NMDA receptor-mediated glutamate toxicity of cultured cerebellar, cortical and mesencephalic neurons: neuroprotective properties of amantadine and memantine

Brain Research, 613 (1993) 143-148 © 1993 Elsevier Science Publishers B.V. All rights reserved 0006-8993/93/$06.00

143

BRES 25666

Short Communications

NMDA receptor-mediated glutamate toxicity of cultured cerebellar, cortical and mesencephalic neurons: neuroprotective properties of amantadine and memantine M i c h a e l W e l l e r *, F r a n ~ o i s e F i n i e l s - M a r l i e r a n d S t e v e n M. P a u l Section on Molecular Pharmacology, Clinical Neuroscience Branch, National Institute of Mental Health, Bethesda, MD 20892 (USA) (Accepted 23 February 1993)

Key words: Amantadine; Memantine; Glutamate; N-Methyl-D-amphetamine neuron; Excitotoxicity; Neurotoxicity

Effects of amantadine and memantine on NMDA receptor-mediated glutamate toxicity were studied in cultured cerebellar, cortical and mesencephalic neurons. Both drugs protected cerebellar and cortical neurons against glutamate toxicity, memantine being consistently more effective than amantadine but less effective than MK-801. Glutamate toxicity of dopaminergic neurons in mesencephalic cultures was only mildly attenuated by memantine but was also only incompletely blocked by MK-801. These findings suggest that adamantanamines act by inhibiting NMDA receptor-mediated excitatory neurotransmission. However, since non-NMDA receptors appear to be principal mediators of glutamate toxicity of dopaminergic mesencephalic neurons, adamantanamines may fail to protect the nigrostriatal neurons which specifically degenerate in Parkinson's disease.

The antiparkinsonian effects of amantadine were attributed to ill-defined dopaminomimetic properties until the amantadine analogue, memantine (1-amino3,5-dimethyladamantane), was found to be a potent antagonist at the N-methyl-D-aspartate (NMDA) type of glutamate receptor ~'~°'tl. Memantine protects cortical neuron cultures from the toxicity of glutamate 4, NMDA 3 and of human immunodeficiency virus (HIV) 1 coat protein gpl20 ~2'18, protects retinal neurones 3'24, and has antihypoxic properties in vitro and in v i v o 26. NMDA antagonistic properties have subsequently been attributed to amantadine, t o o 11'13'14'28. Both amantadine and memantine are used in the therapy of Parkinson's disease and are candidate drugs for other disorders including stroke, neuroleptic malignant syndrome, and AIDS-related neurological d i s t u r b a n c e s 5'9'12'29'3°. To determine whether the beneficial effects of adamantanamines can be attributed to inhibition of glutamate-mediated excitatory neurotransmission in the basal ganglia 5-8'21, we have assessed whether amanta-

dine and memantine attenuate glutamate toxicity of neuronal cultures prepared from rat cerebellum, cerebral cortex, and mesencephalon. Cultures of fetal mesencephalic neurons are of interest for the study of Parkinson's disease because they contain up to 2% dopaminergic neurons. Fluorescein diacetate, ouabain, ATP, poly-D-lysine and poly-L-lysine were obtained from Sigma (St. Louis, MO). Amantadine, glutamate, kainate, NMDA, MK801 and mazindol were purchased from Research Biochemicals, (Natick, MA). Memantine was a gift of Dr. G. Quack (Merz & Co., Frankfurt, Germany). [3H]ouabain, [3H]dopamine and Aquasol were obtained from New England Nuclear (Boston, MA). Culture media and sera were from Gibco (Grand Island, NY). Sprague-Dawley rat pups and pregnant rats were purchased from Taconic Farms (Germantown, NY). Cerebellar granule neurons were prepared from 8day-old Sprague-Dawley rat pups 2°. The seeding density was 1.5 × 10 6 cells per ml. The cultures were

Correspondence: S.M. Paul, National Institute of Mental Health, Clinical Neuroscience Branch, Building 10, Room 4N224, Bethesda, MD 20892, USA. * Present address: Section of Clinical Immunology, Department of Internal Medicine, University of Ziirich, Hiildeliweg 4, CH-8044 Ziirich, Switzerland.

144 maintained by glucose supplements (5 /zM) on day in vitro ( D I V ) 7 and every third day thereafter. Experiments were p e r f o r m e d on D I V 8-10. N e u r o n a l viability was monitored by phase contrast microscopy and assessed by fluorescein diacetate de-esterification at defined time intervals after exposure to N M D A receptor agonists and antagonists t5'3~. Cortical neurons were p r e p a r e d from 18-day-old rat fetuses as by incubation of the dissected hemispheres in 0.05% trypsin/0.53 m M E D T A for 10 min and subsequent mechanical disruption of the tissue in Ca 2÷- and Mg2+-free saline. The cell pellet was resuspended in m e d i u m containing M i n i m u m Essential M e d i u m Eagle (65%), H a n k ' s balanced salt solution (25%), fetal bovine serum (5%), h u m a n serum (5%), and 33 m M glucose. The seeding density was 10 6 ceils per ml. T h e cultures were maintained by replacing a third of the

m e d i u m volume every third DIV. Experiments were p e r f o r m e d on D I V 12-14. N e u r o n a l viability was monitored by phase contrast microscopy daily and by measuring specific [3H]ouabain binding at various time points after drug exposure 16. Mesencephalic n e u r o n s were p r e p a r e d from 15-dayold rat fetuses 2. T h e seeding density was 1.8 × 10 6 cells per ml, m e d i u m exchanges were p e r f o r m e d as in cortical cultures, experiments were p e r f o r m e d on D I V 1113. Overall neuronal viability was assessed by measuring [3H]ouabain binding 16. Viability of dopaminergic n e u r o n s w a s a s s e s s e d by m e a s u r i n g specific [3H]dopamine uptake 2. Neuronal survival was expressed as ratio between n e u r o n counts in treated cultures versus n o n - t r e a t e d control cultures. N e u r o p r o t e c t i o n was expressed as ratio of neurons which were killed by glutamate alone

Fig. 1. Amantadine and memantine attenuate glutamate toxicity of cultured cerebellar granule neurons. The numerous small, bright neurons can easily be differentiated from the few, large astrocytes. Neuronal enrichment in cerebellar cultures is achieved by administration of 10 /xM cytosine arabinoside on DIV 1 which prevents astrocyte proliferation without affecting neuronal viability. Cultures were pretreated with amantadine or memantine for 1 h and then exposed to glutamate for 24 h. Neuronal survival was assessed using fluorescein diacetate staining by counting viable neurons per representative low power field. Memantine is fully protective while amantadine rescues approximately 25% of the neurons. A: control; B: culture exposed to 100 t~M glutamate; C: culture exposed to 100 tzM amantadine and 100/zM glutamate; D: culture exposed to 30 ~M memantine and 100/xM glutamate. Bar = 100/zm.

145 but were rescued when amantadine or memantine were co-administered along with glutamate, versus the total number of neurons killed by glutamate. Exposure of cultured neurons to glutamate for 24 h resulted in a concentration-dependent neurotoxicity with ECs0s of 30 /xM in cerebellar granule neurons and in the range of 50-150 ~M in the other neurons. Cerebellar granule neurons differed from the other neurons in that neuronal survival was only reduced to 20% at glutamate concentrations exceeding the ECs0 several-fold, while toxicity at these concentrations approached > 95% in the other cultures. Excitotoxicity was readily apparent by a reduction of neuron number and loss of neurites (Figs. 1 and 2). Glutamate toxicity of cortical and cerebellar granule neurons but not of mesencephalic neurons was completely blocked by the non-competitive NMDA receptor antagonist MK-801 (Fig. 3). The degree of neuroprotection afforded by amantadine and memantine differed substantially in the dif-

ferent cultures, memantine consistently being more potent than amantadine but less potent than MK-801. Protection was less apparent in mesencephalic cultures, as assessed by [3H]ouabain binding which reflects overall viability of all neurons present in the cultures. Quantitation of [3H]dopamine uptake to specifically measure glutamate toxicity of dopaminergic neurons in these cultures revealed that memantine enhanced the survival of dopaminergic neurons exposed to glutamate only to a moderate degree. Neither a m a n t a d i n e nor m e m a n t i n e interfered with [3H]dopamine uptake in mesencephalic cultures. The effects of MK-801 (Fig. 3) show that, although glutamate toxicity is mediated by NMDA receptors in cortical and cerebellar cultures, non-NMDA receptor activation plays an important role in glutamate toxicity of mesencephalic cultures. The profile of neuroprotection in the different cultures afforded by memantine and amantadine parallels that of MK-801 and confirms that these drugs act through antagonism at the NMDA

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Fig. 2. Memantine provides partial neuroprotection while amantadine fails to modify glutamate toxicity of mesencephalic neurons. Phase contrast microscopy shows that neurons and the neurite networking are destroyed by glutamate. Debris of dead neurons is abundant, e.g. in the left upper quadrants of panels B and C. A: control; B: culture exposed to 200 /zM glutamate; C: culture exposed to 100 /.~M amantadine and 200 /zM glutamate; D: culture exposed to 50/zM memantine and 200 ~tM glutamate. Bar = 50 ~m.

146 receptor. Survival of dopaminergic neurons exposed to glutamate was hardly enhanced by the adamantanamines, suggesting that these neurons are only moderately susceptible to NMDA receptor-mediated glutamate toxicity and more susceptible to non-NMDA receptor activation.

All neuron types uniformly showed toxic changes which were indistinguishable from the features of excitotoxicity at the light microscopic level when exposed to antagonist concentrations exceeding 100-200 /zM for amantadine and 50-100/zM for memantine alone. Glutamate receptor antagonists are evaluated for

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Fig. 3. A - D : neuronal survival (%) assessed as detailed in the text after exposure to glutamate alone or to glutamate after pre-exposure to amantadine, memantine, or MK-801. A, cerebellar granule neurons. B: cortical neurons. C: mesencephalic neurons; D: mesencephalic (dopaminergic) neurons. Data are representative of triplicate experiments and show neuroprotective effects of amantadine and memantine at concentrations which when applied alone did not affect neuronal viability. Asterisks indicate significant difference as compared to glutamate treatment alone by Student's t-test (* P < 0.01, ** P < 0.001).

147 the management of several neurological disorders. Amantadine and memantine are among the few NMDA receptor antagonists that are available for clinical use. Our findings show that adamantanamines attenuate NMDA receptor-mediated glutamate toxicity of early postnatal cerebellar and to a lesser degree fetal mesencephalic neurons in vitro and confirm their neuroprotective properties in cultured fetal cortical neurons 3'4'12. The substantial differences in neuroprotection afforded by amantadine and memantine in the different neuron preparations in vitro are likely due to the diversity of glutamate receptors expressed in the mammalian brain, i.e. among different populations of neurons 19. The neuroprotective potency of both drugs paralleled roughly the potency of MK-801, confirming that the adamantanamines act at the NMDA receptor. In contrast to cortical and cerebellar granule neurons, glutamate toxicity of the mesencephalic neurons including the dopaminergic population in these cultures is mediated by non-NMDA receptors to a significant degree. Although the concentrations of amantadine and memantine required to attenuate glutamate toxicity in vitro probably exceed the brain concentrations achieved during the pharmacotherapy of Parkinson's disease 32, antagonism of NMDA receptors by these compounds could be responsible for their clinical effects for at least two reasons. First, the relative neuroprotective potency of memantine compared to amantadine in our study corresponds to the 20-fold difference in Ki-values for competition with [3H]MK-801 binding in human frontal cortex 11 and to the higher doses of amantadine used in the pharmacotherapy of Parkinson's disease. Second, our in vitro studies only measure neurotoxic effects of overactive excitatory transmission. Thus, higher concentrations of both drugs may be required to enhance neuronal survival. However, in Parkinson's disease less robust or complete antagonism of glutamate overactivity may be sufficient to ameliorate the motor manifestations of reduced dopaminergic nigrostriatal transmission. Since the latter is likely to be achieved by lower concentrations of amantadine or memantine, the antiparkinsonian actions of these compounds may be mediated via inhibition of overactive glutamatergic striatal terminals which originate from cerebral cortex or subthalamic nucleus and which are released from nigrostriatal dopaminergic inhibition. Recently, inhibition of NMDA receptor-mediated excitation of cholinergic striatal interneurons by adamantanamines has been discussed as a specific mode of action of these drugs in Parkinson's disease 13. Although this is a plausible hypothesis, the clinical observation that adamantanamines and conventional anti-

cholinergics like benztropine, trihexyphenidyl or biperiden cannot substitute for each other in the pharmacotherapy of Parkinson's disease points to alternative neuronal populations as targets of adamantanamine therapy. Non-NMDA receptor activation seems to be the principal pathway of glutamate toxicity of dopaminergic nigrostriatal neurons in vitro. The failure of adamantanamines to protect dopaminergic neurons from glutamate toxicity fails to support the notion that attenuation of disease progression in Parkinson's disease can be achieved by application of these agents. Still, indirect neuroprotective actions of adamantanamines for dopaminergic neurons are conceivable in vivo, e.g. attenuation of NMDA receptor-mediated release of glutamate targeting non-NMDA receptors on dopaminergic neurons. Recent in vivo studies have described neurotoxicity induced by the NMDA receptor antagonists MK-801, ketamine, and phencyclidine22'23'27. Our findings confirm that the therapeutic range of NMDA receptor antagonists may indeed be narrow. Future studies utilizing neuronal cultures should facilitate the characterization of NMDA receptor antagonist toxicity prior to the introduction of new drugs into clinical trials.

Acknowledgement. The authors thank Dr. G. Quack (Merz & Co., Frankfurt, Germany) for a generous supply of memantine, and Dr. J. Kornhuber (Wiirzburg, Germany) for valuable discussions during the preparation of this work.

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