Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary...

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: [email protected]

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


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).



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.



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).



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.



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.

References

Amadio S., D’Ambrosi N., Cavaliere F., Murra B., Sancesario G.,

Bernardi G., Burnstock G. and Volonte C. (2002) P2 receptor

modulation and cytotoxic function in cultured CNS neurons.

Neuropharmacology 42, 489–501.

Andsberg G., Kokaia Z. and Lindvall O. (2001) Upregulation of p75

neurotrophin receptor after stroke in mice does not contribute

to differential vulnerability of striatal neurons. Exp. Neurol. 169,

351–363.

Balazs R., Hack N. and Jorgensen O. S. (1992) Neurobiology of exci-

tatory amino acids, in Drug Research Related to Neuroactive

Amino Acids (A. Schousboe, N. H. Diemer and H. Kofod, eds),

pp. 397–410. Munksgaard, Copenhagen.

Barrett G. L. (2000) The p75 neurotrophin receptor and neuronal

apoptosis. Prog. Neurobiol. 61, 205–229.

Barret G. L. and Bartlett P. F. (1994) The p75 nerve growth factor

receptor mediates survival or death depending on the stage of

sensory neuron development. Proc. Natl Acad. Sci. USA 91, 6501–

6505.

Bo X., Zhang Y., Nassar M., Burnstock G. and Schoepfer R. (1995) A

P2X purinoceptor cDNA conferring a novel pharmacological

profile. FEBS Lett. 375, 129–133.

Bobalova J. and Mutafova-Yambolieva V. N. (2001) Presynaptic alpha2-

adrenoceptor-mediated modulation of adenosine 5¢-triphosphateand noradrenaline corelease: differences in canine mesenteric

artery and vein. J. Auton. Pharmacol. 21, 47–55.

Bogdanov Y. D., Wildman S. S., Clements M. P., King B. F. and

Burnstock G. (1998) Molecular cloning and characterization of rat

P2Y4 nucleotide receptor. Br. J. Pharmacol. 124, 428–430.

Bradford M. (1976) A rapid and sensitive method for the quantitation of

microgram quantities of protein utilising the principle of protein-

dye binding. Anal. Biochem. 72, 248–252.

Brake A., Wagenbach M. J. and Julius D. (1994) New structural motif

for ligand-gated ion channels defined by an ionotropic ATP

receptor. Nature 371, 519–523.

Brough D., Le Feuvre R. A., Iwakura Y. and Rothwell N. J. (2002)

Purinergic (P2X7) Receptor activation of microglia induces cell

death via an interleukin-1-independent mechanism. Mol. Cell

Neurosci. 19, 272–280.

Burnstock G. (2002a) Potential therapeutic targets in the rapidly

expanding field of purinergic signalling. Clin. Med. 2, 45–53.

Burnstock G. (2002b) Purinergic and pyrimidinergic signaling I, in

Handbook of Experimental Pharmacology, Vol. 151/1 (Abbracchio

M. P. and Williams M., eds), pp. 89–127. Springer, New York.

Casaccia-Bonnefil P., Carter B. D., Dobrowsky R. T. and Chao M. V.

(1996) Death of oligodendrocytes mediated by the interaction of

NGF with its receptor p75. Nature 383, 716–719.

Cavaliere F., D’Ambrosi N., Ciotti M. T., Mancino G., Sancesario G.,

Bernardi G. and Volonte C. (2001a) Glucose deprivation and



chemical hypoxia: neuroprotection by P2 receptor antagonists.

Neurochem. Int. 38, 189–197.

Cavaliere F., D’Ambrosi N., Sancesario G., Bernardi G. and Volonte C.

(2001b) Hypoglycemia-induced cell death: features of neuropro-

tection by the P2 receptor antagonist basilen blue. Neurochem. Int.

38, 199–207.

Chan C. M., Unwin R. J., Bardini M., Oglesby I. B., Ford A. P.,

Townsend-Nicholson A. and Burnstock G. (1998) Localization of

P2X1 purinoceptors by autoradiography and immunohistochemis-

try in rat kidneys. Am. J. Physiol. 274, F799–F804.

Chizh B. A. and Illes P. (2001) P2X receptors and nociception. Phar-

macol. Rev. 53, 553–568.

Chow S. C., Kass G. E. N. and Orrenius S. (1997) Purines and their roles

in apoptosis. Neuropharmacology 36, 1149–1156.

Collo G., Neidhart S., Kawashima E., Kosco-Vilbois M., North R. A.

and Buell G. (1997) Tissue distribution of the P2X7 receptor.

Neuropharmacology 36, 1277–1283.

Coulson E. J., Reid K., Baca M., Shipham K. A., Hulet S. M., Kilpatrick

T. J. and Bartlet P. F. (2000) Chopper, a new death domain of the

p75 neurotrophin receptor that mediates rapid neuronal cell death.

J. Biol. Chem. 275, 30537–30545.

Courtney M. J., Akerman K. E. and Coffey E. T. (1997) Neurotrophins

protect cultured cerebellar granule neurones against the early phase

of cell death by a two-component mechanism. J. Neurosci. 17,

4201–4211.

D’Ambrosi N., Cavaliere F., Merlo D., Milazzo L., Mercanti D. and

Volonte C. (2000) Antagonists of P2 receptor prevent NGF-

dependent neuritogenesis in PC12 cells. Neuropharmacology 39,

1083–1094.

D’Ambrosi N., Murra B., Cavaliere F., Amadio S., Bernardi G.,

Burnstock G. and Volonte C. (2001) Interaction between ATP and

nerve growth factor signalling in the survival and neuritic out-

growth from PC12 cells. Neuroscience 108, 527–534.

Di Virgilio F., Chiozzi P., Ferrari D., Falzoni S., Sanz J. M., Morelli A.,

Torboli M., Bolognesi G. and Baricordi O. R. (2001) Nucleotide

receptors: an emerging family of regulatory molecules in blood

cells. Blood 97, 587–600.

Frade J. M., Rodriguez-Tebar A. and Barde Y. A. (1996) Induction of

cell death by endogenous nerve growth factor through its p75

receptor. Nature 383, 166–168.

Friedman W. J. (2000) Neurotrophins induce death of hippocampal

neurons via the p75 receptor. J. Neurosci. 20, 6340–6346.

Harrington A. W., Kim J. Y. and Yoon S. O. (2002) Activation of Rac

GTPase by p75 is necessary for c-jun N-terminal kinase-mediated

apoptosis. J. Neurosci. 22, 156–166.

Jo Y. H. and Schlichter R. (1999) Synaptic corelease of ATP and GABA

in cultured spinal neurons. Nat. Neurosci. 2, 241–245.

Juranyi F., Sperlagh B. and Vizi E. S. (1999) Involvement of P2 puri-

noceptors and the nitric oxide pathway in [3H]purine outflow

evoked by short-term hypoxia and hypoglycemia in rat hippo-

campal slices. Brain Res. 823, 183–190.

Kokaia Z., Andsberg G., Martinez-Serrano A. and Lindvall O. (1998)

Focal cerebral ischemia in rats induces expression of P75 neuro-

trophin receptor in resistant striatal cholinergic neurons. Neuro-

science 84, 1113–1125.

Laemmli U.K. (1970) Cleavage of structural proteins during the

assembly of the head of bacteriophage T4. Nature 227, 680–685.

Le K. T., Villeneuve P., Ramjaun A. R., McPherson P. S., Beaudet A.

and Seguela P. (1998) Sensory presynaptic and widespread

somatodendritic immunolocalization of central ionotropic P2X

ATP receptors. Neuroscience 83, 177–190.

Levi G., Aloisi F. and Ciotti M. (1989) Preparation of 98% pure cere-

bellar granule cell cultures, in: A Dissection and Tissue Culture

Manual of the Nervous System. (A. Shahar, J. deVellis, A.

Vernadakis and B. Haber, eds), pp. 211–214 Alan R. Liss, Inc.,

New York.

Lewis C. J. and Evans R. J. (2000) Lack of run-down of smooth muscle

P2X receptor currents recorded with the amphotericin permeabi-

lized patch technique, physiological and pharmacological charac-

terization of the properties of mesenteric artery P2X receptor ion

channels. Br. J. Pharmacol. 131, 1659–1666.

von Lubitz D. K. (1999) Adenosine and cerebral ischemia: therapeutic

future or death of a brave concept? Eur. J. Pharmacol. 371,

85–102.

Lutz P. L. and Kabler S. (1997) Release of adenosine and ATP in the

brain of the freshwater turtle (Trachemys scripta) during long-term

anoxia. Brain Res. 769, 281–286.

Mancino G., Placido R. and Di Virgilio F. (2001) P2X7 receptors and

apoptosis in tuberculosis infection. J. Biol. Regul. Homeost. Agents

15, 286–293.

Merlo D. and Volonte C. (1996) Binding and functions of extracellular

ATP in cultured cerebellar granule neurones. Biochem. Biophys.

Res. Commun. 225, 907–914.

Merlo D., Anelli R., Calissano P., Ciotti M. T. and Volonte C. (1997)

Characterization of an ecto-phosphorylated protein of cultured

cerebellar granule neurons. J. Neurosci. Res. 47, 500–508.

Park J. A., Lee J. Y., Sato T. A. and Koh J. Y. (2000) Co-induction of

p75NTR and p75NTR-associated death executor in neurons after

zinc exposure in cortical culture or transient ischemia in the rat.

J. Neurosci. 20, 9096–9103.

Phillis J. W., O’Regan M. H. P. and Erkins L. M. (1993) Adenosine

5¢-triphosphate release from the normoxic and hypoxic in vivo rat

cerebral cortex. Neurosci. Lett. 151, 94–96.

Picano E. and Abbracchio M. P. (2000) Adenosine, the imperfect

endogenous anti-ischemic cardio-neuroprotector. Brain Res. Bull.

52, 75–82.

Pintor J., Miras-Portugal T. and Fredholm B. B. (2000) Research on

purines and their receptors comes of age. Trends Pharmacol. Sci.

21, 453–456.

Poelchen W., Sieler D., Wirkner K. and Illes P. (2001) Co-transmitter

function of ATP in central catecholaminergic neurons of the rat.

Neuroscience 102, 593–602.

Rabizadeh S., Oh J., Zhong L. T., Yang J., Bitler C. M., Butcher L. L.

and Bredesen D. E. (1993) Induction of apoptosis by the low-

affinity NGF receptor. Science 261, 345–348.

Ramer M. S., Bradbury E. J. and McMahon S. B. (2001) Nerve growth

factor induces P2X(3) expression in sensory neurons. J. Neuro-

chem. 77, 864–675.

Rathbone M. P., Middlemiss P. J., Gysbers J. W., Andrew C., Herman

M. A., Reed J. K., Ciccarelli R., Di Iorio P. and Caciagli F. (1999)

Trophic effects of purines in neurons and glial cells. Prog. Neu-

robiol. 59, 663–690.

Roux P. P., Colicos M. A., Barker P. A. and Kennedy T. E. (1999) p75

neurotrophin receptor expression is induced in apoptotic neurons

after seizure. J. Neurosci. 19, 6887–6896.

Rubio M. E. and Soto F. (2001) Distinct localization of P2X receptors

at excitatory postsynaptic specializations. J. Neurosci. 21, 641–

653.

Sokolova E., Nistri A. and Giniatullin R. (2001) Negative cross talk

between anionic GABAA and cationic P2X ionotropic receptors of

rat dorsal root ganglion neurons. J. Neurosci. 21, 4958–4968.

Stefanis L., Troy C. M., Qi H., Shelanski M. L. and Greene L. A. (1998)

Caspase-2 (Nedd-2) processing and death of trophic factor-

deprived PC12 cells and sympathetic neurons occur independently

of caspase-3 (CPP32)-like activity. J. Neurosci. 18, 9204–9215.

Surprenant A., Rassendren F., Kawashima E., North R. A. and Buell G.

(1996) The cytolytic P2Z receptor for extracellular ATP identified

as a P2X receptor (P2X7). Science 272, 735–738.



Susen K., Heumann R. and Blochl A. (1999) Nerve growth factor sti-

mulates MAPK via the low affinity receptor p75LNTR. FEBS

Lett. 463, 231–234.

Valera S., Hussy N., Evans R. J., Adami N., North R. A., Surprenant A.

and Buell G. (1994) A new class of ligand-gated ion channel defined

by P2X receptor for extracellular ATP. Nature 371, 516–519.

Vizi E. S. and Sperlagh B. (1999) Receptor- and carrier-mediated release

of ATP of postsynaptic origin: cascade transmission. Prog. Brain

Res. 120, 159–169.

Volonte C. and Merlo D. (1996) Selected P2 purinoceptor modulators

prevent glutamate-evoked cytotoxicity in cerebellar granule neu-

rons. J. Neurosci. Res. 45, 183–193.

Volonte C., Ciotti M. T. and Battistini L. (1994) Development of a

method for measuring cell number: application to CNS primary

neuronal cultures. Cytometry. 17, 274–276.

Volonte C., Ciotti M. T., D’Ambrosi N., Lockhart B. and Spedding M.

(1999) Neuroprotective effects of modulators of P2 receptors in

primary culture of CNS neurones. Neuropharmacology 38, 1335–

1342.

Vulchanova L., Riedl M. S., Shuster S. J., Buell G., Surprenant A.,

North R. A. and Elde R. (1997) Immunohistochemical study of

P2X2, P2X3 receptor subunits in rat and monkey sensory neu-

rons and their central terminals. Neuropharmacology 36, 1229–

1242.

Wang X., Bauer J. H., Li Y., Shao Z., Zetoune F. S., Cattaneo E. and

Vincenz C. (2001) Characterization of a p75NTR apoptotic sig-

nalling pathway using a novel cellular model. J. Biol. Chem. 276,

33812–33820.

Webb T. E., Henderson D. J., Roberts J. A. and Barnard E. A. (1998)

Molecular cloning and characterization of the rat P2Y4 receptor.

J. Neurochem. 71, 1348–1357.

Zhang M., Zhong H., Vollmer C. and Nurse C. A. (2000) Co-release of

ATP and ACh mediates hypoxic signalling at rat carotid body

chemoreceptors. J. Physiol. 525, 143–158.



Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary...

Documents

Transcript of Extracellular ATP and nerve growth factor intensify hypoglycemia-induced cell death in primary...