Fred J. Roisen

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.

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.

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.

The Role of Gangliosides in Neurotrophic Interaction in Vitro

Gangliosides are membrane-associated acidic glycolipids that have been implicated in the regulation of cell processes such as differentiation (Moskol et al., 1974; Roisen et al., 1981a, b; Dimpfel et al., 1981; Leon et al., 1982), growth (Fishman et al., 1977; Langenback and Kennedy, 1978; Morgan and Seifert, 1979) and axonal regeneration (Obata et al., 1977; Caccia et al., 1979; Norido et al., 1982; Spirman et al., 1982). Their concentration in the brain is 14 times higher than in liver and nearly 60 times higher than in muscle (Seyfried et al., 1978; Leeden and Yu, 1982). Patterns of ganglioside synthesis have been reported to change during intense periods of neuritogenesis and synaptogenesis (Rosner, 1980; Sonnino et al., 1981). Their abundance, distribution and temporal occurrence in neuronal membranes suggest that gangliosides play a key role in the development of the nervous system.

NEURITE DEVELOPMENT IN VITRO: III. THE EFFECTS OF SEVERAL DERIVATIVES OF CYCLIC AMP, COLCHICINE, AND COLCEMID*

Many cellular phenomena influenced by adenosine 3‘, 5’-monophosphate (CAMP), ie., aggregation,’ contact inhibition,? growth,3 and ~ e c r e t i o n , ~ depend on intact microtubules. In order to examine the relationship between endogenous cAMP levels and microtubule assembly, the colchicine (cokemid)-sensitive neuronal development exhibited by chick embryonic sensory ganglia in culture was studied. These ganglia exhibit a number of easily measured morphological features that appear directly dependent upon microtubule integrity and assembly, e.g. neurite numbers, diameters, lengths, and terminal arborization. This tissue source also provides a system upon which an accurate measure of the radial migration of nonneuronal cells can be made. I t has been shown previously that cAMP and its dibutyryl analog (But) , c A M P enhance sensory neurite These studies have now been extended to include additional derivatives of c A M P as well as other nucleotides. In preliminary experiments with guanosine 3’, 5’-monophosphate ( c G M P ) , neuronal maturation was almost completely inhibited, whereas the development of the nonneuronal cells appeared unaffected. It has been shown further that (But) c A M P can reverse partially or prevent the reduction of microtubule numbers per unit area in neurites treated with colcemid. This effect appears to be independent of protein synthesis, and may be due either to the stabilization of intact tubules or to the mobilization of tubule subunits from a preexisting subunit pool. Colchicine and/or colcemid (affecting microtubules) and cytochalasin B (altering microfilament integrity) have been employed. Effects of these cytoskeletal disruptive agents on the intraneuronal particle movements of in vitro sensory neurites have been examined with differential interference microcinematography.

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.

Ganglioside Induced Surface Activity and Neurite Formation of Neuro-2A Neuroblastoma Cells

These studies demonstrate that while microtubules are essential for BBG-mediated neurite initiation and elongation, they are not involved in microfilament-dependent ganglioside-mediated surface activity. Microfilaments may be more directly altered by exogenous gangliosides than microtubules since they are the major structural elements of microvilli and are required for neurite branching. Our studies suggest that normal neuritogenesis requires a delicately balanced interaction between various cytoskeletal elements. Since there is a close relationship between membrane-associated lipid molecules and submembranous cytoskeletal elements, the incorporation of gangliosides into membranes may alter this balance and result in neurite formation. The use of gangliosides to enhance neurite production provides a unique model for the study of nerve development. We have shown that bovine brain gangliosides stimulate an immediate sequence of surface-related changes as well as microtubule and microfilament dependent neurite formation in Neuro-2a cells. However, the precise molecular events by which gangliosides enhance neuritogenesis await further study.

Diazepam inhibits the spreading of chick embryo fibroblasts

Diazepam (Valium/Roche), a benzodiazepine derivative, reversibly inhibited the spreading of chick embryo fibroblasts in a dose-related manner. This inhibition was accompanied by: 1 Fewer and less organized microfilament bundles. 2 A reduction in myosin fluorescence along these structures. 3 A reduction in the total myosin content of cells compared to corresponding controls. Overall results suggest that myosin plays an active role in the spreading of chick embryo fibroblasts.

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.

A method for studying the three‐dimensional organization of cytoskeletal elements of cells: improvements in the polyethylene glycol technique

A method utilizing polyethylene glycol (PEG) as an extractable embedment for electron microscopy is described. Tissues are fixed according to conventional protocols, embedded in PEG, and sectioned. Sections (ranging from 100 to 500 nm in thickness) are mounted on grids, divested of their PEG matrix, critical‐point‐dried, and examined stereoscopically. This method greatly facilitates studies on the three‐dimensional organization of cytoskeletal and cytoplasmic contractile systems in both muscle and nonmuscle cells.