A role for gangliosides in astroglial cell differentiation in vitro.

Rat cerebral astroglial cells in culture display specific morphological and biochemical behaviors in response to exogenously added gangliosides. To examine a potential function for endogenous gangliosides in the processes of astroglial cell differentiation, we have used the B subunit of cholera toxin as a ganglioside-specific probe. The B subunit, which is multivalent and binds specifically to GM1 ganglioside on the cell surface, induced a classical star-shaped (stellate) morphology in the astroglial cells and inhibited DNA synthesis in a dose-dependent manner. The morphological response was massive and complete within 2 h, with an ED50 of 0.8 nM, and appeared to depend on the direct interaction of the B subunit with GM1 on the cell surface. A B subunit-evoked inhibition of DNA synthesis and cell division (ED50 = 0.2 nM) was observed when the cells were stimulated with defined mitogens, such as epidermal growth factor and basic fibroblast growth factor. Maximal inhibition approached 80% within 24 h. The effects of the B subunit were unrelated to increases in cAMP. These observations, taken together with previous studies, demonstrate that both endogenously occurring plasma membrane gangliosides and exogenously supplied gangliosides can influence the differentiative state (as judged by morphological and growth behaviors) of astroglial cells in vitro.

Excitatory amino acid receptor agonists stimulate membrane inositol phospholipid hydrolysis and increase cytoplasmic free Ca2+ in primary cultures of retinal neurons.

A variety of neurotransmitters are believed to elicit effects through receptor-stimulated inositol phospholipid metabolism. It appears that most major types of retinal neurons receive a direct glutamatergic input. The aim of the present studies was to characterize excitatory amino acid (EAA) receptor-mediated breakdown of inositol phospholipids and changes in Ca2+ homeostasis in primary avian retinal cell cultures. Cell monolayers, prepared from 8-day-old chick embryo neural retina, were labelled with [3H]inositol for 48 h, and used after 7 days in vitro. Kainic acid stimulated the accumulation of inositol phosphates in a time- and dose-dependent manner (ED50 = 30 microM). The EAA receptor agonists glutamate, N-methyl-D-aspartate (NMDA), ibotenate and quisqualate were all active, with the rank order: glutamate greater than kainate greater than NMDA much greater than ibotenate approximately quisqualate. External Ca2+ was required for these effects. Agonist actions were inhibited by type-specific antagonists, and also Mg2+ in the case of glutamate and NMDA. Glutamate, NMDA and kainate also elevated cytosolic free Ca2+ in individual retinal cells loaded with the Ca2(+)-sensitive dye Fura-2, as assessed by digital fluorescence ratio imaging microscopy. The agonist-induced increases in [Ca2+]i were largely dependent on extracellular Ca2+, independent of membrane depolarization and were blocked by Mg2+ for glutamate and NMDA. These results demonstrate that vertebrate retinal cells possess EAA receptors coupled to intracellular signal transduction pathways.

Exogenously administered gangliosides fail to increase in vivo metastatic frequency or in vitro growth of murine neoplastic cells.

The influence of gangliosides on tumor growth and frequency of metastasis in vivo, as well as growth of neoplastic cells in vitro, was tested utilizing the mouse fibrosarcoma cell line MN4. In mice receiving intramuscular tumor transplants, injections of a ganglioside mixture twice daily did not influence the tumor volume or the number of spontaneous metastases per animal. Furthermore, in mice receiving the cells by tail vein injection, injections of a ganglioside mixture once or twice daily did not affect the number of metastatic foci in the lungs. Preincubation of neoplastic cells with the ganglioside mixture decreased the number of metastatic foci in the lungs of mice receiving the cells by tail i.v. injection. The addition of ganglioside mixture to the culture medium for up to a 48-h incubation period had no effect, independently of the cell density utilized, on either the rate of DNA synthesis or the relative numbers of neoplastic cells as compared to controls; at higher ganglioside concentrations, growth was actually reduced. These results are interpreted to indicate that gangliosides, under the present conditions, do not influence tumor growth and metastatic neoplastic capacity in vivo, and growth in vitro.

Corticotropin-releasing factor (CRF) and related peptides confer neuroprotection via type 1 CRF receptors.

Corticotropin-releasing factor (CRF) receptors are members of the superfamily of G-protein coupled receptors that utilise adenylate cyclase and subsequent production of cAMP for signal transduction in many tissues. Activation of cAMP-dependent pathways, through elevation of intracellular cAMP levels is known to promote survival of a large variety of central and peripheral neuronal populations. Utilising cultured primary rat central nervous system neurons, we show that stimulation of endogenous cAMP signalling pathways by forskolin confers neuroprotection, whilst inhibition of this pathway triggers neuronal death. CRF and the related CRF family peptides urotensin I, urocortin, and sauvagine, which also induced cAMP production, prevented the apoptotic death of cerebellar granule neurons triggered by inhibition of phosphatidylinositol kinase-3 pathway activity with LY294002. These effects were negated by the highly selective CRF-R1 antagonist CP154,526. CRF even conferred neuroprotection when its application was delayed by up to 8 h following LY294002 addition. The CRF peptides also protected cortical and hippocampal neurons against death induced by beta-amyloid peptide (1-42), in a CRF-R1 dependent manner. In separate experiments, LY294002 reduced neuronal protein kinase B activity while increasing glycogen synthase kinase-3, whilst CRF (and related peptides) promoted phosphorylation of glycogen synthase kinase-3 without protein kinase B activation. Taken together, these results suggest that the neuroprotective activity of CRF may involve cAMP-dependent phosphorylation of glycogen synthase kinase-3.

Hypoglycemic neurotoxicity in vitro: involvement of excitatory amino acid receptors and attenuation by monosialoganglioside GM1.

Rat cerebellar granule cells, when subjected to a glucose-free environment for 4 h, developed extensive degeneration of neuronal cell bodies and their associated neurite network over the following 24 h. This neuronal damage was quantitated with a colorimetric assay using the metabolic dye 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide. Hypoglycemic neuronal injury could be markedly reduced by the presence of both competitive (3-(+/-)-2-carboxypiperazin-4-yl)-propyl-1-phosphonic acid) and non-competitive (phencyclidine) N-methyl-D-aspartate receptor antagonists, but not by kainate/quisqualate preferring antagonists 6-cyano-7-nitroquinoxaline-2,3-dione and 6,7-dinitroquinoxaline-2,3-dione. Glucose deprivation neuronal injury was also reduced by adding glutamate-degrading enzymes to the incubation medium. Monosialoganglioside GM1, but not its asialo derivative (lacking sialic acid), was also effective in protecting against hypoglycemic neurodegeneration when included during the period of glucose deprivation. These results suggest that the neuronal injury to cerebellar granule cells resulting from glucose deprivation is mediated predominantly by activation of the N-methyl-D-aspartate type of excitatory amino acid receptor, perhaps through the action of endogenously released glutamate. Furthermore, the monosialoganglioside GM1, a member of a class of naturally occurring sialoglycosphingolipids, is able to attenuate this neuronal injury--as already observed for glutamate neurotoxicity and anoxic neuronal death in cerebellar granule cells. Gangliosides may thus prove to be of therapeutic utility in excitatory amino acid-associated neuropathologies.

Mast cells differentially express and release active high molecular weight neurotrophins.

Nerve growth factor (NGF), a target-derived factor for survival and maintenance of peripheral and central neurons, has been implicated in inflammatory processes. Mast cells are the principal effector cells in IgE-dependent hypersensitivity reactions, and also play a role in diseases characterised by inflammation, including those of the nervous system like multiple sclerosis. Mast cells are capable of synthesising and responding to NGF, although the occurrence of other members of the NGF family of neurotrophins and their protein forms have not been described. Immunoblot analysis with highly selective neurotrophin antibodies has now been used to show that rat peritoneal mast cells express a higher molecular weight form (73 kDa) of NGF, but not the monomeric (13 kDa) NGF polypeptide. Mast cells also expressed 73 kDa forms of neurotrophin-4 and neurotrophin-3; brain-derived neurotrophic factor was not detected. Medium conditioned by degranulating peritoneal mast cells contained similar high molecular weight forms of NGF and neurotrophin-4 on Western blots, but no neurotrophin-3. Mast cell-derived neurotrophin immunoreactivities were inhibited by the respective peptide antigen, further demonstrating the specificity of the mast cell-derived neurotrophic protein. Mast cell-released proteins supported the survival of cultured chicken embryonic neural crest- and placode-derived sensory neurons; neurotrophic activities were inhibited by neutralising antibodies for NGF and neurotrophin-4, respectively. High molecular isoforms of neurotrophins have been reported to occur in experimental colitis and in the inflamed gut of patients with Crohn's disease and ulcerative colitis, tissue sites rich in mast cells. The data suggest an important role for neurotrophins in the pathophysiology of inflammatory disease.

Characterization and growth-dependent regulation of the nerve growth factor receptor gp140trk in rat C6 glioma cells.

The glioma cell line C6 was used to study the expression and growth-dependent regulation of the nerve growth factor (NGF) tyrosine kinase receptor gp140trk, which is the mature protein product of the trk proto-oncogene. Chemical cross-linking of 125I-NGF to C6 cells, followed by immunoprecipitation with polyclonal anti-NGF antibodies and separation by polyacrylamide gel electrophoresis, revealed the presence of 90-95 and 150 kDa species. Immunocytochemical staining of C6 cells with antibodies directed against either the low-affinity NGF receptor gp75NGFR or trk proto-oncogene products demonstrated a heterogeneous cellular distribution of both antigens. Brief treatment of C6 cells with NGF led to the tyrosine phosphorylation of 80, 110 and 140 kDa protein species, as detected on anti-phosphotyrosine Western blots. Similar molecular weight species were found with anti-Trk antibodies in the NGF-treated cells. Intracellular localization of Trk-like immunoreactivity in C6 cells released from a growth-arrested state indicated an initial immunostaining of the nuclear periphery, progressing to cytoplasmic vesicles and finally to the plasma membrane. These observations at the light microscopic level were confirmed using immunoelectron microscopy with the same anti-Trk antibodies, and showed clearly the trafficking of Trk-like immunostained particles from the endoplasmic reticulum to the plasmalemma. The cellular localization of trk gene products also appeared to depend on their glycosylation state. Such growth-dependent expression of NGF receptors on glial cells may be important in controlling autocrine regulatory processes of glia to NGF, which these cells produce.

Neuronal protein kinase signaling cascades and excitotoxic cell death.

Perturbation of normal survival mechanisms may play a role in a large number of disease processes. Glutamate neurotoxicity, particularly when mediated by the N-methyl-D-aspartate (NMDA) subtype of glutamate receptors, has been hypothesized to underlie several types of acute brain injury, including stroke. Several neurological insults linked to excessive release of glutamate and neuronal death result in tyrosine kinase activation, including p44/42 mitogen activated protein (MAP) kinase. To further explore a role for MAP kinase activation in excitotoxicity, we used a novel tissue culture model to induce neurotoxicity. Removal of the endogenous blockade by Mg2+ of the NMDA receptor in cultured hippocampal neurons triggers a self perpetuating cycle of excitotoxicity, which has relatively slow onset, and is critically dependent on NMDA receptors and activation of voltage gated Na+ channels. These injury conditions led to a rapid phosphorylation of p44/42 that was blocked by MAP kinase kinase (MEK) inhibitors. MEK inhibition was associated with protection against synaptically mediated excitotoxicity. Interestingly, hippocampal neurons preconditioned by a sublethal exposure to Mg(2+)-free conditions were rendered resistant to injury induced by a subsequently longer exposure to this insult; the preconditioning effect was MAP kinase dependent. The MAP kinase signaling pathway can also promote polypeptide growth factor mediated neuronal survival. MAP kinase regulated pathways may act to promote survival or death, depending upon the cellular context in which they are activated.

Excitotoxicity, oxidative stress, and the neuroprotective potential of melatonin.

The brain consumes large quantities of oxygen relative to its contribution to total body mass. This, together with its paucity of oxidative defense mechanisms, places this organ at risk for damage mediated by reactive oxygen species. The pineal secretory product melatonin possesses broad-spectrum free radical scavenging and antioxidant activities, and prevents kainic acid-induced neuronal lesions, glutathione depletion, and reactive oxygen species-mediated apoptotic nerve cell death. Melatonin's action is thought to involve electron donation to directly detoxify free radicals such as the highly toxic hydroxyl radical, which is a probable end-product of the reaction between NO. and peroxynitrite. Moreover, melatonin limits NO.-induced lipid peroxidation, inhibits cerebellar NO. synthase, scavenges peroxynitrite, and alters the activities of enzymes that improve the total antioxidative defense capacity of the organism. Melatonin function as a free radical scavenger and antioxidant is likely facilitated by the ease with which it crosses morphophysiological barriers, e.g., the blood-brain barrier, and enters cells and subcellular compartments. Pinealectomy, which eliminates the nighttime rise in circulating and tissue melatonin levels, worsens both reactive oxygen species-mediated tissue damage and brain damage after focal cerebral ischemia and excitotoxic seizures. That melatonin protects against hippocampal neurodegeneration linked to excitatory synaptic transmission is fully consistent with the last study. Conceivably, the decreased melatonin secretion that is documented to accompany the aging process may be exaggerated in populations with dementia.

Gangliosides and neurotrophic factors in neurodegenerative diseases: from experimental findings to clinical perspectives.

A large body of experimental data suggests that neurotrophic molecules and/or substances that facilitate their action could be pharmaceutical agents for neurodegenerative pathologies. In particular, it has been demonstrated that nerve growth factor (NGF) exerts a physiological role for forebrain cholinergic neurons, while brain-derived neurotrophic factor (BDNF) seems to play a relevant role in rescuing dopaminergic neurons following damage. In addition, gangliosides are reported to potentiate neurotrophic factor effects in vitro as well as in vivo. In this study we examined the effects of the monosialoganglioside GM1 in different experimental models. The responsiveness of forebrain cholinergic neurons following NGF +/- GM1 was evaluated by assessing choline acetyltransferase (ChAT) activity in hippocampus, septal area and striatum of behaviorally impaired 24-month-old rats. NGF was intracerebroventricularly (i.c.v.) infused for 2 weeks while GM1 was given systemically for 3 weeks, starting from the beginning of NGF infusion. Moreover, the possible protective effects of GM1 were assessed following exposure of cultured cerebellar granule cells and dopaminergic mesencephalic neurons to different doses of 6-OH-DOPA, a metabolite of the dopamine pathway which has excitotoxic properties and has been hypothesized to participate in the pathology of Parkinson's disease. GM1 treatment to aged rats was seen to potentiate the NGF-induced increase of ChAT activity in the striatum ipsilateral to the NGF infusion. Moreover, in the striatum contralateral to the NGF infusion, GM1 increased ChAT activity above the control values, whereas NGF treatment alone did not affect enzymatic activity. GM1 treatment of cerebellar granule cells and mesencephalic neurons counteracted the dose- and time-dependent neurotoxicity of 6-OH-DOPA. These data support the notion that GM1 might prove useful in treating those pathological conditions where trophic factor deficits and/or excitotoxin-related toxicity play an important role.

Intracellular glutathione levels determine cerebellar granule neuron sensitivity to excitotoxic injury by kainic acid.

Glutathione (GSH) is a key component of the cellular defence cascade against injury caused by reactive oxygen species. Kainic acid (KA) is a potent central nervous system excitotoxin. KA-elicited neuronal death may result from the generation of ROS. The present study was undertaken to characterize the role of GSH in KA-induced neurotoxicity. Cultures of cerebellar granule neurons were prepared from 8-day-old rats, and used at 8, 14 and 20 days in vitro (DIV). Granule neurons displayed a developmental increase in their sensitivity to KA injury, as quantified by an ELISA-based assay with the tetrazolium salt MTT. At DIV 14 and 20, a 30-min challenge with KA (500 microM) reduced cell viability by 45% after 24 h, significantly greater (P<0.01) than the 22% cell loss with DIV 8 cultures. Moreover acute (30 min) KA exposure concentration-dependently reduced intracellular GSH and enhanced reactive oxygen species generation (evaluated by 2', 7'-dichlorofluorescein diacetate). In comparison to control, KA (500 microM) lowered GSH levels in DIV 8 granule neurons by 16% (P=0. 0388), and by 36% (P=0.0001) in both DIV 14 and DIV 20 neurons, after 30 min. Preincubation of granule neurons with the membrane permeant GSH delivery agent, GSH ethyl ester (5 mM), for 30 min significantly increased intracellular GSH content. Importantly, GSH ethyl ester reduced the toxic effects of KA, becoming significant at 1 mM (P=0.007 vs. KA-treated group), and was maximal at >/=2.5 mM (P<0.0001). GSH ethyl ester displayed a similar dose-dependence in its ability to counteract KA-induced depletion of cellular GSH. The data strengthen the notion that cellular GSH levels have a fundamental role in KA-induced neurotoxicity.

Morphological modulation of cultured rat brain astroglial cells: antagonism by ganglioside GM1.

Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents thought to activate cyclic AMP-synthesizing systems. Dibutyryl cyclic AMP (dBcAMP), forskolin and cholera toxin promoted, within 2 h, the near-complete conversion of 1-day-old (D1) astroglial cells from a flat, epithelioid morphology to a stellate (star-shaped) morphology. With all 3 agents, cell susceptibility to morphological change declined with culture age, 5-day-old cultures failing to respond altogether. D1 cultures, after 48 h of treatment, had reverted to the flat morphology. Gangliosides reported to stimulate adenylate cyclase were also tested, using purified GM1 X GM1 failed to stimulate the conversion to stellate morphologies. GM1, however, did affect these astroglial cells by causing a block or reversal of their morphological response to dBcAMP, forskolin or cholera toxin. The GM1 response was specific for the intact ganglioside molecule, asialo GM1 and sialic acid having no effect. Gangliosides GD1a, GD1b and GT1b were also active, being effective at ca. 4-fold lower concentrations. The response to GM1 appeared to involve a direct interaction with the astroglial cell, rather than influencing either substratum or medium components.

A semisynthetic glycosphingolipid (LIGA20) reduces 2,4, 5-trihydroxyphenylalanine neurotoxicity in primary neuronal cultures.

The semisynthetic glycosphingolipid derivative II3Neu5-AcGgOse4-2-d-erythro-1,3-dihydroxy-2-chloro-acetamid e-4-trans- octadacene (LIGA20) attenuated injury induced by the excitotoxic L-dopa metabolite 2,4,5-trihydroxyphenylalanine (TOPA) in cultures of rat cerebellar granule cells when presented simultaneously with TOPA (EC50; 9 microM LIGA20). The natural glycosphingolipid ganglioside GM1 up to 200 microM was not neuroprotective as cotreatment, although pretreatment of cells for 2 h was efficacious. This greater potency and speed of LIGA20 action extended to limiting TOPA-induced death of cultured mesencephalic dopaminergic neurons. These data suggest that LIGA20 may have therapeutic potential for the treatment of disorders associated with excitotoxic processes.

Thiokynurenates prevent excitotoxic neuronal death in vitro and in vivo by acting as glycine antagonists and as inhibitors of lipid peroxidation.

Several derivatives of kynurenic and thiokynurenic acids were synthesized and tested for their ability to protect primary cultures of cerebellar granule cells against excitotoxic damage, and to affect the binding of [3H]glycine ([3H]Gly), [3H]alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid ([3H]AMPA), [3H]3-(2-carboxypiperazine-4-yl-)propyl-1-phosphonic acid ([3H]CPP), [3H]kainic acid and [3H]N-[1-(2-thienyl)cyclohexyl]-3,4-piperidine ([3H]TCP) to rat cortical membranes. Kynurenic and thiokynurenic acid derivatives with one or two halogens in position 5 or 7 were selective glycine antagonists, failing to affect N-methyl-D-aspartate (NMDA), kainate or AMPA sites at micromolar concentrations. 7-Cl-kynurenic, 7-Cl-thiokynurenic, 5,7-diCl-kynurenic and 5,7-diCl-thiokynurenic acids had similar IC50s for displacing [3H]Gly from its strychnine-insensitive site and for reducing the stimulated (0.5 microM NMDA and 1 microM glycine) [3H]TCP binding to cortical membranes. However, 7-Cl-thiokynurenic acid was particularly potent to prevent excitotoxic neuronal death in cultured cerebellar granule cells. This action may be ascribed to inhibition of lipid peroxidation, a property which was demonstrated for the 5- or 7-Cl derivatives of thiokynurenic acid. Furthermore, 7-Cl-thiokynurenic acid reduced excitotoxic damage caused by the injection of quinolinic acid in the rat striatum. Thus, 7-Cl-thiokynurenic acid appears to be a new compound with interesting antiexcitotoxic properties both in vitro and in vivo.

Basic fibroblast growth factor modulates sensitivity of cultured hippocampal pyramidal neurons to glutamate cytotoxicity: interaction with ganglioside GM1.

Basic fibroblast growth factor (bFGF), a polypeptide originally identified as a mitogen for a variety of cells including astroglial cells, also exhibits neurotrophic (survival) effects on a number of neuronal populations, among the latter being hippocampal pyramidal cells. The present study investigated the effects of bFGF on the sensitivity of pyramidal neurons to the excitatory neurotransmitter, glutamate, and possible modulation by monosialoganglioside GM1. Cultures were generated from embryonic day 18 rat hippocampus, and first treated with bFGF at 4-5 days in vitro. Twenty-four hours later, cells were exposed to glutamate (100 microM-1 mM) for a further 24 h in the continued presence of bFGF. The cytotoxic action caused by 200-500 microM glutamate, which normally is present at this culture stage, was reduced by bFGF in a concentration- and time-dependent manner. GM1 (100 microM), given alone 2 h prior to glutamate, also limited this neuronal loss by 50-80%. At lower concentrations, neither bFGF (0.3 ng/ml) nor GM1 (1-10 microM) alone for 24 h was effective in altering neuronal sensitivity to glutamate. However, given together for 24 h these levels of bFGF and GM1 were almost as efficacious as bFGF alone at 3-10 ng/ml. Similar results were obtained with more mature (12 day) cultures. The ability of GM1 to modulate trophic factor actions towards excitatory amino acids makes gangliosides useful tools in the study of central nervous system plasticity and repair processes.

Phospholipids can influence the interconversion of flat epithelial-like and stellate process-bearing astroglial cells in culture: relationships between molecular structure and biological activity.

Secondary cultures of neonatal rat astroglial cells, maintained in a serum-free, chemically defined medium were treated with several agents known to elevate intracellular cyclic AMP levels in these cells. Earlier studies had shown such drugs to induce a process-bearing (stellate) morphology in the astroglial cells, a response that is antagonized or reversed by the presence of exogenously added gangliosides. As a next step in understanding the basis for such an influence on cell morphologics, we have examined in more detail the molecular specificity of this response. In particular, a variety of phospholipids have been used in substitution of GM1 ganglioside. Natural phosphatidic acid (PA), which physicochemically displays lipophilic and hydrophilic bipolarity as does GM1, was fully active in mimicking the effects of GM1. The ED50 for the morphologic effect of PA was 10 microM, similar to that of GM1. Synthetic PAs (oleic, stearic, palmitic, myristic) had no effect up to 50 microM. Relatively long fatty acid chains were thus required for a PA effect. Other phospholipids including phosphatidylserine could not replace PA. Exposure of the cells to phospholipase D to generate endogenous PA from other phospholipids elicited the morphological response as well. These results indicate that the ability of exogenously supplied lipid molecules to modulate astroglial cell behaviors can be assigned, in functional terms, to a class of molecules having the appropriate balance (which includes PA and GM1) between their hydrophobic and hydrophilic domains.

Ganglioside GM1 prevents death induced by excessive excitatory neurotransmission in cultured hippocampal pyramidal neurons.

Rat hippocampal pyramidal neurons in culture, exposed 30 min to Mg(2+)-free, glycine-supplemented medium undergo a selective (about 35%) degeneration over the next 24 h. This neuronal injury appeared to result from excitatory synaptic transmission and subsequent activation of N-methyl-D-aspartate (NMDA) receptors, as cell death could be blocked by tetrodotoxin and NMDA, but not non-NMDA, receptor antagonists. Ganglioside GM1, which has recently been described to protect against excitotoxin-induced damage, also prevented the death of hippocampal neurons associated with the above phenomenon. Gangliosides may be a novel therapeutic tool for brain injury associated with epileptic-like activity.

Gangliosides attenuate the delayed neurotoxicity of aspartic acid in vitro.

The neurotoxic effects of L-aspartate were evaluated in rat cerebellar granule cell cultures. Acute (15 min) exposure to L-aspartate produced a time-dependent, delayed degeneration of neuronal cell bodies and neurites (LD50 about 40 microM) 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 GM1, were also effective in attenuating the cytotoxicity of L-aspartate. The neurotoxic potential of L-aspartate may thus contribute to pathologies involving the action of endogenous excitatory amino acids.

Ganglioside GM1 cooperates with brain-derived neurotrophic factor to protect dopaminergic neurons from 6-hydroxydopamine-induced degeneration.

Brain-derived neurotrophic factor (BDNF) has recently been shown to enhance the survival of dopaminergic neurons in cultures derived from the embryonic rat mesencephalon. In the present study BDNF was found to protect cultured dopaminergic neurons from injury induced by acute exposure to the dopaminergic-selective neurotoxin 6-hydroxydopamine. The BDNF effect was concentration (ED50 approximately 10 ng/ml) and time-dependent, as determined by tyrosine hydroxylase immunocytochemistry. More importantly, subthreshold amounts of BDNF were rendered efficacious in the presence of ganglioside GM1: loss of tyrosine hydroxylase positive cells was reduced from 80% to only 20%. Thus GM1 may provide a fruitful treatment strategy for disorders of dopamine function such as Parkinson's disease.