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Protective action of idebenone against excitotoxic degeneration in cultured cortical neurons
ELSEVIER
Neuroscience Letters 178 (1994) 193-196
HEUROSCIENCE LETTERS
Protective action of idebenone against excitotoxic degeneration in cultured cortical neurons Valeria Bruno a, Giuseppe Battaglia a, Agata Copani a, Maria Angela Sortino a, Pier Luigi Canonico b, Ferdinando Nicoletti c'* alnstitute of Pharmacology, School of Medicine, University of Catania, Catania, Italy bChair of Pharmacology, School of Dentistry, University of Pavia, Pavia, Italy 'Department of Experimental Medicine and Bioehemical Sciences, Pharmacology Section, University of Perugia, Perugia, Italy Received 11 May 1994; Revised version received 11 July 1994; Accepted 11 July 1994
Abstract
The novel free radical scavenger and electron-trapping agent, idebenone, protects cultured cortical neurons against necrotic degeneration induced by either a brief exposure to N-methyl-o-aspartate (NMDA) or a prolonged exposure to kainate. As opposed to the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-iminehydrogen maleate (MK801), idebenone rescued cortical neurons even when applied 30 min after the NMDA pulse, suggesting that the drug interferes with the chain of toxic reactions triggered by an excessive stimulation of excitatory amino acid receptors. Key words: NMDA; Kainate; Excitotoxicity; Idebenone; Free radical; Cortical culture
An excessive stimulation of excitatory amino acid (EAA) receptors has been implicated in the pathophysiology of neuronal degeneration in a variety of acute and chronic degenerative disorders [3,17,10]. The toxic action of EAAs is mediated by an enhanced infux of extracellular Ca 2÷ which ultimately leads to neuronal degeneration as a result of a sustained activation of Ca2+-dependent enzymes [4]. Formation of reactive oxygen species is generally considered as a common final path in the intracellular mechanisms responsible for excitotoxic neuronal degeneration [15]. Hydroxyl radicals represent the most dangerous radical species, i.e., they react with high speed with the majority of intracellular molecules, including DNA, carbohydrates and membrane lipids [6]. Free radical scavengers or chain-breaking antioxidant inhibitors of lipid peroxidation have been widely used to prevent or attenuate the toxic action of free radicals [12]. However, some of the most common antioxidants, including
*Corresponding author. Address: Institute of Pharmacology, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Fax: (39) (95) 333219. 0304-3940194l$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 5 4 7 - 8
ascorbic acid, several flavonoids and a-tocopherol, can reduce Fe 3+ into Fe 2+, thus, increasing hydroxyl radical formation from hydrogen peroxide by the Fenton's reaction [6]. Through these mechanisms, free radical scavengers may paradoxically exacerbate free radical reactions. New classes of drugs that attenuate free radical-mediated cell damage include iron chelators, recombinant superoxide dismutase, spin and electron-trapping agents. The latter are able to trap the electrons that are anomalously transferred from the mitochondrial respiratory chain (in particular from coenzyme Q and cytochrome b566) to oxygen under pathological conditions. The use of electron-trapping agents is promising in Alzheimer's disease where it has been suggested that a reduced activity of cytochrome oxidase contributes to free radical production and, therefore, to the pathophysiology of neuronal damage [7]. 6-(10-Hydroxydecyl)-2,3-dimetoxy-5-methyl1,4-benzoquinone (idebenone) is a new neuroprotective agent that acts both as a radical scavenger and electrontrapping agent [13,16]. We now report that idebenone is extremely potent in protecting cultured cortical neurons against excitotoxininduced degeneration, supporting its potential therapeu-
194
I~ Brum~ cl a/. I N e u r o s c i c n c c l, ettcr.s 17,~' ~ 1~94) 193 196
A
B
120
E ~
100
o
80
•~
60
-o
.~
40
2o
// -
log M [idebenone]
Fig. 1. Idebenone protects cortical cultures against NMDA-induced toxicity. A: phase-contrast microscopy (200x magnification) of cortical neurons exposed to N M D A lbr 10 min in the presence or absence of 10 ,uM idebenone, a: control; b: N M D A 100 ,uM; and c: N M D A + idebenone. Photomicrographs were taken 24 h after the N M D A pulse. Bar = 33 ,urn. B: concentration-protection relationship for idebenone: cortical cultures were exposed to 100 ,uM N M D A for 10 min after 30 min pretreatment with the indicated concentrations of idebenone. Trypan blue-staining was performed 24 h later. Data represent the number of cells stained with Trypan blue and are expressed as % of N M D A - i n d u c e d neuronal degeneration (mean _+ S.E.M., n = 3). All the values obtained in the presence of idebenone were significantly different from N M D A alone (P < 0.05 by Fisher PLSD test). Identical results were obtained by measuring L D H activity released from damaged neurons in the incubation medium. L D H values (mean _+ S.E.M., n = 6 ) : basal 35_+9; N M D A 500 /,tM, 530_+22: N M D A 100,uM, 230 + 16; N M D A 100 ,uM + idebenone l0 ,uM, 66 _+ 7; idebenonc I 0 / t M , 40 -+ 7.
tical use in acute and chronic neurodegenerative disorders. Cell culture. Murine cortical primary cultures containing both neurons and astroglia were prepared as described by Choi et al. [2]. In brief, cortical cells were
obtained by 14-day-old mouse embryos, dissociated by 0.09% trypsme and plated in MEM~ containing 25 mM HCO~, 21 mM glucose, 2 mM glutamine, 5% horse serum and 5% fetal calf serum onto a monolayer of astrocytes grown in 24 multiwell plates [14]. Cytosine-flarabinofuranoside (Ara-C, 10/~M) has been added after 3 5 days in vitro to prevent glial proliferation over the monolayer. After additional 3 days, cultures were shifted in the same medium containing 10% horse serum without fetal calf serum. Cultures at 13 14 days in vitro were used in all experiments. Estimation (~f slow and fast excitotoxicity. N-methyl-Daspartate (NMDA) toxicity was carried out by incubating the cultures for 10 rain ('fast toxicity') at room temperature in HEPES-buffered balanced salt solution containing submaximal concentrations of N M D A (100/~M). ldebenone was added to the cultures for 30 min and then washed out before the addition of the excitotoxin. At the end of the N M D A pulse, cultures were returned to the medium lacking serum and glutamine (medium stock) and toxicity was evaluated 24 h later. Kainate toxicity was carried out by incubating cultures with submaximal concentrations of kainate (60 ~M) in the absence or presence of idebenone for 24 h in the incubator ('slow toxicity') in medium stock containing 10/,tM MK-801 to prevent any secondary activation of N M D A receptors. Overall neuronal cell injury was estimated by phase-contrast microscopy, Trypan blue exclusion or by measuring lactate dehydrogenase (LDH) activity released by damaged neurons in the incubation medium. Materials, N M D A and kainate were purchased from Sigma (St Louis, MO); MK801 was obtained from RBI (Natick, MA) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) from Tocris Neuramin (Bristol, UK). N M D A (10-rain exposure) and kainate (24-h exposure) induced necrotic degeneration of cultured cortical neurons in a concentration-dependent fashion, with apparent EC~,, values of 100 ~M for N M D A and 60/~M l\)r kainate, and maximal neuronal damage at concentra-
195
V. Bruno et al./Neuroscience Letters 178 (1994) 193-196
tions of higher than 150 and 100 gM, respectively (see legends of Figs. 1 and 2). The toxic actions of NMDA and kainate were totally prevented by MK-801 (10 gM) (not shown) and DNQX, respectively (30 gM) (Fig. 2). Idebenone was highly potent (IC5o = 300 nM) in protecting cultured cortical neurons against degeneration induced by both NMDA and kainate (Fig. 1). When applied alone for 30 min (in the paradigm of fast toxicity), idebenone did not influence either the morphology or the release of LDH at all concentrations tested (up to 100 /.tM, see legend of Fig. 1). However, the highest concentration of idebenone tested (100/zM) induced neuronal degeneration over a 24-h period of exposure (see legend of Fig. 2). Idebenone was still neuroprotective when applied 30 min after the NMDA pulse, suggesting that the drug interferes with the cascade of intracellular reactions triggered by NMDA receptor activation (Table 1). Excessive activation of NMDA or kainate receptors has long been known to induce necrotic degeneration of vulnerable neurons either in vivo or in culture (for a review, see Ref. 3). The role of excitotoxic degeneration in the pathophysiology of acute and chronic degenerative disorders has encouraged the search for neuroprotective agents which limit the toxic effects of EAA receptor activation. N M D A receptor antagonists have been found to protect vulnerable neurons in models of focal ischemia, providing that they are administered before or immediately after the ischemic period [1]. Receptor antagonists, however, often induce psychotomimetic side effects and hamper the physiological activation of NMDA receptors which contributes to the regulation of synaptic plasticity [9]. For these reasons, drugs that act downstream the receptor level have a great potential for clinical use. Formation of free radicals has been associated to an excessive stimulation of NMDA or kainate receptors [8] and antioxidants, such as ubiquinone, or 21-amino steroids are known to rescue cultured neurons from excitotoxic damage [5,11]. Idebenone acts both as free radical scavenger and electron-trapping agent [13,16]. To test the neuroprotective action of idebenone, we have used cultured cortical neurons grown on a monTable 1 Delayed rescue of N M D A - i n d u c e d toxicity by idebenone % n e u r o n a l death N M D A 100/.tM + idebenone 10 g M (30 min) + MK801 10 g M (30 min) + idebenone 10/,tM (2 h) + MK801 1 0 g M (2 h)
100 + 7.9 58.7 + 4.5* 7 9 + 14 78.1 + 7.3 83+8
Cortical cultures were exposed to N M D A for 10 rain; 30 rain and 2 h after the N M D A wash-out, cultures were rescued with the indicated concentration of idebenone or MK801. Trypan blue staining was performed 24 h later. Values represent m e a n + SEM, n = 3, scaled to the value present in the cultures exposed to N M D A without rescue (= 100). *P < 0.05 by Fisher P L S D test.
120 I00 80 ~
6O
Tt. ,~.Zt .,efj f.g'j ff/ ,¢ .,¢ /
U
•~
40
f i t
/.¢/ [// i l l
2O
f// ///!
S
f// f//
r/'/
0 KA 6 0 laM
+100nM +l/aM +I0~aM -- Idebenone --
+ DNQX 30 pM
Fig. 2. Kainate toxicity is prevented in a concentration-dependent manner by idebenone. Cortical cultures were exposed to kainate for 24 h in the presence or absence of different concentrations of idebenone or to 3 0 / t M D N Q X . Concentrations o f idebenone of higher than 10 g M were toxic when cultures were exposed for 24 h even in the absence of kainate. Data represent mean _+ S.E.M., n = 3. *P < 0.05 by Fisher PLSD test. L D H values (mean + S.E.M., n = 6): basal 54 + 8; K A 300 ~ M , 6 0 0 + 1 8 ; K A 60ktM, 3 6 0 + 3 5 ; K A 6 0 g M + M K 8 0 1 10/~M, 2 4 0 + 15; K A 60 / I M + M K 8 0 1 10 / . t M + D N Q X 30 /tM, 8 0 + 9 ; K A 6 0 / t M + MK801 10 g M + idebenone 10 ,uM, 65 + 6.
olayer of astrocytes, a model that has been extensively characterized in the past. Idebenone displayed a great potency in protecting cultured cortical neurons against the toxic action of NMDA or kainate and rescued cortical neurons even when applied 30 min after the NMDA pulse, suggesting that free radicals are still produced or still reactive at this time after receptor activation. This suggests to search for a late neuroprotective activity of idebenone in experimental models of ischemia where NMDA receptor antagonists are mostly active when administered before the induction of ischemia or immediately after the onset of postischemic recirculation [1]. Idebenone may attenuate the chronic degeneration of vulnerable neurons in Alzheimer's patients by limiting the consequences of NMDA receptors activation (as suggested by the present study) and by reducing the anomalous flow of electrons from the mitochondrial respiratory chain by virtue of its electron-trapping activity. Idebenone was kindly provided by Takeda Italia Farmaceutici. [1] Albers, G.W., Goldberg, M.P. and Choi, D.W., Do N M D A antagonists prevent neuronal injury? Yes, Arch. Neurol., 49 (1992) 418420.
196
V. Bruno et al. / Neuroseience Letters 178 (1994) 193 196
[2] Choi, D.W., Maulucci-Gedde, M.A. and Kriegstein, A.R., Glutamate neurotoxicity in cortical cell culture, J. Neurosci., 7 (1987) 357-368. [3] Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623 634. [4] Choi, D.W., Excitotoxic cell death, J. Neurobiol., 23 (1992) 1261 t276 [5] Favit, A., Nicoletti, F., Scapagnini, U. and Canonico, EL., Ubiquinone protects cultured neurons against spontaneous or excitotoxin-induced degeneration, J. Cer. Blood Flow Metaboh, 12 (1992) 638 -645. [6] Halliwell, B., Reactive oxygen species and the central nervous system, J. Neurochem., 59 (1992) 1609 1623. [7] Kish, S.J., Bergeron, C., Rajput, A. et ah, Brain cytochrome oxidase in Alzheimer's disease, J. Neurochem., 59 (1992) 776 779. [8] Lafon-Cazal, M., Pietri, S., Culcasi, M. and Bockaert, J., NMDAdependent superoxide production and neurotoxicity, Nature (London), 364 (1993) 535 537. [9] Lipton, S.A., Prospects for clinically tolerated NMDA antagonists: open-channel blockers and alternative redox states of nitric oxide, Trends Neurosci., 16 (1993) 527 532. [10] Maragos, W.F., Greenamyre, J.T., Penney, J.B. and Young, A.B., Glutamate dysfunction in Alzheimer's disease: an hypothesis. Trends Neurosci., 10 (1987) 65 68.
[ll] Monyer, H., Hartley, D.M. and Choi, D.W., 21-Aminosteroids attenuate excitotoxic neuronal injury in cortical cell cultures, Neuron, 5 (1990) 121 126, [12] Miyamoto, M., Murphy, T.H,, Schnaar, R.L. and Coyle, J.T.. Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line, J. Pharmacol. Exp. Ther., 260 (1989) 1132 1140. [13] Miyamoto, M. and Coyle, J.T., Idebenone attenuates neuronal degeneration induced by intrastriatal injection of excitotoxins, Exp. Neurol., 108 (1990) 3845. [14] Rose, K., Goldberg, M.P. and Choi, D.W., Cytotoxicity in murine neocortical cell culture. In, Methods in Toxicology, Vol. 1, Academic Press, New York, NY, 1993, pp.46-60. [15] Siesjo, B.K., Free radicals and brain damage, Cerebrovasc. Brain Metab. Rev., 1 (1989) 165 211. [16] Suno, M. and Nagaoka, A., Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition, Biochem. Biophys. Res. Commun., 125 (1984) 1046 1052. [17] Young, A.B., Greenamare, J.T., Hollingsworth, Z., Albin, R., D'Amato, C., Shoulson, I. and Penney, J.B., NMDA receptor losses in putamen from patients with Huntington's disease, Science, 241 (1988) 98l 983.
Neuroscience Letters 178 (1994) 193-196
HEUROSCIENCE LETTERS
Protective action of idebenone against excitotoxic degeneration in cultured cortical neurons Valeria Bruno a, Giuseppe Battaglia a, Agata Copani a, Maria Angela Sortino a, Pier Luigi Canonico b, Ferdinando Nicoletti c'* alnstitute of Pharmacology, School of Medicine, University of Catania, Catania, Italy bChair of Pharmacology, School of Dentistry, University of Pavia, Pavia, Italy 'Department of Experimental Medicine and Bioehemical Sciences, Pharmacology Section, University of Perugia, Perugia, Italy Received 11 May 1994; Revised version received 11 July 1994; Accepted 11 July 1994
Abstract
The novel free radical scavenger and electron-trapping agent, idebenone, protects cultured cortical neurons against necrotic degeneration induced by either a brief exposure to N-methyl-o-aspartate (NMDA) or a prolonged exposure to kainate. As opposed to the NMDA receptor antagonist (+)-5-methyl-10,11-dihydro-5H-dibenzo[a,d]-cyclohepten-5,10-iminehydrogen maleate (MK801), idebenone rescued cortical neurons even when applied 30 min after the NMDA pulse, suggesting that the drug interferes with the chain of toxic reactions triggered by an excessive stimulation of excitatory amino acid receptors. Key words: NMDA; Kainate; Excitotoxicity; Idebenone; Free radical; Cortical culture
An excessive stimulation of excitatory amino acid (EAA) receptors has been implicated in the pathophysiology of neuronal degeneration in a variety of acute and chronic degenerative disorders [3,17,10]. The toxic action of EAAs is mediated by an enhanced infux of extracellular Ca 2÷ which ultimately leads to neuronal degeneration as a result of a sustained activation of Ca2+-dependent enzymes [4]. Formation of reactive oxygen species is generally considered as a common final path in the intracellular mechanisms responsible for excitotoxic neuronal degeneration [15]. Hydroxyl radicals represent the most dangerous radical species, i.e., they react with high speed with the majority of intracellular molecules, including DNA, carbohydrates and membrane lipids [6]. Free radical scavengers or chain-breaking antioxidant inhibitors of lipid peroxidation have been widely used to prevent or attenuate the toxic action of free radicals [12]. However, some of the most common antioxidants, including
*Corresponding author. Address: Institute of Pharmacology, University of Catania, Viale A. Doria 6, 95125 Catania, Italy. Fax: (39) (95) 333219. 0304-3940194l$7.00 © 1994 Elsevier Science Ireland Ltd. All rights reserved SSD1 0 3 0 4 - 3 9 4 0 ( 9 4 ) 0 0 5 4 7 - 8
ascorbic acid, several flavonoids and a-tocopherol, can reduce Fe 3+ into Fe 2+, thus, increasing hydroxyl radical formation from hydrogen peroxide by the Fenton's reaction [6]. Through these mechanisms, free radical scavengers may paradoxically exacerbate free radical reactions. New classes of drugs that attenuate free radical-mediated cell damage include iron chelators, recombinant superoxide dismutase, spin and electron-trapping agents. The latter are able to trap the electrons that are anomalously transferred from the mitochondrial respiratory chain (in particular from coenzyme Q and cytochrome b566) to oxygen under pathological conditions. The use of electron-trapping agents is promising in Alzheimer's disease where it has been suggested that a reduced activity of cytochrome oxidase contributes to free radical production and, therefore, to the pathophysiology of neuronal damage [7]. 6-(10-Hydroxydecyl)-2,3-dimetoxy-5-methyl1,4-benzoquinone (idebenone) is a new neuroprotective agent that acts both as a radical scavenger and electrontrapping agent [13,16]. We now report that idebenone is extremely potent in protecting cultured cortical neurons against excitotoxininduced degeneration, supporting its potential therapeu-
194
I~ Brum~ cl a/. I N e u r o s c i c n c c l, ettcr.s 17,~' ~ 1~94) 193 196
A
B
120
E ~
100
o
80
•~
60
-o
.~
40
2o
// -
log M [idebenone]
Fig. 1. Idebenone protects cortical cultures against NMDA-induced toxicity. A: phase-contrast microscopy (200x magnification) of cortical neurons exposed to N M D A lbr 10 min in the presence or absence of 10 ,uM idebenone, a: control; b: N M D A 100 ,uM; and c: N M D A + idebenone. Photomicrographs were taken 24 h after the N M D A pulse. Bar = 33 ,urn. B: concentration-protection relationship for idebenone: cortical cultures were exposed to 100 ,uM N M D A for 10 min after 30 min pretreatment with the indicated concentrations of idebenone. Trypan blue-staining was performed 24 h later. Data represent the number of cells stained with Trypan blue and are expressed as % of N M D A - i n d u c e d neuronal degeneration (mean _+ S.E.M., n = 3). All the values obtained in the presence of idebenone were significantly different from N M D A alone (P < 0.05 by Fisher PLSD test). Identical results were obtained by measuring L D H activity released from damaged neurons in the incubation medium. L D H values (mean _+ S.E.M., n = 6 ) : basal 35_+9; N M D A 500 /,tM, 530_+22: N M D A 100,uM, 230 + 16; N M D A 100 ,uM + idebenone l0 ,uM, 66 _+ 7; idebenonc I 0 / t M , 40 -+ 7.
tical use in acute and chronic neurodegenerative disorders. Cell culture. Murine cortical primary cultures containing both neurons and astroglia were prepared as described by Choi et al. [2]. In brief, cortical cells were
obtained by 14-day-old mouse embryos, dissociated by 0.09% trypsme and plated in MEM~ containing 25 mM HCO~, 21 mM glucose, 2 mM glutamine, 5% horse serum and 5% fetal calf serum onto a monolayer of astrocytes grown in 24 multiwell plates [14]. Cytosine-flarabinofuranoside (Ara-C, 10/~M) has been added after 3 5 days in vitro to prevent glial proliferation over the monolayer. After additional 3 days, cultures were shifted in the same medium containing 10% horse serum without fetal calf serum. Cultures at 13 14 days in vitro were used in all experiments. Estimation (~f slow and fast excitotoxicity. N-methyl-Daspartate (NMDA) toxicity was carried out by incubating the cultures for 10 rain ('fast toxicity') at room temperature in HEPES-buffered balanced salt solution containing submaximal concentrations of N M D A (100/~M). ldebenone was added to the cultures for 30 min and then washed out before the addition of the excitotoxin. At the end of the N M D A pulse, cultures were returned to the medium lacking serum and glutamine (medium stock) and toxicity was evaluated 24 h later. Kainate toxicity was carried out by incubating cultures with submaximal concentrations of kainate (60 ~M) in the absence or presence of idebenone for 24 h in the incubator ('slow toxicity') in medium stock containing 10/,tM MK-801 to prevent any secondary activation of N M D A receptors. Overall neuronal cell injury was estimated by phase-contrast microscopy, Trypan blue exclusion or by measuring lactate dehydrogenase (LDH) activity released by damaged neurons in the incubation medium. Materials, N M D A and kainate were purchased from Sigma (St Louis, MO); MK801 was obtained from RBI (Natick, MA) and 6,7-dinitroquinoxaline-2,3-dione (DNQX) from Tocris Neuramin (Bristol, UK). N M D A (10-rain exposure) and kainate (24-h exposure) induced necrotic degeneration of cultured cortical neurons in a concentration-dependent fashion, with apparent EC~,, values of 100 ~M for N M D A and 60/~M l\)r kainate, and maximal neuronal damage at concentra-
195
V. Bruno et al./Neuroscience Letters 178 (1994) 193-196
tions of higher than 150 and 100 gM, respectively (see legends of Figs. 1 and 2). The toxic actions of NMDA and kainate were totally prevented by MK-801 (10 gM) (not shown) and DNQX, respectively (30 gM) (Fig. 2). Idebenone was highly potent (IC5o = 300 nM) in protecting cultured cortical neurons against degeneration induced by both NMDA and kainate (Fig. 1). When applied alone for 30 min (in the paradigm of fast toxicity), idebenone did not influence either the morphology or the release of LDH at all concentrations tested (up to 100 /.tM, see legend of Fig. 1). However, the highest concentration of idebenone tested (100/zM) induced neuronal degeneration over a 24-h period of exposure (see legend of Fig. 2). Idebenone was still neuroprotective when applied 30 min after the NMDA pulse, suggesting that the drug interferes with the cascade of intracellular reactions triggered by NMDA receptor activation (Table 1). Excessive activation of NMDA or kainate receptors has long been known to induce necrotic degeneration of vulnerable neurons either in vivo or in culture (for a review, see Ref. 3). The role of excitotoxic degeneration in the pathophysiology of acute and chronic degenerative disorders has encouraged the search for neuroprotective agents which limit the toxic effects of EAA receptor activation. N M D A receptor antagonists have been found to protect vulnerable neurons in models of focal ischemia, providing that they are administered before or immediately after the ischemic period [1]. Receptor antagonists, however, often induce psychotomimetic side effects and hamper the physiological activation of NMDA receptors which contributes to the regulation of synaptic plasticity [9]. For these reasons, drugs that act downstream the receptor level have a great potential for clinical use. Formation of free radicals has been associated to an excessive stimulation of NMDA or kainate receptors [8] and antioxidants, such as ubiquinone, or 21-amino steroids are known to rescue cultured neurons from excitotoxic damage [5,11]. Idebenone acts both as free radical scavenger and electron-trapping agent [13,16]. To test the neuroprotective action of idebenone, we have used cultured cortical neurons grown on a monTable 1 Delayed rescue of N M D A - i n d u c e d toxicity by idebenone % n e u r o n a l death N M D A 100/.tM + idebenone 10 g M (30 min) + MK801 10 g M (30 min) + idebenone 10/,tM (2 h) + MK801 1 0 g M (2 h)
100 + 7.9 58.7 + 4.5* 7 9 + 14 78.1 + 7.3 83+8
Cortical cultures were exposed to N M D A for 10 rain; 30 rain and 2 h after the N M D A wash-out, cultures were rescued with the indicated concentration of idebenone or MK801. Trypan blue staining was performed 24 h later. Values represent m e a n + SEM, n = 3, scaled to the value present in the cultures exposed to N M D A without rescue (= 100). *P < 0.05 by Fisher P L S D test.
120 I00 80 ~
6O
Tt. ,~.Zt .,efj f.g'j ff/ ,¢ .,¢ /
U
•~
40
f i t
/.¢/ [// i l l
2O
f// ///!
S
f// f//
r/'/
0 KA 6 0 laM
+100nM +l/aM +I0~aM -- Idebenone --
+ DNQX 30 pM
Fig. 2. Kainate toxicity is prevented in a concentration-dependent manner by idebenone. Cortical cultures were exposed to kainate for 24 h in the presence or absence of different concentrations of idebenone or to 3 0 / t M D N Q X . Concentrations o f idebenone of higher than 10 g M were toxic when cultures were exposed for 24 h even in the absence of kainate. Data represent mean _+ S.E.M., n = 3. *P < 0.05 by Fisher PLSD test. L D H values (mean + S.E.M., n = 6): basal 54 + 8; K A 300 ~ M , 6 0 0 + 1 8 ; K A 60ktM, 3 6 0 + 3 5 ; K A 6 0 g M + M K 8 0 1 10/~M, 2 4 0 + 15; K A 60 / I M + M K 8 0 1 10 / . t M + D N Q X 30 /tM, 8 0 + 9 ; K A 6 0 / t M + MK801 10 g M + idebenone 10 ,uM, 65 + 6.
olayer of astrocytes, a model that has been extensively characterized in the past. Idebenone displayed a great potency in protecting cultured cortical neurons against the toxic action of NMDA or kainate and rescued cortical neurons even when applied 30 min after the NMDA pulse, suggesting that free radicals are still produced or still reactive at this time after receptor activation. This suggests to search for a late neuroprotective activity of idebenone in experimental models of ischemia where NMDA receptor antagonists are mostly active when administered before the induction of ischemia or immediately after the onset of postischemic recirculation [1]. Idebenone may attenuate the chronic degeneration of vulnerable neurons in Alzheimer's patients by limiting the consequences of NMDA receptors activation (as suggested by the present study) and by reducing the anomalous flow of electrons from the mitochondrial respiratory chain by virtue of its electron-trapping activity. Idebenone was kindly provided by Takeda Italia Farmaceutici. [1] Albers, G.W., Goldberg, M.P. and Choi, D.W., Do N M D A antagonists prevent neuronal injury? Yes, Arch. Neurol., 49 (1992) 418420.
196
V. Bruno et al. / Neuroseience Letters 178 (1994) 193 196
[2] Choi, D.W., Maulucci-Gedde, M.A. and Kriegstein, A.R., Glutamate neurotoxicity in cortical cell culture, J. Neurosci., 7 (1987) 357-368. [3] Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system, Neuron, 1 (1988) 623 634. [4] Choi, D.W., Excitotoxic cell death, J. Neurobiol., 23 (1992) 1261 t276 [5] Favit, A., Nicoletti, F., Scapagnini, U. and Canonico, EL., Ubiquinone protects cultured neurons against spontaneous or excitotoxin-induced degeneration, J. Cer. Blood Flow Metaboh, 12 (1992) 638 -645. [6] Halliwell, B., Reactive oxygen species and the central nervous system, J. Neurochem., 59 (1992) 1609 1623. [7] Kish, S.J., Bergeron, C., Rajput, A. et ah, Brain cytochrome oxidase in Alzheimer's disease, J. Neurochem., 59 (1992) 776 779. [8] Lafon-Cazal, M., Pietri, S., Culcasi, M. and Bockaert, J., NMDAdependent superoxide production and neurotoxicity, Nature (London), 364 (1993) 535 537. [9] Lipton, S.A., Prospects for clinically tolerated NMDA antagonists: open-channel blockers and alternative redox states of nitric oxide, Trends Neurosci., 16 (1993) 527 532. [10] Maragos, W.F., Greenamyre, J.T., Penney, J.B. and Young, A.B., Glutamate dysfunction in Alzheimer's disease: an hypothesis. Trends Neurosci., 10 (1987) 65 68.
[ll] Monyer, H., Hartley, D.M. and Choi, D.W., 21-Aminosteroids attenuate excitotoxic neuronal injury in cortical cell cultures, Neuron, 5 (1990) 121 126, [12] Miyamoto, M., Murphy, T.H,, Schnaar, R.L. and Coyle, J.T.. Antioxidants protect against glutamate-induced cytotoxicity in a neuronal cell line, J. Pharmacol. Exp. Ther., 260 (1989) 1132 1140. [13] Miyamoto, M. and Coyle, J.T., Idebenone attenuates neuronal degeneration induced by intrastriatal injection of excitotoxins, Exp. Neurol., 108 (1990) 3845. [14] Rose, K., Goldberg, M.P. and Choi, D.W., Cytotoxicity in murine neocortical cell culture. In, Methods in Toxicology, Vol. 1, Academic Press, New York, NY, 1993, pp.46-60. [15] Siesjo, B.K., Free radicals and brain damage, Cerebrovasc. Brain Metab. Rev., 1 (1989) 165 211. [16] Suno, M. and Nagaoka, A., Inhibition of lipid peroxidation by a novel compound (CV-2619) in brain mitochondria and mode of action of the inhibition, Biochem. Biophys. Res. Commun., 125 (1984) 1046 1052. [17] Young, A.B., Greenamare, J.T., Hollingsworth, Z., Albin, R., D'Amato, C., Shoulson, I. and Penney, J.B., NMDA receptor losses in putamen from patients with Huntington's disease, Science, 241 (1988) 98l 983.