Nicergoline, a drug used for age-dependent cognitive impairment, protects cultured neurons against β-amyloid toxicity

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Brain Research 1047

Research report

Nicergoline, a drug used for age-dependent cognitive impairment,

protects cultured neurons against h-amyloid toxicity

Filippo Caracia,1, Mariangela Chisarib,1, Giuseppina Frascab, Pier Luigi Canonicoc,

Angelo Battagliad, Marco Calafiorea, Giuseppe Battagliae, Paolo Boscof,

Ferdinando Nicolettie,g, Agata Copania,h, Maria Angela Sortinob,*

aDepartment of Pharmaceutical Sciences, University of Catania, Viale A. Doria, 6, 95125, Catania, ItalybDepartment of Experimental and Clinical Pharmacology, University of Catania, Viale A. Doria, 6, 95125, Catania, Italy

cDISCAFF, University of Piemonte Orientale, ItalydCNS Medical Department, Pfizer Italia, Rome, Italy

eI.N.M. Neuromed, Pozzilli, ItalyfIRCCS Oasi Maria SS, Troina, Italy

gDepartment of Human Physiology and Pharmacology, University of Rome ‘‘La Sapienza’’, ItalyhIBB, CNR, Catania, Italy

Accepted 1 April 2005

Available online 10 May 2005

Abstract

Nicergoline, a drug used for the treatment of Alzheimer’s disease and other types of dementia, was tested for its ability to protect neurons

against h-amyloid toxicity. Pure cultures of rat cortical neurons were challenged with a toxic fragment of h-amyloid peptide (hAP25–35) andtoxicity was assessed after 24 h. Micromolar concentrations of nicergoline or its metabolite, MDL, attenuated hAP25–35-induced neuronal

death, whereas MMDL (another metabolite of nicergoline), the a1-adrenergic receptor antagonist, prazosin, or the serotonin 5HT-2 receptor

antagonist, methysergide, were inactive. Nicergoline increased the basal levels of Bcl-2 and reduced the increase in Bax levels induced by

h-amyloid, indicating that the drug inhibits the execution of an apoptotic program in cortical neurons. In mixed cultures of rat cortical cells

containing both neurons and astrocytes, nicergoline and MDL were more efficacious than in pure neuronal cultures in reducing h-amyloid

neurotoxicity. Experiments carried out in pure cultures of astrocytes showed that a component of neuroprotection was mediated by a

mechanism of glial–neuronal interaction. The conditioned medium of cultured astrocytes treated with nicergoline or MDL for 72–96 h

(collected 24 h after drug withdrawal) was neuroprotective when transferred to pure neuronal cultures challenged with h-amyloid. In cultured

astrocytes, nicergoline increased the intracellular levels of transforming-growth factor-h and glial-derived neurotrophic factor, two trophic

factors that are known to protect neurons against h-amyloid toxicity. These results raise the possibility that nicergoline reduces

neurodegeneration in the Alzheimer’s brain.

D 2005 Elsevier B.V. All rights reserved.

Theme: Disorders of the nervous system

Topic: Degenerative disease: Alzheimer’s—neuropharmacology and neurotransmitters

Keywords: h-Amyloid toxicity; Cortical neurons; Nicergoline; Glial-neuronal interaction

0006-8993/$ - see front matter D 2005 Elsevier B.V. All rights reserved.

doi:10.1016/j.brainres.2005.04.004

* Corresponding author. Fax: +39 095 7384228.

E-mail address: [email protected] (M.A. Sortino).1 These authors contributed equally to this work.

1. Introduction

Nicergoline (10a-methoxy-1,6-dimethylergoline-8h-methanol-5-bromonicotinate) is an ergot alkaloid derivative

that is clinically used for the treatment of cognitive impair-

ment associated with various types of dementia, including

(2005) 30 – 37

F. Caraci et al. / Brain Research 1047 (2005) 30–37 31

primary degenerative, vascular, and mixed dementia

[13,22,30,40]. Similarly to other ergoline derivatives, nicer-

goline improves brain hemodynamics and metabolism

[24,28,37], increases energy production in injured mitochon-

dria [31], and blocks a1-adrenergic receptors [21]. In

addition, nicergoline enhances cholinergic transmission [7]

and polyphosphoinositide turnover [8], two effects that

support the use of the drug in Alzheimer’s disease (AD).

The evidence that nicergoline stimulates protein kinase C-

mediated a-secretase processing of the amyloid precursor

protein (APP) [10] suggests that the drug limits the formation

of pathological cleavage products of APP. It becomes

particularly interesting to establish whether nicergoline is

also able to protect neurons against the degeneration cascade

occurring in the AD brain. In vivo studies have shown that a

nicergoline treatment supports the survival of forebrain

cholinergic neurons, preventing the age- and colchicine-

induced reduction in choline acetyl transferase mRNA and

increasing the content of nerve growth factor and brain-

derived neurotrophic factor in the brain of aged rats [17].

Although these effects are relevant to the AD pathology, no

evidence is yet provided that nicergoline affects neurotoxicity

induced by h-amyloid peptide (hAP), the main constituent of

the amyloid plaques in the AD brain. hAP is recognized

among the major factors involved in neurodegeneration

associated with AD and is largely used to induce neuronal

death in vitro [20]. We now report that nicergoline protects

cultured cortical neurons against hAP toxicity and that part ofthis effect is mediated by a mechanism of glial–neuronal

interaction.

2. Materials and methods

All animal experimental procedures were carried out in

accordance with the directives of the Italian and EU

regulations for care and use of experimental animals

(DL116/92) and approved by the Italian Ministry of Health.

2.1. Drugs

Nicergoline, 1-methyl-10-alpha-methoxy-9,10-dihydro-

lysergol (MMDL), and 10-alpha-methoxy-9,10-dihydroly-

sergol (MDL) were a kind gift of Pharmacia Italia, Nerviano,

Italy. The hAP fragment, hAP25–35, was purchased from

Bachem Feinchemikalien AG (Bubendorf, Switzerland).

2.2. Cultures of cortical neurons

Cultures of pure cortical neurons were obtained from rats

at embryonic day 15 (Morini, s.a.s., Reggio Emilia, Italy).

Briefly, cortices were dissected in Ca++/Mg++-free buffer and

mechanically dissociated. Cortical cells were plated at a

density of 2 � 106/dish on 35 mm dishes (Nunc, Rochester,

NY) precoated with 0.1 mg/ml poly-d-lysine (Sigma-Aldrich

S.r.l., Milan, Italy) in DMEM/Ham’s F12 (1:1) medium

supplemented with the following components: 10 mg/ml bo-

vine serum albumin, 10 Ag/ml insulin, 100 Ag/ml transferrin,

100 AM putrescine, 20 nM progesterone, 30 nM selenium,

2 mM glutamine, 6 mg/ml glucose, 100 U/ml penicillin, and

100 Ag/ml streptomycin. Cytosine-d-arabinofuranoside

(10 AM) was added to the cultures 18 h after plating to avoid

the proliferation of non-neuronal elements and was kept for

3 days before medium replacement. This method yields

>99% pure neuronal cultures, as judged by immunocytoche-

mistry for glial fibrillary acidic protein and flow cytometry

for neuron-specific microtubule-associated protein 2 [11].

For mixed cortical cultures, cells were grown into

DMEM/F12 (1:1) supplemented with 10% horse serum,

10% fetal calf serum (FCS), 2 mM glutamine, 6 mg/ml

glucose. After 7–10 days in vitro, glial cell division was

halted by exposure to 10 AM cytosine-d-arabinoside for 3

days and cells were shifted into a maintenance serum-free

medium. Mature cultures contained about 35–40% neurons.

2.3. Pure cultures of cortical astrocytes

Cortical glial cells were prepared from 1- to 3-day-old

Sprague–Dowley rats (Morini, s.a.s.). After removal of

meninges and isolation of cortices, cells were dispersed by

mechanical and enzymatic dissociation using a 0.25%

solution of trypsin (Invitrogen, S.r.l., Milan, Italy). Cells

were plated onto 75 mm2 flasks and maintained in DMEM

culture medium, supplemented with 10% FCS, penicillin/

streptomycin (100 U/ml–100 Ag/ml), and glutamine

(2 mM). All medium constituents were from Invitrogen,

and all plastic materials were from Corning Life Sciences

(Acton, MA). Confluent cultures at 8–10 DIV were

shaked overnight at 37 -C to remove microglia and

oligodendrocytes. Astrocytes were collected by trypsin

digestion, seeded onto 35 or 100 mm dishes and used for

experiments 6–8 days after re-plating.

2.4. Handling of bAP

Different lots of hAP25–35 were tested and the same

batch was used throughout the entire study to rely on a

consistent profile of toxicity. Peptides were solubilized in

sterile, double distilled water at an initial concentration of

2.5 mM, and stored frozen at �20 -C. hAP(25–35) was usedat a final concentration of 25 AM in the presence of the

glutamate receptor antagonists MK-801 (10 AM) and

DNQX (30 AM) to avoid the potentiation of endogenous

glutamate toxicity.

2.5. MTT assay

Cells were incubated with MTT (0.9 mg/ml final

concentration, Sigma) for 2 h at 37 -C. A solubilization

solution containing 20% SDS was then added for an

additional 1 h, and formazan production was evaluated in

a plate reader (absorbance = 560 nm).

F. Caraci et al. / Brain Research 1047 (2005) 30–3732

2.6. Evaluation of apoptotic neuronal death

Apoptotic death was assessed by FACS analysis of

prediploid DNA. Mature cortical neurons at 7–8 DIV were

harvested with 0.25% trypsin, fixed in 70% ethanol, and

stored at �20 -C until use. After repeated washing, cells

were incubated for 1 h with 100 Ag/ml RNase and stained

with 50 Ag/ml propidium iodide. DNA content and ploidy

was assessed by a Coulter Elite Flow Cytometer. The

prediploid peak was evaluated as an index of apoptosis.

2.7. Western blot analysis

Cells were harvested in lysis buffer containing a cocktail

of protease inhibitors including bestatin, aprotinin, leupep-

tin, and sodium EDTA. After sonication, an aliquot of the

sample was processed for protein concentrations according

to the method of Bradford. Samples were concentrated and

boiled for 5 min. Proteins were separated electrophoretically

on polyacrylamide gel (30 mA/h) using 60–80 Ag of cell

proteins per lane. Proteins were transferred to nitrocellulose

membranes (Hybond ECL, Amersham Biosciences Europe

GmbH, Milan, Italy) at room temperature using a transblot

semidry transfer cell. After blocking, the membranes were

incubated with rabbit anti-Bax or anti-Bcl-2 (1:200, Santa

Cruz Bioctechnology, Santa Cruz, CA) overnight at 4 -C.Membranes were then thoroughly washed and incubated

with HRP-conjugated secondary antibodies. Specific bands

were visualized using the SuperSignal chemiluminescent

detection system (Pierce Biotechnology, Rockford, IL).

2.8. RT-PCR

Astrocytes were treated with 10 AM nicergoline for 18 h.

Total RNA was extracted with the Tryzol reagent (Sigma).

Two micrograms of total RNA was used for cDNA

synthesis, using oligo(dT) primers and 10 mM deoxynu-

cleotide triphosphate mix (Invitrogen) and M-MLV Reverse

Transcriptase (RT, 200 U/reaction; Promega, Madison, WI)

at 37 -C for 50 min. Two microliters of cDNA was used in

each subsequent PCR amplification, in an automatic

thermocycler, using 2.5 U/reaction of Platinum Taq DNA

polymerase and corresponding primers. Reaction conditions

were 95 -C for 5 min followed by 33 cycles of 95 -C, 56 -C,and 72 -C each for 1 min. The final extension step was 7

min at 72 -C. PCR product were analyzed electrophoreti-

cally on a 1.8% agarose gel run in Tris–acetate/EDTA

buffer.

Primers used were as follows: TGF-h1 (f 5V-TGGACCG-CAACAACGCCATCTATGAGAAAACC-3V; r 5V-TGGAG-CTGAAGCAATAGTTGGTATCCAGGGCT-3V); IL-6(f 5V-CAAGAGACTTCCAGCCAGTTGC-3V; r 5V-TTGCCGAG-TAGACCTCATAGTGACC-3V); GDNF (f 5V-ATGAAGTT-ATGGGATGTCGTGGCTG-3V; r 5V-CCTTTTACGCGGA-ATGCTTTCTTAG-3V); GAPDH (f 5V-GCCGCCTGGTCAC-CAGGGCTG-3V; r 5V-ATGGACTGTGGTCATGAGCCC-3V).

3. Results

Initially, we examined the neuroprotective activity of

nicergoline against hAP toxicity using pure cultures of rat

cortical neurons, which were virtually devoid of astrocytes

or other contaminating cells [11]. Cultures were treated

with hAP25–35 in the presence of ionotropic glutamate

receptor antagonists (1 AM MK-801 + 10 AM DNQX) to

limit endogenous excitotoxicity. Under these conditions,

neurons exposed to hAP die showing an apoptotic

phenotype [26]. Neurotoxicity was assessed by combining

the MTT assay and FACS analysis of pre-diploid DNA.

Exposure to hAP25–35 (25 AM) was toxic in a time-

dependent manner, inducing apoptotic death in about 40–

50% of neuronal population at 24 h, and 60–70% at 48 h.

All experiments with nicergoline were carried out in

cultures exposed to hAP for 24 h. Nicergoline co-applied

with hAP reduced neuronal toxicity in a concentration-

dependent fashion and was maximally protective (about

60% of neuronal rescue) at 10 AM (Figs. 1a and b).

Nicergoline alone did not affect neuronal viability in a

concentration range of up to 10 AM (not shown). We

compared the action of nicergoline to that of its major

metabolites, MDL and MMDL, which contribute to the

overall therapeutic efficacy of nicergoline in humans [1].

MDL, applied at equimolar concentrations, mimicked the

neuroprotective action of nicergoline, whereas MMDL was

virtually inactive (Figs. 1c and d). As nicergoline is known

to interact with serotonin 5-HT2 and a-1 adrenergic

receptors, specific antagonist were tested to rule out the

possibility that the protective effect of nicergoline was due

to its interaction with these receptors. However, the mixed

5-HT2 receptor antagonist, methysergide (0.1 AM), and the

selective a1-receptor antagonist, prazosin (1 AM), did not

affect hAP toxicity (Fig. 1c).

When applied to cortical neurons challenged with the

apoptotic agent staurosporine (2 AM for 24 h) nicergoline

failed to exert any neuroprotective activity (Table 1).

Searching for a molecular correlate of neuroprotection, we

examined the levels of the pro-apoptotic protein Bax by

immunoblotting. As expected [9,12], a 24-h exposure to

hAP increased the intracellular levels of Bax more than 3-

fold in cortical neurons. This effect was substantially

reduced in cultures treated with 5 AM nicergoline or MDL

(Fig. 2a), whereas MMDL was inactive (not shown).

Neither nicergoline nor its metabolites induced changes in

Bax levels when applied alone (not shown). In contrast,

nicergoline alone increased the expression of the anti-

apoptotic protein Bcl-2 after 24 h of exposure. This is

consistent with the evidence that nicergoline activates

protein kinase C (PKC) in brain tissue [6], and that activated

PKC induces the expression of the anti-apoptotic protein,

Bcl-2 [25]. Exposure to hAP for 24 h induced a 50%

reduction in Bcl-2 levels. When hAP was combined with

nicergoline, Bcl-2 expression was still higher than in

untreated cultures (Fig. 2b).

Fig. 1. Protection by nicergoline and its metabolite MDL against h-amyloid toxicity in pure cultures of cortical neurons is shown by the MTT assay (a and c) or

by cytofluorometric analysis of pre-diploid DNA (b and d). In b, nicer = nicergoline; In c, nicergoline (nicer), MDL, and MMDL were applied at concentrations

of 5 AM; prazosin (praz) = 1 AM; methysergide (met) = 0.1 AM. Cultures were exposed to hAP25 – 35 for 24 h. Values are expressed as percent of h-amyloid

toxicity in panels a and c, or as percent of apoptotic neurons (neurons with pre-diploid DNA) in panels b and d. Values were calculated from 6 to 9

determinations from 2 to 3 independent experiments. *P < 0.05 (one-way ANOVA + Fisher’s PLSD) vs. hAP alone.

F. Caraci et al. / Brain Research 1047 (2005) 30–37 33

We extended the study to mixed cultures of cortical cells

containing both neurons and astrocytes, as detected by

immunocytochemistry with antibodies for the neuronal

marker, MAP-2, and for the astrocyte marker, GFAP (not

shown). Here, hAP toxicity was assessed by counting the

neurons stained with trypan blue. Using this model, neuro-

toxicity showed a slower kinetics, with a substantial neuronal

death (about 60%) being detected after 72 h of exposure to

hAP. Nicergoline (co-applied with hAP and maintained in

the medium for the following 72 h) completely prevented

Table 1

Effect of nicergoline on neuronal death induced by a 24 h-exposure to 2 AMstaurosporine

Neuronal survival (% of control)

Control 100.0 T 2.8

Staurosporine 71.5 T 4.2*

Nicergoline 102.2 T 4.7

Staurosporine + nicergoline 62.6 T 3.7*

Pure cortical cultures were treated with 5 AM nicergoline and 2 AMstaurosporine for 24 h prior to evaluation of neuronal viability by the MTT

assay.

Values are mean T SE of 8 values obtained in two independent experiments.

* P < 0.01 vs. respective control.

hAP neurotoxicity even at concentrations (3 AM) that

showed only a partial protection in pure neuronal cultures

(Fig. 3). The action of nicergoline was mimicked by MDL,

whereas MMDL showed only a partial neuroprotective

activity (Fig. 3). This suggested that the presence of

astrocytes enabled a full neuroprotective activity of nicer-

goline and MDL. To assess whether nicergoline could have a

direct action on glial cells, we treated pure cultures of rat

cortical astrocytes (GFAP+ cells > 95%) with nicergoline or

its metabolites, and the collected medium was transferred to

cultures of pure cortical neurons challenged with hAP.Experiments were carried out as follows: glial cultures were

exposed to nicergoline, MDL, or MMDL for 24 or 72 h. At

the end of this treatment the medium was replaced with a

fresh medium lacking nicergoline or metabolites, and this

new medium was collected after additional 24 h and

transferred to pure cultures of cortical neurons. This strategy

was adopted to minimize the amount of nicergoline (or

metabolites) present in the glial medium. Addition of

conditioned medium from untreated glial cultures did not

affect hAP toxicity in cultured cortical neurons. Similar

results were obtained by transferring the medium collected

from glial cultures 24 h after a 24-h exposure to nicergoline.

Fig. 2. Western blot analysis of Bax (a) and Bcl-2 (b) in pure cultures of cortical neurons exposed to hAP25 – 35 and/or nicergoline (nicer) for 24 h. In panel a theeffect of MDL on hAP-increased Bax levels is also shown. Nicergoline and MDL were applied at concentrations of 5 AM. Values of densitometric analysis are

means T SEM of 3 determinations. *P < 0.05 (one-way ANOVA + Fisher’s PLSD) vs. control and *P < 0.05 vs. hAP alone.

F. Caraci et al. / Brain Research 1047 (2005) 30–3734

In contrast, the medium collected 24 h after a 72-h exposure

to nicergoline or MDL (but not MMDL) was highly

neuroprotective (Figs. 4a and b), suggesting that nicergoline

and MDL induced a slow release of a neurotrophic/neuro-

protective factor into the glial medium. We focused on

transforming-growth factor-h1 (TGF-h1) because this factoris known to protect neurons against hAP toxicity [11,34]. We

found that cultured astrocytes treated with nicergoline

showed a transient increase in the intracellular levels of

TGF-h1, which was detectable at 24 h of exposure by

cytofluorometric assessment of intracellular TGF-h1 protein

(Fig. 4c). These data were supported by an increase of TGF-

h1 mRNA expression by RT-PCR analysis (Fig. 4d).

Interestingly, glia-derived neurotrophic factor (GDNF)

Fig. 3. Nicergoline and MDL protect mixed cultures of cortical cells

against h-amyloid toxicity. Cultures were exposed to hAP25 – 35 alone or

combined with nicergoline (nicer, 3 AM), MDL (3 AM), or MMDL (3 AM)

for 72 h. Neuronal toxicity was assessed by trypan blue staining. Values

are means T SEM of 6 determinations from 2 independent experiments.

*P < 0.01 (one-way ANOVA + Fisher’s PLSD) vs. hAP alone.

expression in response to nicergoline exhibited a profile

very similar to that of TGF-h1 as by flow cytometry and RT-

PCR (Figs. 4c and d). In contrast, the levels of another

putative neurotrophic agent, IL-6, were not modified by

nicergoline treatment (Figs. 4c and d).

4. Discussion

Multiple mechanisms may account for the beneficial

effects of nicergoline in AD, including an improvement of

brain metabolism [24,28,31,37] and an enhancement of

cholinergic transmission [7]. Our results provide the first

evidence that nicergoline protects neurons against hAPtoxicity, a process that may contribute to the pathophysiology

of AD [20]. At least a component of neuroprotection was

mediated by a direct action of nicergoline on neurons, as

shown by the experiments carried out in pure neuronal

cultures. Neuroprotection by nicergoline cannot be recon-

ducted to its classical interaction witha1-adrenergic [21] or 5-

HT2 serotonergic [19] receptors, because prazosin and

methysergide were inactive against hAP toxicity. This is

noteworthy because pharmacological blockade – or the

absence – of a1-adrenergic receptors is protective in other

models, including methamphetamine neurotoxicity in mice

[3]. The finding that nicergoline affected both constitutive

and hAP-reduced Bcl-2 levels and decreased the expression

of Bax in hAP-treated neurons suggests that the drug

interferes with the execution of the apoptotic program. This

is particularly relevant to our model because cultured cortical

neurons exposed to hAP combined with a cocktail of

ionotropic glutamate receptor antagonists show an apoptotic

phenotype [11,12,26]. Markers of apoptotic death are also

found in degenerating neurons of the AD brain [32,35]. Other

Fig. 4. (a and b) The conditioned medium collected from cultured astrocytes exposed to nicergoline or MDL for >24 h protects cultured neurons against

h-amyloid toxicity. Cultured astrocytes were treated with drugs for 24–96 h and then extensively washed. The medium was collected 24 h after drug

withdrawal. Values are means T SEM of 6–9 determinations from 2 to 3 independent experiments. *P < 0.05 (one-way ANOVA + Fisher’s PLSD) vs. hAPalone. Intracellular levels of TGF-h1, GDNF, or IL-6 in cultured astrocytes exposed to 5 AM nicergoline are shown in panel c, where values are means T SEM

of 3 determinations. RT-PCR of TGF-h1, GDNF, and IL-6 in cultured astrocytes treated with hAP for 24 h is shown in panel d. *P < 0.05 (one-way ANOVA +

Fisher’s PLSD) vs. control values.

F. Caraci et al. / Brain Research 1047 (2005) 30–37 35

studies have shown a protective role of nicergoline against

apoptosis in PC12 cells deprived of nerve growth factor [5],

GT1-7 cells depleted of glutathione [33], or B50 cells

challenged with hydrogen peroxide [23]. An antioxidant

activity of nicergoline may contribute to its protective effects

because perturbation of Ca2+ homeostasis and oxidative

stress are central to the neurotoxic cascade induced by hAP inneurons [2,18,27]. Nicergoline is known to inhibit lipid

peroxidation and free radical generation and reduces auto-

oxidation processes in brain tissue homogenates [36].

However, the evidence that both MDL and MMDL share

the antioxidant properties of nicergoline [38] – whereas only

MDL was substantially active in our model – suggests that

additional mechanisms are involved in the neuroprotective

activity of nicergoline against hAP toxicity. Among these,

activation of PKC [10] might be particularly relevant because

PKC iota protects neural cells against apoptosis induced by

hAP [39], and activated PKC enhances Bcl-2 expression in

neuroblastoma cells [25]. Studies with isoform-selective

inhibitors of PKC are necessary to address this issue. We

were particularly intrigued by the finding that nicergoline

showed a higher neuroprotective activity in mixed cortical

cultures, and that the medium collected from cultured

astrocytes exposed to nicergoline or MDL was protective

when transferred to cultured neurons challenged with hAP. Itis unlikely that the conditioned glial medium contained an

amount of nicergoline sufficient to account for neuroprotec-

tion because (i) astrocytes were extensively washed 24 h

before collecting the medium, and (ii) we observed protection

when astrocytes were exposed to nicergoline for 72 h, but not

for 24 h, although nicergoline levels in the medium are

expected to be higher after 24 than 72 h. The long exposure

time required for the protective activity of the glial medium

suggested that nicergoline or MDL induced the synthesis and

release of neurotrophic/neuroprotective factors in cultured

astrocytes. We focused on TGF-h and GDNF because both

factors are known to protect neurons against hAP toxicity

[11,16,29,34] and activation of glial metabotropic glutamate

receptors protects neighbor neurons against excitotoxic death

through the formation of TGF-h1 and -h2 [4,14]. Exposure tonicergoline was found to increase the intracellular levels of

both TGF-h1 and GDNF. In our experience [4,14,34] an

increase in intracellular levels of at least TGF-h is always

associated with its secretion in cultured astrocytes. We could

F. Caraci et al. / Brain Research 1047 (2005) 30–3736

not detect extracellular TGF-h or GDNF because levels were

too low to be quantified by ELISA. Although the relative

contribution of TGF-h or GDNF to the overall neuro-

protection remains to be determined, this finding suggests

an entirely novel mechanism of action of nicergoline based

on glial–neuronal interaction. Interestingly, an increased

production of TGF-h1 in astrocytes has recently been

implicated in the neuroprotective activity of h-estradiolagainst hAP toxicity [34]. More intriguing is the recent

observation that GDNF protects neurons against hAP by

reducing the activation of ERK kinases [16], an intracellular

pathway that we have shown is involved in the induction of

cell cycle by hAP [15].

In conclusion, the evidence that nicergoline protects

neurons against hAP toxicity raises the interesting possi-

bility that the drug slows the progression of AD by

limiting the death of neurons surrounded by amyloid

deposits.

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