154
NeuroscienceLetters, 117 (1990) 154-159 Elsevier Scientific Publishers Ireland Ltd.
NSL 07117
Gangliosides attenuate the delayed neurotoxicity of aspartic acid in vitro Stephen D. Skaper, Laura Facci and Alberta Leon Fidia Research Laboratories, Department of CNS Research, Abano Terme (Italy)
(Received 3 April 1990; Revised version received 9 May 1990;Accepted 14 May 1990) Key words." Aspartate; Neuronal death; N-methyl-D-aspartate receptor; Cerebellar granule cell; Ganglio-
side; Neuroprotection The neurotoxic effects of L-aspartate were evaluated in rat cerebellar granule cell cultures. Acute (15 min) exposure to e-aspartate produced a time-dependent, delayed degeneration of neuronal cell bodies and neurites (LDs0 about 40 gM) over 24 h. Aspartate neurotoxicity was prevented by competitive and non-competitive N-methyl-D-aspartate (NMDA) antagonists, but not by non-NMDA antagonists, suggesting a major involvement of NMDA receptors in this neuronal injury. Gangliosides, including GM 1, were also effective in attenuating the cytotoxicity of L-aspartate. The neurotoxic potential of e-aspartate may thus contribute to pathologies involving the action of endogenous excitatory amino acids.
E n d o g e n o u s excitatory amino acid ( E A A ) neurotransmitters are believed to play an i m p o r t a n t role in the pathogenesis associated with a n u m b e r o f neurological diseases, including ischemia/hypoxia, hypoglycemia and epilepsy [2, 7, 22]. These excitatory transmitters mediate their action by interacting with at least three receptor subtypes, as defined by the selective agonists N-methyl-D-aspartate ( N M D A ) , kainate and quisqualate [11, 17]. The E A A transmitters L-glutamate and L-aspartate are present ubiquitously in the central nervous system (CNS). Their tissue and extracellular concentrations increase during ischemia [3, 4] and hypoglycemia [2, 5]. While m u c h attention has been focused on glutamate neurotoxicity, there have been few reports on aspartate neurotoxicity, either in vivo [20] or in vitro [8]. The potential for aspartate to contribute significantly to E A A neurotoxicity should not be overlooked, as in hypoglycemia, for example, aspartate accumulation can exceed that o f glutamate [2, 5]. Using cerebellar granule cell cultures as a model for studying E A A - t y p e neurotoxicity [10, 25, 26], the present experiments were designed to examine aspartate neurotoxicity and possible neuroprotective effects o f gangliosides.
Correspondence: S.D. Skaper, Fidia Research Laboratories, Department of CNS Research, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
155
Granule cell cultures were prepared from day 8 postnatal Sprague-Dawley rat cerebella, as previously described [25, 26]. Dissociated granule cells were plated in 35 mm tissue culture plastic dishes (Falcon) coated with 10 gg/ml poly-L-lysine (77,000 MW; Sigma) (3.0 × 106 cells/dish) in basal modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (Seromed), gentamycin (50 gg/ml), penicillin (100 U/ml), and 25 mM KCI. Cultures were kept at 37°C in a humidified 5% CO2air atmosphere. After 18-20 h, I0 gM cytosine arabinoside was added to inhibit nonneuronal cell growth. Medium glucose was replenished every 3-4 days by adding 5 mM glucose (from a 0.5 M stock in water). Cultures were used for study at 11-13 days in vitro, without medium change. Exposure to aspartate, either alone or in the presence of antagonists, was carried out at 22-24°C in Locke's solution with the following composition (in mM): NaC1 154, KC1 5.6, CaC12 2.3, MgC12 1.0, NaHCO3 3.6, glucose 5.6, HEPES 5 (pH 7.4). In some cases, MgCI2 was omitted. After 15 min, the test solution was removed, the
Fig. I. L-Aspartate neurotoxicity. Phase contrast photomicrographs of representative fields of cerebellar granule cells before (A) and 24 h after (B-D) a 15 min exposure to 250/~M L-aspartate at 22°C. In C, Mg 2+ was included with aspartate. In D, cells were treated with 100/zM GMI ganglioside for 2 h, prior to aspartate. Bar = 25/zM.
156
dishes washed 3 times with Locke's solution and the original culture medium added back. Incubation was continued for another 24 h prior to assessment of cell vitality. Neuronal cell injury was estimated by microscopic examination under phase contrast optics at X200. The neurotoxic effects of EAA exposure are gross enough to be apparent in this manner [6, 23, 25]. Granule cell death following acute aspartate treatment was largely complete after 24 h. Neuronal survival was quantitated with a colorimetric assay using the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) [18]. This index accurately reflects the number of cerebellar granule cells in culture [25], and has been adapted to assess glutamate cytotoxicity in these neurons [26]. The MTT technique has been shown equivalent to lactate dehydrogenase release in the measurement of EAA-mediated neuronal cytotoxicity [2t]. Because absolute values for MTT color yield differed somewhat (+15%) between culture platings, the MTT values obtained are expressed relative to the mean MTT value (= 100) in untreated sister cultures. Statistical analyses were performed by one-way analysis of variance followed by Duncan's post hoc test. Exposure of cerebellar granule cells to 250/~M L-aspartate (--Mg 2÷) was rapidly followed by swelling and darkening of neuronal cell bodies. The neurons began to undergo degeneration over the next 3-4 h; by the next day, a large number (but not all) of neurons and the associated neurite network had disintegrated, being replaced
'2° t 100 80
U
C O N T - M g 2+ Mg 2+
PeP
CPP
GAM$ DNQX
Fig. 2. Pharmacology of L-aspartate neurotoxicity. Granule cells were exposed to 250/~M L-aspartate (--Mg 2~) for 15 min (22°C) in the presence of the indicated antagonists, and survival assessed 24 h later with MTT. CONT, control Locke's solution; - M g : * , aspartate without Mg2÷; Mg 2+, 1 mM MgCI~; PCP (phencyclidine), 10 pM; CPP (3-(+)-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid), 250 /tM; GAMS (},-D-glutamylaminomethyl sulfonate), 1 mM; DNQX (6,7-dinitroquinoxaline-2,3-dione), 5/IM. Values are means + S.D. (n = 3). *Significant difference from aspartate ( - Mg 2+) at P < 0.01.
157
by debris (Fig. la, b). The LDs0 for L-aspartate-induced cytotoxicity was approximately 40/tM (not shown). Inclusion of Mg 2+ during the acute pulse of aspartate prevented this neuronal injury (Fig. lc). The pharmacology of L-aspartate neurotoxicity was examined by adding maximal concentrations of EAA antagonists together with aspartate (Fig. 2). Aspartate neurotoxicity was essentially eliminated by both competitive (CPP; 3-(_)-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid) [9] and non-competitive (PCP; phencyclidine) [14] NMDA antagonists as well as Mg 2+, the latter blocking the NMDA receptorgated ion channel [19]. In contrast, 6,7-dinitroquinoxaline-2,3-dione (DNQX) [12] and y-o-glutamylaminomethyl sulfonate (GAMS) [17], EAA antagonists which may be preferentially active at non-NMDA receptors, were ineffective in reducing Laspartate neurotoxicity (Fig. 2). Kainic acid (250 ~tM), which produced an approximate 50% loss of granule cells, as already reported [10], was fully protected against by DNQX (5 pM) and GAMS (1 mM) thus confirming their activity (not shown). Gangliosides, a class of naturally occurring sialoglycosphingolipids [27] have recently been described to limit glutamate and kainate toxicity [10] and anoxic neuronal injury [25] in cerebellar granule cells, as well as exogenous excitotoxin damage in vivo [15, 16]. These agents were therefore tested for an effect on L-aspartate neurotoxicity. Granule cells were pretreated with 100 pM GM1 ganglioside for 2 h, followed by washing with serum and acute exposure to 250/~M aspartate. This protocol provides for maximal protection against glutamate neurotoxicity [10]. GMI clearly attenuated the morphologic evidence of aspartate neuronal injury (Fig. ld). The
O lO .- . 120.
O l O-
A
80-
120-
~
B
80-
~.
60-
~
60-
0
40il ~
.
~ 4020
0
50
100 150 200 250
GM1 (~M)
0
CONT ASP GM1 S I A G G D l b
GA
AS.
GM1
Fig. 3. Gangliosides attenuate the loss of cerebellar granule neurons induced by L-asparate. Cells were pretreated 2 h with (a) 5-250/zM GMI or (b) 100/IM of the following gangliosides: GM1; GM1 inner ester, siagoside (SLAG); GD 1b; ganglioside mixture (GA); asialo GM 1 (AS.GMI). After serum wash, cells were exposed to 250 pM aspartate - M g 2+ (ASP) (22°C). MTT values after 24 h are the mean +S.D. (n = 6). Gangliosides were prepared from bovine brain (> 99% purity). The mixture contains GMI (21%), GDla (40%), GDIb (16%) and GTlb (19%). Significant difference from ASP at P < 0.01 (*); P < 0.05 (**).
158 neuroprotective effect of G M I was concentration-dependent, with an EDs0 of approximately 35/tm (Fig. 3a), as confirmed by the corresponding increase in cellular vitality determined by MTT. This G M 1 effect could not be overcome by increasing concentrations of aspartate. Ganglioside action in reducing aspartate neurotoxicity was not G M 1-specific, as the inner ester form of G M 1 (siagoside), G D 1b and a mixture of bovine brain gangliosides were all equally effective at 100/IM (Fig. 3b); the mixture was fully effective also at 50/~M. Asialo G M 1, which lacks sialic acid was, however, inactive. The protective effect of G M 1 was reversible, with a half-life of about 12 h following the 2 h of pretreatment; post-aspartate treatment of the granule cells with G M 1 failed to afford protection (not shown). The present findings demonstrate that L-aspartate is a potent neurotoxin for cerebellar granule cells in vitro, destroying the majority of neurons over a 24 h period following transient exposure. This neurodegeneration was not immediate, but rather started to become evident only several hours following aspartate removal. Such delayed neurotoxicity in vitro has been described for glutamate acutely applied to cortical [6], hippocampal [23] and cerebellar [10] neurons. The pharmacological profile of aspartate neurotoxicity in granule cells suggests a selective activation of N M D A receptors, as reported for cortical neurons [8]. L-aspartate neurotoxicity, therefore, can be expected to contribute to the pathophysiology of diseases where EAAs are released in excessive amounts. In addition, the protection afforded by gangliosides against aspartate neurotoxicity is of potential clinical interest. Pharmacologic strategies that aim to prevent the delayed neuronal death accompanying cerebrovascular insufficiencies represent an area of intense investigation. Ganglioside reduction of both glutamate [10] and aspartate neurotoxicity in vitro and excitotoxin damage in vivo [15, 16], and their postEAA receptor/channel site of action [10] make these compounds attractive candidates for application to CNS disorders associated with dysfunction of EAA systems [1, 13, 24].
I Argentino, C., Sacchetti, M.L., Toni, D., Savoini, G., D'Arcangelo, E., Erminio, F., Federico, F., Ferro Milone, F., Gallai, V., Gambi, D., Mamoli, A., Ottonello, G.A., Ponari, O., Rebucci, G., Senin, U. and Fieschi, C., GM1 gangliosidetherapy in acute ischemicstroke, Stroke, 20 (1989) 1143 1149. 2 Auer, R.N., Progress review: hypoglycemicbrain damage, Stroke, 17 (1986) 69%708. 3 Benveniste,H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis,J. Neurochem., 43 (1984) 1369-1374. 4 Benveniste, H., Jorgensen, M.B., Sandberg, M., Christensen, T., Hagberg, H. and Diemer, N.H., lschemic damage in hippocampal CA 1 is dependent on glutamate release and intact innervation from CA3, J. Cereb. Blood Flow Metab., 9 (1989) 629 639. 5 Butcher, S.P., Sandberg, M., Hagberg, H. and Hamberger, A., Cellular origins of endogenous amino acids released into the extracellular fluid of the rat striatum during severe insulin-induced hypoglycemia, J. Neurochem., 48 (1987) 722-728. 6 Choi, D.W., Ionic dependence of glutamate neurotoxicity, J. Neurosci., 7 (1987) 369-379. 7 Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system,Neuron, 1 (1988) 623-634. 8 Choi, D.W., Viseskul, V., Amirthanayagam, M. and Monyer, H., Aspartate neurotoxicity on cultured cortical neurons, J. Neurosci. Res., 23 (1989) 116-121.
159 9 Davies, J., Evans, R.H., Herding, P.L., Jones, A.W., Olverman, H.J., Pook, P. and Watkins, J.C., CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for [3H]D-AP5 binding sites on brain membranes and anticonvulsant activity, Brain Res., 382 (1986) 169-173. 10 Favaron, M., Manev, H., Alho, H., Bertolino, M., Ferret, B., Guidotti, A. and Costa, E., Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7351-7355. 11 Foster, A.C. and Fagg, A.G., Acidic amino acid binding sites in neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 12 Honor6, T., Davies, S.N., Drejer, J., Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, F., Quinoxalinediones: potent non-NMDA glutamate receptor antagonists, Science, 241 (I 988) 701-703. 13 Karpiak, S.E. and Mahadik, S.P., Ganglioside reduction of CNS ischemic injury. CRC Crit. Rev. Neurobiol., in press. 14 Kemp, J.A., Foster, A.C. and Wong, E.H.F., Non-competitive antagonists of excitatory amino acid receptors, Trends Neurosci., I0 (1987) 294-298. 15 Lipartiti, M., Mazzari, S., Lazzaro, A., Zanoni, R., Seren, M.S. and Leon, A., Monosialogangliosides reduce NMDA neurotoxicity in neonatal rats, Soc. Neurosci. Abstr., 15 (1989) 764. 16 Lombardi, G., Zanoni, R. and Moroni, F., Systemic treatments with GM 1 ganglioside reduce quinolinic acid-induced striatal lesions in the rat, Eur. J. Pharmacol., 174 (1989) 123-125. 17 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365-402. 18 Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Meth., 65 (1983) 55~3. 19 Nowak, L., Bregestovski, P., Ascher, P., Herbert, A. and Prochiantz, A., Magnesium gates glutamate activated channels in mouse central neurones, Nature, 307 (1984) 462-465. 20 Olney, J.W., Ho, O.L. and Rhee, V., Cytotoxic effects of acidic and sulfur containing amino acids on the infant mouse central nervous system, Exp. Brain Res., 14 (1971) 61-76. 21 Patel, J., Zinkand, W.C., Thompson, C., Keith, R. and Salama, A., Role of glycine in the N-methyl-Daspartate-mediated neuronal cytotoxicity, J. Neurochem., 54 (1990) 849-854. 22 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 23 Rothman, S.M., Thurston, J.H. and Hauhart, R.E., Delayed neurotoxicity of excitatory amino acids in vitro, Neuroscience, 22 (1987) 471-480. 24 Seren, M.S., Lazzaro, A., Rubini, R., Zanoni, R., Skaper, S.D. and Leon, A., Cerebral ischemia and excitatory amino acid transmitter-induced neurodegeneration: effects of monosialoganglioside treatment. In J. Krieglstein (Ed.), Pharmacology of Cerebral Ischemia 1988, CRC Press, Boca Raton, 1989, pp. 285-288. 25 Skaper, S.D., Facci, L., Milani, D. and Leon, A., Monosialoganglioside GM1 protects against anoxiainduced neuronal death in vitro, Exp. Neurol., 106 (1989) 297-305. 26 Skaper, S.D., Facci, L., Milani, D., Leon, A. and Toffano, G., Culture and use of primary and clonal neural cells. In P.M. Conn (Ed.), Methods in Neurosciences, Vol. 2, Academic Press, Orlando, 1990, pp. 17-33. 27 Svennerholm, L., Chromatographic separation of bovine brain gangliosides, J. Neurochem., 10 (1963) 613~23.
NeuroscienceLetters, 117 (1990) 154-159 Elsevier Scientific Publishers Ireland Ltd.
NSL 07117
Gangliosides attenuate the delayed neurotoxicity of aspartic acid in vitro Stephen D. Skaper, Laura Facci and Alberta Leon Fidia Research Laboratories, Department of CNS Research, Abano Terme (Italy)
(Received 3 April 1990; Revised version received 9 May 1990;Accepted 14 May 1990) Key words." Aspartate; Neuronal death; N-methyl-D-aspartate receptor; Cerebellar granule cell; Ganglio-
side; Neuroprotection The neurotoxic effects of L-aspartate were evaluated in rat cerebellar granule cell cultures. Acute (15 min) exposure to e-aspartate produced a time-dependent, delayed degeneration of neuronal cell bodies and neurites (LDs0 about 40 gM) over 24 h. Aspartate neurotoxicity was prevented by competitive and non-competitive N-methyl-D-aspartate (NMDA) antagonists, but not by non-NMDA antagonists, suggesting a major involvement of NMDA receptors in this neuronal injury. Gangliosides, including GM 1, were also effective in attenuating the cytotoxicity of L-aspartate. The neurotoxic potential of e-aspartate may thus contribute to pathologies involving the action of endogenous excitatory amino acids.
E n d o g e n o u s excitatory amino acid ( E A A ) neurotransmitters are believed to play an i m p o r t a n t role in the pathogenesis associated with a n u m b e r o f neurological diseases, including ischemia/hypoxia, hypoglycemia and epilepsy [2, 7, 22]. These excitatory transmitters mediate their action by interacting with at least three receptor subtypes, as defined by the selective agonists N-methyl-D-aspartate ( N M D A ) , kainate and quisqualate [11, 17]. The E A A transmitters L-glutamate and L-aspartate are present ubiquitously in the central nervous system (CNS). Their tissue and extracellular concentrations increase during ischemia [3, 4] and hypoglycemia [2, 5]. While m u c h attention has been focused on glutamate neurotoxicity, there have been few reports on aspartate neurotoxicity, either in vivo [20] or in vitro [8]. The potential for aspartate to contribute significantly to E A A neurotoxicity should not be overlooked, as in hypoglycemia, for example, aspartate accumulation can exceed that o f glutamate [2, 5]. Using cerebellar granule cell cultures as a model for studying E A A - t y p e neurotoxicity [10, 25, 26], the present experiments were designed to examine aspartate neurotoxicity and possible neuroprotective effects o f gangliosides.
Correspondence: S.D. Skaper, Fidia Research Laboratories, Department of CNS Research, Via Ponte della Fabbrica 3/A, 35031 Abano Terme, Italy.
0304-3940/90/$ 03.50 © 1990 Elsevier Scientific Publishers Ireland Ltd.
155
Granule cell cultures were prepared from day 8 postnatal Sprague-Dawley rat cerebella, as previously described [25, 26]. Dissociated granule cells were plated in 35 mm tissue culture plastic dishes (Falcon) coated with 10 gg/ml poly-L-lysine (77,000 MW; Sigma) (3.0 × 106 cells/dish) in basal modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum (Seromed), gentamycin (50 gg/ml), penicillin (100 U/ml), and 25 mM KCI. Cultures were kept at 37°C in a humidified 5% CO2air atmosphere. After 18-20 h, I0 gM cytosine arabinoside was added to inhibit nonneuronal cell growth. Medium glucose was replenished every 3-4 days by adding 5 mM glucose (from a 0.5 M stock in water). Cultures were used for study at 11-13 days in vitro, without medium change. Exposure to aspartate, either alone or in the presence of antagonists, was carried out at 22-24°C in Locke's solution with the following composition (in mM): NaC1 154, KC1 5.6, CaC12 2.3, MgC12 1.0, NaHCO3 3.6, glucose 5.6, HEPES 5 (pH 7.4). In some cases, MgCI2 was omitted. After 15 min, the test solution was removed, the
Fig. I. L-Aspartate neurotoxicity. Phase contrast photomicrographs of representative fields of cerebellar granule cells before (A) and 24 h after (B-D) a 15 min exposure to 250/~M L-aspartate at 22°C. In C, Mg 2+ was included with aspartate. In D, cells were treated with 100/zM GMI ganglioside for 2 h, prior to aspartate. Bar = 25/zM.
156
dishes washed 3 times with Locke's solution and the original culture medium added back. Incubation was continued for another 24 h prior to assessment of cell vitality. Neuronal cell injury was estimated by microscopic examination under phase contrast optics at X200. The neurotoxic effects of EAA exposure are gross enough to be apparent in this manner [6, 23, 25]. Granule cell death following acute aspartate treatment was largely complete after 24 h. Neuronal survival was quantitated with a colorimetric assay using the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) [18]. This index accurately reflects the number of cerebellar granule cells in culture [25], and has been adapted to assess glutamate cytotoxicity in these neurons [26]. The MTT technique has been shown equivalent to lactate dehydrogenase release in the measurement of EAA-mediated neuronal cytotoxicity [2t]. Because absolute values for MTT color yield differed somewhat (+15%) between culture platings, the MTT values obtained are expressed relative to the mean MTT value (= 100) in untreated sister cultures. Statistical analyses were performed by one-way analysis of variance followed by Duncan's post hoc test. Exposure of cerebellar granule cells to 250/~M L-aspartate (--Mg 2÷) was rapidly followed by swelling and darkening of neuronal cell bodies. The neurons began to undergo degeneration over the next 3-4 h; by the next day, a large number (but not all) of neurons and the associated neurite network had disintegrated, being replaced
'2° t 100 80
U
C O N T - M g 2+ Mg 2+
PeP
CPP
GAM$ DNQX
Fig. 2. Pharmacology of L-aspartate neurotoxicity. Granule cells were exposed to 250/~M L-aspartate (--Mg 2~) for 15 min (22°C) in the presence of the indicated antagonists, and survival assessed 24 h later with MTT. CONT, control Locke's solution; - M g : * , aspartate without Mg2÷; Mg 2+, 1 mM MgCI~; PCP (phencyclidine), 10 pM; CPP (3-(+)-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid), 250 /tM; GAMS (},-D-glutamylaminomethyl sulfonate), 1 mM; DNQX (6,7-dinitroquinoxaline-2,3-dione), 5/IM. Values are means + S.D. (n = 3). *Significant difference from aspartate ( - Mg 2+) at P < 0.01.
157
by debris (Fig. la, b). The LDs0 for L-aspartate-induced cytotoxicity was approximately 40/tM (not shown). Inclusion of Mg 2+ during the acute pulse of aspartate prevented this neuronal injury (Fig. lc). The pharmacology of L-aspartate neurotoxicity was examined by adding maximal concentrations of EAA antagonists together with aspartate (Fig. 2). Aspartate neurotoxicity was essentially eliminated by both competitive (CPP; 3-(_)-2-carboxypiperazin-4-yl)-propyl-l-phosphonic acid) [9] and non-competitive (PCP; phencyclidine) [14] NMDA antagonists as well as Mg 2+, the latter blocking the NMDA receptorgated ion channel [19]. In contrast, 6,7-dinitroquinoxaline-2,3-dione (DNQX) [12] and y-o-glutamylaminomethyl sulfonate (GAMS) [17], EAA antagonists which may be preferentially active at non-NMDA receptors, were ineffective in reducing Laspartate neurotoxicity (Fig. 2). Kainic acid (250 ~tM), which produced an approximate 50% loss of granule cells, as already reported [10], was fully protected against by DNQX (5 pM) and GAMS (1 mM) thus confirming their activity (not shown). Gangliosides, a class of naturally occurring sialoglycosphingolipids [27] have recently been described to limit glutamate and kainate toxicity [10] and anoxic neuronal injury [25] in cerebellar granule cells, as well as exogenous excitotoxin damage in vivo [15, 16]. These agents were therefore tested for an effect on L-aspartate neurotoxicity. Granule cells were pretreated with 100 pM GM1 ganglioside for 2 h, followed by washing with serum and acute exposure to 250/~M aspartate. This protocol provides for maximal protection against glutamate neurotoxicity [10]. GMI clearly attenuated the morphologic evidence of aspartate neuronal injury (Fig. ld). The
O lO .- . 120.
O l O-
A
80-
120-
~
B
80-
~.
60-
~
60-
0
40il ~
.
~ 4020
0
50
100 150 200 250
GM1 (~M)
0
CONT ASP GM1 S I A G G D l b
GA
AS.
GM1
Fig. 3. Gangliosides attenuate the loss of cerebellar granule neurons induced by L-asparate. Cells were pretreated 2 h with (a) 5-250/zM GMI or (b) 100/IM of the following gangliosides: GM1; GM1 inner ester, siagoside (SLAG); GD 1b; ganglioside mixture (GA); asialo GM 1 (AS.GMI). After serum wash, cells were exposed to 250 pM aspartate - M g 2+ (ASP) (22°C). MTT values after 24 h are the mean +S.D. (n = 6). Gangliosides were prepared from bovine brain (> 99% purity). The mixture contains GMI (21%), GDla (40%), GDIb (16%) and GTlb (19%). Significant difference from ASP at P < 0.01 (*); P < 0.05 (**).
158 neuroprotective effect of G M I was concentration-dependent, with an EDs0 of approximately 35/tm (Fig. 3a), as confirmed by the corresponding increase in cellular vitality determined by MTT. This G M 1 effect could not be overcome by increasing concentrations of aspartate. Ganglioside action in reducing aspartate neurotoxicity was not G M 1-specific, as the inner ester form of G M 1 (siagoside), G D 1b and a mixture of bovine brain gangliosides were all equally effective at 100/IM (Fig. 3b); the mixture was fully effective also at 50/~M. Asialo G M 1, which lacks sialic acid was, however, inactive. The protective effect of G M 1 was reversible, with a half-life of about 12 h following the 2 h of pretreatment; post-aspartate treatment of the granule cells with G M 1 failed to afford protection (not shown). The present findings demonstrate that L-aspartate is a potent neurotoxin for cerebellar granule cells in vitro, destroying the majority of neurons over a 24 h period following transient exposure. This neurodegeneration was not immediate, but rather started to become evident only several hours following aspartate removal. Such delayed neurotoxicity in vitro has been described for glutamate acutely applied to cortical [6], hippocampal [23] and cerebellar [10] neurons. The pharmacological profile of aspartate neurotoxicity in granule cells suggests a selective activation of N M D A receptors, as reported for cortical neurons [8]. L-aspartate neurotoxicity, therefore, can be expected to contribute to the pathophysiology of diseases where EAAs are released in excessive amounts. In addition, the protection afforded by gangliosides against aspartate neurotoxicity is of potential clinical interest. Pharmacologic strategies that aim to prevent the delayed neuronal death accompanying cerebrovascular insufficiencies represent an area of intense investigation. Ganglioside reduction of both glutamate [10] and aspartate neurotoxicity in vitro and excitotoxin damage in vivo [15, 16], and their postEAA receptor/channel site of action [10] make these compounds attractive candidates for application to CNS disorders associated with dysfunction of EAA systems [1, 13, 24].
I Argentino, C., Sacchetti, M.L., Toni, D., Savoini, G., D'Arcangelo, E., Erminio, F., Federico, F., Ferro Milone, F., Gallai, V., Gambi, D., Mamoli, A., Ottonello, G.A., Ponari, O., Rebucci, G., Senin, U. and Fieschi, C., GM1 gangliosidetherapy in acute ischemicstroke, Stroke, 20 (1989) 1143 1149. 2 Auer, R.N., Progress review: hypoglycemicbrain damage, Stroke, 17 (1986) 69%708. 3 Benveniste,H., Drejer, J., Schousboe, A. and Diemer, N.H., Elevation of the extracellular concentrations of glutamate and aspartate in rat hippocampus during transient cerebral ischemia monitored by intracerebral microdialysis,J. Neurochem., 43 (1984) 1369-1374. 4 Benveniste, H., Jorgensen, M.B., Sandberg, M., Christensen, T., Hagberg, H. and Diemer, N.H., lschemic damage in hippocampal CA 1 is dependent on glutamate release and intact innervation from CA3, J. Cereb. Blood Flow Metab., 9 (1989) 629 639. 5 Butcher, S.P., Sandberg, M., Hagberg, H. and Hamberger, A., Cellular origins of endogenous amino acids released into the extracellular fluid of the rat striatum during severe insulin-induced hypoglycemia, J. Neurochem., 48 (1987) 722-728. 6 Choi, D.W., Ionic dependence of glutamate neurotoxicity, J. Neurosci., 7 (1987) 369-379. 7 Choi, D.W., Glutamate neurotoxicity and diseases of the nervous system,Neuron, 1 (1988) 623-634. 8 Choi, D.W., Viseskul, V., Amirthanayagam, M. and Monyer, H., Aspartate neurotoxicity on cultured cortical neurons, J. Neurosci. Res., 23 (1989) 116-121.
159 9 Davies, J., Evans, R.H., Herding, P.L., Jones, A.W., Olverman, H.J., Pook, P. and Watkins, J.C., CPP, a new potent and selective NMDA antagonist. Depression of central neuron responses, affinity for [3H]D-AP5 binding sites on brain membranes and anticonvulsant activity, Brain Res., 382 (1986) 169-173. 10 Favaron, M., Manev, H., Alho, H., Bertolino, M., Ferret, B., Guidotti, A. and Costa, E., Gangliosides prevent glutamate and kainate neurotoxicity in primary neuronal cultures of neonatal rat cerebellum and cortex, Proc. Natl. Acad. Sci. U.S.A., 85 (1988) 7351-7355. 11 Foster, A.C. and Fagg, A.G., Acidic amino acid binding sites in neuronal membranes: their characteristics and relationship to synaptic receptors, Brain Res. Rev., 7 (1984) 103-164. 12 Honor6, T., Davies, S.N., Drejer, J., Fletcher, E.J., Jacobsen, P., Lodge, D. and Nielsen, F., Quinoxalinediones: potent non-NMDA glutamate receptor antagonists, Science, 241 (I 988) 701-703. 13 Karpiak, S.E. and Mahadik, S.P., Ganglioside reduction of CNS ischemic injury. CRC Crit. Rev. Neurobiol., in press. 14 Kemp, J.A., Foster, A.C. and Wong, E.H.F., Non-competitive antagonists of excitatory amino acid receptors, Trends Neurosci., I0 (1987) 294-298. 15 Lipartiti, M., Mazzari, S., Lazzaro, A., Zanoni, R., Seren, M.S. and Leon, A., Monosialogangliosides reduce NMDA neurotoxicity in neonatal rats, Soc. Neurosci. Abstr., 15 (1989) 764. 16 Lombardi, G., Zanoni, R. and Moroni, F., Systemic treatments with GM 1 ganglioside reduce quinolinic acid-induced striatal lesions in the rat, Eur. J. Pharmacol., 174 (1989) 123-125. 17 Monaghan, D.T., Bridges, R.J. and Cotman, C.W., The excitatory amino acid receptors: their classes, pharmacology, and distinct properties in the function of the central nervous system, Annu. Rev. Pharmacol. Toxicol., 29 (1989) 365-402. 18 Mosmann, T., Rapid colorimetric assay for cellular growth and survival: application to proliferation and cytotoxicity assays, J. Immunol. Meth., 65 (1983) 55~3. 19 Nowak, L., Bregestovski, P., Ascher, P., Herbert, A. and Prochiantz, A., Magnesium gates glutamate activated channels in mouse central neurones, Nature, 307 (1984) 462-465. 20 Olney, J.W., Ho, O.L. and Rhee, V., Cytotoxic effects of acidic and sulfur containing amino acids on the infant mouse central nervous system, Exp. Brain Res., 14 (1971) 61-76. 21 Patel, J., Zinkand, W.C., Thompson, C., Keith, R. and Salama, A., Role of glycine in the N-methyl-Daspartate-mediated neuronal cytotoxicity, J. Neurochem., 54 (1990) 849-854. 22 Rothman, S.M. and Olney, J.W., Glutamate and the pathophysiology of hypoxic-ischemic brain damage, Ann. Neurol., 19 (1986) 105-111. 23 Rothman, S.M., Thurston, J.H. and Hauhart, R.E., Delayed neurotoxicity of excitatory amino acids in vitro, Neuroscience, 22 (1987) 471-480. 24 Seren, M.S., Lazzaro, A., Rubini, R., Zanoni, R., Skaper, S.D. and Leon, A., Cerebral ischemia and excitatory amino acid transmitter-induced neurodegeneration: effects of monosialoganglioside treatment. In J. Krieglstein (Ed.), Pharmacology of Cerebral Ischemia 1988, CRC Press, Boca Raton, 1989, pp. 285-288. 25 Skaper, S.D., Facci, L., Milani, D. and Leon, A., Monosialoganglioside GM1 protects against anoxiainduced neuronal death in vitro, Exp. Neurol., 106 (1989) 297-305. 26 Skaper, S.D., Facci, L., Milani, D., Leon, A. and Toffano, G., Culture and use of primary and clonal neural cells. In P.M. Conn (Ed.), Methods in Neurosciences, Vol. 2, Academic Press, Orlando, 1990, pp. 17-33. 27 Svennerholm, L., Chromatographic separation of bovine brain gangliosides, J. Neurochem., 10 (1963) 613~23.
