19 ± 5
Hypo 3 h 91 ± 5 50 ± 6 62 ± 4
Hypo 3 h + NGF 86 ± 6 14 ± 6 95 ± 9
Hypo 3 h + ATP 85 ± 4 64 ± 3 14 ± 5
Glut 93 ± 2 100 ± 2 22 ± 1
K5/S– 83 ± 7 17 ± 3 100 ± 7
CGN at 8 DIV were maintained in the absence of glucose for 3 h, in
the presence or absence of exogenous NGF (50 ng/mL) or 500 lM
ATP; 1 mg/mL glucose was added to the control conditions (CTRL).
Cell death was evaluated as described in Materials and methods. The
percentage of LDH release and Hoechst-fragmented nuclei were,
respectively, compared with controls obtained with 100 lM glutamate
(Glut) or 5 mM potassium and serum deprivation (K5/S–). Statistical
differences were evaluated by t-test analysis of the data, with
0.001 < p < 0.05.
Dying by glucose starvation: P2 and NGFRp75 receptors 1133
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1129–1138
BB, which instead inhibited the induction of P2X7 and P2Y4,
highly induced by hypoglycemia. Because CGN simulta-
neously express several P2X and P2Y proteins (Amadio
et al. 2002), we believe that the different receptor subtypes
must either act in synergism or sustain diverse physiological
and/or pathological functions. The present data do point to
P2X7 and P2Y4 as preferred candidates for modulating cell
death. Indeed, during hypoglycemia, the P2 antagonist BB
reverts the up-regulation of only P2X7 and P2Y4 and
simultaneously switches the cell fate of CGN from death to
survival. Moreover, P2X7 and P2Y4 are the same subtypes
also induced by cytotoxic extracellular ATP (Amadio et al.
Fig. 4 Extracellular NGF and ATP interfere with the features of cell
death. CGN were maintained for 3 h in the presence (a and e) or
absence (b and f) of glucose; 50 ng/mL NGF was added both 30 min
before as well as during 1 h of glucose deprivation (c and g). In
(d and h), 500 lM ATP was added only during glucose starvation.
Morphological analysis was performed by contrast microscopy (a–d)
or fluorescence microscopy after staining with Hoechst 33258 (e–h).
Apoptotic shrunk cell bodies are indicated by black arrows; necrotic
cell bodies are indicated by white arrows (b–d). Scale bar is 40 lm
(a–d) or 90 lm (e–h).
(a)
(b)
Fig. 5 NGFRp75 protein expression: effect of extracellular ATP. CGN
were incubated for 60 min in the presence (1–3) or absence (4–6) of
glucose, and with 500 lM ATP (2, 5) or 50 ng/mL NGF (3, 6). (a) Total
protein was collected and western blot performed as described in
Materials and methods. Specific bands were then quantified (b).
Similar results were obtained in three independent experiments. Sta-
tistical differences were evaluated by t-test analysis of the data
(**,***versus control condition, ##versus hypoglycemia).
Fig. 6 P2 receptor protein expression: effect of extracellular NGF and
ATP. CGN were treated for 30 (P2X7) or 60 min (P2Y4) in the pres-
ence or absence of 1 mg/mL glucose, and with or without different
compounds. Total protein was extracted and western blot analysis
performed with both P2X7 and P2Y4 antisera. Densitometric analysis
was performed as previously described and only statistically significant
immunoblot band intensity data are reported. Statistical differences
were evaluated by t-test analysis of the data (*,**,***versus control
condition; #,##,###versus hypoglycemia; §,§§§versus hypoglycemia +
NGF).
1134 F. Cavaliere et al.
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1129–1138
2002) and, not least, P2X7 is well known to mediate cell
death in many different cell types and under several
experimental conditions (Chow et al. 1997; Mancino et al.
2001; Brough et al. 2002). Nevertheless, we cannot com-
pletely exclude that P2X4 and P2Y1 play a certain role in cell
death or that a more constant receptor expression (P2X1,
P2X2 and P2X3) would necessarily mean lack of involve-
ment, especially because BB might not exclusively elicit
antagonistic effects on P2 receptors. Different combinations
of P2 receptors might even function at different steps of the
degenerative process, either as initiators, propagators or
ending signals for cell death, inasmuch we described that
P2X2,3,4 and P2Y2 share a permissive role in ATP- or NGF-
dependent neurite regeneration from PC12 cells (D’Ambrosi
et al. 2000, 2001).
In general, we are inclined to consider the up-regulation of
P2 receptors by hypoglycemia as a means for augmenting the
ATP-binding capacity of CGN and for increasing neuronal
responsiveness to toxic extracellular ATP. This also occurs
under physiological conditions, as the increased release of
ATP from CGN that takes place as a function of neuronal
maturation in culture was similarly coupled to increased
overall binding of ATP to CGN (Merlo and Volonte 1996;
Merlo et al. 1987), to up-regulation of selected P2X and
P2Y receptor proteins and to increased responsiveness to
ATP-evoked cell death (Amadio et al. 2002). Consistently,
ATP released by neurons under metabolism impairment (Vizi
and Sperlagh 1999) was shown here to have a direct
detrimental function during hypoglycemic cell death. Exo-
genous addition of ATP in fact increases cell loss and drives
cell death toward more marked necrotic features (increased
cellular swelling and outflow of LDH but decreased Hoechst-
fragmented nuclei, effects all prevented by the inhibitor of
ATP release, guanethidine).
In conclusion, our results support the involvement of P2X7and P2Y4 in cell death, they confirm and extend the
detrimental role played by ATP in CNS primary cultures
(Amadio et al. 2002) and provide a biological function for
(a)
(b) (c)
Apoptosis
Necrosis
Fig. 7 Schematic hypothesis of interaction
between NGFRp75 and P2 receptors dur-
ing hypoglycemic cell death. Up-regulation
of both NGFRp75 and P2 receptors is the
mutual consequence of glucose deprivation
giving rise to both necrotic and apoptotic
cell death (a). Over-stimulation of P2
receptors by exogenous ATP in the
absence of glucose drives cell death toward
a more necrotic phenotype and up-regu-
lates NGFRp75 protein levels (b). Equally,
exogenous NGF in the absence of glucose
generates more apoptotic features and
up-regulates only P2X7 and P2Y4, but not
NGFRp75 (c). Note that simultaneous acti-
vation of P2X7, P2Y4 and NGFRp75 is
needed for hypoglycemic death, whereas
NGF per se does not up-regulate NGFRp75
and is not toxic to CGN.
Dying by glucose starvation: P2 and NGFRp75 receptors 1135
� 2002 International Society for Neurochemistry, Journal of Neurochemistry, 83, 1129–1138
the extracellular ATP massively released during in vivo
ischemic episodes (Lutz and Kabler 1997). Finally, they
support the neuroprotection provided by several P2 receptor
antagonists against various different insults, in particular
glucose starvation (Cavaliere et al. 2001a,b).
An additional receptor that regulates cell death is
NGFRp75 (Barrett 2000), known to be involved in the
biological mechanisms triggered by metabolism impairment
in cholinergic striatal neurons (Kokaia et al. 1998; Andsberg
et al. 2001). By means of NGFRp75, NGF stimulates
ceramide production and MAPK activation (Susen et al.
1999) but, in the absence of the high affinity TrkA receptor,
NGF can also be deleterious, playing a direct role in
apoptotic mechanisms (Casaccia-Bonnefil et al. 1996; Barret
2000; Coulson et al. 2000; Harrington et al. 2002). The
results presented here reinforce the importance of this
receptor in neuronal damage caused by metabolism impair-
ment, demonstrating that glucose starvation not only aug-
ments the extracellular release of NGF from CGN, but also
transiently up-regulates NGFRp75 protein expression. This
suggests that P2 and NGFRp75 receptors might both be
ligand-activated and act similarly during glucose starvation,
with regard to ligand release, receptor expression and
detrimental consequences. Indeed, we demonstrated that
not only ATP but also extracellular NGF can be functional to
metabolism impairment, exacerbating and driving hypo-
glycemic cell death towards a more clear apoptotic pheno-
type (increased cellular shrinkage and Hoechst-positive
fragmented nuclei, reduced LDH release). An additional
consideration emerging from our data is that extracellular
NGF or ATP sustain features of apoptotic and necrotic
hypoglycemic cell death, respectively, by overexpressing
NGF the heterologous P2X7/P2Y4 receptors (the effect is
counteracted by the presence of the NGF-antiserum) and
ATP the NGFRp75 protein (effect susceptible to inhibition
by guanethidine). This would suggest a measure of interac-
tion between NGF and ATP receptors (as schematically
depicted in Fig. 7), that could range from heterologous
receptor activation to partial overlapping of the downstream
signal transduction pathway(s).
Further experiments will now endeavor to explain how
extracellular ATP can drive necrotic hypoglycemic cell death
by further up-regulating the apoptotic NGFRp75 receptor,
and why extracellular NGF does not require the up-regula-
tion of the NGFRp75 receptor in order to drive apoptotic
hypoglycemic cell death.
In conclusion, our results prove that ATP and NGF released
under conditions of metabolism impairment can both be
included among the concomitant aggravating causes of
hypoglycemic neurodegeneration targeting the CNS. ATP
per se appears to be necessary and sufficient to provoke cell
death [up-regulating P2X7, P2Y4 (Amadio et al. 2002) and
NGFRp75 (this work)], whereas NGF seems necessary but
not sufficient (up-regulating only P2X7 and P2Y4, not
NGFRp75). A current challenge in this field is now to define
these receptor mechanisms and to shed light on the regulatory
and signaling cascades sustained by hypoglycemic cell death.
Acknowledgements
The research presented was supported by the Italian Health Ministry,
project number RA00.86 V and by Cofinanziamento MURST 2001.
Our grateful thanks go to Dr Delio Mercanti for providing the
blocking aNGF antiserum. The professional style editing of Mrs
Catherine Wrenn is gratefully acknowledged.
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