Volume 28, number 1-3

MOLECULAR • CELLULAR BIOCHEMISTRY

D e c e m b e r 14, 1979

GROWTH REGULATION BY NERVE GROWTH FACTOR Takeyuki I K E N O & G o r d o n G U R O F F

Section on Intermediary Metabolism, Laboratory of Developmental Neurobiology, National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, Maryland 20205 (Received July 23, 1979)

Smumary Although a great deal of descriptive information has been obtained about the actions of nerve growth factor on its target tissues, its structure, its receptors, and even its biosynthesis, there is no clear understanding, as yet, of the intracellular events mediating its transcriptional involvements. Work in this laboratory over the past five years has uncovered a number of nerve growth factor-initiated intracellular changes in sympathetic neurons and other nerve growth factor-sensitive systems, and has provided a framework into which they might fit. This article is written in an attempt to collect the data in a single communication and to suggest at least one mechanism by which the nerve growth factor may work.

Introduction The series of studies, in the late 1940's, which led up to the concept of a "nerve growth factor" has been described in several early reviews 1'2. Briefly, the introduction of a tumor, the Sarcoma 180, into a chick embryo by BUEKER 3"4 led to the overgrowth of the sympathetic nervous system and the innervation of the tumor by these nerve fibers. Subsequent studies by LEvI-MONTALCINI and HAMBURGER5'6, in which the tumor was placed outside the chorioallantoic membrane, showed the same response, and led to the conclusion that the factor causing the outgrowth was diffusable. The observations on outgrowth allowed the develop-

ment of the dorsal root ganglion " h a l o " assay, still a standard quantitative methodology, in which the ganglion is placed in a plasma clot and the unknown amount of nerve growth factor is rated against a series of standards for its ability to induce neurite outgrowth 7. Using this assay, an attempt was made by CoFrEN8'9 to purify the factor from the tumor. When the lytic agent snake venom was used to remove the nucleic acids it was observed that the nerve growth factor activity of the venom was greater than that of the tumor s . Keyed by this observation, a search for a better source of the factor was initiated, and the richest source was found to be the salivary gland of the mature male mouse. Although the significance of the inordinately high concentration of nerve growth factor in this tissue is not known, the gland remains the source of choice for studies on the nerve growth factor. The biological importance of nerve growth factor was established by studies which showed that the antibody produced to nerve growth factor destroyed the sympathetic nervous system of young rats 1°. Now, after more than 30 years of effort in many laboratories, a number of things are known about the factor. It is, of course, a protein and occurs as a dimer, each chain of which has a molecular weight of 13,259 daltons. The protein is quite basic and can be isolated from the salivary gland as a molecule with a sedimentation of 2.5811. The nerve growth factor can also be isolated in a high molecular weight form, the 7S 12, which contains three different subunits 13 and has a molecular weight

Dr. W. Junk b.v. Publishers- The Hague, the Netherlands

67

of 130,000. The a-subunit has no known function, the 7-subunit is a protease which may participate in the post-translational processing of some larger precursor to the mature nerve growth factor. The/3-subunit contains the total nerve growth factor activity of the molecule and is the same as the 2.5S described above, except that the 2.5S undergoes some minor proteolytic modification during purification14. The 7S molecule contains Z n 2+ 15 and it appears that t h i s Z n 2+ is critical for the stability of the complex 16. Earlier data indicated that, molecule for molecule, the 7S was more active than the 2.5S. This led to the suggestion that the combination of the 13-subunit with the o~- and the 7-subunits somehow enhanced its activity. Now the weight of evidence is in the opposite direction, i.e., the two forms appear to have equal activity, molecule for molecule. The function of the 7S molecule is not known, but it is suspected that the combination with the o~- and 7-subunits may serve to protect and store the nerve growth factor. BRADSHAWand his colleagues have determined the linear sequence of the nerve growth factor and have shown that it bears substantial homology to the sequence of proinsulin T M . Further studies have indicated that the tertiary structure of the molecule as well may resemble that of insulin 19. Studies by others, however, have suggested that there may be some differences in the secondary structure of these two molecules2°. Nevertheless, this work has provided a new insight into the relationship between the various peptide effectors, and has suggested that the mode of action of the two materials in their respective targets may be analogous. It seems fair to say that one of the major stumbling blocks to rapid progress in the studies on nerve growth factor has been the difficulty in executing and interpreting the quantitative assays. The original outgrowth assay7, using chick dorsal root ganglia, is semi-quantitative at best and requires an experienced individual to carry it out. Modifications based on the use of dissociated cell Cultures have been presented 21. A number of immunochemical methods have been published, including one-site 22, two-site 23, and complement fixation assays24. These various methods have been less than satisfactory due to the presence in all sera of low affinity, nerve 68

growth factor-binding proteins, and because of the lack of reproducibility in the commercially available reagents. Also in use are a phage immunoassay25, a recombination assay26, and a "rocket immunoelectrophoresis" method 27. Perhaps the most promising recent development has been the publication of a carefully reworked immunoassay28 based on the use of purified anti-nerve growth factor antibody29"3°. It seems reasonable to hope that the use of this new tool will clear up some of the inconsistencies in the nerve growth factor literature, especially that literature dealing with clinical problems. Although the salivary gland, at least in the mouse, contains the highest concentration of nerve growth factor known, there is evidence that many types of cells can make nerve growth factor. Indeed, the wide variety of cells which have been shown to elaborate the factor into culture medium would suggest that the synthesis is carried out in most, if not all, cells of the body. Fibroblasts, L-cells, muscle cells in culture, and a number of tumor lines are found to secrete nerve growth factor 25'3x-34. C-6 glioma cells produce nerve growth factor and this production is stimulated by/3-adrenergic agonists 35 and by 17-/3-estradio136. The most complete studies have been carried out by SHOOTER and his colleagues who have studied explants of mouse salivary gland37'38. These experiments have shown that the gland will make nerve growth factor in vitro, and that the synthesis is stimulated by testosterone, as one would expect from the observation that glands from mature male mice contain much more factor than those from females. Further, these data provide evidence that nerve growth factor, like so many other peptide effectors, is made in a high molecular weight form and processed to its active state after translation. Classically, the targets of the nerve growth factor have been thought to be sympathetic and sensory neurons. The former appear to be responsive to the factor throughout their life, the latter seem to need the factor only during embryonic development. However, there are many reports in the literature of responses to nerve growth factor by a wide variety of other normal and malignant cells. Adrenal medullary cells, early in their development, can be shown to respond morphologically both in 1)ivo 39 and

in vitro4°; nerve growth factor also produces increases in the activity of specific enzymes in these cells 41. When treated with nerve growth factor, cells of the optic tectum show changes in cellular adhesiveness42, chondrocytes exhibit an increased mucopolysaccharide synthesis43, and cells in the central nervous system show some morphological and histological responses 44-46. The PC12 clone of rat pheochromocytoma, a most useful model for both nerve growth factor action and neuronal differentiation, when treated with nerve growth factor, elaborates neurites, develops excitable membranes, and stops dividing47-49. Another clone of pheochromocytoma shows a selective induction of tyrosine hydroxylase5°. In addition, there are several reports of responses ranging from simple receptor interaction to morphological and biochemical alterations in various other tumor lines sl-5a. So, although nerve growth factor is clearly required for the survival and development of sympathetic and sensory neurons, there is growing evidence that it also acts on a wide variety of other cells. There are at least three different sites on the cell at which nerve growth factor might bind. Some years ago, it was found that specific receptors for the peptide were present on the plasma membranes of responsive cells 54-56. These receptors were of high affinity and specific for nerve growth factor. At about the same time it was observed that nerve growth factor injected into target organs of the superior cervical ganglia would move in a retrograde direction up the axon to the ganglia and that other proteins with similary physical properties would not 57-59. These data, while having a number of important implications, certainly suggest that specific and high-affinity receptors are present on the synaptic membrane. Finally and most recently, it has been shown that the nerve growth factor, like insulin, has receptors on the nuclei of responsive cells 6°'61. It is presently not known which of these receptors is involved in which of the various actions of the factor. The meaning of the retrograde transport data seems clear. There is abundant evidence from many sources that the vitality and the integrity of the target organs influences the development and the characteristics of the innervating cells. In spite of the wealth of data suggesting this, the mechanism by which a target could in-

fluence its eventual innervation remains unknown. One particularly attractive mechanism would be for the target organ to elaborate a material which would be sent in some fashion to the cell body and evoke a biochemical response. In the case of targets innervated by sympathetic arid sensory neurons, this signal could be nerve growth factor. The actual demonstration by HENDRY, TnOENEN and their colleagues that this peptide could travel through the axon back to the cell body provided evidence for such a mechanism. The further fact that the transport was specific, even among other effector peptides, or among peptides of similar molecular weight and isoelectric point, but of different biological properties, supported the postulate. Studies in which the biological activity of the nerve growth factor was destroyed by oxidation showed that the retrograde movement and the biological activity depended upon similar structural principles. The demonstration that the nerve growth factor moving to the cell body was both biologically active and structurally intact strengthened the theory. Overall, then, although retrograde movement is not the only way in which nerve growth factor acts on a cell, these studies have provided a mechanism by which a target organ can direct and nourish its incoming innervation. The actions of nerve growth factor on its neurons are bewilderingly various. Indeed, the variety of responses that it evokes has complicated rather than aided investigations into its mechanism of action. Included in its actions on cells are hypertrophy and possibly hyperplasia, increases in a whole host of anabolic responses, such as protein, RNA and lipid synthesis, a stimulation of membrane transport systems, neurite production, and the induction of certain specific enzymes. This latter response, the induction of the transmitter-synthesizing enzymes in sympathetic neurons, first observed by THOENEN and his colleagues 62, seemed a reasonable place to initiate investigations on the mechanism. Specifically, their observation was that administration of/~g quantities of nerve growth factor to young rats led, after a period of several days to large increases in the specific activity of tyrosine hydroxylase and dopamine-/3-hydroxylase (Fig. 1), two of the enzymes in the biosynthetic pathway to the neurotransmitter, norepinephrine. The increase is quite specific in that the 69

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Fig. 1. The effect of nerve growth factor on enzymes involved in the synthesis and degradation of norepinephrine in rat superior cervical ganglia. Newborn rats were treated daily with 10 ~g/g of nerve growth factor for 10 days. The specific activity of all the enzymes studied is expressed as a percent of the control activity. From the work of THOENEN et al. 62

other enzyme in the same pathway (Fig. 2), D O P A decarboxylate, and related enzymes, such as monoamine oxidase, are not increased. Indeed, even the enzyme which couples pteridine reduction to tyrosine hydroxylation, the dihydropteridine reductase, is unaffected 63. A m o n g the several reasons for hoping that this pathway might lend itself to useful studies on the mechanism were that the induction of tyrosine hydroxylase represents a most specific and discrete action of the nerve growth factor, that the chemistry of the reaction catalyzed by tyrosine hydroxylase is well understood, that the characteristics of the enzyme itself are known, and that the process of enzyme induction in eukaryotes is understood to some extent. These considerations seemed to make the system a favorable one and to promise some advantage over the dorsal root ganglia, for example, where even the nature of the transmitter is not yet known, or the process of neurite outgrowth about which little is understood in a molecular sense. There seemed to be a logical series of questions which could be posed about this

70

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action of nerve growth factor. First, is nerve growth factor, in fact, an inducer of the enzyme. That is, can one show that the differential rate of tyrosine hydroxylase synthesis increases in response to nerve growth factor. Second, what are the intervening intracellular events which mediate the combination of peptide with receptor and the eventual enzyme induction. Third, which of these intracellular events are specific for the induction and which are common to the many actions of nerve growth factor. Finally, through which of the three receptors, membrane, synaptic, or nuclear does nerve growth factor act to exert its action on tyrosine hydroxylase.

Experimental procedures Most of the experiments to be described were done using superior cervical ganglia under a standard set of conditions 64. Ganglia were removed from 5-day or 21-day old rats; they were cleaned, decapsulated, and placed in cold medium until used. Incubations were done in serum-free B G J b medium containing 0.1% bovine serum albumin, ascorbic acid, and glutamine, but with no phenol red indicator.

The ganglia were kept at 37 ° in an atmosphere of 95% 0 2 and 5% C O 2. Tyrosine hydroxylase activity was estimated by the method of NAGATSU et al. 65 as modified by OESCR et al. 66 The de n o v o synthesis of tyrosine hydroxylase was measured in comparable organ cultures of superior cervical ganglia or adrenal medullae using radioactive leucine67'68. Following the incubation the tissues were homogenized in 5 rnM Tris, pH 7.4, containing 0.1% Triton X-100 and centrifuged. The supernatant fraction was brought to 1% Triton X-100 and 1% freshly prepared deoxycholate and carrier tyrosine hydroxylase and monospecific antiserum added in a total volume of 1 ml. The precipitation was allowed to proceed for 1 hour at 30 ° and for 16 hours at 4 °. Then the sample was layered onto 0.5 ml of 1 M sucrose containing 150 naM NaC1, 1% Triton X-100, and 10 mM sodium phosphate, pH 7.4. The sample was centrifuged at 16,000 g for 15 minutes. The precipitate was washed thoroughly, dissolved, and placed on a 7.5% SDS-polyacrylamide gel by the procedure of WEBER et al. 69 After the gels had been removed from the tubes they were sliced into 1.2 mm sections. Each section was dissolved in hydrogen peroxide containing 1% ammonium hydroxide and counted. Experiments were done in order to measure the response of cAMP levels to nerve growth factor 7°. The ganglia from 21-day old animals were incubated as described above, rinsed in ice-cold 0.25 M sucrose for 2 minutes, blotted on filter paper, and homogenized in 0.6 ml of ice-cold 5% trichloroacetic acid. After 15 minutes the homogenate was centrifuged and the supernatant portion was washed three times with 2 volumes of ether. The aqueous phase was assayed in quadruplicate by the competitive binding assay described by GILMAN71. Each experiment included an identical set of duplicate samples with an internal standard of 0.5 pmol of cAMP. Measurements of ornithine decarboxylase activity72-74 have been done by the method of PE60 and WILLIAMS--AsHMAN75 as modified by OKA and PERRY76. Briefly, the method involves the formation of (14CO2) from (1-14C)-ornithine. The enzyme reaction was carried out in rubberstoppered 15 × 100-ram glass tubes that were fitted with a hanging center well which con-

tained 0.2 ml of Hyamine hydroxide. The 0.5 ml reaction mixture contained 75 mM Tris, pH 7.5, 7.5 mM dithiothreitol, 60/~M pyridoxal phosphate, 6 mM EDTA, 55.5/ZM (1-14C)-ornithine monohydrochloride (2.5 tzCi), and a portion of the ganglia supernatant. Samples were incubated at 37 ° for 1 hour after which the reaction was terminated by injecting 0.5 ml of 2.5 M H 2 S O 4. The samples were allowed to stand overnight at room temperature. Then the center well was removed and the radioactivity of the trapped 14CO2 was measured in Liquifluor. In studies on the phosphorylation of nuclear proteins 77 intact ganglia have been incubated in the presence of radioactive phosphate, following various periods of treatment, either in vitro or in vivo, with nerve growth factor or other agents. After the incubation the ganglia were removed and rinsed in 0.32 M sucrose containing 3 mM CaC12, 1 mM MgC12, and 1 mM sodium phosphate, pH 6.5. The ganglia were then homogenized in 1 ml of the same buffer. The homogenates were centrifuged at 800 g for 10 minutes, the pellet resuspended in 1 ml of the same buffer containing 0.2% Triton X-100, and the centrifugation repeated. The pellet was suspended once again in 1 ml of the original buffer and layered on a 3.9 ml discontinuous sucrose gradient composed of 1.3 ml portions of 0.8 M, 1.2 M, and 2.4 M sucrose, all brought to the ionic composition of the original buffer, and centrifuged at 58,400 g for 1 hour. The nuclear pellet was then prepared for gel electrophoresis. For SDS gels the nuclear pellet was suspended in 150/zl of SDS sample buffer (0.0625 M Tris-HC1, pH 6.8, containing 10% glycerol, 5% /3-mercaptoethanol, and 2.3% SDS) and the suspension heated for 10 minutes at 90 °. The samples were centrifuged, counted, and portions of the supernatant fractions were placed in sample wells of a 10% SDSpolyacrylamide slab gel. The gel was run in SDS running buffer (0.025 M Tris base, 0.19 M glycine, and 0.1% SDS) at 100 volts for 3 hours. After electrophoresis, the gels were removed, stained for 15 hours with 0.5% Coomassie blue in 45% methanol-9% acetic acid, and destained in 40% ethanol-7% acetic acid followed by 7% acetic acid. The gels were dried and then exposed to X-ray film for 15 hours or more. The films were developed and 71

fixed by standard techniques and scanned with a densitometer. Acid-urea gels (15%) were done according to the procedure of PANYIMand CHALKLEY78. The stacking gel (3%) was prepared by mixing 1 volume of 43.2% glacial acetic acid (v/v)-4% TEMED, 0.4 volumes of 50% acrylamide (w/v) in 10 M urea, and 1.6 volumes of water. The staining and destaining of acid-urea gels was similar to that used for the SDS gels. Experiments on RNA polymerase79 were done with partially purified nuclei by a modification of the procedure of AUSTOKERet aL s°, originally applied to brain nuclei. Briefly, ganglia were homogenized in pairs in 0.8 ml of a solution containing 0.32 M sucrose, 1 mM MgC12, 3 mM CaC12, and 1 m~ potassium phosphate buffer, pH 6.8. Homogenization was done carefully in a ground glass homogenizer with mechanical pestle rotation at low speed. First, the ganglia were crushed in the bottom of the homogenizer and then they were dispersed with 3 to 4 vertical strokes. The homogenate was centrifuged at 12,000 g for 15 minutes. The supernatant portion was removed from the nuclei-containing pellet and the pellet was resuspended in 100/zl of assay medium by scraping it from the side of the tube and stirring the suspension briefly. The assay medium contained 40 mu Tris-HC1 buffer, pH 8.6, ATP, UTP, and CTP, 0.1 mM each, 1/zCi of (3H)GTP (0.2/XM), a creatine phosphate-creatine kinase ATP regenerating system, and KC1, 70 raM. MgC12, 5 raM, was added for polymerase I assay, and (NH4)2SO4, 200 raM, and MnC12, 0.6 raM, for polymerase II assays, as described by BANKSand JOHNSON81. The reaction mixture was incubated for 10 minutes at 30 ° with shaking. The assay was stopped by adding 200/zl of 10% trichloroacetic acid and allowing the tubes to cool in an ice bath. After 30 minutes the precipitate was deposited in a Yeda filtration apparatus on a Whatman GF/C filter disc which had been previously soaked in 5% trichloroacetic acid containing 0.1% pyrophosphate. The filter was then washed four times with 2 ml portions of 5% trichloroacetic acid containing 0.1% pyrophosphate. The precipitate was then washed four more times with 2 ml portions and two times with 5 ml portions of the same solution. Finally, the filtration top was removed and the paper washed around the edges with 72

the suction applied. The filter and the precipitate were digested with NCS for 1 hour at 55 °, cooled, and counted in 10 ml of Liquifluor. Certain experiments 82 were conducted with PC12 cells, a nerve growth factor-responsive clone from a rat pheochromocytoma developed by GREENE and his colleagues47-49. These cells were maintained in Dulbecco's Modified Eagle Medium (GIBCO) medium containing 15% fetal calf serum, glutamine, and a penicillinstreptomycin mixture. The cells were kept in Falcon flasks at 37 °. Ornithine decarboxylase activity and nuclear phosphorylation were measured as described above for ganglia. In preparation for such measurements, cells were washed carefully with fresh medium, shaken vigorously to detach them from the flask, and centrifuged to collect them. They were usually frozen in medium and then homogenized in an all-glass homogenizer to prepare a cell-free extract. Adhesion assays were done essentially by the procedure of SCHUBERTand WHITLOCK83. Exponentially growing cells were labelled overnight with 2 tzCi per ml of (3H)-leucine. The cells were washed three times with serum-free medium, detached with vigorous agitation, and collected by centrifugation. The labeled cells were then placed in new flasks with fresh medium and allowed to attach for 25 minutes. After this time the flasks were swirled in a standardized manner and the medium poured off. The adherent cells were shaken off in fresh medium, and the radioactivity in each fraction was measured. In most experiments, trichloroacetic acid precipitation of the fractions was also done to check that all residual free leucine had been removed. Measurement of epidermal growth factor receptors on these cells was done in a standard manner. Briefly, epidermal growth factor, prepared from the salivary glands of mature male mice 84, was iodinated by the procedure of CUATRECASAS85 using 1251, chloramine T, and talcum. PC12 cells were grown in Falcon dishes, washed gently to remove the medium, rinsed once with fresh medium without serum, and then exposed to iodinated epidermal growth factor in fresh serum-flee medium. Non-specific binding was evaluated by adding an excess of unlabeled epidermal growth factor to the incubation. The experiments on the action of nerve

growth factor in the central nervous system73"74 involved the intraventricular administration of txg quantities of the 2.5S form to adult rats. The nerve growth factor was dissolved in acetate buffer, 0.05 M, pH 5.0. The animals were placed under light ether anesthesia and the injections made in a volume of 5/xl or less with a Hamilton syringe. Tissues were removed after various time intervals and assayed for ornithine decarboxylase activity as described above. The 2.5S form of nerve growth factor was used in all experiments. It was prepared by the method of BOCCHINIand ANGELETr111. Antiserum to nerve growth factor was raised in sheep by standard methodology and purified over an affinity column as previously described 3°. Protein determinations were done by the method of LowRY et al. 86 All chemicals were obtained from commercial sources. Further details of the methodology and exact sources for chemicals and radioisotopes can be obtained by consulting the previous publications from this laboratory which have been referred to in the appropriate paragraphs above.

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Fig. 3. T h e time course of changes in the specific activity of tyrosine hydroxylase in rat superior cervical ganglia in organ culture without nerve growth factor and in the presence of 2.5S nerve growth factor at a concentration of 1 ~g/ml. For further details see Y u et al. 64

Thus, this system did provide a tool by which the exact rate of tyrosine hydroxylase synthesis could be measured. By incubating the ganglia for 16-18 hours in the presence of radioactive leucine, precipitating the enzyme with a monospecific antibody, and examining the precipitated

Results 4013

Superior cervical ganglia

These studies on the action of nerve growth factor on tyrosine hydroxylase synthesis were designed to determine if the increases in tyrosine hydroxylase specific activity observed by THOENEN et al. 62 did, in fact, reflect an increase in the synthesis of the enzyme, that is, a true induction. The initial experiments 67 were done using an explant system in which the levels of tyrosine hydroxylase were maintained by nerve growth factor (Fig. 3). Indeed, in most experiments, including the one illustrated, there was a small increase in the specific activity of tyrosine hydroxylase, but not nearly as much as that seen in vivo. Certainly most of the difference in tyrosine hydroxylase activity seen between nerve growth factor-containing and nerve growth factor-lacking explant cultures is due to the precipitous loss of activity in the latter samples because of the death of nerve growth factor-dependent sympathetic neurons. This difference is, however, dose-dependent (Fig. 4), specific for nerve growth factor, and can be used to evaluate the in vitro action of nerve growth factor on the ganglia.

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73

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Fig. 5. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis profile of immunoprecipitated tyrosine hydroxylase synthesized in superior cervical ganglia in culture. Ganglia from 7-day old rats were maintained in culture for 16 hours in the presence of 1/zg/ml of nerve growth factor and 50/zCi of (3H)-Leucine. Direction of migration is from left to right. For further details see MAcDONN~LL et al. 67

product on an SDS-polyacrylamide gel (Fig. 5), the amount of tyrosine hydroxylase synthesized under various conditions could be evaluated. Such experiments showed that the ganglia and the adrenal medulla synthesized the enzyme, the latter at four to five times the rate found in the ganglia (Table I). Further, other tissues not exhibiting tyrosine hydroxylase activity, as expected, did not synthesize the enzyme. These methods also allowed for the first time, an estimate of the subunit molecular weight of the

enzyme (Fig. 6) and, also for the first time, the clear-cut demonstration that the enzyme contains phosphate (Fig. 7). However, ganglia treated this way, as might have been predicted from the measurements of specific activity, did not show a marked increase in the differential rate of tyrosine hydroxylase synthesis upon addition of nerve growth factor (Table II). Under these conditions there was, at most, a two-fold increase, and that only at very high levels of nerve growth factor in the medium. Thus, the in vitro system, while providing a reasonable amount of information about tyrosine hydroxylase synthesis, did not offer much in the way of information about nerve growth factor action. A much more satisfying answer was obtained by adopting an in vivo-in vitro protocol 68. In these experiments the nerve growth factor was given in vivo and after 24 hours the ganglia were removed and cultured with radioactive leucine in order to measure tyrosine hydroxylase synthesis. The experiment showed that when nerve growth factor is administered to young rats the differential rate of tyrosine hydroxylase synthesis is increased on the order of four-fold (Table III). Further, the increase in the synthesis of the enzyme is probably due to an increase in the synthesis of tyrosine hydroxylase messenger RNA since the concomitant administration of actinomycin D prevents the nerve growth factor-induced increase in enzyme

Table I Synthesis of Tyrosine hydroxylase in rat tissues in culture Radioactivity in: Soluble protein TH (cpm/mg protein) x 10 -3 Superior cervical ganglia Nodose ganglia Liver Kidney Adrenal medulla

25,752 12,852 8,475 14,066 10.226

69.8 0 0 0 124.8

% Radioactivity in TH 0.27 0 0 0 1.22

Tissues from 5-7-day-old rats were cultured for 16-18 h in BGJ b media containing 1/zg/ml of 2.5S nerve growth factor (SCG and nodose ganglia only) and 50/zCi [3H]leucine (all tissues). The concentration of leucine in BGJ b medium is 0.38 mM; thus, the specific activity of [3H]leucine is 0.44 Ci/mmol. Tissues were homogenized in 5 mM-Tris, pH 7.4, containing 0.1% Triton X-100, and centrifuged at either 20,000 g or 105,000 g. Carrier tyrosine hydroxylase was added to aliquots of each supernatant fluid to a final concentration of 1.3-2.5 Units and was immunologically precipitated with a 2-4-fold excess of antiserum (500/zl) and purified by SDS-polyacrylamide gel electrophoresis. For further details see I~cDONNELL el al. 67

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synthesis. It should be noted that nerve growth factor was present in all the cultures to avoid the problems of differential neuronal survival in vitro. The maximal rate of tyrosine hydroxylase synthesis in the ganglia, even with repeated administration of nerve growth factor, is about 3200

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0.5% (Table IV). Thus, the nerve growth factor is, in fact, an inducer of the enzyme tyrosine hydroxylase. In order to understand the intracellular events mediating the induction of the enzyme by nerve growth factor studies on the possible involvement of cyclic nucleotides in the induction were done. At the time that these studies were initiated there was evidence in the literature that in dorsal root ganglia, at least, the addition of nerve growth factor did not lead to increases in cAMP content ss'89. On the other hand, the work of CosTa and his colleagues 9°'91 had strongly implicated cAMP in the induction of tyrosine hydroxylase in the adrenal medulla by stress, drugs, or transmitter substances. It seemed reasonable to reinspect the possible involvement of cAMP levels in the action of nerve growth factor on tyrosine hydroxylase induction in sympathetic ganglia. When such studies were done 64 it was found that cAMP derivatives had the same effect as did nerve growth factor on tyrosine hydroxylase levels in the culture system (Fig. 8). Cyclic GMP derivatives, on the other hand, did not. Further, when ganglia from 21-day old rats were preincubated for 60 minutes in medium, to stabilize as well as lower the endogenous levels of cAMP, the addition of nerve growth factor increased these levels several-fold within a few minutes 7° (Fig. 9). Similar nerve growth factorinduced increases in cAMP levels have now been observed in dorsal root ganglia 92'93 and in PC12 cells 83, although other investigators have failed to find them in superior cervical ganglia using methods similar to ours 94'95. If cAMP is involved in the action of nerve growth factor in sympathetic ganglia, it is reasonable to expect a rise in ornithine decarboxylase activity. The induction of this enzyme, which is rate-limiting for the synthesis of the polyamines, is frequently linked with increases in cAMP levels or with stimuli which also impinge on the cyclic nucleotide system96'97. Increases in this enzyme have been found to be associated with malignant transformation, rapid cellular growth, and target cell responses to many different hormones. Such increases in ornithine decarboxylase activity are known to be true enzyme inductions and to depend on the synthesis of RNA. When superior cervical ganglia are exposed to 75

Table II The effect of nerve growth factor on the de novo synthesis of tyrosine hydroxylase in superior cervical ganglia in culture

NGF Concentration (/zg/ml)

Radioactivity in: Soluble protein TH (cpm/mg protein x 10 -3)

0 0.05 0.33 1.00 3.00 10.00

16,862 17,668 22,176 29,955 23,762 16,802

25.2 36.5 36.0 44.2 50.0 50.5

% Radioactivity in enzyme 0.15 0.18 0.16 0.16 0.21 0.30

Ganglia, in groups of 3, maintained in culture for 20 h in the presence of 50/~Ci [3H]leucine and the indicated concentration of 2.5S nerve growth factor. For further details see MACDONNELL et al. 67

Table III Effect of in vivo administration of nerve growth factor and of actinomycin D given in vivo or in vitro on the in vitro synthesis of tyrosine hydroxylase Differential rate of tyrosine hydroxylase synthesis

I n vivo

I n vitro

Radioactivity in: Tyrosine Soluble proteins hydroxylase

Vehicle NGF NGF+ actinomycin D NGF

NGF NGF NGF

cpm x 10-3/rag protein 184,965+13,438 203+59 140,702+ 1,223 586+86 153,628 + 4,951 227±56

% 0.114-0.03 0.42±0.06 0.15±0.03

113,059+ 3,230

0.63±0.07

Treatment

NGF+ actinomycin D

708±71

Animals were injected subcutaneously with vehicle or with 2.5S NGF (10/~g/g of body weight). Actinomycin D was either injected (1/~g/g of body weight) 1 h before the NGF or included (0.2/~g/ml) in the cultures. After 24 h the animals were killed and the ganglia were cultured for 22 h with 250/zCi of [3H]leucine and NGF (1/~g/ml). Values represent the mean±S.D. For further details see MACDONNELL et al. 6a

Table IV Effect of repeated administration of nerve growth factor on the in vitro synthesis of tyrosine hydroxylase

Treatment In vivo

In vitro

Vehicle NGF

NGF NGF

Radioactivity in: Tyrosine soluble proteins hydroxylase cpm x 10-3/ganglion 2,217+411 2.75+0.33 3,411 + 514 17.10 ± 3.50

Differential rate of tyrosine hydroxylase synthesis %

0.13+0.02 0.50 + 0.03

The animals were injected with 2.5S NGF (10/xg/g of body weight) every day for 3 days and killed 24 h after the last injection. For further details see MACDOr,~ELL et al. 68

76

200 1-

..T.,.

X O el--r-

100

-r

8~ < ,--r i

t Control

NGF 0.1btg/ml

dbcAMP 10"3M ..p

THEOPH

dbcGMP 10-3M +

THEOPH

Fig. 8. Effects of dibutyryl cyclic AMP and dibutyryl cyclic GMP on the specific activity of tyrosine hydroxylase in rat superior cervical ganglia in vitro. Theophylline (2 raM) was added and pre-incubated with the ganglia for 30 minutes prior to the addition of the cyclic nucleotides or of nerve growth factor. For further details see Yu et al. 64

nerve growth factor, either in vitro (Fig. 10) or in vivo (Table V), there is an exuberant increase in ornithine decarboxylase activity 72. This increase in specific to nerve growth factor (Table VI) and seems to be mediated by cAMP (Table VII). Dorsal root ganglia also exhibit this nerve growth factor-dependent increase in ornithine decarboxylase activity (Table VIII), but any number of other tissues which are not nerve growth factor targets, do not show an increase of this enzyme either in culture or in vivo. Since the increased tyrosine hydroxylase activity certainly depends upon increased R N A synthesis, and since many of the other nerve growth factor-initiated changes probably do also, we asked whether changes in R N A polymerase activity in the cells was increased by treatment with nerve growth factor. Perhaps as expected, it is possible to show modest increases in the activity of the R N A polymerases in the ganglia following treatment with nerve growth factor either in vivo (Fig. 11) or in vitro 79. From these data it is possible to postdate a chain of events in the ganglia leading from the binding of nerve growth factor to its receptor, through increases in cAMP levels, alterations in cellular protein kinases, induction of ornithine decarboxylase, and increases in R N A polymerase activity, to the induction of tyrosine

hydroxylase mRNA, the increased synthesis of tyrosine hydroxylase, and the increase in tyrosine hydroxylase specific activity. This chain of events resembles, in outline if not in detail, the sequence described by COSTA and his colleagues leading to trans-synaptic induction of tyrosine hydroxylase in the adrenal medulla 9°, and also the general sequence of action of a trophic agent postulated by RUSSELL98. The evidence for and against these changes being obligatory in the action of nerve growth factor on its targets will be summarized in the Discussion. Transcriptional events are clearly needed for the increased synthesis of tyrosine hydroxylase and ornithine decarboxylase, so one could logically look for nerve growth factor-induced alterations in the chemistry of the nuclear proteins. 2.0

1.8

1.6

.....

1.4

Control NGF

tO

1.2

C m 0

E o.

1.0

0.8' \

0.6/

I

----O

'1¢/ 0.4

0.2

13

I

I

I

5

10

30

INCUBATION (mino)

Fig. 9. The effect of nerve growth factor on cAMP levels in rat superior cervical ganglia (60 minute preincnbation). 2.5S Nerve growth factor was added at a concentration of 3 ~g/ml. A comparable amount of the appropriate buffer was added in the control experiments. For further details see

NIKODIJEVIC

et al. 7°

77

20

I----

15

¢,o

--IZ " t~

5 O

0

2

4 6 8 HOURS IN CULTURE

10

Fig. 10. The in vitro time course of the increase in ornithine decarboxylase activity in superior cervical ganglia. Ganglia from 6-day old rats were cultured either with (O) or without (O) nerve growth factor (1/~g/ml). For further details see MAcDONNELL et al. 72

Table V I n vivo increase in ornithine decarboxylase

activity in superior cervical ganglia

Treatment

Ornithine decarboxylase activity, pmol/hr per ganglion

Saline, 6hr NGF, 6 hr

0.63± 0.25 17.52, 12.47

Saline, 8 hr NGF, 8hr

0.05± 0.09 7.68, 4.03

Six-day-old rats were injected subcutaneously with saline or with NGF (10 ~g/g of body weight), and ornithine decarboxylase activity was estimated 6 and 8 hr later. Values represent the mean±S.D, of three experiments or, where individual values are given, of separate experiments. For further details see MAcDoNNELL et al. 7a

78

In addition, since these transcriptional events are linked to cAMP, and possibly to protein kinase, the alterations should be in the phosphate metabolism of the nuclear proteins. Accordingly, we have looked for such changes in the nuclei of the cells of the sympathetic ganglia. Treatment of ganglia from 5-day old rats with nerve growth factor in vitro leads to an increase in the amount of radioactive phosphate found in at least one specific nuclear protein 77 (Fig. 12). This increase can be estimated by scanning the gels in a densitometer (Fig. 13) or by cutting the gel and digesting and counting the radioactivity present in each slice. The increase amounts to about 70 to 100%. The same increase can be seen if rats are given nerve growth factor in vivo and the ganglia subsequently cultured in the presence of radioactive phosphate. The increase is both dose and time dependent. As little as 100 ng of nerve growth factor per ml gives a response and 1/xg per ml is maximal. The response is evident in as little as one hour and is complete in four. The protein is in the nuclei and is, in fact, chromatin-bound. It is in the neurons since ganglia from animals treated with 6-hydroxydopamine to destroy the sympathetic neurons do not show an increase in the phosphorylation of this protein. The increased phosphate content is not due to an increase in the overall synthesis of the protein since cycloheximide has no effect on the increase in labeling. In addition, when (3H)-leucine incorporation was measured under the same conditions there was no increase in the incorporation of amino acid into this protein. Comparable changes in labeling are seen in nerve growthfactor-treated dorsal root ganglia. The migration of this protein on SDS gels coincides with that of the H1 histone (Fig. 14). When the particulate fraction is extracted with acid, instead of with the SDS buffer, and the proteins are chromatographed in an acid-urea system, increased amounts of phosphate also occur in the band migrating with HI (Fig. 15). On this gel system there appears to be a second enriched band as well, migrating slower than the H1 (SMP). This band represents a protein of the same molecular weight as the H~ since elution of it from acid-urea gels and rechromatography on SDS shows that it moves to the H1 position. The identity of these two proteins is not sure

Table VI Specificity of the effect of nerve growth factor on ornithine decarboxylase activity in superior cervical ganglia

Additions to media

Concentration, nM

Ornithine decarboxylase activity, pmol/hr per ganglion

-40 40 40 40 40 100

1.68 -4-0.22 14.40 -4-2.44 1.91 ± 0.05 1.16 ± 0.18 3.34 ± 1.44 1.53 -4-1.52 0.99 -4-0.56

None NGF Bovine growth hormone Thyroxine Insulin Glucagon Dexamethasone

Ganglia from 6-day-old rats were cultured for 6 hr with the appropriate hormones. Values represent the m e a n ± S.D. of three experiments. For further details see MACDONNELL et al. 72 Table VII Effect of cyclic nucleotides and phosphodieste,rase inhibitors on the nerve growth factor-mediated increase in ornithine decarboxylase activity in superior cervical ganglia

Additions to culture

Concentrations

None NGF Bt2cAMP Bt2GMP Adenosine Butyrate IBMX IBMX + Bt2cAMP NGF +Bt2cAMP NGF + IBMX NGF + Bt2cAMP + IBMX

-1/zg/ml 10 mM 10 m ~ 10 m ~ 10 mta 0.5 mM 0.5 mM 10 rnM 1/z g/ml 10 mM 1/~ g/m! 0.5 mM 1/z g/ml 10 mra 0.5 mM

Ornithine decarboxylase activity, pmol/hr per ganglion 1 9 5 + 0.26 8.77 -4-2.93 6.26-4- 1.55 0.68 1.68 1.99 5.75 ± 2.74

(3) (4) (3)

% Control

(4)

100 450 + 150 (4) 321.4. 80 (3) 35 86 102 295 -4-110 (4)

4.30 + 0.62 (4)

221 ± 32 (4)

25.92-4- 2.00 (3)

1329± 103 (3)

20.84 ± 6.25 (4)

1069 ± 321 (4)

30.28 -4-7.04 (4)

1553 ± 361 (4)

Ganglia from 6-day-old rats were cultured for 6 hr with the indicated concentrations of the compounds. Ornithine decarboxylase activity is expressed as the mean-a-S.D., with the number of experiments in parentheses, except were single experiments were performed. The ornithine decarboxylase value from uncultured ganglia ("0" time) is 0 . 4 2 ± 0.4316. For further details see MACDONNELL et al. 72

I n vitro

Table VIII increase in ornithine decarboxylase activity in dorsal root ganglia from 16 day fetal rats Ornithine decarboxylase activity (pmol/hr per mg protein)

Addition to culture None N G F (0.5/~g/ml)

Expt. ]

Expt. 2

2164 6005

984 2736

79

200

o

150

o "0

~

100

¢-

._=

.~ "6

50

0

I

I

I

I

I

150

a_ z rr'

0

I

I

I

I

I

8

16

24

32

40

TIME FOLLOWING NGF INJECTION (hours) Fig. 11. Time course of R N A polymerase activity following nerve growth factor administration. For further details see HUFF et al. TM

as yet. They are, however, nuclear, even chromosomal proteins occurring in sympathetic neurons, into which increased amounts of phosphate are rapidly incorporated after the ganglia are exposed to nerve growth factor in vivo or in vitro. Whatever the identity of these proteins there is now some evidence that the increased phosphorylation is an integral part of the action of nerve growth factor. Recent experiments show that 12-0-tetradecanoylphorbol-13-acetate (TPA), a tumor promoter, and a material which has recently been shown to inhibit, albeit temporarily, the nerve growth factor-induced outgrowth of sympathetic ganglia99 also inhibits the increased phosphorylation of these nuclear proteins. PC12 The PC12 clone is an interesting and important model for the study of the nerve growth f a c t o r 47-49. It exhibits marked changes in properties upon contact with nerve growth factor. These changes en toto appear to be a differentiation into sympathetic neurons and include outgrowth of neurites, development of excitable membranes, formation of synapses with muscle cells in culture, and cessation of multiplication. One of the most felicitous aspects of the PC12 is that they do not die in the absence of nerve growth factor, as do sympathetic neurons. Thus they can be inspected in vitro for nerve growth factor-induced change without concern that any changes seen are due simply to the survival of the cells. They do not, however, exhibit any

Fig. 12. The phosphorylation of nuclear proteins from rat superior cervical ganglia cultured in the absence (left) or in the presence (right) of 2.5S nerve growth factor (300 ng/ml) for 4 hours. For further details see Y u et al. 77

80

100

-

~

NGF --- CONTROL

80

60

40 i

20

(-)

(+)

Fig. 13. Densitometer tracing of gels in Figure 12.

STAINED GEL

AUTORADIOGRAM

BSA }'-globulin (H chain) HI

?'-globulin (L chain)

Fig. 14. Comparison of stained gels with appropriate markers with autoradiograms of sodium dodecyl sulfate-gels of detergent-extracted, phosphate-labeled nuclear proteins from rat superior cervical ganglia cultured with (+) and without ( - ) nerve growth factor. 6

81

STAINED GEL

AUTORADIOGRAM

SMP H1

H1

H2B H4

Fig. 15. Comparison of stained gels with appropriate markers with autoradiograms of acid-urea gels of acid-extracted, phosphate-labeled nuclear proteins from rat superior cervical ganglia cultured with (+) and without (-) nerve growth factor. nerve growth factor-dependent induction of tyrosine hydroxylase; in this way they are not the same as sympathetic neurons. It was of interest to determine, which, if any, of the nerve growth factor-induced intracellular changes seen in sympathetic neurons also occur in the PC12. By such a strategy it may be possible to associate certain intracellular events with, for example, tyrosine hydroxylase induction and others with neurite outgrowth. It has been shown by others s3 that a small, transient, increase in cAMP occurs in these cells after nerve growth factor is added to the cultures. This increase is comparable in magnitude and duration to that seen in the ganglia explants. 82

Certainly there is a substantial increase in ornithine decarboxylase levels in these cells 82'1°°. Finally, increases in nuclear phosphorylation have been observed. So the intracellular chain of events initiated by nerve growth factor in sympathetic ganglia seems to take place in the PC12 cells also, although the details of the nuclear phosphorylation remain to be worked out. The PC12 system has shown some nerve growth factor-dependent responses which have not beeen seen, or have not been seen yet, with normal sympathetic neurons. A m o n g these are increases in cellular adhesiveness 82's3, and changes in the metabolism of specific membrane

glycoproteins 1°1. One of the most intersting is the response to epidermal growth factor 82. E p i d e r m a l growth factor 1°2-1°4 is, in m a n y systems, a potent mitogen. It is found, like the nerve growth factor, in large quantities in the salivary gland of the m a t u r e male mouse. It is also, like the nerve growth factor, found in association with another protein which has proteolytic activity and which m a y aid in the processing of the peptide. However, the epidermal growth factor is a much smaller peptide than the nerve growth factor and has no apparent structural relationship to it. In PC12 cells epidermal growth factor produces a m a r k e d increase in the activity of ornithine decarboxylase, perhaps 30 to 4 0 % as great as that produced by nerve growth factor. It also enhances cellular adhesiveness as does nerve growth factor. On the other hand, it does not inhibit D N A synthesis nor block cell division. Indeed, it stimulates the incorporation of thymidine into D N A in these cells whereas nerve growth factor inhibits it. Finally, epidermal growth factor does not produce neurite outgrowth. The P C 1 2 cells have specific, high-affinity receptors for epidermal growth factor. These receptors do not bind nerve growth factor, nor do high levels of nerve growth factor directly alter the binding of epidermal growth factor. It would a p p e a r that these are the first " n e u r o n a l " cells shown to have epidermal growth factor receptors. Since nerve growth factor induces a "terminal differentiation", that is, it conveys differentiated neuronal properties and stops cell division, and epidermal growth factor in most of its targets is a potent mitogen and stimulates thymidine incorporation in PC12, it seemed of interest to investigate the interaction of these two factors. Specifically, experiments were designed to determine if differentiation by nerve growth factor changed the ability of the ceils to respond to epidermal growth factor. Cells treated continuously for three days with nerve growth factor exhibited a much reduced ornithine decarboxylase activity in response to the addition of epidermal growth factor than did controls 82 (Table IX). T h r e e days of t r e a t m e n t was chosen in order to allow the ornithine decarboxylase response to nerve growth factor itself to return to baseline 1°°. Continuous treat-

Table IX The effect of epidermal growth factor on ornithine decarboxylase levels in nerve growth factor-treated and control PC12 cells Ceils

Treatment

Ornithine decarboxylase activity

Control

None EGF

nmol/hr per mg protein 1.22 7.56

NGF-treated

None EGF

1.01 2.31

PC12 cells were placed in 25 cm/ Falcon flasks in 7 ml of DMEM+ 15% fetal calf serum. Three days later the medium was changed; the control cells received fresh DMEM- 15% fetal calf serum, the treated ceils received comparable medium containing 2.5S nerve growth factor (10 ng/ml). This medium was removed and fresh control or NGF-containing medium added two days later. Twenty-four hours later epidermal growth factor was added (100 ng/ml). The flasks were kept at 37° for 5 hours and then the medium was removed. The cells were collected in 4.5 ml of cold 0.32 M sucrose-0.01 M Tris pH 7.4. They were centrifuged, resuspended in 4.5 ml of fresh sucrose-Tris, centrifuged again, and finally dispersed in 0.2 ml of ornithine decarboxylase homogenizing buffer and frozen on dry ice. The next day the cells were thawed, homogenized in an all glass homogenizer, centrifuged, and the supernatant fraction assayed for ornithine decarboxylase activity and for protein. For further details see Hum: and GuRo~ s3. m e n t with nerve growth factor was necessary since in this system it is known that the effects of nerve growth factor are reversible 47. Nonspecific refractoriness of the ornithine decarboxylase response due to the presence of nerve growth factor does not seem to be the explanation for this result since the differentiated cells exhibit normal ornithine decarboxylase induction in response to the addition of dibutyryl cAMP. T h e r e also is a nerve growth factor-induced disappearance of epidermal growth factor receptors. Cells treated continuously for three days with nerve growth factor bind less than 20% as much labeled epidermal growth factor as do controls s2 (Table X). The nerve growth factortreated cells are larger than the controls, but even on a per cell basis the apparent loss of epidermal growth factor receptors is greater than 60%. It would seem logical then to conclude that one possible reason that the differentiated cells do not respond to epidermal growth factor with an induction of ornithine decarboxylase is that they have lost epidermal growth factor receptors. W h e t h e r or not this 83

Table X The binding of 125I-epidermal growth factor to nerve growth factor-treated and control PC12 cells Specific binding of epidermal growth factor

Cells

Control NGF-treated

cpm//~g protein Experiment 1 Experiment 2 762 416 131 81

PC12 cells were grown as described in Table IX. Epidermal growth factor (4/zg) was iodinated by the chloramine T-talcum procedure. Labeled epidermal growth factor (6 × 106 cpm per flask, 1.6 ng/ml) was added to the cells in fresh medium. The cells were kept at 24 ° for 40 minutes, rinsed rapidly once with cold D M E M and once with cold phosphate-buffered saline and then detached from the surface by vigorous agitation in 5 ml of phosphate-buffered saline. Specific binding was evaluated by subtracting the counts bound to the cells using 125I-epidermal growth factor after a 10 minute preincubation in the presence of 350 ng/ml of unlabeled epidermal growth factor from those bound in its absence. In the presence of this excess of epidermal growth factor control cells bound 265 cpm//zg protein and NGFtreated cells bound to 254 cpm//~g protein. For further details see HUFF and GUROFF82.

loss of receptors is the direct cause of the loss of ornithine decarboxylase responsiveness, the fact remains that one effect of nerve growth factor in this system is to diminish the receptors for another and perhaps opposing growth factor. The mechanism of this loss of receptors is not known. It is possible that receptor synthesis is repressed and that fewer receptors are present. Alternatively, surface alterations may change the affinity of existing receptors for epidermal growth factor. It is of interest, independent of the mechanism, to consider whether a similar response occurs in normal sympathetic neurons at some early stage in their development. Central nervous system

Over the last several years some evidence has accumulated indicating that nerve growth factor has a role in the damaged or even the normal brain. This evidence includes a number of different observations. First, the well known action of nerve growth factor on catechoiaminergic neurons in the peripheral nervous system suggests that the factor might interact with catecholaminergic neurons in the central nervous system. Second, in experiments 84

in which lesions have been created in the catecholaminergic tracts, the regrowth, or axonal sprouting is stimulated by nerve growth f a c t o r 44'46. Third, behavior altered by such lesions appears to return to normal more quickly when nerve growth factor is administered 1°5. Fourth, nerve growth factor receptors are known to be present in the brain 1°6-1°9. Finally, there are persistent reports that nerve growth factor occurs, albeit in small amounts, in the brain z2,2a'26,11°. Since little or no biochemistry had been done on the effects of nerve growth factor in the central nervous system, and since the ornithine decarboxylase response appears to be a facile probe for nerve growth factor effects, it seemed reasonable to investigate the effects of nerve growth factor on ornithine decarboxylase levels, in brain. It had been shown in one previous study 1~1 that nerve growth given intraventriculady would raise ornithine decarboxylase levels, but the amounts used were very high, and no details of the induction had been presented. Work in this laboratory 73 showed that ornithine decarboxylase levels in the brains of mature rats could be elevated several-fold by the injection of /xg quantities of 2.5S nerve growth factor into the ventricles. As little as 400 ng gave a measurable response, and this response was fairly specific for nerve growth factor (Table XI), though not as specific as the response in the ganglia. Surprisingly, this response was anatomically diffuse, occurring in all parts of the brain to an approximately equal extent (Table XII). Also surprisingly, the response in young animals was less than that in adults, although the basal levels in young animals were much higher than in the adults. Finally, and again surprisingly, the response was not specific to the neurons, the glia being affected at least equally (Fig. 16). Systemic injections of comparable quantities of nerve growth factor are without effect on the ornithine decarboxylase levels of the brain, so the effects are not due to escape of the peptide from the brain and its subsequent effect on the periphery. On the other hand, intraventricular injection of nerve growth factor raises not only brain ornithine decarboxylase activity, but liver, kidney, and adrenal levels of the enzyme as well (Fig. 17). Since this effect is clearly not systemic, it seems likely that the peripheral tissue

Table XI Specificity of the effect of nerve growth factor on ornithine decarboxylase induction in adult rat brain

Treatment

Ornithine decarboxylase activity nmol/hr per g tissue

Expt. 1 Buffer N G F (0.468/~g) N G F (2.34 tzg) Insulin (2.5/zg) Bovine growth h o r m o n e (20/zg)

0.62-4-0.10 9.60±2.38 10.08 -4-0.85 0.98 + 0.14 1.35 ± 0.46

(9) (3) (9) (3) (3)

Expt. 2 Buffer NGF Thyroxine Insulin Glucagon Bovine s e r u m albumin Cytochrome c

0.269 -4-0.087 3.598 ± 2.729 0.220 ± 0.083 0 . 4 1 9 ± 0.087 0.369 ± 0.149 0.195 m 0.020 0.255 ± 0.036

In experiment 1 adult male rats were injected intraventricularly with the various materials. T h e animals were killed 7 hr later. Values represent the mean-4- SEM, with the n u m b e r of animals used given in parentheses. In experiment 2 ten microliters of buffer (0.05 M Na acetate, p H 5.0) alone or containing N G F (0.20 nmol, 4.68 tzg), thyroxine, insulin, glucagon, bovine s e r u m albumin or cytochrome c (0.40 nmole each) were injected intraventrically. Rats were killed 4.5 hr later and ornithine decarboxylase activity was measured. Results are expressed as m e a n s ± S E M for three rats per group. For futher details see LEWIS et al. 73 and NAGAIAH et al. TM

Table XII Regional distribution of ornithine decarboxylase induction in adult rat brain Ornithine decarboxylase activity, n m o l / h r per g tissue Brain region Limbie forebrain Striatum Cerebral h e m i s p h e r e s Cerebellum Diencephalon Brainstem

Control

NGF

0.99 ± 0.13 0.71 ± 0.10 0.98 -4-0.14 0.84 + 0.29 1.48 + 0.23 1.56 ± 0.17

3.47 -4-0.27 1,76 ± 0.21 2,14 -4-0.22 1,80 + 0.18 4.15 + 0.48 3.93 + 0.18

A d u l t male rats were injected intraventricularly with 2.3/xg of N G F in 10//,1 of 0.05 M acetate, p H 5.0, or with acetate buffer alone. T h e animals were killed 4.5 hr later and ornithine decarboxylase activity was measured. Values represent the m e a n + S E M for four animals in each group, 85

25 z

_o jc.) D a z

20

q

15

UA

BRAIN

NEURONAL BODIES

GLIA

Fig. 16. T h e induction of ornithine decarboxylase by nerve growth factor in neuronal and glial-enriched fractions from adult rat brain. A d u l t male rats were injected intraventricularly with 2 . 3 / z g of 2.5S nerve growth factor in 10/zl of 0.05 M acetate, p H 5.0. T h e animals were killed 7 hours later and the whole brains m i n u s cerebellum and medulla oblongata were fractionated. Controls received acetate alone. For further details see LEWIS et al. 73

levels are being raised by some neurohumoral response to nerve growth factor action on the brain. Support for this suggestion is found in experiments in which the animals are hypophysectomized, pituitary stalk sectioned, or adrenalectomized (Table XIII). In each of these cases the peripheral response is much reduced. Further evidence that the nerve growth factor injection is activating the hypothalmohypophyseal-adrenal axis comes from experiments in which the level of cortical steroids was measured. In these experiments it was found that intracerebral injections of nerve growth factor produced a rapid rise in the levels of cortical steroids in the blood (Fig. 18). The exact mode of action of nerve growth factor on the hypothalamus is not known. One line of study shows, however, that the actionmay be modulated by catecholaminergic pathways. Specifically, a tyrosine hydroxylase inhibitor, a-methyltyrosine, inhibited the action of nerve growth factor on ornithine decarboxylase, as did a neurotoxin which is rather specific 86

for the catecholaminergic neurons, 6hydroxydopamine. The catecholamine receptor antagonists haloperidol and phenyoxybenzamine also inhibited the increase in activity. These data are consistent with the concept that catecholamines modulate the central action of nerve growth factor. Certainly it would not be surprising if catecholamines were involved in the action of an agent on the hypothalamus. It is well known that the release of many of the hypothalamic factors, and through them, the pituitary hormones, is altered by monoaminergic stimuli. In any case, the action of nerve growth factor on the central nervous system, mirrored in the changes in ornithine decarboxylase levels, seems to be an indirect one, initiated by the action of the factor on the hypothalamus. The details of this action are unknown, but the rapidity of it would seem to suggest that it is a different kind of action than the classical action of nerve growth factor on its other targets.

Discussion Evidence from this laboratory suggests that at least one chain of intracellular events initiated

76• I-

7.5 ==

Liver

L

6.0 4.5 3.0

-5

2O

1.5 0

10

1'.5 3'.o 4 ,s 6. o Hours

0

1.5 3.0 4,5 6.0 Hours

t~

375

"

~

125

30C

10C

=._

225

7E

E

15C

5C

7E

2E

O

1.5 a'.o 4'.s 61o Hours

0

Adrenal

1.5 3.0 4.5 6.0 Hours

Fig. 17. Ornithine decarboxylase activity in various rat tissues after the intraventricular administration ot nerve growth factor (4.68/zg in 10/zl of 0.05 M acetate, p H 5.0). For further details see NAGAIAH et al. TM

Table XIII Effect of hypophysectomy, pituitary stalk section, or adrenalectomy on the NGF-mediated induction of ornithine decarboxylase in central and peripheral tissues

Treatment Operation Injection

Sham Hypophysectomy Sham Pituitary stalk section Sham Adrenalectomy

Ornithine decarboxylase activity, nmol/hr per g of tissue Liver Kidney

Brain

Adrenal

Buffer NGF Buffer NGF

0.309±0.014 (4) 4.2414-0.750(5) 0.3194-0.026 (4) 4A664-0.762 (5)

1.274± 0.161 (4) 96.7434-17.555(5) 0.8284- 0.062(4) 4.4224- 1.618 (6)

71.5314-28.935 (4) 345.7794-78.330(5) 3.4184- 1.416 (4) 8.026± 2.803 (5)

Buffer NGF Buffer NGF

0.3354-0.019 6.1664-0.749 0.3874-0.071 4.8924-0.805

1.0724-0.174 (8) 79.8294- 7.278 (9) 0.471± 0.093 (8) 32.7744- 9.131 (11)

35.2204- 7.494 189.6714-26.180 10.5474- 6.151 58.2284-15.919

Buffer NGF Buffer NGF

0.3554-0.030 (4) 5.126±0.277(4) 0.502 + 0.091 (4) 1.6094-0.239 (4)

2.936± 1.046 (4) 76.577± 3.802(4) 1.074-4-0.149 (4) 0.8784- 0.148 (4)

89.8874- 11.263 (4) 355.160±68.022(4) 33.528 ± 10.093 (4) 24.8134- 9.538 (4)

(8) (11) (8) (11)

(8) (10) (8) (11)

1.5934- 0.262 (4) 94.1984-13.098(5) 0.2704- 0.162(4) 8.0254- 1.488 (6) 3.601± 0.588 110.8504-13.898 0.8824- 0.371 43.8774-12.387

(8) (10) (8) (11)

Rats were sham-operated or surgically altered 1 week before use. Ten microliters of acetate buffer alone or containing N G F (0.20 nmol, equivalent to 4.68 tzg) was injected intraventrically. Rats were killed 4.5 hr after the injection and ornithine decarboxylase activity was measured. Results are expressed as means 4-SEM; the number of animals is shown in parentheses. For further details see NAGAIAH et al. TM I

I

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Fig. 18. Plasma cortieosterone levels after the intraventricular administration of nerve growth factor in three groups of rats: O anesthetized but not injected; • anesthetized and given acetate buffer (10/~1 of 0.05 M acetate, pH 5.0); ZX anesthetized and given nerve growth factor (4.68/xg in 10/xl of the acetate buffer). For further details see NAGAIAH et al. TM

by nerve g r o w t h factor in its target n e u r o n s starts with an increase in c A M P concentration, and is followed by increases in the p h o s p h o r y l a tion of certain nuclear proteins, increases in ornithine decarboxylase levels, increases in the activity of the R N A polymerases, and finally, increases in the synthesis of m R N A for tyrosine hydroxylase and of tyrosine hydroxylase itself. T h e evidence that c A M P increases are a general aspect of the action of nerve growth factor include observations of similar changes in dorsal r o o t ganglia 92'93 and in P C 1 2 cells 83 u p o n addition of nerve g r o w t h factor. O t h e r experim e n t s have shown that derivatives of c A M P maintain tyrosine hydroxylase levels in explanted ganglia, as does nerve g r o w t h factor 64, mimics nerve g r o w t h factor in increasing the adhesiveness of P C 1 2 cells 83, and p r o d u c e s o u t g r o w t h of dorsal r o o t ganglia in culture 88. This latter response, h o w e v e r , is said to differ m o r p h o l o g i c a l l y f r o m that p r o d u c e d by nerve g r o w t h factor. In fairness, it m u s t be p o i n t e d o u t that at least two groups have failed to o b s e r v e increases in c A M P levels in dorsal r o o t ganglia 88'89 and two others in superior cervical ganglia t r e a t e d with nerve g r o w t h factor u n d e r conditions similar to ours 94'95. T h e evidence that these intracellular changes are related to one a n o t h e r include a n u m b e r of 87

observations. The increased phosphorylation of nuclear proteins cannot be produced by any of a number of other proteins, but dBcAMP will mimic the effect77. The ornithine decarboxylase increases, in our hands at least, can also be produced by increasing the cAMP content of the ganglia as well as by nerve growth factor 72, and there is a great deal of data supporting the concept that at least one mechanism for inducing ornithine decarboxylase is cAMPdependent. Again, in fairness, there is one report that the increased ornithine decarboxylase levels produced by nerve growth factor in PC12 cells were not accompanied by increases in cAMP, and so concluded that the induction was cAMP-independent 112. Our data lead to the suggestion that the increase in the specific activity of tyrosine hydroxylase produced by nerve growth factor in vivo is due to the increased synthesis of mRNA for tyrosine hydroxylase68. This would be consistent with the occurrence of nuclear alterations, and the increases in RNA polymerase activities. It is based on the observation that the administration of actinomycin D prevents the nerve growth factor-induced increase in the rate of synthesis. More recently, experiments have been presented in which this question has been reinspected under somewhat different conditions 1~. Using ganglia from adult animals some increase in tyrosine hydroxylase synthesis has been observed in vitro and this increase is not prevented by inhibition of RNA synthesis. Thus, the final answer to this question is not yet at hand, but it is our feeling that the small increases in tyrosine hydroxylase levels and in the rate of tyrosine hydroxylase synthesis observed in vitro are not sufficient to explain the increases produced in vivo and so the characteristics of this small increase are not relevant to the action of nerve growth factor on tyrosine hydroxylase. Our data clearly show that the action of nerve growth factor on increasing the rate of synthesis of tyrosine hydroxylase in vivo is dependent on the synthesis of R N A . The overall conclusion, albeit with some reservations, is that the increases in cAMP, the nuclear phosphorylations, and the ornithine decarboxylase inductions are general aspects of nerve growth factor action and are related. The evidence that the intracellular events described here are obligatory for any of the actions of 88

nerve growth factor has been more difficult to obtain. Indeed, no single action of the nerve growth factor on its target cells has been found to depend on any specific intracellular event. On the contrary, some specific responses of the cell have been clearly dissociated from these events. In this laboratory it has been shown that the levels of nerve growth factor which are optimum for increasing cAMP levels are higher than those needed for survival of neurons and the maintenance of tyrosine hydroxylase levels in the explanted gangliaTM. In another experiment it has been shown that the rise in the cAMP content of the cells does not commit the cells to later increases in tyrosine hydroxylase content; addition of nerve growth factorantiserum even several hours after the addition of nerve growth factor still prevents some of the ability of nerve growth factor to maintain the levels of tyrosine hydroxylase, and this addition of antiserum comes long after the cAMP level has returned to base-line. Similar kinds of experiments have been done with the ornithine decarboxylase response. Using PC12 cells it has been clearly shown that inhibition of the increases in ornithine decarboxylase activity does not influence the subsequent ability of the cells to elaborate processes 1°°. Unpublished data from this laboratory shows that, in superior cervical ganglia, the inhibition of ornithine decarboxylase activity or of ornithine decarboxylase induction does not interfere with the maintenance of tyrosine hydroxylase activity in vitro. So, at this point, although it seems clear that nerve growth factor initiates a chain of events starting with increases in cAMP, there is little evidence that this increase or any of its sequalae are obligatory for the final actions of nerve growth factor on the cell. The first such evidence may come from current studies with the tumor promoter TPA. It has been reported that this agent interferes with the ability of nerve growth factor to produce outgrowth99. The data is a bit difficult to interpret because this interference is only temporary; the ganglia "escape" eventually and produce processes. Nevertheless, TPA prevents the increased phosphorylation of nuclear proteins in the ganglia due to nerve growth factor. Its effects on cAMP levels, ornithine decarboxylase induction, or the eventual synthesis of tyrosine hydroxylase have not yet been

explored. The initial studies, however, would tend to relate the nuclear phosphorylations to the eventual outgrowth of processes produced by nerve growth factor in the superior cervical ganglia. Studies with the PC12 system and, indeed, with the library of nerve growth factorresponsive cells lines now becoming available, offer the possibility of linking discrete cellular responses to specific intracellular events. For example, PC12 cells respond to nerve growth factor in a number of ways including the formation of processes, development of excitable membranes, and the cessation of cell d i v i s i o n 47-49. They do not, however, induce tyrosine hydroxylase. Another clone, recently described s°, shows an induction of tyrosine hydroxylase, but no morphological response. Yet other cells show increased adhesivenessla4; some simply have nerve growth factor receptors 51'52. In the PC12 cells, at least, the intracellular events, i.e., cAMP, nuclear phosphorylation, ornithine decarboxylase, seem to be comparable to the events seen in the superior cervical ganglia. These data would seem to indicate that the intracellular events observed are general consequences of the action of nerve growth factor action and are not specifically involved with the induction of tyrosine hydroxylase. It is of interest that preliminary studies with the tyrosine hydroxylase-inducing clone of pheochromocytoma and with the F98 glioma which exhibits some morphological alterations and an increased adhesiveness 1~4 with nerve growth factor, showed that neither of these have a marked ornithine decarboxylase response. Thus, these cell lines may serve as tools to dissect the responses seen in the neurons. They may also serve to point the way to responses not yet seen in neurons. The studies on the response to epidermal growth factor suggest that these cells, and perhaps by analogy, sympathetic neurons at some early state of development, can respond to mitogens. Clearly, one of the differentiating effects of nerve growth factor on these cells, and, by analogy, on normal sympathetic neurons, could be to decrease or obliterate the response to the mitogen. It is interesting to speculate that this action causes or at least participates in the cessation of cell division in PC12 and perhaps in normal differentiating neurons as well.

The response in the central nervous system is the most puzzling because it is the least typical. It is quite rapid, cortical steroids being elaborated within 30 minutes; it is the least specific, vasopressin 1~5, growth hormone and others having a similar action. Evidence from this laboratory, reviewed above, suggests that nerve growth factor is causing an activation of the hypothalamus. Evidence from other laboratories supports this suggestion. Systemic injection of nerve growth factor into intact animals produces an increase in the cAMP levels in the adrenal cortex 94, but in hypophysectomized animals nerve growth factor is without effect. Injection of nerve growth factor into the brain produces changes in behavior reminiscent of those produced by the elaboration of pituitary peptides la6. These observations suggest several possibilities. First, that the action of nerve growth factor on the central nervous system may be primarily an action on the hypothalamus. Second, that this action on the hypothalamus could be a "non-classical" action in which the nerve growth factor acts as a neurotransmitter or as a pharmacological agent. Third, that this specific action on the hypothalamus, and its attendant widespread but indirect effects, may explain or help to explain the previous puzzling histochemical and behavioral effects of nerve growth factor on the catecholaminergic tracts in the brain. In conclusion, it seems possible that the studies on the sympathetic ganglia and especially those on the specific phosphorylations in the nucleus could lead to at least one of the molecular mechanisms by which nerve growth factor impinges on the cell. The experiments with the PC12 could help to dissect the several responses and to point the way to actions of the factor as yet uncovered due perhaps to the difficulty of obtaining neurons at such early stages of decision. The experiments on the central nervous system may help uncover yet another action of the nerve growth factor and one quite different than the ones known for peripheral neurons. References 1. Levi-Montalcini, R., 1966. Harvey Lect. 60, 217-259. 2. Levi-Montalcini, R. and Angeletti, P. U., 1968. Physiol. Rev. 48, 534-569.

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