Nerve growth factor and its mode of action

EXPERIMENTAL

NEUROLOGY

Nerve

48,

Growth

NO.

3,

PART

Factor SILVIO

Drpat’tmeut

of Biology,

2, 75-92

(1975)

and Its Mode

of Action

VARON

School of hfedicirkc, San Diego 92037

Utkivcrsity

of Califorraia,

CONTENTS Introduction ................................................ The NGF Proteins from Mouse Submaxillary Gland ............. Sources of NGF Mouse submaxillary gland and snake venoms ............... Tumoral tissue ......................................... Neuroglia .............................................. NGF Target Cells .......................................... Association Between NGF and Target Cells NGF binding sites ...................................... NGF internalization ..................................... Functional association ................................... Tubulin involvement ..................................... NGF Effects on Traditional Cell Targets The role of NGF ....................................... RNA and protein synthesis ............................... NGF and the cell membrane ..............................

75 76 78 79 79 80 82 83 83 83 84 85 87

INTRODUCTION One of the most exciting landmarks in the progress made by developmental neurobiology over the last two decades has been the discovery by Drs. Levi-Montalcini and Hamburger of a diffusible factor which specifically promotes growth and differentiated functions in selected classes of neurons. The term Nerve Growth Factor (NGF) , originally assigned to the agent discovered in certain mesenchymal tumors, has been retained to cover several proteins from different sources, which share immunochemical and functional properties even though they may differ in their 7.5 Copyright All rights

1975 by Academic Press, Inc. o3 reproduction in any form reserved.

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detailed chemical structure : The NGF attention on several grounds :

VARON

phenomenon

has drawn

increasing

(i) the unique occurence of NGF as a defined, neuron-directed protein agent ; (ii) the apparent restriction of its responsive cells to sympathetic ganglionic neurons, and to one of the two neuronal populations of dorsal root ganglia (DRG) over a limited developmental period ; (iii) the possibility that NGF constitutes but one representative of an entire family of neuron-directed protein agents, and can serve as a model for such other hypothetical factors; (iv) the potential use of NGF as a probe into the operations of a nerve cell and the control mechanisms to which they are subjected; and (v) the role that other tissues, acting as sources or storage sites for NGF, may play in neuronal development, maintenance and/or regeneration. Much of the information collected over several years on the NGF phenomenon has been the object of a number of extensive reviews (39, 40, 58, 63, 71). The present paper, therefore, will survey in detail only the most recent data and focus on the several issues still open at the current stage of the NGF investigation. At the same time, it will attempt to provide a sense of the shifts in perspective which the NGF phenomenon may be undergoing as knowledge increases about its many aspects. THE

NGF

PROTEINS

FROM GLAND

MOUSE (SM)

SUBMAXILLARY

In the SM extract, NGF occurs in a high molecular weight form (7s NGF, about 140,000 dalton). 7s NGF is a complex of Alpha, Beta and Gamma subunits (presumed composition = c&s) with similar sizes (approximately 25,000 dalton) , but distinct electrochemical properties. The 7s complex can be isolated in pure form at neutral pH, and dissociates reversibly into component subunits at both acid and alkaline pHs (61). The Beta subunit, a basic polypeptide, is the one endowed with NGF activity @NGF). In several in vitro systems, it is 3-4 times more potent when presented in combination with the other two subunits than in the isolated form. The Gawwna subunit displays arginine esteropeptidase activity and shares with other such enzymes the ability to promote proliferation of fibroblasts in “contact-inhibited” (confluent) cultures (25, 26). The enzymatic activity of Gamma is fully inhibited in the 7s complex. The Alpha sztbunit, a highly acidic polypeptide, has been shown to interact with ganglion cell membranes as revealed by a reduced cell breakage upon mechanical dissociation of the source tissue (62). The “protection” afforded by the Alpha protein is increased 50-100 times by the concurrent

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presence of the other two subunits in the correct stoichiometric proportions. The extreme specificity with which the three subunits select one another from a heterogenous mix of proteins (54), the precise stoichiometry with which they occur in the original complex or with which they regenerate it after separation, and the quantitative changes that their stoichiometric coexistence imposes on the individual activities of each subunit support the view that the 7s complex is a distinct entity with its own importance. However, its biological significance remains to be properly identified (62). One should note that other tissue-specific factors present in the SM gland have also been found to occur as complexes with at least an esteropeptidase subunit. Two procedures are now widely used for the purification of the NGF polypeptide, although they yield slightly different products (47). Both procedures involve dissociation of the 7s complex at acidic pH and chromatographic separation of the NGF subunit on CM-cellulose columns, but they differ in the stage at which the 7S complex is dissociated. One procedure (57)) first isolates the 7s complex in pure form and then derives from it the PNGF, as well as the other two subunit proteins. The other procedure (9), splits the complex at an intermediate stage of purification and isolates from it what is now called 2% NGF (2). The two species are obtained essentially in the same amounts and at the same final potency (5 x lo-lo M = concentration which elicits optimal fiber outgrowth from 8-day chick embryo dorsal root ganglia in explant culture) (63). Both species have been extensively characterized and sequenced. They consist of tzero polypeptide chains, held by noncovalent bonds (the three disulfide bridges of each chain are all intrachain), and separable upon treatment of the dkeer NGF protein with SDS, urea or guanidine. However, significant differences have been found in the chains involved in the /3 and the 2.5s NGF dimers (47). The native chain (A chain) consists of 11s amino acids (MW = 13,250 dalton), with an N-terminal serine and a C-terminal arginine. Carboxypeptidase B and a similar enzyme in the SM extract cleave off the arginine, leaving the chain (des-argihe c/z&) with a C-terminal threonine. Another SM enzyme specifically removes an octapeptide from the N-terminal, leaving the shorter chain (B chak) with an N-terminal methionine. Both arginine and octapeptide cleavages are prevented when the dimer is in the 75 complex form, and therefore, may occur more extensively when the 7s complex is dissociated before its complete isolation from other SM constituents. Thus, 2.5s NGF contains more desarginine chains (30 versus 10%) and more B chains (50% versus none) than the PNGF. The different chains have no apparent effects on dimer formation or on NGF activity, but either chain reduction limits the dimer’s ability to regenerate a 7S complex with Alpha and Gamma subunits. The

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preference of the Gamma esteropeptidase for arginine groups and the arginine C-terminal of the /&VGF chains have raised the possibility of an enzyme-product type of relationship between the two subunits, i.e., that Gamma be required for the cleavage of the active NGF from a larger protein precursor (47, 62). A provocative outcome of these structural studies has been the recognition, by Frazier, et al. (20), of substantial analogies between the NGF and the proinsulin/insulin polypeptides with regard to their amino acid sequences and polymer formation. The suggestion of an evolutionary relationship between these two protein “hormones” drew further encouragement from the resemblances noted among some of the metabolic responses elicited from their target cells. Finally, additional analogies are being uncovered with regard to zinc involvement in insulin (20) and NGF (52) polymerization. SOURCES

OF NGF

Mouse Submaxillary Gland and Snake Venoms. The high NGF content of the mouse SM gland makes this organ the choice source for the preparation of. purified NGF. However, the physiological significance of this occurrence remains uncertain. High NGF storage occurs only in the SM gland of the adult male mouse, where it coincides with a tubular component of the gland whose development is strictly dependent on testosterone. Also paralleling the tubular development are several other special proteins of the SM, such as enzymes (esteropeptidases and proteases) and growth- or differentiation-promoting factors (epidermal growth factor, mesenchymal growth factor, thymotropic factor, etc.), the biological functions of which are only partially explored (26, 41, 62). The occurrence of synthesis, as well as storage, of NGF in the male SM gland (though not necessarily in its tubular component) is indicated by several observations (40), such as the precipitation of radiolabeled antigen, by antiserum to NGF, from SM glands injected in z&o or incubated in vitro with radio-amino acids, or the inability of circulating antiserum to prevent the NGF build-up in the SM glands of female mice under testosterone administration. Whether other salivary glands are able to synthesize NGF while lacking a storage mechanism has not been investigated, On the other hand, minute amounts of NGF activity or NGF antigen have been demonstrated in most tissues and body fluids, including serum, of several species ; sympathetic ganglia (one of the known NGF target tissues) were noteworthy by their several-fold higher NGF content (28, 36, 40). Finally, removal of the SM gland in adult male mice causes a decline in serum levels of NGF antigen, which is followed by a return to normal within the next 2 mo (29), indicating the occurrence of at least alternate sources of NGF in the organism. A variety of snake venoms have also been found to con-

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tain considerable amounts of NGF, initially isolated as a ZO-22,000 dalton, basic protein (16). Snake NGF is currently the object of detailed investigation in several laboratories and appears to have very close, though not identical, properties when compared to the mouse NGF protein (53). Tunzoral ?‘issz4e. Following the discovery of NGF in mouse tumors implanted into chick embryos (38, 39), several studies suggested a correlation between NGF activity and a mesenchymal origin of the tumors (sarcomata, granulomatous tissue), as well as their invasiveness (40). This intriguing relationship has recently resurfaced in the course of culture studies of L cells (a clonal line of mesenchymal cells derived from a chemically-induced mouse tumor). Oger, et al. (50) noted that chick embryo dorsal root ganglia explanted on monolayer cultures of L cells develop a halo of nerve fiber outgrowth, (the classical response to NGF), and in L cell-conditioned media they could demonstrate an NGF activity which was blocked by antiserum to mouse NGF. T\iGF-antigen was also found in media conditioned over 3T3 fibroblasts and primary embryonic chick fibroblasts. These investigators propose that “fibroblast” cells (i.e., mesenchymal elements) may be a generalized source of NGF itz z&o. If so, the ubiquitous occurrence of NGF in body tissues could reflect a limited storage in NGF-producing connective cells. Neuroglia. A role of glial cells in the supply of NGF or NGF-like support to the ganglionic neurons is indicated by another set of recent investigations (11, 64, 65). Dissociation of newborn mouse dorsal root ganglia (DRG) provided a cell suspension consisting almost exclusively of neurons and glial elements; this suspension was susceptible to fractionation into neuronal and glial cell populations. Despite the apparent lack of a response to NGF by the intact postnatal DRG, ifz viva or in vitro (see section on NGF target cells), cultures of unfractionated cell suspensions revealed that most neurons depended on the availability of NGF in the medium for their attachment, neurite production, and survival. However, the same maximal performance was elicited in the absence of exogenous NGF if the cultures were supplemented with glial cells from the same DRG source. Under such conditions, no further effects were observed with esogenous NGF. The neuronal performance could be set at any intermediate level by intermediate supplementation with ganglionic glia. or submaximal concentrations of NGF in the medium, or a combination of both. DRG neurons from chick embryo or postnatal rat, like those from the newborn mouse, could also be supported in the absence of exogenous NGF when supplemented with their homologous non-neuronal cells (in these C;LSCS, a mixture of glial and fi~JrdhStic elements), and so did neurons from sympathetic ganglia of the same animals. Of some twenty neural and non-neural cell types tested on the purified mouse DRG

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neurons, only DRG-derived non-neuronal elements showed this NGF-like support capability in the absence of exogenous NGF. However, crosstesting neurons and non-neurons from DRG of different species, or from mouse DRG of different ages, indicated that NGF-like competence was most effectively displayed toward strictly homologous neuronal partners, raising the possibility of some differences in the nature or in the mode of delivery of the glial support. From these several observations, the hypothesis was proposed that neuronal support in intact DRG and sympathetic ganglia is provided indigenously by their own glia both in vitro and in z&o, and that exogenous NGF is required-and, thus, demonstrably effective-only when the glial supportive competence is subjected to developmental, experimental or possibly pathological restrictions. The glial support to ganglionic neurons was found to involve an NGFlike antigen (66, 67). The performance of mouse DRG neurons in a culture exclusively supported by homologous glia is blocked by the presence in the medium of antibody against ,8NGF, and so is the performance of chick embryo DRG neurons in a similar situation, The same interference with the mouse DRG performance can be achieved by treating with the antibody the glial supplement before it is presented to the neurons. The essential involvement of an NGF-like antigen in culture systems in which no known NGF has been introduced is consistent with a production of the antigen by the glial cells themselves. Johnson, et al. (37) have suggested an NGF production by satellite cells in explanted rat sympathetic ganglia. Longo and Penhoet (43) have reported that extracts from a rat glioma grown in viva contain both NGF activity and protein constituents that closely resemble mouse NGF in their antigenic and physicochemical properties. However, an alternate possibility yet to be ruled out is that glial cells capture and activate a serum-borne inactive NGF precursor. Such an interpretation would have the merit to accommodate a mesenchymal origin of the presumptive NGF precursor, since “mesenchymal” NGF (50) h as b een recognized by immune criteria, or by activity tests on glia-containing systems. The production and/or supply of NGF-like proteins by glial cells, as well as other data (24, 46, 56) with a possible bearing on neuron-directed glial factors, are further discussed in the Workshop on Culture Techniques and Glial-Neuronal Interrelationships ivt vitro (see report by Varon and Saier) . NGF

TARGET

CELLS

NGF has traditionally (38-40) been described to act exclusively on sympathetic ganglionic neurons (at all ages, although with progressively restricted effects) and on DRG neurons (but only one of their two neuronal populations, and only for a limited period of embryonic life). If

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these age restrictions on ganglionic responses to exogenous NGF reflected the progressive effectiveness of an NGF-like supportive ganglionic glia (11)) other neurons might be found responsive to NGF when tested under conditions of restricted “indigenous” support. In recent years, the list of NGF-responsive cells (target cells) has been steadily increasing. Pcripherd Ganglia. Postnatal DRG neurons, from both mouse and rat, have been shown to respond to NGF in terms of adhesive properties, neurite production and survival in dissociated cell cultures. In monolayer cultures from trigeminal ganglia of lo-day chick embryos, NGF increases neuronal numbers and long-term survival, neurite development and glianeuron relationships (55). NGF has been reported to increase neurite elongation from trigeminal ganglia in explant cultures (27). It should be pointed out, even in the traditional target tissues, that because NGF effects were detected or sought at the neuronal level the untested assumption has always been that the action of NGF is directly on the responsive neurons. Evidence to support this tacit assumption has now been provided by the mouse DRG studies and even more explicitly by the NGF-dependent survival of sympathetic neurons cultured in the near-complete absence of non-neuronal cells of any kind (10). CNS Neurons. Explant cultures from early head neural crest (as well as trunk neural crest, the main source of both DRG and sympathetic ganglia) exhibit earlier and more numerous catacholamine-containing cells when supplied with exogenous NGF (6). In adult rat CNS, transection of the medial forebrain bundle is followed by regeneration of catecholamine-containing axons that will selectively innervate intracerebral transplants of tissue normally supplied with sympathetic nerve fibers. Both axonal regeneration and implant innervation are enhanced by intracerebral or intraventricular injection of NGF (8)) and inhibited by similar administration of antiserum to mouse NGF (7). Behavioral recovery after hypothalamic lesions has also been reported to be enhanced by intracerebra1 injection of NGF (5). Non-Neural Cells. Chick embryo cartilage rudiments, in explant culture, synthesize a chondromucoprotein characteristic of cartilage matrix, and NGF has been reported to inhibit specifically the synthesis of this constituent (19). Young and Arnason’s group (44) may also have evidence for a stimulatory effect of NGF on proliferation of cultured macrophages, although one must await further details on both the purity of the NGF used (in view of possible esteropeptidase contamination) and the macrophage response. The inclusion of non-neural cells in the list of NGF targets may be disturbing for a concept of NGF as a neuroll-specific agent, but cottld reveal exciting-and, thus far, ~unsusl)ectetl--llew facets of the NGF phenomenon. It will be critical to distinguish, among cells that interact with NGF, those that are actual targets from those that may be involved

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in production and/or storage of an NGF-like protein. Also to be considered is the possibility of NGF as a neural crest-directed agent, in view of the expanding list of neural crest derivatives (including cartilage and other mesenchymal elements) (17). ASSOCIATION

BETWEEN

NGF

AND

TARGET

CELLS

The first step of an NGF action must involve its physical association with the plasma membrane of its target cells, either as a binding to receptor sites (if the site of action is restricted to the outer cell membrane) or for the internalization of the agent (if the site of action is intracellular). There is considerable evidence that both relationships can occur between NGF and ganglionic cells. NGF Binding Sites. Several independent studies have firmly established the occurrence of binding sites for NGF on dissociated chick embryo DRG cells (23, 31) and sympathetic ganglionic cells (42)) and on membrane subcellular fractions from young rabbit sympathetic ganglia (4). The binding sites have a high affinity for NGF (lOe-lO1o l/~), which matches the range of optimal NGF activity in various test systems. Substrate-specificity is indicated by the lack of interference from any other protein tested (including basic proteins, and the structurally-related insulin), and further supported by the finding that chemically altered NGF lost binding ability in parallel with its bioactivity. Cell-specificity, however, remains very much in doubt. Frazier, et al. (23) reported that DRG cells bind NGF with two apparent affinity ranges ( lo6 as well as 10’ l/~), and that both sets of binding sites were found in all other cell types examined, both neural and non-neural. The number of binding sites did vary considerably with age of ganglionic cells or type of peripheral tissue, generally correlating with the traditional responsiveness of the ganglia or the expected sympathetic innervation of the tissue, respectively. For binding sites to be viewed as truly functional receptors, two criteria should be verified : (a) NGF prevented from entering the cell should elicit the full spectrum of expected responses, and (b) no responses should be elicited by NGF if its binding sites are blocked. With DRG, strong evidence has been provided toward the first criterion by the successful elicitation of neurite outgrowth in vitro upon presentation of NGF that had been covalently coupled to agarose (21) or to T4-bacteriophase (SO). One must recognize, however, the inherent hazard of such approaches since free NGF could be released at or near the target cells (e.g., by lytic enzymes), that would escape the most careful examination. Evidence pertaining to the second criterion is much more circumstantial, The higher number of binding sites in recognized target tissues only speaks for an involvement of the binding sites but does not define their role. The parallel

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loss of NGF activity and binding ability in chemically treated NGF demonstrates their common dependence on certain molecular structures but does not subordinate one to the other. NGF Intemalizatioul. In vivo injection of radio-NGF is followed by its rapid disappearance from the plasma and a differential accumulation in tissues: accumulation was several-fold higher in superior cervical ganglia, and markedly and selectively reduced there on prior destruction of sympathetic terminals (6-OH-dopamine treatment), suggesting their involvement in the accumulation process (3). Injection of radio-NGF into one eye of rats led to the appearance of labeled material along the sympathetic fibers and its progressive accumulation in the neuronal bodies of the corresponding superior cervical ganglion, further supporting the view of a direct uptake of NGF by sympathetic terminals followed by retrograde axonal transport to the soma (30). 1~ vitro studies with intact and dissociated DRG also have provided evidence for an endocytotic internalization of exogenous NGF (12, 49). The process required the binding of NGF to substrate-specific sites (with an apparent affinity in the lo6 l/nr range), was enhanced rather than antagonized by insulin, and caused accumuIation of NGF that retained at least part of its biological activity. Similar binding sites, with a similar involvement in an internalization of NGF, were observed in all other cells examined, denying any noticeable cell-specificity to the phenomenon. Functional .4ssocintio~~.The occurrence of both modalities of NGF association with recognized target tissue raises the question whether both “routes” are in fact involved in the action of NGF and, if so, whether they may be involved in different sets of cellular responseselicited by the agent (e.g., metabolism versus neurite production). Moreover, the reported occurrence of both modalities in all cells tested raises again the question of the target specificity of NGF action and the related question of the nature of the involvement with NGF by other cell populations, Specific responsivenessmust involve other aspects than the occurrence of an association mechanism, for example the extent to which association occurs (possibly including a threshold concept), or the presence or absence of a follow-up machinery to transduce the association into meaningful cellular alterations. Nonresponsive NGF-binding cells could be involved as general buffer systems (regulating the levels of circulating NGF), or to map pathways for NGF-dependent outgrowing axons (directional guidance to the appropriate peripheral territories), or as NGF-producing cells (exocytotic externalization of membrane-bound product). Finally, some of these cells may be genuine targets for NGF action, resulting in cellular responsesas yet undetected. T&&z I?/v&e+~L~/lf. Tntriguing suggestions have beet1 recently offerctl by Levi-Montalcini, et al. (42) on the roles that tubulin (the microtubule

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subunit protein) may play in the association between NGF and target cells, as well as some of the cellular responses elicited by NGF. Mouse neuroblastoma cells, which have failed to exhibit a response to NGF in several investigations, were found to bind radio-NGF or NGF-coated sheep red blood cells (with a 107-lo8 l/~ affinity), as well as to internalize the bound erythrocytes at 37 C. Binding sites became unmasked at specified stages of the mitotic cell cycle to reach maximal numbers (late G1-early S phases) equal to those observed in normal sympathetic ganglia. Antiserum to tubulin caused lysis of neuroblastoma cells in the presence of complement (indicating “tubulin-like” antigens on the cell surface), and also blocked the binding of NGF-coated red blood cells. Moreover, NGF was found to precipitate tubulin from soluble extracts of brain or neuroblastoma cells, indicating the occurrence of selective NGF-tub&n binding and, possibly, a promotion by NGF of tubulin polymerization. These investigators propose that tubulin molecules on the membrane surface may themselves be the NGF binding sites, while also operating as transducers systems for the NGF action on the cells. NGF

EFFECTS

ON THE

TRADITIONAL

TARGET

CELLS

The Role of NGF. In dissociated cell cultures, a large number of neurons from DRG and practically all neurons from sympathetic ganglia will die unless they are supplied with NGF (40) or NGF-like supportive cells (11, 65). A similar massive death of neurons also occurs within the intact ganglia in explant or free-floating cultures (41, 68). That NGF is equally essential to the survival of its target neurons in tivo cannot be demonstrated unless all sources of NGF could be shut down. However, the well documented destruction of sympathetic neurons upon administration of antiserum to NGF (the so-called immunosympathectomy) (41, 58, 71) is at least susceptible of such an interpretation, although it may reflect a complement-dependent immunocytolytic effect rather than an NGF deprivation. The increasing recognition of a survival role of NGF raises the question whether all other cellular responses to NGF may not reflect the normal, or even an optimized, performance of the healthy neuron rather than distinctive traits that are selectively promoted by the factor. As Levi-Montalcini pointed out (38)) the “potentialities of (embryonic nerve) cells far exceed their growth range under normal conditions.” If their performance in z&o reflects not their potential but the level of NGF or NGFlike support available to them-as the in vitro experiments indicate-exogenous administration of additional NGF would merely lead the target neurons to anticipate or even exceed the performance they would otherwise diSlJkIy at their normal maturation stage.

Indeed, no unequivocal information describes neuronal responses to NGF that are abnormal except in terms of earlier and/or greater expressions of normal traits. Neuronal numbers are increased only to the extent that mitotic capabilities are still present in the ganglionic populations (18). Neuronal size and general anabolic and energy-yielding activities are increased by NGF in a dose-dependent manner, as befits the view of a quantitative rather than qualitative action of the factor (IS, 41). Selective biochemical changes have been reported for NGF-treated ganglia in studies concerning glucose utilization and lipid or protein synthesis (40), as has been an NGF-induced increase in the specific activity of tyrosine hydrosylase and dopamine-p hydroxylase, the most characteristic enzymes of sympathetic neurons (59). These studies, however, suffer from two major complications : the undetermined contributions by the non-neuronal elements of ganglionic tissue and (at least ill vitro) the different2 survival of neurons in treated and untreated ganglia. In the absence of similar studies with purified neuronal populations, the results may only indicate an increased contribution by NGF-dependent neurons to the overall performance of the ganglia, rather than actual NGF-induced selective changes within the neurons themselves. All available information also points to NGF being continuously required for the neuronal performances attributed to its action ill vitro (1.5, 45) and, possibly, its zi2ro (antiserum studies). Furthermore, it appears that NGF plays its role over a much longer span of the target cell history than a differentiation-inducing agent would. Sensory and sympathetic neuroblasts are already subject to (and possibly in need of) NGF, since neuronal generation is increased by the factor during the replicating stages of the ganglia (40). A very early involvement of NGF is also suggested by the study of catecholamine-containing cells in explant cultures from early trunk and head neural crest (6). Norr (48) has described the interactions among neural crest, somite and ventral neural tube in the course of sympathetic differentiation in z&o. Neural tube was involved at two sequential levels, the first to confer competence to the somite tissue for its induction of neural crest into sympathetic elements, and the second to permit survival of the induced sympathetic cells. NGF could substitute for neural tube in its survival, but not its induction role. The graded action of NGF, the requirement for its continuous presence, and the long developmental span over which it appears to operate fit the definition of NGF as a modulation factor (with cell death as a limit case) rather than a differentiation factor, as it has often been described. RNA and Protein .‘?~l~ltl?csis. The early view of NGF as a differentiation factor focused attention to transcriptional events in NGF-treated ganglia, id’.,

tllc

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LvtJlll~[

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to reside. Angeletti et al. (1 j examined the accumulation of labeled RNA and protein in S-day chick embryo DRG incubated (free-floating) with radioprecursors in the presence or absence of NGF. The treated ganglia had a twofold higher level of labeled RNA by 2 hr and a 30% higher level of labeled protein by 6 hr. Blocking RNA synthesis with actinomycin eliminated the protein difference, while a puromycin block of protein synthesis did not abolish the RNA difference. The authors, therefore, proposed that RNA synthesis may be the primary site of NGF action and that all other responses may be secondary to the RNA response. Several later studies (13, 41, Sl), however, showed that neurite production could still be elicited by NGF in actinomycin-treated DRG and sympathetic ganglia. Moreover, sympathetic ganglia in explant cultures (51) , as well as unattached DRG or DRG cells (13), revealed a progressive decline in RNA and protein labeling capabilities in the absence of NGF and no better than a maintenance of the initial rates in its presence. The inadequacy of the untreated ganglia as a “control” material drew attention to (i) the possible role of other supportive agents, and (ii) the consequences of NGF deprivation. Insulin has been also reported to stimulate anabolic performances of DRG (39) and sympathetic ganglia (51), and several anabolic responses of 3T3 fibroblasts to insulin [the so-called “positive pleiotypic response” (32)] resemble those displayed by ganglionic tissue under NGF treatment. The possibility that the two agents might elicit similar responses through similar mechanisms was further strengthened by the uncovered similarities of structure between the insulin and the NGF polypeptides (20). Burnham et al. (14) described RNA and protein labeling in S-day chick embryo DRG (intact or dissociated) that were treated with NGF, insulin, concanavalin A or serum. All four agents, applied individually, maintained or improved the initial incorporation rates. However, combined treatments (with each agent at its optimal dose) led to different effects, ranging from mutually inhibitory to potentiating interactions, and pointing to the involvement of different processes or of different subpopulations of cells. In particular, NGF and insulin had additive effects, clearly separating the actions of the two agents on DRG cells. The consequences of NGF deprivation were studied with dissociated DRG cells, supplied with NGF after increasing incubation times and tested with l-hr pulses of radioprecursors (34, 3.5, 60). An early set of events develops over the first 6 hr without NGF, revealed by a decline in the labeling of RNA but not protein. The decline was fully reversed by NGF, however late it was presented in this period. The reversal took place within 10 min of the NGF presentation, the first example of a biochemical response elicited by NGF with a very short latency and against a reliable control. A later set of events takes place if NGF deprivation is prolonged

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beyond 6 hr [as already morphologically recognized by several invcstigatars (41 ) 1. It comprised irreversible declines in RNA labeling and protein labeling, developing in precise coincidence with each other, and a progressive degradation of prelabeled RNA and protein. Presentation of NGF before 6 hr prevented the occurrence of this entire set of events, between 6 and 15 hr interrupted or delayed its progression, and after 15 hr could no longer affect it. Further examination of the early and reversible events (60) revealed that accumulation of acid-soluble radioactivity (upon 1-hr pulse of radiouridine) at 6 hr was also lower in the NGF-deprived cells and was rapidly raised by the factor presented at that time. The NGF-induced increase in soluble radioactivity occurred even after pretreatment with actinomycin or cycloheximide, demonstrating its independence from any NGF consequences on RNA or protein synthesis. Converseiy, the rise in soIuble radioactivity was adequate to explain the coincidental rise in RNA labeling: the ratio of the two events was fairly constant in all measurements, the soluble radiopools consisted mainly of uridine and its three nucleotides with the same relative proportions in NGF-deprived and NGFtreated cells, and the amounts of radio UTP (the direct precursor to labeled RNA) were small enough to impose corresponding differences to the specific activity of UTP total pools in the two cell preparations. Finally, accumulation of labeled Z-deoxyglucose and cY-isoaminobutyrate (nonmetabolizable analogs of sugar and amino acids, respectively) was also lower in NGF-deprived cells, and was rapidly raised by delayed presentation of NGF even after actinomycin or cyclohesimide pretreatments. NGF and the Cell Membrane. The presumption that NGF operates on the outer surface of the target cell membrane forces attention to changes occurring within the membrane and most closely reflecting the interaction between the factor and its receptor sites. Recent investigations have ruled out a direct involvement of cyclic AMP as a “second message” relaying the NGF action to other segments of the cell machinery (22, 45)) although a possible role of other cyclic nucleotides remains to be explored. On the other hand, the data described in the preceding section suggests that a basic consequence of the NGF action could be a modulation of membrane permeation properties. The rise in intracelIular accumulation of labeled exogenous substrates can be demonstrated within minutes of the NGF presentation and in the absence of transcriptional and translational events, and must involve changes in transport mechanisms or retention mechanisms, either of which would reside in the plasma membrane. Thus, of all the responses to NGF presently known, this is the closest in both time and space to the association between NGF and the target cell membrane. Changes in substrate accumulation appears adequate to explain the early

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chauges in RNA labeling and, because of the wide spectrum of substrates iuvolved, may readily be responsible for the later set of RNA and protein changes, the ultimate degeneration of the target neurons or, conversely, the survival and optimized performance of these neurons when properly supplied with NGF. The effects of NGF on plasma membrane, of course, need not be restricted to pernreation changes. There are several indications that membrane adhesiveness may also be altered, leading to better neuronal attachment to a culture surface (64, 65), stronger interaction between neurons and non-neurons (55, 68)) and greater aggregation of early embryonic ganglionic cells (69). Furthermore, the neuronal membrane is critically involved in neurite elongation. Promotion of neurite outgrowth is the most classical NGF effect, and yet the mechanisms involved in this NGF action have been perhaps least amenable to experimental investigation. Thus far, attention has been essentially limited to the involvement of microtubules in neurite elongation, but attempts to uncover an NGF effect on the synthesis of tubulin have only led to conflicting results (33, 70). However, studies on the general modalities of neurite outgrowth (see “Neurons and glia in neural cultures: a review,” and the Workshop on “Culture techniques and glial-neuronal interrelationships in vitro”) indicate that production and peripheral delivery of neurite constituents may be only subsidiary events, and that control mechanisms probably reside in membrane phenomena such as adhesion and mobility of the growth cone and/or the net balance between membrane assembly and resorption. Thus, NGF might elicit neurite elongation through a primary alteration of such membrane events, and support it by a secondary promotion of synthesis and supply of the necessary materials. Much remains to be learned with respect to the actions of NGF at the membrane level: what membrane activities reflect such actions, what modalities underlie each membrane response, how they are to be sequentially ranked and, ultimately, which is the first NGF-induced molecular alteration that may direct them all. The tubulin involvement recently postulated by Levi-Montalcini et al. (42) may be a key component, particularly in view of the several relationships noted between tubulin polymerization and membrane permeabilities to ions, sugars and other substrates. Intake or retention of essential substrates or of extrinsic regulatory agents, regulation of the intracellular ionic environment, control of microtubule and/or microfilament polymer structures are other possibilities to be further investigated. A prior determination that NGF acts exclusively on the cell membrane (or membranes), and a precise recognition of the alterations it may cause within the confines of cell membranes will immensely enhance the value of NGF as a “dissecting” tool for a further under-

NERVE

GROWTH

stallding of the nerve cell machinery operate 011 it.

FACTOR

and the regulatory

89

mecliallisms

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REFERENCES 1. AN(;ELETTI, I’. U., D. G~\NIJINI-ATTAK~I, G. TOSCHI, M. L. SALVI, and R. LEVIMONTALCINI. lY65. Metabolic aspects of the effect of Nerve Growth Factor on aynllrathrtic and stllscjr-y ganglia ; l’rotciu au,1 rihonucleic acid synthesis. llioc/lir,l. Hiopllys. AL.tll 95 : 11 l-120. 2. ANGELETTI, K. H., and K. A. BKADSHAW. 1071. Nerve Growth Factor from mouse submaxillary gland: amino acid sequence. Proc. Nut. Acnd. Sci USA 68 : 2417-2420. 3. ANGELETTI, R. H., P. U. ANGELETTI, and R. LEVI-M• NTALCINI. 1972. Selective accumulation of l”I-labeled Nerve Growth Factor in sympathetic ganglia. Urni~ lies. 46 : 421-425. 4. BANERJEE, S. P., S. H. SNYDER, P. CU~~TRECASAS, and L. GREENE. 1973. Binding of Nerve Growth Factor receptor in sympathetic ganglia. Proc. Nut. Acad. Sci. USA 70 : 2519-2523. 5. BERGER, B. D., B. C. WISE, and L. STEIN. 1973. Nerve Growth Factor: Enhanced recovery of feeding after hypothalamic damage. Science 180: 506-508. 6. BJERRE, B., and A. BJ~RKLUND. 1973. The production of catecholamine-containing cells in vitro by young chick embryos: Effects of Nerve Growth Factor (NGF) and its antiserum. NewobiologJl 3 : 140-161. 7. BJERRE, B., A. BJ~RKLUKD, and U. STENEVI. 1974. Inhibition of the regenerative growth of central nora&-energic neurons by intracerebrally administered antiNGF serum. Brain Kcs. 74 : l-18. 8. BJORKLUND, A., and U. STENEIT. 1972. Nerve Growth Factor: Stimulation of regenerative growth of central noradrenergic neurons. Scic~~ce 17.5 : 1251-1253. 9 BOCCHINI, V., and P. U. ANGELETTI. 1969. The Nerve Growth Factor: Purification as a 30,000 molecular weight protein. Proc. Nat. Acad. Sri. US.4 64: 787-794. 10. BRAY, D. 1970. Surface movements during the growth of single explanted neurons. Proc. Nat. ilcad. Sci. USA 65: 905-910. 11. BURNHAN, P. A., C. RAIBORN, and S. VARON. 1972. Replacement of Nerve Growth Factor by ganglionic non-ncuronal cells for the survival in vitro of dissociated ganglionic neurons. Proc. Nat. Acad. Sri. USA 69: 3556-3560. 12. Bu~wr~~znr, 1’. A., and S. VARON. 1973. In vitro uptake of active Nerve Growth Factor in dorsal root ganglia of embryonic chick. Neurobiology 3: 232-245. 13. BURNHAX~, P. A., and S. VARON. 1974. Biosynthetic activities of dorsal root ganglia in vitro and the influence of Nerve Growth Factor. Neurobiology 4: 57-70. 14. BURNHA~I, P. A., J. SILVA, and S. VARON. 1974. Anabolic responses of embryonic dorsal root ganglia to Nerve Growth Factor, insulin, concanavalin A or serum in vitro. J. Ncurochcm 23 : 689-697. 15. COHEN, S. 1958. A nerve growth-promoting protein pp. 665-675. In “Chemical Basis of Development.” W. D. McElroy and B. Glass [Eds.]. Johns Hopkins Press, Baltimore, Md. 16. COHEN, S. 1959. Purification and metabolic effects of a nerve growth-promoting protein from snake venom. 1. Biol. Cheer. 234: 1129-1137. 17. COULOMBRE. A., M. JOHNSON, and J. WESTON. 1974. Conference on neural crest in normal and abnormal tmbryogenesis. Dnvlop. Biol. 36, Fl-F5.

90 18. 19. 20. 21. 22.

23.

24. 25. 26.

SILVIO

VARON

J. D. 1967. Molecular and cellular interactions in development, pp. 241247. I>c “The Neurosciences: A Study Pragram.” G. C. Quarton, T. Melnechuck, and F. 0. Schmitt [Eds.]. Rockefeller University Press, New York. EISENBARTH, G. S., M. K. DHEZNEK, and H. E. LEBOVITZ. 1975. Inhibition of chondromucoprotein synthesis : an extra-neuronal effect of Nerve Growth Factor. J. Pharnzacol. Exp. Therap. In press. FRAZIER, W. A., R. H. ANGELETTI, and R. A. BRADSHAW. 1972. Nerve Growth Factor and insulin. Science 176 : 482-488. FRAZIER, W. A., L. F. BOYD, and R. A. BRADSHAW. 1973. Interaction of Nerve Growth Factor with surface membranes : Biological competence of insolubilized Nerve Growth Factor. Proc. Nat. Acod. Sci. USA 70: 2931-2935. FRAZIER, W. A., C. E. OHLENDORF, L. F. BOYD, L. ALOE, E. M. JOHNSON, J. A. FERRENDELLI, and R. A. BRADSHAW. 1973. Mechanism of action of Nerve Growth Factor and cyclic AMP on neurite outgrowth in embryonic chick sensory ganglia : Demonstration of independent pathways of stimulation. Proc. Nat. Acad. Sri. USA 70: 2448-2452. FRAZIER, W. A., L. F. BOYD, A. SZUTOWICZ, M. W. PULLMAN, and R. A. BRADSHAW. 1974. Specific binding sites for 1251-Nerve Growth Factor in peripheral tissues and brain. Biochem. Biophys. Res. Comm. 57: 1096-1103. GARBER, B. B., and A. A. MOSCONA. 1972. Reconstruction of brain tissue from cell suspensions. II. Specific enhancement of aggregation of embryonic cells by supernatant from homologous cell cultures. Devel. Biol. 27 : 235-243. GREENE, L. A., E. M. SHOOTER, and S. VARON. 1969. Subunit interactions and enzymatic activity of mouse 7s Nerve Growth Factor. Biochemistry 8: 37353741. GREENE, L. A., J. T. TOMITA, and S. VARON. 1971. Growth-stimulating activities of mouse submaxillary esteropeptidases on chick embryo fibroblasts in vitro. EBERT,

Exp.

Cell

Res. 64 : 387-395.

27. HAAS, D. C., D. B. HIER, B. G. W. ARNASON, and M. YOUNG. 1972. On a possible relationship of cyclic AMP to the mechanism of action of Nerve Growth Factor. Proc. Sot. Exp. Biol. Med. 140: 45-47. 28. HENDRY, I. A. 1972. Developmental changes in tissue and plasma concentrations of the biologically active species of Nerve Growth Factor in the mouse, by using a two-site radioimmunoassay. Biochem J. 128: 1265-1272. 29. HENDRY, I. A., and L. L. I~ERSEN. 1973. Reduction in the concentration of Nerve Growth Factor in mice after sialectomy and castration. Nature (London) 243 : 500-504. 30. HENDRY, I. A., K. STOCKEL, H. THOENEN, and L. L. IVERSEN. 1974. The retrograde axonal transport of Nerve Growth Factor. Brain Res. 68: 103-121. 31. HERRUP, K., and E. M. SHOOTER. 1973. Properties of the fi Nerve Growth Factor receptor of avian dorsal root ganglia. Proc. Nat. Acad. Sci. USA 70 : 3884-3888. 32. HERSHKO, A., P. MAMONT, R. SHIELDS, and G. M. TOMKINS. 1971. Pleiotypic response. Nature New Biol. 232 : 206-211. 33. HIER, D. B., B. G. W. ARNASON, and M. YOUNG. 1972. Studies on the mechanism of action of Nerve Growth Factor. Proc. Nat. Acad. Sci. USA 69: 2268-2272. 34. HORII, Z. I., and S. VARON. 1974. Rapid activation by Nerve Growth Factor of ganglionic RNA synthesis in vitro. Proc. 5th Meeting, Amer. Sot. Neurochem. (March 1974), p. 76. 35. HORII, Z. I., and S. VARON. 1975. Nerve Growth Factor induction of rapid radiolabeling of RNA in dorsal root ganglionic dissociates from the chick embryo. Submitted.

NERVE

GROWTH

FACTOR

91

.%I.JOII NSON, b. fi., I’. 37.

38.

39. 40. 41.

42.

43. 44. 45.

46.

47.

~UJON, and I. J. I
48. Nonlt, S. C. 1973. In vitro analysis of sympathetic neuron differentiation from chick neural crest cells. Dez*eZop. Biof. 34: 16-38. 49. NORR, S. C., and S. VARON. 1975. Dynamic, temperature-sensitive association of ?-Nerve Growth Factor in vitro with ganglionic and non-ganglionic cells from embryonic chick. Neurobiology. In press. 50. OGER, J., B. G. W. ARNASON, N. PANTAZIS, J. LEHRICH, and M. YOUNG. 1974. Synthesis of Nerve Growth Factor by L and 3T3 cells in culture. Proc. Nat. Acad. Sci. USA 71: 15541558. 51. PARTLOW, L. M., and M. G. LARRABEE. 1971. Effects of a Nerve Growth Factor, embryo age and metabolic inhibitors on growth of fibers and on synthesis of ribonucleic acid and protein in embryonic sympathetic ganglia. J. Newochem. 18 : 2101-2118. 52. PATTISON, S. E., and M. F. Dun-~. 1974. Relationship of Zn++ to the structure and function of Nerve Growth Factor. Bioche,nistry/Biophysics 1974 Meeting Abstracts. 53. PEARCE, F. L., B. A. E. BANKS, D. V. BANTHORPE, A. R. BERRY, H. P. S. DAVIES, and C. A. VERNON. 1973. The isolation and characterization of Nerve Growth Factor from the venom of Vipcra rzlsselli, pp. 4-18. In “Nerve Growth

92

SILVIO

Factor London. 54,

56.

57 58. 59.

60.

61. 62.

63.

64.

65.

66.

67.

68.

69.

70.

71.

Its

Antiserum.”

E. Zaimis

and

J. Knight

[Eds.].

Athlone

Press,

and E. bl. SHUOTEH. 1974. Subunit athnities in the 7S Ncrvc complex. Nrzrrohio/o~/y 4 : 197-209. RIESKE, Einfluss eines spezifischen Nervenwachstumsfaktors (NGF) auf des Ganglion Trigeminale. 2. Zellforsch. 95: 54~567. SENSENBRENNER, M., N. SPRINGER, J. BOOHER, and P. MANDEL. 1972. Histochemical studies during the differentiation of dissociated nerve c,ells cultivated in the presence of brain extracts. Neurobiology 2: 49-60. SMITH, A., S. VARON, and E. M. SHOOTER. 1%8. Multiple forms of the Nerve Growth Factor protein and its subunits. Biochemistry 7: 3259-3268. STEINER, G., and E. SCHONBAUM. [Eds.]. 1972. “Immunosympathectomy,” Elsevier, New York. THOENEN, H., P. U. ANGELETTI, R. LEVI-M• NTALCINI, and R. KETTLER. 1971. Selective induction by Nerve Growth Factor of tyrosine hydroxylase and dopamine+hydroxylase in the rat superior cervical ganglion. Proc. Nut. Acad. Sci. USA 68 : 1598-1602. VARON, S., and Z. I. HORII. 1975. Effects of Nerve Growth Factor on the accumulation of exogenous substrates in dorsal root ganglionic cells. Proc. 6th Meetizzg, Amer. Sot. Neurochem. (March 1975). VARON, S., J. NOMURA, and E. M. SHOOTER. 1968, Reversible dissociation of the mouse Nerve Growth Factor into different subunits. Biockelnistry 7 : 1296-1303. VARON, S., and C. RAIBORN. 1972. Protective effect of mouse 7s Nerve Growth Factor protein and its Alpha subunit on embryonic sensory ganglionic cells during dissociation. Neurobiology 2 : 183-196. VARON, S., J. NOMURA, J. R. PEREZ-POLO, and E. M. SHOOTER. 1972. The isolation and assay of the Nerve Growth Factor proteins, pp. 203-229. In “Methods and Techniques of Neurosciences.” R. Fried [Ed.]. M. Dekker, New York. VARON, S., C. RAIBORN, and P. A. BURNHAM. 1974. Comparative effects of Nerve Growth Factor and ganglionic non-neuronal cells on purified mouse ganglionic neurons in culture. J. Nezrrobiol. 5: 355-371. VARON, S., C. RAIBORN, and P. A. BURNHAM. 1974. Selective potency of homologous ganglionic non-neuronal cells for the support of dissociated ganglionic neurons in culture. Neurobiology 4: 231-252. VARON, S., C. RAIBORN, and P. A. BURNHAM. 1974. Implication of a Nerve Growth Factor-like antigen in the support derived by ganglionic neurons from their homologous glia in dissociated cultures. Nezlrobiology 4: 317-327. VARON, S., C. RAIBORN, and S. C. NORR. 1974. Association of antibody to Nerve Growth Factor with ganglionic non-neurons (glia )and consequent interference with their neuron-supportive action. E.zp. Cell Res. 88: 247-256. WEIS, P. 1970. The in vitro effect of the Nerve Growth Factor on chick embryo spinal ganglia-a light-microscopic evaluation. J. Enzbryol. Exp. Morph. 24 : 381-392. WESTON, J. A. 1972. Cell interaction in neural crest development, pp. 286-xxx In “Cell Interactions” (3rd Lepetit Colloquium). L. G. Silvestri [Ed.]. Elsevier, New York. YAMADA, K. M., and N. K. WESSELS. 1971. Axon elongation: Effect of Nerve Growth Factor on microtubule protein. Exp. Cell Res. 66: 346-352. ZAIMIS, E., and J. KNIGHT. [Eds.]. 1972. “Nerve Growth Factor and Its Antiserum.” Athlone Press (University of London), London. PEREZ-POLO,

Growth

55.

and

VARON

J. R.,

Factor E. 1969. Zellkulturen