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S D Skaper

Publications and source records attributed to S D Skaper.

At least 91 records · Page 5Linked to original sources
PubMed1990

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.

Animals↗
PubMed1990

Excitatory amino acid neurotoxicity in cultured retinal neurons: involvement of N-methyl-D-aspartate (NMDA) and non-NMDA receptors and effect of ganglioside GM1.

Cultures of chicken day 8 embryo retinal cells, essentially free of contaminating non-neuronal elements, were used to examine the neurotoxicity of various excitatory amino acid transmitter receptor agonists. At 7 days in vitro, N-methyl-D-aspartate (NMDA), following 24 hr exposure to 0.1-1.0 mM, destroyed 60-70% of the multipolar neurons, but apparently spared photoreceptors. The cytotoxic effect of NMDA was prevented by extracellular Mg2+ or phencyclidine, suggesting a role for the NMDA ion channel; competitive NMDA antagonists were also neuroprotective. The mixed excitatory amino acid receptor agonist glutamate (0.1-1.0 mM) was also neurotoxic (approximately 70% loss of multipolar neurons) and strongly blocked by NMDA (but weakly by non-NMDA) antagonists and Mg2+, indicating a major action at NMDA receptors. As with NMDA, glutamate did not appear to affect photoreceptors. The neurotoxic action of kainate against multipolar retinal neurons, as reported by others, was confirmed here. Kainate neuronal injury was sensitive to the quinoxalinedione non-NMDA antagonists 6,7-dinitroquinoxaline-2,3-dione (DNQX) and 6-cyanoquinoxaline-2,3-dione (CNQX), but not to Mg2+ or phencyclidine. Ibotenate and quisqualate, even at millimolar concentrations, were not neurotoxic. The monosialoganglioside GM1 was also effective in reducing NMDA and non-NMDA agonist neurotoxicity to retinal neurons. Maximal ganglioside benefit required 1-2 hr of pretreatment with 100-200 microM GM1. The percentage of multipolar neurons remaining after the neurotoxin insult approximately doubled with GM1 treatment. Gangliosides may thus have a therapeutic potential in excitatory amino acid-initiated neuropathologies.

Amino Acids↗
PubMed1990

Large scale purification and immunological characterization of human placental nerve growth factor.

Nerve growth factor (NGF) is a protein which plays a critical role in the development and survival of not only peripheral neurons, but possibly also cholinergic brain neurons. The present study describes a procedure for large scale isolation of human NGF of placental origin, and its immunological characterization. A protein species of approximately 26 kDa was obtained, which cross-reacted with antibodies to mouse NGF. Polyclonal and monoclonal anti-mouse NGF antibodies appeared to recognize different bands within this human NGF preparation. Although these polyclonal antibodies recognized both the dimeric and monomeric forms of mouse NGF, the monoclonal antibody recognized only a band corresponding to the dimeric form of mouse NGF.

Antibodies↗
PubMed1990

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.

6-Cyano-7-nitroquinoxaline-2,3-dione↗
PubMed1990

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.

Amino Acids↗
PubMed1990

Hypoxic-ischemic damage and the neuroprotective effects of GM1 ganglioside.

In vitro studies have shown that monosialoganglioside GM1 reduces excitatory amino acid-related neurotoxicity by limiting the downstream consequences of abusive excitatory amino acid receptor stimulation. Systemic administration of GM1 appears to be efficacious in reducing acute neuronal damage and in facilitating medium- and long-term functional recovery after brain injury. We propose that GM1 protective effects in the acute injury phase results from attenuation of excitotoxicity, whereas the functional recovery seen at longer term could reflect GM1 potentiation of neuronotrophic factors. The potential therapeutic efficacy of GM1 administration in humans is suggested by clinical studies demonstrating improved neurologic outcome in stroke patients.

Amino Acids↗
PubMed1989

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.

Animals↗
PubMed1989

Activation of polyphosphoinositide metabolism as a signal-transducing system coupled to excitatory amino acid receptors in astroglial cells.

Excitatory amino acids (EAA) are known to induce an increase in the breakdown of polyphosphoinositides (PI) in brain slices and in dispersed cultures of neurons. We have now used astroglia cultured from newborn rat cerebra to demonstrate that glutamate provokes, in [3H]inositol-labeled cells, an accumulation of inositol phosphates in a time- and concentration-dependent manner. The ED50 value for glutamate was 40 microM. Quisqualate, ibotenate, and kainate were also active, with their relative potencies in the order of quisqualate greater than ibotenate much greater than kainate. No effect was detected with N-methyl-D-aspartate and quinolinic acid in the absence of Mg2+. The nonselective glutamate receptor antagonist gamma-D-glutamylglycine fully inhibited glutamate agonist-induced PI breakdown. A brief pretreatment of the astroglial cells with phorbol esters negated these effects of EAA receptor agonists, suggesting a feedback role for protein kinase C in phospholipase C action. Glutamate also elevated cytosolic free Ca2+ in Fura-2-loaded astroglial cells, as assessed by digital fluorescence imaging microscopy. Since a close metabolic partnership is known to exist between neurons and glia, these findings may have important functional consequences for neural cells in vivo.

Animals↗
PubMed1989

Ganglioside function in the development and repair of the nervous system. From basic science to clinical application.

Gangliosides play important roles in the normal physiological operations of the nervous system, in particular that of the brain. Changes in ganglioside composition occur in the mammalian brain not only during development, but also in aging and in several neuropathological situations. Gangliosides may modulate the ability of the brain to modify its response to cues or signals from the microenvironment. For example, cultured neurons are known to respond to exogenous ganglioside with changes characteristic of cell differentiation. Gangliosides can amplify the responses of neurons to extrinsic protein factors (neuronotrophic factors) that are normal constituents of the neuron's environment. The systemic administration of monosialoganglioside also potentiates trophic actions in vivo and improves neural responses following various types of injury to the adult mammalian central nervous system. The possible molecular mechanism(s) underlying the ganglioside effects may reflect an action in modulating ligand-receptor linked transfer of information across the plasma membrane of the cell.

Animals↗
PubMed1989

Basic fibroblast growth factor promotes the survival and development of mesencephalic neurons in culture.

Neuronotrophic effects of basic fibroblast growth factor (bFGF) have been reported for some central nervous system neurons. Here we report that also rat mesencephalic neurons are responsive to bFGF. bFGF produces a significant increase in the number of neurite-bearing cells, as well as in the degree of their fiber network. The present findings also provide the first evidence that bFGF can affect, in a concentration-dependent manner, at least two defined CNS neuronal populations, i.e., dopaminergic and gabaergic neurons. This effect was quantified by assessing the specific [3H]dopamine and [gamma-14C]aminobutyric acid uptakes with time in culture. Stimulation of uptake was more pronounced for dopaminergic neurons, suggesting a relative specificity in the actions of bFGF. These effects of bFGF were completely blocked by affinity-purified polyclonal antibodies. The possibility that bFGF plays a key role in normal nervous system development or function is discussed.

Animals↗
PubMed1989

Monosialoganglioside GM1 protects against anoxia-induced neuronal death in vitro.

Excitatory dicarboxylic amino acid neurotransmitters, particularly glutamate, have been implicated in mediating neuronal cell injury in brain ischemia-anoxia, epilepsy, and stroke. Glutamate neurotoxicity has been demonstrated in several in vitro models, as well as its prevention by a variety of agents, including several sialic acid-containing glycosphingolipid species, gangliosides. We have now examined ganglioside effects in anoxic exposed cultures of granule cells from Postnatal Day 8 rat cerebellum. Cells between 10 and 12 days in vitro were placed into an anoxic atmosphere or subjected to a chemical model of anoxia by a pulse exposure to rotenone. Widespread neuronal degeneration of neuronal cell bodies and their associated neurite network was seen the following day. These effects on cell vitality at the morphological level were quantitatively confirmed by measuring the photometric reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide to a blue formazan product. This neuronal injury was abolished by the specific N-methyl-D-aspartate receptor noncompetitive antagonists Mg2+, phencyclidine and MK-801, suggesting that this subtype of glutamate receptor is involved in the pathogenesis of anoxic granule cell injury. Pretreatment for 30 to 60 min or more or concurrent treatment with ganglioside GM1 largely prevented the ensuing neuronal death (ED50 = 25 microM), even 4 days later. Degeneration induced by exogenous glutamate was equally reduced. Asialo GM1 (lacking sialic acid) was ineffective. These results are consistent with the observed beneficial effects of the gangliosides in ischemic brain injury models in vivo.

Animals↗
PubMed1988

Peroxidative block of glucose utilization and survival in CNS neuronal cultures.

The search for neuronotrophic factors addressing CNS neurons requires CNS neuronal cell cultures to quantitate putative effects on neuronal survival. Investigation of neurons dissociated from several embryonic CNS tissues have shown that their short-term survival requires supplementation of the culture medium with either pyruvate or the enzyme catalase. Pyruvate can be replaced with alpha-ketoglutarate or oxaloacetate, or with amino acids capable to transaminate to these three metabolites in the presence of exogenous alpha-ketoacid acceptors. Experiments were designed to evaluate the ability of cultured CNS neurons to utilize glucose as their primary source. We show that: (1) catalase requires the availability of glucose in the medium in order to exert its neuronal maintenance effect, (2) in the absence of catalase, the cells are unable to metabolize glucose through the tricarboxylic acid cycle, (3) catalase restores the neuronal ability to utilize glucose for oxydative metabolism, and renders redundant the use of other sources such as glutamate conversion to alpha-ketoglutarate, (4) graded concentrations of glucose in the medium affect in parallel these metabolic activities and the viability of the cultured neurons, and (5) anti-oxidant agents other than catalase mimic the catalase effects. We conclude that dissociated embryonic CNS neurons suffer from a block in glucose utilization which results from an imbalance between free radical attack and cellular defenses to it and speculate on a more general involvement of peroxidation damage in the trophic requirements for neuronal survival.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Inhibition of DNA synthesis in C6 glioma cells following cellular incorporation of GM1 ganglioside and choleragenoid exposure.

The B subunit of cholera toxin, which is multivalent and binds specifically to GM1 ganglioside on the cell surface, has previously been used as a ganglioside-specific probe to regulate DNA synthesis in thymocytes and fibroblasts. To explore in more detail this growth-regulatory action of gangliosides, C6 glioma cells (which are GM1 ganglioside deficient) were used as a model system. When cultures of C6 cells were first treated with GM1, followed by exposure to the B subunit, proliferation was inhibited, as measured by 3H-labeled thymidine incorporation into DNA. Pretreatment of the cells with 50 microM GM1 for 15 min (followed by washing with fetal calf serum) and incubation with 1 microgram/ml of B subunit for 21 h was sufficient to reduce DNA synthesis to 15% of control values (and confirmed by autoradiographic analysis), although maximal inhibition could be achieved with as little as 30 min exposure to B, followed by washing. Furthermore, the B subunit inhibited the response of the C6 cells to basic fibroblast growth factor only following GM1 pretreatment. The B subunit-induced inhibition of DNA synthesis was specific for the ganglioside GM1, and was unrelated to increases of cyclic AMP. These results demonstrate that cell-incorporated GM1 ganglioside may act as a receptor capable of undergoing a specific ligand interaction, subsequently affecting molecular processes at the nuclear level.

Animals↗
PubMed1988

Growth factors.

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Animals↗