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.

Ganglioside modulation of neural cell adhesion molecule and N-cadherin-dependent neurite outgrowth.

We have used monolayers of control 3T3 cells and 3T3 cells expressing transfected human neural cell adhesion molecule (NCAM) or chick N-cadherin as a culture substrate for PC12 cells. NCAM and N-cadherin in the monolayer directly promote neurite outgrowth from PC12 cells via a G-protein-dependent activation of neuronal calcium channels. In the present study we show that ganglioside GM1 does not directly activate this pathway in PC12 cells. However, the presence of GM1 (12.5-100 micrograms/ml) in the co-culture was associated with a potentiation of NCAM and N-cadherin-dependent neurite outgrowth. Treatment of PC12 cells with GM1 (100 micrograms/ml) for 90 min led to trypsin-stable increases in both beta-cholera toxin binding to PC12 cells and an enhanced neurite outgrowth response to N-cadherin. The ganglioside response could be fully inhibited by treatment with pertussis toxin. These data are consistent with exogenous gangliosides enhancing neuritic growth by promoting cell adhesion molecule-induced calcium influx into neurons.

Hypoxanthine-guanine phosphoribosyltransferase mutant glioma cells: diminished monamine oxidase activity.

A defective capability of cultured rat glioma cells to reutilize purine bases (hypoxanthine-guanine phosphoribosyltransferase deficiency) was associated with a reduced capacity to oxidatively deaminate serotonin and tryptamine. The mutant glioma cells were also more sensitive to the cytotoxic effects of serotonin than were normal cells

Cholera toxin-peroxidase: changes in surface labeling of glioblastoma cells with increased time in tissue culture.

Cholera toxin coupled to peroxidase yielded a highly specific ultrastructural marker of plasma membrane monosialogangliosides. Studies with cultures of brain and brain tumors suggested that long-term culture of tissue in monolayers results in eventual loss of surface monosialogangliosides.

Brain injury causes a time-dependent increase in neuronotrophic activity at the lesion site.

A cavity was made in the brain (entorhinal cortex) of developing or adult rats, and a small piece of Gelfoam was emplaced to collect fluid secreted into the wound. The neuronotrophic activity of the fluid was assayed with sympathetic and parasympathetic neurons in culture. The results show that wounds in the brain of developing or adult rats stimulate the accumulation of neuronotrophic factors and that the activity of these factors increases over the first few days after infliction of the damage.

Nerve growth factor: from neurotrophin to neurokine.

Nerve growth factor (NGF) is largely known as a target-derived factor responsible for the survival and maintenance of the phenotype of specific subsets of peripheral neurones and basal forebrain cholinergic nuclei during development and maturation. However, NGF also exerts a modulatory role on sensory, nociceptive nerve physiology during adulthood that appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. Other NGF-responsive cells are now recognized as belonging to the haemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. The concentration of NGF is elevated in a number of inflammatory and autoimmune states in conjunction with an increased accumulation of mast cells. Mast cells and NGF appear to be involved in neuroimmune interactions and tissue inflammation, with NGF acting as a general 'alert' molecule capable of recruiting and priming tissue defence processes following insult as well as systemic defensive mechanisms. Moreover, mast cells themselves produce NGF, suggesting that alterations in normal mast cell behaviours can provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states. This review discusses recent discoveries involving novel and diverse biological activities of this fascinating molecule.

Cell signalling cascades regulating neuronal growth-promoting and inhibitory cues.

During development of the nervous system, neurons extend axons over considerable distances in a highly stereospecific fashion in order to innervate their targets in an appropriate manner. This involves the recognition, by the axonal growth cone, of guidance cues that determine the pathway taken by the axons. These guidance cues can act to promote and/or repel growth cone advance, and they can act either locally or at a distance from their place of synthesis. The directed growth of axons is partly governed by cell adhesion molecules (CAMs) on the neuronal growth cone that bind to CAMs on the surface of other axons or non-neuronal cells. In vitro assays have established the importance of the CAMs (N-CAM, N-cadherin and the L1 glycoprotein) in promoting axonal growth over cells, such as Schwann cells, astrocytes and muscle cells. Strong evidence now exists implicating the fibroblast growth factor receptor tyrosine kinase as the primary signal transduction molecule in the CAM pathway. Cell adhesion molecules are important constituents of synapses, and CAMs appear to play important and diverse roles in regulating synaptic plasticity associated with learning and memory. Negative extracellular signals which physically direct neurite growth have also been described. The latter include the neuronal growth inhibitory proteins Nogo and myelin-associated glycoprotein, as well as the growth cone collapsing Semaphorins/neuropilins. Although less well characterised, evidence is now beginning to emerge describing a role for Rho kinase-mediated signalling in inhibition of neurite outgrowth. This review focuses on some of the major themes and ideas associated with this fast-moving field of neuroscience.

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.

Ca2+ channels and intracellular Ca2+ stores in neuronal and neuroendocrine cells.

Changes in [Ca2+]i are essential in modulating a variety of cellular functions. In no other cell type does the regulation of [Ca2+]i reach the level of sophistication observed in cells of neuronal origin. Because of its physicochemical characteristics, the fluorescent Ca2+ indicator Fura-2 has become extremely popular among neuroscientists. The use of this probe, however, has generated a number of problems, in particular, extracytosolic trapping and leakage from intact cells. In the first part of this contribution we briefly discuss the practical application of Fura-2 to the study of [Ca2+]i in primary cultures of neurons and astrocytes. In the second part, we review some recent data (mainly from our laboratories) obtained in neurons and neuroendocrine cells, concerning the regulation of different types of Ca2+ channels and the role and mechanism of intracellular Ca2+ mobilization. The experimental evidence supporting the existence of a previously unrecognised organelle, the calciosome, that we hypothesize represents the functional equivalent in non-muscle cells of sarcoplasmic reticulum, will also briefly be discussed.

Colocalization of low- and high-affinity NGF receptors on PC12 cells, C6 glioma cells and dorsal root ganglion neurons.

The biological responsiveness of neural cells to nerve growth factor (NGF) appears to require expression and ligand binding to both the low-affinity NGF receptor (LNGFR) and the proto-oncogene product trk, the latter being a receptor tyrosine kinase. Immunolocalization of the LNGFR and the high-affinity component of the NGF receptor, trk (HNGFR) was studied by electron microscopic morphometric analysis on cultured PC12 pheochromocytoma cells, C6 glioma cells and neonatal rat dorsal root ganglia neurons using a double immunogold labeling technique. Two receptor-specific antibodies, anti-LNGFR monoclonal antibody 192-IgG and a polyclonal antibody against the 14 carboxy-terminal amino acids of the Trk protein, were utilized in conjunction with immunoglobulin conjugated to colloidal gold particles of different sizes. All cells treated with NGF (50 ng/ml) displayed significant colocalization of LNGFR/HNGFR-like immunoreactivity. Gold particles associated with LNGFR (LNGFR-like immunoreactivity) were frequently seen near 2 or 3 (or more) particles delineating the HNGFR on all cell surfaces. Positive Trk-like immunoreactivity (HNGFR) thus seems to localize in close proximity to LNGFRs in at least these cell types.

Nerve growth factor: a neurokine orchestrating neuroimmune-endocrine functions.

Nerve growth factor (NGF) is widely recognized as a target-derived factor responsible for the survival and maintenance of the phenotype of specific subsets of peripheral neurons and basal forebrain cholinergic nuclei during development and maturation. Other NGF-responsive cells are now known to belong to the hemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. The concentration of NGF is elevated in a number of inflammatory and autoimmune states in conjunction with increased accumulation of mast cells. Mast cells and NGF appear to be involved in neuroimmune interactions and tissue inflammation. Mast cells themselves are capable of producing and responding to NGF, suggesting that alterations in mast cell behavior may trigger maladaptive neuroimmune tissue responses, including those of an autoimmune nature. Moreover, NGF exerts a modulatory role on sensory nociceptive nerve physiology in the adult, and appears to correlate with hyperalgesic phenomena occurring in tissue inflammation. NGF can thus be viewed as a multifactorial modulator of neuroimmune-endocrine functions.

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.

Synthesis of the biologically active beta-subunit of human nerve growth factor in Escherichia coli.

The gene (NGFB) encoding the beta subunit of mature human nerve growth factor (hNGFB) was subcloned into the pJLA503 expression vector under the control of bacteriophage promoters PR and PL, and expressed in Escherichia coli. The recombinant protein represented approximately 3% of the total cellular protein. Biologically active hNGFB was solubilized (0.2% total NGFB) and purified by cation-exchange chromatography and it yielded two bands on polyacrylamide-gel electrophoresis under nonreducing conditions, corresponding to the monomeric (14 kDa) and homodimeric (26.5 kDa) forms of the molecule. Both hNGFB forms were immunopositive on Western blots with rabbit anti-NGFB antibodies; however, following additional purification, only the species corresponding to the hNGFB homodimer was biologically active on cultured chicken dorsal root ganglion neurons. These results demonstrate the feasibility of synthesizing the biologically active form of hNGFB in E. coli.

Quercetin protects cutaneous tissue-associated cell types including sensory neurons from oxidative stress induced by glutathione depletion: cooperative effects of ascorbic acid.

Oxidation reactions are essential biological reactions necessary for the formation of high-energy compounds used to fuel metabolic processes, but can be injurious to cells when produced in excess. Cutaneous tissue is especially susceptible to damage mediated by reactive oxygen species and low-density lipoprotein oxidation, triggered by dysmetabolic diseases, inflammation, environmental factors, or aging. Here we have examined the ability of the flavonoid quercetin to protect cutaneous tissue-associated cell types from injury induced by oxidative stress, and possible cooperative effects of ascorbic acid. Human skin fibroblasts, keratinocytes, and endothelial cells were cultured in the presence of buthionine sulfoximine (BSO), an irreversible inhibitor of glutathione (GSH) synthesis. Depletion of intracellular levels of GSH leads to an accumulation of cellular peroxides and eventual cell death. Quercetin concentration-dependently (EC50: 30-40 microM) reduced oxidative injury of BSO to all cell types, and was also effective when first added after BSO washout. BSO caused marked decreases in the intracellular level of GSH, which remained depressed in quercetin-protected cells. Ascorbic acid, while by itself not cytoprotective synergized with quercetin, lowered the quercetin EC50 and prolonged the window for cytoprotection. The related flavonoids rutin and dihydroquercetin also decreased BSO-induced injury to dermal fibroblasts, albeit less efficaciously so than quercetin. The cytoprotective effect of rutin, but not that of dihydroquercetin, was enhanced in the presence of ascorbic acid. Further, quercetin rescued sensory ganglion neurons from death provoked by GSH depletion. Direct oxidative injury to this last cell type has not been previously demonstrated. The results show that flavonoids are broadly protective for cutaneous tissue-type cell populations subjected to a chronic intracellular form of oxidative stress. Quercetin in particular, paired with ascorbic acid, may be of therapeutic benefit in protecting neurovasculature structures in skin from oxidative damage.

The complete mature bovine prion protein highly expressed in Escherichia coli: biochemical and structural studies.

According to the 'protein only' hypothesis, modification of the 3-dimensional fold of the constituent cellular protein, PrP(C), into the disease-associated isoform, PrP(Sc), is the cause of neurodegenerative diseases in animals and humans. Here we describe the high-level synthesis in Escherichia coli, and purification in the monomeric form, of a histidine-tagged full-length mature PrP (25-249) of bovine brain, termed His-PrP. Based on biochemical and spectroscopic data, His-PrP displays characteristics expected for the PrP(C) isoform. The reported expression system should allow the production of quantities of bovine PrP(C) sufficient to permit 3-dimensional structure determinations.

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.

Poly(ADP-ribosyl)ation enzyme-1 as a target for neuroprotection in acute central nervous system injury.

Poly(ADP-ribose) polymerase 1 (PARP-1) protects the genome by functioning in the DNA damage surveillance network. In response to stresses that are toxic to the genome, PARP-1 activity increases substantially, an event that appears crucial for maintaining genomic integrity. Massive PARP-1 activation, however, can deplete the cell of NAD(+) and ATP, ultimately leading to energy failure and cell death. The discovery that cell death may be suppressed by PARP inhibitors or by deletion of the parp-1 gene has prompted a great deal of interest in the process of poly(ADP-ribosyl)ation. Suppression of PARP-1 is capable of protecting against cerebral and cardiac ischemia, 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine-induced parkinsonism, traumatic spinal cord injury, and streptozotocin-induced diabetes. The secondary damage of initially surviving neurons in brain stroke accounts for most of the volume of the infarcted area and the subsequent loss of brain function. Microglial migration is strongly controlled in living brain tissue by expression of the integrin CD11a, which is regulated in turn by PARP-1, proposing that PARP-1 downregulation may therefore be a promising strategy in protecting neurons from this secondary damage, as well. As PARP-1 is now recognised as playing a role also in the regulation of gene transcription, this further increases the intricacy of poly(ADP-ribosyl)ation in the control of cell homeostasis and challenges the notion that energy collapse is the sole mechanism by which poly(ADP-ribose) formation contributes to cell death. PARP(s) might regulate cell fate as essential modulators of death and survival transcriptional programs with relation to NF-kappaB and p53, proposing that inhibitors of poly(ADP-ribosyl)ation could therefore prevent the deleterious consequences of neuroinflammation by reducing NF-kappaB activity.

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.

Nerve growth factor and autoimmune diseases.

The initiation of a humoral immune response to a foreign antigen is a complex biologic process involving the interaction of many cell types and their secreted products. Autoimmune diseases, which are characterized by an abnormal activation of the immune system, probably result from the failure of normal self-tolerance mechanisms. The etiology of such illnesses, however, is far from being understood. While there have been extensive studies on the participation of the immune and endocrine systems in autoimmune diseases, few have dealt with nervous system-mediated immunoregulation in such situations. Evidence continues to grow suggesting that nerve growth factor (NGF), first identified for its activity in promoting the growth and differentiation of sensory and sympathetic neurons, may exert a modulatory role on neuroimmunoendocrine functions of vital importance in the regulation of homeostatic processes. Newly detected NGF-responsive cells belong to the hemopoietic-immune system and to populations in the brain involved in neuroendocrine functions. NGF levels are elevated in a number of autoimmune states, along with increased accumulation of mast cells. NGF and mast cells both appear to be involved in neuroimmune interactions and tissue inflammation. Moreover, mast cells themselves synthesize, store, and release NGF, proposing that alterations in normal mast cell behaviors may provoke maladaptive neuroimmune tissue responses whose consequences could have profound implications in inflammatory disease states, including those of an autoimmune nature. This review focuses on these cellular events and presents a working model which attempts to explain the close interrelationships of the neuroendocrinoimmune triade via a modulatory action of NGF.