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Biomedical subjects

A H Futerman

Publications and source records attributed to A H Futerman.

At least 37 records · Page 2Linked to original sources
PubMed1999

Up-regulation of neutral glycosphingolipid synthesis upon long term inhibition of ceramide synthesis by fumonisin B1.

In a previous study we observed that long term (5 days) incubation with fumonisin B1 (FB1), an inhibitor of acylation of sphingoid long chain bases to (dihydro)ceramide, resulted in morphological and biochemical changes in 3T3 fibroblasts (Meivar-Levy, I., Sabanay, H., Bershadsky, A. D., and Futerman, A. H. (1997) J. Biol. Chem. 272, 1558-1564). Among these were changes in the profile of synthesis of sphingolipids (SLs) and glycosphingolipids (GSLs). Whereas [3H]globotriaosylceramide ([3H]Gb3) comprised 1.9% of the total [3H]SLs and [3H]GSLs synthesized in control cells, it comprised 16. 5% in FB1-treated cells. We now demonstrate by in vitro analysis that inhibition of ceramide synthesis by FB1 for 5 days results in up-regulation of the activities of three enzymes in the pathway of Gb3 synthesis, namely glucosylceramide, lactosylceramide, and Gb3 synthases; up-regulation is due to an increase in Vmax, with no change in Km values toward lipid substrates. Moreover, molecular analysis (reverse transcriptase-polymerase chain reaction) of glucosylceramide synthase indicated that this enzyme is up-regulated at the transcriptional level. No changes in either the Vmax or Km values of sphingomyelin or of GM3 synthase were detected after FB1 treatment. Analysis of SL and GSL synthesis in cultured cells using [4,5-3H]sphinganine as a metabolic precursor demonstrated that at low substrate concentrations, Gb3 synthesis is favored over GM3 synthesis and glucosylceramide synthesis is favored over sphingomyelin synthesis, whereas the opposite is true at high substrate concentrations. These data demonstrate that GSL synthesis and in particular Gb3 synthesis are tightly regulated in fibroblasts, presumably so as to maintain constant levels of Gb3 on the cell surface.

3T3 Cells↗
PubMed1999

Autoantibodies to the glutamate receptor kill neurons via activation of the receptor ion channel.

Antibodies to the glutamate/AMPA receptor subunit 3 (GluR3), are found in a human epilepsy, Rasmussen's encephalitis [RE], and were hypothesized as the major cause for the neuronal loss, chronic inflammatory changes and epileptic seizures characteristic of the disease. To establish the pathogenic potential and mechanism of action of such antibodies, we raised murine antibodies against specific peptides of the GluR3 protein and studied their ability to bind, activate, and kill neurons. Mice were immunized with two GluR3 specific peptides: GluR3A (amino acids 245-274) and GluR3B (amino acids 372-395), and with a scrambled GluR3B peptide for control. High levels of antibodies to each of these peptides were obtained, with no cross reactivity between them. Antibodies to the GluR3B peptide were found to bind to cultured neurons, evoke GluR ion channel activity, and kill neurons. In contrast, antibodies against GluR3A peptide bound to neurons but failed to activate the receptor or kill neurons. Anti-scrambled-GluR3B antibodies had no effect. Both the activation of the GluRs and the neuronal death induced by anti-GluR3B antibodies were blocked by CNQX, a specific glutamate/AMPA receptor antagonist; killing was independent of complement. This indicates a mechanism of excitotoxicity-neuronal death due to over-activation of the receptor, a phenomenon known to be caused by excess of glutamate. Purified anti-GluR3B IgGs retained the neuronal killing capacity, and killing was completely and specifically blocked by preincubation with the GluR3B peptide. Excitotoxic neuronal death induced by anti-GluR3B antibodies took place primarily via apoptosis. Taken together, these results show that antibodies to a specific peptide of the GluR can kill neurons by an excitotoxic mechanism, thus mimicking the effects of excess of glutamate. This is the first example that antibodies can lead to neuronal death in a non-classical complement-independent manner, via activation of a membranal neurotransmitter receptor.

Amino Acid Sequence↗
PubMed1998

The diffusion of molecules in axonal plasma membranes: the sites of insertion of new membrane molecules and their distribution along the axon surface.

The neuronal cell surface consists of two domains, the somatodendritic and axonal plasma membranes. Each domain serves different functions, and has a different complement of membrane molecules. Since membrane molecules are able to diffuse in the plane of the plasma membrane lipid bilayer, with diffusion coefficients ranging from 10-8 cm 2 s-1 for lipids to 10-10 cm 2 s-1 for proteins, mechanisms must exist to prevent as intermixing of membrane molecules from each domain by diffusion. Presented here is a theoretical analysis of the distribution of axonal molecules in both growing and non-growing axons based on two models for the insertion of these molecules into the axonal membrane, namely insertion exclusively at the distal end of the axon, or insertion with equal probability all along the axon. In all cases, assuming that the molecules have a finite half-life in the axonal membrane, compositional differences between the axonal and somatodendritic membranes can be obtained that are similar to those observed in other polarized cells, even in the absence of a physical barrier to prevent the intermixing of membrane molecules. Moreover, our analyses demonstrate that the diffusion of membrane molecules in the plane of the axonal lipid bilayer is a sufficiently slow process to preclude the possibility that membrane molecules are inserted into axonal membranes at a site remote from their final location, and then move to their final locations for diffusion. Thus, in long axons, for membrane molecules that are localized all along the length of the axon, mechanisms must exist for their insertion in the axonal membrane at sites all along the axon, and not just at the distal end.

Animals↗
PubMed1998

Regulatory roles for sphingolipids in the growth of polarized neurons.

Over the past few years, our laboratory has focused on defining the regulatory roles of sphingolipids at various stages of neuronal development. These studies have been performed using primary cultures of hippocampal neurons, which are unique among neuronal cultures inasmuch as they develop by a well-characterized and stereotypic sequence of events that gives rise to fully differentiated axons and dendrites. The data demonstrate that sphingolipids play at least three distinct roles in regulating neuronal development, namely (1) that ceramide enhances the formation of minor neuronal processes from lamellipodia, (2) that glucosylceramide synthesis is required for both normal and accelerated axon growth, and (3) that at both of these stages of development, ceramide induces apoptotic cell death at high concentrations. These observations are consistent with the possibility that minor process formation and apoptosis are regulated by ceramide-dependent signaling pathways, whereas axonal growth may require GlcCer synthesis to support an intracellular transport pathway.

Animals↗
PubMed1998

Inhibition of sphingolipid synthesis, but not degradation, alters the rate of dendrite growth in cultured hippocampal neurons.

Axonal growth can be disrupted by various treatments that inhibit the synthesis of membrane components or their delivery by microtubule-based transport. In cultured hippocampal neurons, a direct correlation exists between the synthesis of sphingolipids, and particularly the simplest glycosphingolipid, glucosylceramide, and the ability of growth factors to stimulate axonal growth [S. Boldin, A.H. Futerman, J. Neurochem. 68 (1997) 882-885]. We now demonstrate that dendritic growth in hippocampal neurons also requires ongoing sphingolipid synthesis. Upon incubation with fumonisin B1 (FB1), an inhibitor of acylation of sphingoid long-chain bases, dendritic growth rates are approximately 25% slower than those of control cells, resulting in neurons with shorter dendritic arbors and less dendritic branch points per cell, and readily apparent differences in morphology compared to control cells after 10-14 days in culture. In contrast, FB1 had no effect on the initial growth of the minor processes, which are destined to become dendrites, even in cells in which FB1 affected the rate of axon growth. Inhibition of sphingolipid degradation, by incubation with conduritol-B-epoxide (an inhibitor of glucosylceramide degradation) had no effect on dendrite or minor process growth at any stage of development, and no aberrant neurite or ectopic dendrite formation was observed. Together, these data demonstrate that normal dendrite growth in hippocampal neurons requires sphingolipid synthesis, although the molecular requirements for sphingolipid synthesis may differ from those in axons.

Animals↗
PubMed1998

1-Methylthiodihydroceramide, a novel analog of dihydroceramide, stimulates sphinganine degradation resulting in decreased de novo sphingolipid biosynthesis.

1-Methylthiodihydroceramide (10 microM) decreased de novo ceramide biosynthesis by about 90% in primary cultured cerebellar neurons. Accordingly, de novo formation of sphingomyelin and of glycosphingolipids, all of which contain ceramide in their backbone, was reduced in a time- and concentration-dependent manner by up to 80%. Complex sphingolipid synthesis was restored upon addition of dihydroceramide or ceramide, in micromolar concentrations, to the culture medium, suggesting that none of the glycosyltransferases involved in glycosphingolipid biosynthesis is inhibited by this analog. Assays of the enzymes catalyzing sphinganine biosynthesis, as well as its N-acylation to form dihydroceramide, revealed that they were also not affected. In contrast, there was a 2.5-fold increase in the activity of sphinganine kinase. Reduction of de novo sphingolipid biosynthesis by 1-methylthiodihydroceramide is therefore due to its ability to deplete cells of newly formed free sphinganine. As a consequence of depletion of sphinganine levels, 1-methylthiodihydroceramide disrupted axonal growth in cultured hippocampal neurons in a manner similar to that reported for direct inhibitors of sphingolipid synthesis; thus, there was essentially no axon growth after incubation with 1-methylthiodihydroceramide between days 2 and 3, and co-incubation with short acyl chain analogs of ceramide (5 microM) antagonized the inhibition of growth. Interestingly, the D-erythro and the L-threo isomere were equally effective, but the corresponding free base as well as other structurally related compounds did not affect either sphingolipid biosynthesis or neuronal growth.

Animals↗
PubMed1998

The roles of ceramide in the regulation of neuronal growth and development.

Ceramide can be formed by the activity of two general metabolic pathways, the anabolic pathway, in which ceramide is formed by acylation of a sphingoid long chain base, and the catabolic pathway, in which ceramide is formed by the degradation of either glycosphingolipids or of sphingomyelin (SM). The anabolic reactions take place in the early compartments of the secretory pathway (the endoplasmic reticulum and the Golgi apparatus) and the catabolic reactions take place either in lysosomes or at the plasma membrane. Work from our and other laboratories has shown that neuronal growth and development can be regulated by manipulating ceramide metabolism. Thus, synthesis of glucosylceramide from ceramide is required for axonal growth in cultured hippocampal neurons, but the formation of ceramide from SM, by a sphingomyelinase activity, stimulates the earliest stages of development in these cells, namely the formation of minor neuronal processes and the initial formation of the axon. Thus, ceramide and its metabolites play distinct roles in the same neuron, depending on the intracellular site of generation of ceramide and on the stage of neuronal development.

Animals↗
PubMed1998

Distinct roles for sphingolipids and glycosphingolipids at different stages of neuronal development.

Studies on the roles of sphingolipids (SLs) and glycosphingolipids (GSLs) at distinct stages of neuronal development have been performed using primary cultures of hippocampal neurons, which are unique among neuronal cultures inasmuch as they develop by a well-characterized and stereotypic sequence of events that gives rise to fully differentiated axons and dendrites. Our data demonstrate that SLs and GSLs play at least three distinct roles in regulating neuronal development, namely: (i) ceramide enhances the formation of minor neuronal processes from lamellipodia and the subsequent stage of axonogenesis; (ii) glucosylceramide synthesis, but not the synthesis of higher-order GSLs, is required for normal axon growth and for accelerated axonal growth upon stimulation by growth factors; and (iii) at both of these stages, ceramide at high concentrations can induce apoptotic cell death. Together, these observations are consistent with the possibility that minor process formation and apoptosis are regulated by ceramide-dependent signaling pathways, whereas axonal growth requires glucosylceramide synthesis, perhaps to support an intracellular transport pathway.

Animals↗
PubMed1997

Isolation of gangliosides by cloud-point extraction with a nonionic detergent.

A rapid, simple, and efficient chloroform/methanol-free method of isolating gangliosides is described. The nonionic polyoxyethylene detergent, hexaethyleneglycol mono-n-tetradecyl ether (C14EO6), forms clear micellar solutions in water, but two-phase separation can be achieved by centrifugation in the presence of ammonium sulfate at room temperature. A mixture of pure gangliosides, metabolically labeled gangliosides obtained from cultured hippocampal neurons, and gangliosides from rat cerebellar tissue were quantitatively recovered in the detergent-rich upper (coacervate) phase, with a partition coefficient K > 60. Gangliosides were subsequently separated from the detergent using an Iatrobead column prior to analysis by thin-layer chromatography. The procedure described here is as efficient as other methods of ganglioside extraction, such as that using chloroform/methanol/water/pyridine, but is less time-consuming inasmuch as extraction, purification, and TLC analysis can be completed within 1 day.

Animals↗
PubMed1997

Distinct roles for ceramide and glucosylceramide at different stages of neuronal growth.

Sphingolipids (SLs) are important structural and regulatory components of neuronal plasma membranes. Previous studies using fumonisin B1, an inhibitor of the synthesis of ceramide, the precursor of all SLs, demonstrated that ceramide synthesis is required to sustain axonal growth in hippocampal neurons (; ) and dendritic growth in cerebellar Purkinje cells (). We now show that ceramide plays distinct roles at different stages of neuronal development. (1) During axon growth, ceramide must be metabolized to glucosylceramide (GlcCer) to sustain growth. Thus, whereas D-erythro-ceramide, which is metabolized to GlcCer, is able to antagonize the disruptive effects of fumonisin B1 on axon growth, L-threo-ceramide, which is not metabolized to GlcCer, is ineffective. (2) The formation of minor processes from lamellipodia can be stimulated by incubation with short-acyl chain analogs of ceramide that are active in ceramide-mediated signaling pathways, or by generation of endogenous ceramide by incubation with sphingomyelinase. However, GlcCer synthesis is not required for this initial stage of neuronal development. (3) During minor process formation and during axon growth, incubation with high concentrations of ceramide or sphingomyelinase, but not dihydroceramide, induces apoptosis. Together, these observations are consistent with the possibility that minor process formation and apoptosis can be regulated by ceramide-dependent signaling pathways and that the decision whether to enter these diametrically opposed pathways depends on intracellular ceramide concentrations. In contrast, axonal growth requires the synthesis of GlcCer from ceramide, perhaps to support an intracellular transport pathway.

Animals↗
PubMed1997

Parallel increases in [alpha-125I]bungarotoxin binding and alpha 7 nicotinic subunit immunoreactivity during the development of rat hippocampal neurons in culture.

Previous studies have shown that hippocampal neurons cultured at high density express alpha-bungarotoxin binding sites and have alpha 7 nicotinic acetylcholine receptor subunit immunoreactivity [Barrantes, G.E., Rogers, A.T., Lindstrom, J. and Wonnacott, S., Brain Res., 672 (1995) 228-236]. We now examine both of these parameters in well-characterized hippocampal neurons cultured at sufficiently low densities to resolve individual neurons and their processes. The specific binding of [alpha-125I]bungarotoxin is first detectable after 3 days in culture and increases during the next 12 days in culture, reaching a maximum of approximately 30,000 binding sites per cell. This is accompanied, over the same timecourse, by an increase in immunoreactivity for two antibodies that specifically bind to the alpha 7 subunit. Both cell bodies and processes were labelled by 9 days in culture. The timecourse of alpha 7-type nicotinic receptor expression resembles that previously described for synapse formation in hippocampal cultures.

Animals↗
PubMed1997

The role of sphingolipids in the maintenance of fibroblast morphology. The inhibition of protrusional activity, cell spreading, and cytokinesis induced by fumonisin B1 can be reversed by ganglioside GM3.

Previous studies demonstrated that inhibition of sphingolipid synthesis by the mycotoxin fumonisin B1 (FB1) disrupts axonal growth in cultured hippocampal neurons (Harel, R., and Futerman, A. H. (1993) J. Biol. Chem. 268, 14476-14481) by affecting the formation or stabilization of axonal branches (Schwarz, A., Rapaport, E., Hirschberg, K., and Futerman, A.H. (1995) J. Biol. Chem. 270, 10990-10998). We now demonstrate that long term incubation with FB1 affects fibroblast morphology and proliferation. Incubation of Swiss 3T3 cells with FB1 resulted in a decrease in synthesis of ganglioside GM3, the major glycosphingolipid in 3T3 fibroblasts and of sphingomyelin. The projected cell area of FB1-treated cells was approximately 45% less than control cells. FB1 had no affect on the organization of microtubules or intermediate filaments, but fewer actin-rich stress fibers were observed, and there was a loss of actin-rich lamellipodia at the leading edge. Three other processes involving the actin cytoskeleton, cytokinesis, microvilli formation, and the formation of long processes induced by protein kinase inhibitors, were all disrupted by FB1. All the effects of FB1 on cell morphology could be reversed by addition of ganglioside GM3 even in the presence of FB1, whereas the bioactive intermediates, sphinganine, sphingosine, and ceramide, were without effect. Finally, FB1 blocked cell proliferation and DNA synthesis in a reversible manner, although ganglioside GM3 could not reverse the effects of FB1 on cell proliferation. Together, these data suggest that ongoing sphingolipid synthesis is required for the assembly of both new membrane and of the underlying cytoskeleton.

3T3 Cells↗
PubMed1997

Glucosylceramide synthesis is required for basic fibroblast growth factor and laminin to stimulate axonal growth.

To test the hypothesis that neuronal growth requires the synthesis and supply of new membrane components to the growing neurite, we have examined the relationship between the synthesis of sphingolipids and the ability of two growth factors, basic fibroblast growth factor (bFGF) and laminin, to stimulate axonal growth in cultured hippocampal neurons. Both bFGF and laminin stimulate axonal growth by approximately fourfold; but the stimulatory effects of both factors can be abolished completely by two inhibitors of sphingolipid synthesis, fumonisin B1 and D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol. By using these inhibitors, together with two stereoisomers of short acyl chain derivatives of ceramide, only one of which is metabolized to glucosylceramide, we demonstrate that ongoing synthesis of glucosylceramide, the simplest glycosphingolipid, is a prerequisite for both bFGF and laminin to stimulate axon growth. These data imply that the ability of a growth factor to stimulate neuronal growth is dependent on the synthesis of an essential membrane lipid.

4-Chloro-7-nitrobenzofurazan↗
PubMed1997

Determination of the localization of gangliosides using anti-ganglioside antibodies: comparison of fixation methods.

Many studies have examined the localization of gangliosides using anti-ganglioside antibodies, although widely differing conclusions have been reached. We now demonstrate that the apparent localization of gangliosides can be greatly influenced by the fixation method. Using monoclonal antibody (MAb) A2B5 (which reacts with a variety of gangliosides), hippocampal neurons were labeled at the cell surface when incubated with the antibody before fixation, but when incubated after fixation the cells displayed a variety of labeling patterns, depending on the fixation method. Biochemical analysis demonstrated that some of the fixatives (particularly acetone and methanol) significantly reduced or completely depleted cellular gangliosides, implying that the immunoreactivity observed with A2B5, and with other antibodies, was not due to gangliosides. When neurons were incubated with an anti-GD1b antibody prefixation, uniform labeling of the plasma membrane was observed, but after ganglioside depletion using biochemical inhibitors of ganglioside synthesis no cell surface labeling was detected. However, even in cells depleted of gangliosides, labeling of both the cell surface and intracellular compartments was observed when the anti-GD1b antibody was applied after fixation. Moreover, after fixation, antibodies to GM4 and GD2 reacted with hippocampal neurons, although these gangliosides are absent from these neurons. In contrast, the JONES antibody (which reacts with 9-O-acetylated GD3) labeled neurons with a similar pattern, essentially irrespective of the fixation method. These observations demonstrate that great care must be taken in assigning gangliosides to specific cell populations or to intracellular locations solely on the basis of use of anti-ganglioside antibodies, and suggest that optimal fixation conditions must be established for each anti-ganglioside antibody.

Animals↗