Ganglioside stimulation of axonal sprouting in vitro.

Bovine brain gangliosides were applied to primary and established neuronal cultures to examine the role of gangliosides in neuronal development. Media containing gangliosides enhanced the degree of axonal elongation exhibited by sensory ganglia neurons and increased the length and number of Neuro-2a neuroblastoma cell processes. Ganglioside-supplemented media caused a twofold increase in ornithine decarboxylase activity in both culture systems. These experiments suggest that gangliosides function as acceptor molecules for growth-promoting substances in embryonic and tumor-derived neurons.

Dibutyryl cyclic adenosine monophosphate stimulation of colcemid-inhibited axonal elongation.

Adenosine 3',5'-monophosphate (cyclic AMP) and its dibutyryl derivative induce a variety of morphological changes, including those associated with in vitro axonal maturation. Established sensory ganglia treated with dibutyryl cyclic AMP show significant increases in average axonal length and number in comparison with controls; those treated with maintenance doses of Colcemid show no increases in either parameter; simultaneous treatment with both agents results in growth statistically similar to that produced by dibutyryl cyclic AMP alone. The data are consistent with our hypothesis that cyclic AMP promotes axonal elongation by stimulating microtubule assembly from a preexisting subunit pool.

Cyclic adenosine monophosphate stimulation of axonal elongation.

Elevated concentrations of adenosine 3',5'-monophosphate induce a variety of cell movements. The role of adenosine 3',5'-monophosphate in promoting those movements associated with growth prompted our study of in vitro microtubule-dependent axonal elongation. Ganglia treated with adenosine 5'-monophosphate show no enhancement over controls; treatment with adenosine 3', 5'monophosphate or its dibutyryl derivative significantly enhances elongation, as measured by increases in both axonal numbers and length. Our study suggests that adenosine 3',5'-monophosphate promotes elongation by stimulation of microtubule assembly.

The dynamic distribution of fluoro-gold and its interrelation with neural nitric oxide synthase following intracerebroventricular injection into rat brain.

We mapped the dynamic distribution of fluoro-gold (FG) within rat brain following intracerebroventricular (icv) injection into the lateral ventricle and observed its interrelation with neural nitric oxide synthase (nNOS) using FG fluorescent microphotography combined with nNOS immunohistochemistry. We also detected the amount of icv administered FG entering the peripheral circulation using a fluorescence microplate assay. The degree of periventricular penetration of FG was significantly increased over time. At 2 min after icv injection, FG primarily labeled the choroid plexus in the lateral and third ventricles, with limited penetration into the ependyma and the subependyma of the same ventricles. Some FG/nNOS-double labeled cerebrospinal fluid-contacting neurons were observed in these ventricles as well. At 15 and 30 min, FG penetrated mainly into forebrain ventricular organs and parenchymal structures. Many FG/nNOS double labeled neurons were found at each of these sites. In addition, at 30 min intense FG labeling was found in the hypophysis, while limited periventricular penetration of FG was detected in the hindbrain circumventricular areas. In the peripheral circulation, a low concentration of FG was detected 2 min after icv injection. The concentration increased slowly, peaked at 20 min, then gradually decreased until the end of the experiment at 30 min. These findings indicate that dynamic penetration of icv administrated agents into the periventricular tissues and peripheral circulation should be considered when designing icv experiments.

Improved immunoelectron microscopic method for localizing cytoskeletal proteins in Lowicryl K4M embedded tissues.

We have modified the Lowicryl K4M low-temperature dehydration and embedding procedure for immunoelectron microscopy to provide improved ultrastructural detail and facilitate the localization of actin and tubulin in isolated rat adrenocortical cells, chick spinal cord with attached dorsal root ganglia (SC-DRG), and cultured dorsal root ganglia (DRG). Cells and tissues were fixed for immunocytochemistry either in a mixture of 2% paraformaldehyde and 0.25% glutaraldehyde (0.1 M PIPES buffer, pH 7.3) or in a mixture of 0.3% glutaraldehyde and 1.0% ethyldimethylaminopropylcarbodiimide (0.1 M phosphate buffered saline, pH 7.3). Dehydration was in ethanol at progressively lower temperatures to -35 degrees C. Infiltration at -35 degrees C was followed by ultraviolet polymerization at -20 degrees C. Comparable samples were fixed in glutaraldehyde and osmium tetroxide and embedded in Epon 812 or Epon-Araldite. Post-embedding immunostaining of thin sections utilized commercially available monoclonal antibodies to tubulin and actin followed by the protein A-gold technique (Roth et al., Endocrinology 108:247, 1981). Actin immunoreactivity was observed at the periphery of mitochondria and between mitochondria and lipid droplets in rat adrenocortical cells and at the periphery of neuronal cell processes of SC-DRG. Tubulin immunoreactivity was associated with microtubules throughout neurites of cultured DRG. Our modified technique allows preservation of ultrastructural details as well as localization of antigens by immunoelectron microscopy.

Lysosomes and proteolytic enzyme activities in cultured striated muscle cells.

Primary cell cultures prepared from chick embryonic skeletal muscle and the rat myogenic line L6 were examined morphologically and biochemically during several stages of development. The L6 cells were cultured to provide three morphologically distinct populations: prefusion, postfusion, and a subclone of cells that did not fuse even at high density. Ultrastructural studies revealed the characteristic morphology of healthy myoblasts. Acridine orange staining and cytochemical localization of acid phosphatase suggest the presence of presumptive lysosomal material. Enzymatic studies of lysosomal cathepsins B, D, H, and L revealed unusually high enzyme specific activities in these homogeneous myoblast populations. No activity was detected for the two nonlysosomal enzymes Ca2+-proteinase and serine proteinase. It is suggested that the lysosomal apparatus and its complement of enzymes play a significant role in the differentiation of muscle myotubes.

The effects of dimethyl sulfoxide on neurite development in vitro.

In these studies, DMSO at concentration above 1% was shown to be a potent inhibitor of neurite maturation in vitro. The inhibition was not reversible, and occurred in both newly formed and established neurites. In the presence of DMSO, the addition of NGF resulted in a general shift of the dose-response curve. The data indicate that at levels of DMSO below 5%, DRG neurons are capable of responding to NGF stimulation. It has been suggested that DMSO blocks a specific process associated with neurite extension. These studies suggest that the inhibitory action of membranous organelles in the developing neurite, a finding consistent with previous speculation that DMSO is responsible for an increased intracellular disorganization.

Ultrastructure of a new microtubule-neurofilament coupler in nerves.

A new structure associated with the surfaces of neuronal microtubules is described which connects microtubules to neurofilaments in the axonal processes of cultured chick sensory ganglia. These couplers consist of a spherical core particle (15 nm in diameter) from which radiate several thin filaments (4 nm in diameter). Connection of adjacent microtubules and neurofilaments is achieved by thin filaments radiating from core particles positioned between these cytoskeletal elements. Couplers are most conspicuous in regions of axonal processes containing widely separated microtubules and neurofilaments. The structure and distribution of these couplers suggests that they are directly involved in intra-axonal organelle movements, possibly by modulating the spatial separation of adjacent microtubules and neurofilaments, thereby allowing the passage of transported organelles.

Neuro-2a neuroblastoma cells form neurites in the presence of taxol and cytochalasin D.

We have examined the role of microtubules and microfilaments in neurite outgrowth by chemically modifying their interaction in Neuro-2a neuroblastoma cells. Cells exposed to taxol (1 microM), an agent that promotes microtubule polymerization and stabilization, did not form neurites over a 24 h period. Similarly, cells exposed to cytochalasin D (4 microM), an agent which promotes microfilament depolymerization, did not develop neurites. However, cells treated simultaneously with taxol (1 microM) and cytochalasin D (4 microM) produced long (50 microns) thin, unbranched neurites. Neurites formed during this simultaneous treatment grew in a circular pattern, lacked typical growth cones, were packed densely with microtubules and were deficient in microfilaments. Untreated cells maintained in control medium for 24 h formed short (15 microns), thick, highly branched neurites containing a dense meshwork of microtubules, microfilaments and neurofilaments. These results demonstrate that taxol does not block neurite outgrowth from Neuro-2a cells maintained under microfilament-limiting conditions. They suggest further that microtubules may provide the major cytoskeletal framework for neurite elongation.

Ganglioside-mediated enhancement of the cytoskeletal organization and activity in neuro-2a neuroblastoma cells.

Our previous studies have demonstrated that a mixture of bovine brain gangliosides ( BBG ) applied to Neuro-2a neuroblastoma cells markedly increased the degree and rate of neurite formation. In the present study, the cytoskeletal basis for BBG -mediated neurite outgrowth was investigated by comparing cells grown in the presence or absence of BBG (250 micrograms/ml). After 24-48 h, neurite morphology and the distribution of cytoskeletal components were analyzed with correlative whole-cell transmission electron microscopy, thin-section transmission electron microscopy and scanning electron microscopy. BBG treatment enhanced markedly the organization of the microfilamentous system, and had a less pronounced effect on the number and organization of microtubules. The most prominent changes in microfilament organization were in the distal segment of the neurite and the growth cone. BBG -treated cells had a complex cytoskeletal consisting of numerous bundles of microfilaments. These filament bundles were distributed into the secondary and teritary neuritic branches. Cells grown in serum-depleted medium to stimulate neurite outgrowth, lacked these bundles of microfilaments, suggesting that the formation of microfilament bundles was not required for non- BBG -mediated neuritogenesis . The role that the cytoskeletal components play in BBG -induced neurite outgrowth was examined following disruption of microtubules or microfilaments with Colcemid and cytochalasin D, respectively. Simultaneous treatment of cells with BBG and Colcemid (0.25 microgram/ml) at the time of plating resulted in cells with numerous spine-like projections which did not extend neurites. In contrast, the simultaneous treatment of cells with BBG and cytochalasin D (2 micrograms/ml) at the time of plating resulted in cells devoid of spines, but exhibiting anomalous neurite outgrowth consisting of many long, thin, unbranched neurites. These neurites lacked characteristic flattened growth cones and had a tendency to grow in a circular fashion. These results demonstrate that neurite outgrowth under microfilament-limiting conditions results in reduced neuritic branching while growth under microtubule-limiting conditions allows initiation, but prevents significant elongation. The different neuritic growth patterns induced by serum deprivation, ganglioside treatment or the various cytoskeletal disruptive agents reflect changes in the organization of microtubules and microfilaments. Our studies suggest that the organizational state and activity of these cytoskeletal elements determine neurite morphology. Microfilaments appear to be the primary determinants in ganglioside-mediated growth.

Neuritogenic and metabolic effects of individual gangliosides and their interaction with nerve growth factor in cultures of neuroblastoma and pheochromocytoma.

The 4 major ganglioside species, GM1, GD1a, GD1b and GT1b (200 micrograms/ml), were tested individually for the ability to stimulate neuronal trophic responses. The growth parameters measured were: morphologic changes, quantitated by computer-assisted morphometry of neurite length and number per soma, and metabolic changes, indicated by alterations in ornithine decarboxylase activity (ODC). In addition, the interaction of each ganglioside with nerve growth factor (NGF) was investigated with an NGF-responsive pheochromocytoma PC12 cell line and NGF-insensitive neuroblastoma Neuro-2a cultures. PC12 cells responded to gangliosides only in the presence of NGF (20 micrograms/ml): GM1 produced the greatest morphologic response, but did not alter metabolic levels; GT1b increased both parameters. The presence (5 micrograms/ml) or absence of NGF did not have an effect on the ganglioside-mediated morphologic responses of Neuro-2a cells to each species: GD1b elicited the greatest increase in neurite length, while GD1a and GT1b stimulated both length and number. In contrast, while GT1b alone was able to elevate ODC activity independently of NGF, the simultaneous exposure of Neuro-2a cultures to NGF and GM1 or GD1a resulted in a stimulation of cellular metabolism. These results indicate that each ganglioside species has a specific target action in the stimulation of different trophic responses and that performance in one category is not a predictor of the result in another. In addition, it is possible to confer a sensitivity to NGF by simultaneous treatment with specific gangliosides. This indicates that membrane gangliosides may modulate the actions of neurotrophic factors.

Chemical traumatization of adult mouse olfactory epithelium in situ stimulates growth and differentiation of olfactory neurons in vitro.

This study demonstrates that ZnSO4-induced chemical trauma results in an in situ regeneration of the olfactory epithelium which, when maintained in vitro, provides an enriched population of olfactory neurons. Therefore, the ability of the olfactory epithelium to respond to chemical trauma with increased mitotic activity can be used to increase growth of neurons in culture. Tissue obtained from normal or vehicle-treated adult mice produced few olfactory neurons, when maintained in culture, compared to cultures established from tissue following an in situ ZnSO4 trauma. Maximal neuronal yields were obtained in cultures established from tissue that was removed 4-6 days following chemical trauma. The morphological appearance and the presence of cell specific intermediate filament proteins were used to classify the cell types in these olfactory epithelial cultures. Single cells and aggregates of cells which were immunopositive for keratin, but immunonegative for neurofilament protein and GFAP, were identified as epithelioid. Flattened polygonal cells immunopositive for GFAP were identified as glia. A small population of flattened cells was immunonegative for all of the antibodies used in this study. Cells that had processes were immunonegative for GFAP and keratin. Some were immunopositive for 200 kDa and 160 kDa neurofilament proteins but immunonegative for the 68 kDa neurofilament protein. A few of these cells showed positive immunoreactivity with the olfactory marker protein (OMP) antibody and most likely represented the most mature olfactory neurons in the cultures. This trauma-induced culture model using olfactory tissue from adult mice can serve as a source of CNS neurons for comparison with cultured embryonic neurons.

Gangliosides prevent MPTP toxicity in mice--an immunocytochemical study.

The role of gangliosides in preventing neuronal degeneration was examined in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP)-induced mouse parkinsonian model. Intraventricular injections of a ganglioside mixture prior to MPTP treatment reduced MPTP's toxicity on tyrosine hydroxylase-positive neurons in the substantia nigra. This raises the interesting possibility that early ganglioside administration may be beneficial in the treatment of neurodegenerative disorders.

Comparison of epi-GM3 with GM3 and GM1 as stimulators of neurite outgrowth.

A variety of naturally occurring ganglioside structures were previously shown to be effective agents for inducing neurite outgrowth of primary neurons and neuroblastoma lines. We report here the results of similar experiments with a synthetic epimer of GM3 (epi-GM3) possessing a neuraminidase-resistant beta-ketosidic linkage. This substance was found to enhance neuritogenesis toward two transformed cell lines (neuro-2A, PC-12) and one primary neuronal tissue (dorsal root ganglia). The results indicate that the stereochemistry of the ketoside linkage is not critical and that metabolism of exogenous ganglioside by the treated cells is not involved directly in the neuritogenic phenomenon.

Inhibition of conditioned media-mediated neuritogenesis of sensory ganglia by monoclonal antibodies to GM1 ganglioside.

The monosialoganglioside GM1 can potentiate the neuritogenic activity of media conditioned by several cell types: neonatal glia, C6 glioma, embryonic chick heart or skeletal muscle and the rat myogenic line L6. To probe further the neuritogenic activity of conditioned media (CM), 5 mouse monoclonal antibodies (mAbs) against GM1, designated B6, C3, C4h2, D1 and D3 were incorporated individually into nutrient medium (NM) supplemented with CM prior to incubation with sensory ganglia. Nine-day embryonic chick dorsal root ganglia were explanted onto collagen-coated coverslips and incubated at 35 degrees C for 5 h in NM supplemented with 150 micrograms/ml GM1. After washing with NM, the explants were re-fed with NM + CM containing 20% mAb and cultured for an additional 43 h. The resultant neuritogenesis was evaluated microscopically by determining mean neurite number and length of randomly mixed cultures. The 5 antibodies differed in their capacities to inhibit CM-mediated neuritogenesis of these primed target cells. D1 and D3 were most effective in reducing neurite length and number produced by all sources of the CM, while C3 and C4h2 were intermediate in their inhibition of neurite initiation (number). The effect of B6 on neurite initiation and elongation was the least. The ability of these mAbs to inhibit neuritogenic activity of CM derived from both glial and myogenic tissue suggests that gangliosides play a basic role in neuronal development. The differing responses elicited by the individual mAbs may reflect a relationship between the structural complexity of the GM1 molecule and the neuritogenic mechanisms.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult human olfactory stem cells.

The location of stem cells within the adult CNS makes them impractical for surgical removal and autologous transplantation. Their limited availability and histocompatibility issues further restrict their use. In contrast, olfactory neuroepithelium (ONe) located in the nasal passageways has a continuous regenerative capability and can be biopsied readily. To investigate the potential of human ONe to provide viable populations of pluripotent cells, ONe was harvested from cadavers 6-18 h postmortem, dissociated, plated and fed every 3-4 days. Heterogeneous populations of neurons, glia, and epithelia were identified with lineage-specific markers. After several weeks, 5-10% of the cultures produced a population of rapidly dividing cells, which in turn, produced neurospheres containing at least two subpopulations based on neuronal and glial specific antigens. Most contained one or more neuronal markers; a few were positive for A2B5 and/or GFAP. To determine if growth modulators would affect the neurosphere forming cells, they were exposed to dibutyryl-cAMP. The nucleotide reduced cell division and increased process formation. Although the cells had been passaged more than 70 times, their viability remained constant as shown by the MTT viability index. Donor age or sex were not limiting factors, because neurospheres have been established from cadavers of both sexes from 50 to 95 years old at time of death. The ex vivo expansion of these cells will provide a patient-specific population of cells for immunological, genetic and pharmacological evaluation. Our long-term goal is to determine the utility of these cells to facilitate CNS repair.

Gangliosides induce microfilament-dependent changes in membrane surface activity of Neuro-2a neuroblastoma cells.

Exogenous mixtures of bovine brain gangliosides (BBG) initiated a rapid series of sequential changes in surface activity in Neuro-2a neuroblastoma cells. These changes were characterized by the formation of microvilli, ridge-like projections and ruffled membranes within l min of BBG exposure. A maximum number of microvilli was reached after 10 min exposure and disappeared by 30 min. Ruffled membranes were most prominent within 5 min, but were still present after 60 min. These surface changes were accompanied by the formation of a thick subcortical meshwork of microfilaments which formed the cores of the microvilli and ruffled membranes. The simultaneous treatment of cells with BBG and an agent known to disrupt microfilaments (cytochalasin D) inhibited the formation of both microvilli and ruffles. In contrast, the simultaneous application of BBG and agents affecting microtubule polymerization (Colcemid and taxol) did not inhibit the formation of these surface structures. Treatment of Neuro-2a cells with cyclic AMP (cAMP) analogs (dibutyryl cAMP and 8-bromo cAMP) induced a similar series of surface changes which were inhibited by cytochalasin D. Furthermore, these surface changes may be specific to cAMP since neither cGMP analogs (dibutyryl cGMP and 8-bromo cGMP) nor 5'AMP induced surface changes. We propose that the initial membrane response to exogenous gangliosides is mediated by a microfilament-dependent mechanism. The basic similarities in surface responses elicited by BBG and cAMP analogs suggest that the effects of gangliosides on surface activity may be induced in part by a cAMP-dependent mechanism.

Ganglioside potentiation of NGF-independent conditioned medium enhancement of neuritic outgrowth from spinal cord and ciliary ganglia explants.

Culture medium conditioned (CM) by embryonic chick skeletal muscle or RN22 Schwannoma cells enhanced dramatically the neuritic development of chick embryonic spinal cord slices explanted onto a collagen substratum. The addition of a mixture of bovine brain gangliosides (BBG) or the monosialoganglioside GM1 to this medium potentiated the nerve growth factor (NGF)-independent CM-mediated neuritogenesis. A 3-4 fold increase in spinal cord outgrowth was due to increased neurite number, length and branching. The ability of the gangliosides to potentiate the positive neuritogenic action of CM was not limited solely to spinal cord cultures since similar results were obtained in parallel studies employing organized cultures of embryonic chick ciliary ganglia. These studies demonstrate the ability of gangliosides to enhance the trophic action of factor(s) present in CM. They suggest further that gangliosides may play a modulatory role in the development of the nervous system.

Promotion of neurite outgrowth by protein kinase inhibitors and ganglioside GM1 in neuroblastoma cells involves MAP kinase ERK1/2.

To investigate mechanisms of neurite outgrowth, murine Neuro-2a neuroblastoma cells were exposed to ganglioside GM1 in the presence or absence of specific protein kinase inhibitors. Isoquinolinesulfonamide (H-89), an inhibitor of cyclic AMP dependent protein kinase A (PKA), and bisindolylmaleimide I (BIM), which inhibits protein kinase C, each stimulated neurite outgrowth in a dose-dependent manner in the absence of exogenous GM1. Minimally effective (threshold) concentrations of H-89 or BIM potentiated outgrowth when they were used in combination with GM1. To search for a shared component in the mechanisms of GM1, H-89 and BIM, phosphorylation of ERK1/2 was examined. Inhibition of the activation of extracellular signal regulated kinases (ERK1/2) by U0126, prevented neuritogenesis of Neuro-2a by all the three agents. Pretreatment of serum-depleted Neuro-2a cultures with GM1 or BIM enhanced ERK1/2 phosphorylation when the serum level was restored to 10%. In contrast, H-89 did not alter the serum-mediated response. In cells exposed to GM1 or BIM without additional serum, a transitory decrease in ERK phosphorylation occurred. These data suggest that GM1 influences two neuritogenic pathways, one modulated by PKC and the other regulated by PKA. Therefore, GM1 may have the potential to stimulate alternate pathways resulting in outgrowth.

Taurine-induced neuronal differentiation: the influence of calcium and the ganglioside GM1.

Taurine-induced differentiation was examined in the murine neuroblastoma Neuro-2a cell line in the presence or absence of the monosialoganglioside GM1 and under conditions in which Ca2+ levels were manipulated. Taurine (4 mM), GM1 (200 micrograms/ml), or taurine with GM1 were applied to culture media that contained either various concentrations of Ca2+ or the Ca2+ ionophore A23187. Taurine or GM1 and taurine with GM1 increased the number of cells emitting neurites above that found for controls. A significant interaction was found between treatment (taurine, GM1 or taurine + GM1) and the manipulations of Ca2+ levels, affecting the number of neurites and producing changes on the neuritic and perikaryal surfaces. Treatment with both taurine and taurine + GM1 and the various concentrations of Ca2+ resulted in a significant increase in neurite elongation. The Ca2+ ionophore A23187 in the presence of taurine or taurine + GM1 caused neurites to grow longer than observed in media containing Ca2+, either in a low concentration (about 125 microM) or at 1-2 mM. Taurine-treated cultures in the presence of extracellular Ca2+ or A23187 were characterized by surfaces with numerous microvillar, spine-like projections. This effect was enhanced with GM1 and was less pronounced in the medium containing low levels of Ca2+. Transmission electron microscopy of the taurine-stimulated neurons revealed an excessive number of clear-core vesicles (40-200 nm in diameter) in perikarya, neurites and neuritic varicosities and growth cones. In addition, numerous aggregates of intermediate filaments were seen. They were most abundant in the taurine + GM1 treated cultures. The taurine + A23187 cultures also exhibited numerous microtubules within the elongated processes. The different neuritic patterns induced by taurine under conditions in which Ca2+ levels were manipulated and/or when cells were exposed to exogenous GM1 suggest that taurine's actions depend in part on Ca2+ flux.