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Ornithine decarboxylase in motoneurons during regeneration
EXPERIMENTAL
NEUROLOGY
Ornithine
89,679-688
(1985)
Decarboxylase in Motoneurons during Regeneration
WOLFRAM TETZLAFF AND GEORG
W. KREUTZBERG’
Max-Planck-Institute for Psychiatry, Am Klopferspitz 18a. 8033 Planegg-Martinsried, Federal Republic of Germany Received April 29, 1985 The activity of omithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, was assayed in the isolated facial nucleus of the rat at various times after axotomy of the facial nerve. In addition, it was measured 24 h after the second of a series of two lesions (conditioning lesion design) with various times between the first and second operations. Ornithine decarboxylase activity was found to increase 8 h atIer nerve transection and was maximum after 24 h (300% of control). Thereafter the activity declined to subnormal levels where it remained for several weeks. Omithine decarboxylase activity did not increase again when a second axotomy was made 2 weeks after the first lesion. However, omithine decarboxylase did respond to the second axotomy if it was carried out 3 weeks after the first lesion. Histochemical localization of omithine decarboxylase demonstrated that the increase in enzyme activity was mainly confined to the perikarya of the motoneurons. These data suggest that this enzyme is somehow involved in triggering the “regeneration program” and clearly indicate that at least some aspects of the neuronal response to axotomy are not further stimulated by a conditioning lesion. o 1985~csdemic PIUS,IIN.
INTRODUCTION
The interruption of the continuity of peripheral nerve axons leads to a number of morphologic, metabolic, and physiologic changes in the nerve cell body known as chromatolysis or “axonal reaction” [reviewed in (10, 17)]. Among the earliest changes observed so far, an increase in omithi* decarboxylase (ODC) activity has been reported in the superior cervical ganglion of the rat (1, 9) and in the goldfish retina (16). Omithine Abbreviations: ODC-omithine decarboxylase; a-DPMO-a-ditluoromethylomithine. ’ The authors thank Dr. M. Reddington for critical reading of the manuscript. Please address correspondence to Professor Kreutzberg.
679 00144886/85
$3.00
Copyright 8 1985 by Academic Ress Inc. All riglm of npmdwlion in any form reserved
680
TETZLAFF
AND
KREUTZBERG
decarboxylase catalyzes the conversion of omithine to the diamine, putrescine (27), which is the rate-limiting step in the synthesis of the polyamines, spermidine and spermine [reviewed in (4, 22)]. High levels of ODC activity and polyamines are found in embryonic, proliferating, and regenerating tissues [for review see (4, 22, 3 1, 34)]. They are presumed to be involved in gene expression by regulating DNA replication and RNA synthesis (4, 37). This function could be of interest in the switching of the nerve cell from its normal function to the metabolic program typical of regeneration. In the present study we investigated the activity of ODC for the first time in regenerating motoneurones. We also applied the conditioning lesion paradigm and studied the response of this enzyme to a repeated lesion. MATERIALS
AND
METHODS
Male Sprague-Dawley rats weighing 230 to 280 g were kept in a 12 h light:12 h dark cycle and fed a standard diet and water ad libitum. Surgery. Design A. Under deep ether anesthesia the left facial nerve was transected at the stylomastoid foramen and the right facial nerve was shamoperated, i.e., exposed but not mobilized. The rats were killed 3, 5, 8, or 24 h, 2, 3, 7, 12, 14, or 20 days after the operation. Design B. The left facial nerve was crushed 3 mm distal to the stylomastoid foramen and the right facial nerve was sham-operated. In a second operation, both the right and the left facial nerve were transected. The interval between the first and the second operations was 14, 20, or 30 days. The rats were killed 24 h after the second operation. Biochemical assays. The isolated facial nuclei from six rats were pooled at each timepoint; the 24-h experiment was conducted twice. A control group of three untreated rats, i.e., six combined left and right facial nuclei, was included in each assay series. The brain stems were frozen and the facial nuclei were punched out with a blunt canula of 1.1 mm inner diameter. Omithine decarboxylase (E.C. 4.1.1.17) activity was assayed in supernatants (13.000 g/10 min) of the homogenated facial nuclei using L(1-14C)-labeled omithine and counting the cleaved radioactive CO;! after 1 h or 2 h of incubation (27). The assay medium contained 5 mMdithiothreito1, 0.3 mM pyridoxal phosphate, 0.1 mM nonradioactive L-omithine in 50 mM Tris/HCl, pH 7.2. To start the assay 0.25 &i L-[l-‘4C]omithine (56 mCi/mmol) was added to 1 ml of the medium/supematant. The reaction was stopped with 0.5 ml 40% trichloroacetic acid and 2 h later the KOH-moistened (200 ~1, 5 N) filters were removed from the center of the rubber-stoppered vials and counted in Supersolve (Zinsser). Low ODC activity made it necessary to pool six facial nuclei for each duplicate measurement. The ODC activities were related to the protein content of the homogenates assayed according to the method of Lowry et al. (20). The
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activities were expressedas a percentage of the unoperated control group which typically displayed activities of ca. 536 + 34 dpm/mg protein/h. Histochemistry For histochemical localization of ODC activity, unfixed cryostat sectionsof the brain stem (20 pm) were used 24 h after facial nerve transection. The unlixed sections were incubated with biotinylated (rdifluoromethylornithine (cy-DFMO), an irreversible inhibitor of ODC, according to the technique of Gilad and Gilad (8). The ODC-DFMO-complex was visualized with avidin-coupled horseradish peroxidase (avidin-HRP) that was processedwith diaminobenzidine. Chemicals. If not mentioned otherwise, all chemicals were purchased from Sigma except avidin-HRP (Vector-Chemicals), L( 1-‘4C)-omithine (Amersham), and Supersolve (Zinsser). Biotinylated cr-DFMO was a gift from Dr. G. Gilad (Weizmann Institute, Rehovot). RESULTS There was no obvious difference in ODC activity between the control facial nuclei, i.e., pooled left and right sidesfrom three unoperated animals, and those contralateral to the operated sides (design A) on days 1, 7, and 12. The unoperated contralateral values of days 2 and 3 were higher than in the control groups (122 and 146% of control values, respectively). In the axotomized facial nuclei the ODC activity started to increase 5 h after axotomy and was twice the control values after 8 h (Fig. 1). The maximum was found after 24 h when the activity was about 300% of controls. On the 2nd and 3rd postoperative days,the ODC activity declined and was at subnormal levels 1 week after operation at which it remained for several weeks.
IQ”,
I;’ days
. 2bd
post OP
FIG. 1. BiochemicaUy measured omithine decarboxylase (ODC) activity in the facial nucleus of the rat at various time intervals after facial nerve transection. The ODC activities are expressed as a percentage of a control group of normal rats (unoperated). Bach symbol represents duplicate measurements of six pooled facial nuclei. The time scale (abscissa) is interrupted.
682
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To investigate how this ODC activity reacts to a second axotomy, experiments were conducted according to design B (see Materials and Methods). The ODC activity was measured 24 .h after the second operation as this was the timepoint of maximal activity after single axotomy. When the second axotomy was carried out 14 days after the first, no reincrease of the ODC activity was detectable when measured 24 h later. By that time most axons had already reached the peripheral targets again, as indicated by the reonset of whisker movement on day 12. However, axonal maturation was not yet accomplished. Twenty or thirty days after the first axotomy the facial nucleus responded to a second axotomy by a second increase of ODC activity. This second increase was somewhat smaller compared with the first increase: after an operation interval of 30 days, this second increase was about 250% of control whereas the first increase after a single lesion was about 300% of the unoperated control group (Fig. 2). Histochemistry was carried out 24 h after axotomy in order to localize the increasing ODC activity. The activity was rather low in the facial nucleus and the biochemically measured differences were not as pronounced in histochemistry using an irreversibly binding enzyme inhibitor (a-DFMO) for enzyme visualization. The increasing ODC activity was localized in the perikarya of the facial motoneurons (Fig. 3a). There was also some activity in the neuropil whereas the bundles of nerve fibers that cross the facial nuclei appeared blank. The contralateral facial nuclei displayed a positive neuropil but the perikarya were not visible as they were on the axotomized side (Fig. 3b). Control i’ncubations also displayed a slightly positive neuropil, which was less intense than in the unoperated contralateral sides. This indicated that the increasing
1 all data
24h
POI~ 2nd
OP
OP-Interval
FIG. 2. ODC activity in the facial nucleus of the rat 24 h after a second axotomy (CL). The interval between the first (conditioning) and the second (testing) lesion was varied (OP-interval). The ODC increase 24 h after single lesion (SL) is indicated at the left margin of the diagram. Each bar represents a group of six pooled facial nuclei, expressed as a percentage of a control group.
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FIG. 3. Histochemistry of ODC activity in normal (3b) and in axotomized facial nucleus 24 h after nerve transection (3a). Unfixed cryostat sections were incubated with biotinylated (Ydilluoromethylomithine, which binds irreversibly to ODC. This was coupled to avidin-HRP that was visualized with diaminobenzidine.
ODC activity is localized mainly motoneurons.
in the perikarya of the axotomized
facial
DISCUSSION The main finding of the present study is an early and transient increase in ODC activity in the axotomized motoneurons, which did not increase
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again if the neurons were axotomized for a second time during the regeneration period according to the conditioning lesion paradigm. The increase of ODC activity in the axotomized facial nucleus is the earliest sign known of the neuronal response to axotomy in a motoneuron system. An increase in ODC activity, which represents the rate-limiting step in polyamine synthesis, can be found in various tissues and tissue cultures [reviewed in (4, 22)] including the nervous system (5, 6, 15, 19, 21, 28) as a response to a variety of stimuli such as hormones (5, 28), growth factors (11, 19, 21), &hernia (6, 15), and tissue damage (6). At present it is not possible to distinguish whether a de nova synthesis or a posttranslational regulation mechanism is involved in this ODC activation of the facial motoneurons. Both mechanisms have been discussed in a variety of experiments (22, 25). Generally the latency of the ODC increase takes 2 to 3 h. It is mediated by CAMP (29) and in some systems it has been shown to be sensitive to actinomycin D or cycloheximide [reviewed in (22, 29)]. This is of interest with respect to the observation that actinomycin D inhibits the chromatolysis of mouse facial motoneurons when applied within the first 9 h after axotomy; later applications (15 h) are no longer effective (39). These experiments indicate that at least some RNA species must be synthesized within a few hours after injury before the nerve cell body can respond to axotomy. As is known from other systems, polyamines can be involved in the regulation of DNA and RNA synthesis (4, 34, 37) and the ODC molecule itself can stimulate RNA polymerase I (30). Thus it is possible that the increase in ODC activity in the axotomized motoneurons is somehow linked to these early changes in RNA metabolism [see Austin, for recent review (2)]. These changes give rise to the formation of a group of polypeptides and proteins that are either specific for regeneration or have a highly stimulated turnover during regeneration (12, 36). Preliminary studies indicate that such specific changes in the protein pattern also occur in the facial nucleus of the rat (in preparation). Whether or not the increase in ODC activity in the axotomized motoneurons is a prerequisite for these changes in RNA and protein metabolism and thus for the initiation of the regeneration program can be only speculated on at present. The results of the conditioning lesion experiment support the view that the ODC increase may be involved in the initiation of the regeneration program. The ODC activity of the facial nucleus did not increase again when a second axotomy was performed 14 days after nerve crush. At this time the neuronal metabolism is still in the regeneration mode and a second initiation of this program, which includes an ODC activation, probably does not take place. It might therefore be expected that a second
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increase of OLX would occur only after nerve regeneration is accomplished and the neurons are again at normal metabolism. This would agree with our observation of triggering an ODC increase only 20 days after the first lesion. We assume that the second lesion is signaled to the nerve cell body because the facial nucleus reacts by further decreasing acetylcholinesterase activity and by increasing 5’ nucleotidase activity (38). However, a simple amplification of the original response to axotomy does not take place. The signal after a second axotomy could be different or differently processed. Also, after a second axotomy the signal may arrive at the cell body while the cell is in a different metabolic state. This different state could mean that the pool of polyamines formed during the early transient increase in ODC activity still persists and causes a negative feed-back inhibition of ODC activity. Polyamines can have half-lives of several days (3 1, 33, 35) and can induce the formation of a complexing protein that inactivates ODC activity (7, 13, 14). In contrast to the facial nucleus, there is a second increase of ODC activity in the goldfish visual system (32). This second ODC increase is even enhanced by 60% in those retinal explants whose optic nerves had been crushed 2 weeks previously. This is correlated with a stimulated fiber outgrowth from precrushed retinae (18, 32) that can be inhibited by CXDPMO, an irreversible ODC inhibitor (32). In vivo, the response to a second axotomy is more pronounced in the goldfish visual system than in the facial nerve. In the goldfish the velocity and amount of axonal transport are stimulated by about 50% and axonal regeneration is accelerated by more than 90% (23). This contrasts to the faint stimulation of regeneration velocity (23 to 30%) by a conditioning lesion in the rat, as was measured in the sciatic nerve (3, 24, 26). Thus, in the goldfish the enhanced ODC response after a preceding crush of the optic nerve seems to be related to a stimulation of the regenerative response of the retinal ganglion cells. The above considerations prompt us to speculate whether the missing increase of ODC in the facial nucleus reflects a lack of further stimulation of the “regenerative response” of the facial motoneurons aher a conditioning lesion. At least the early changes in RNA metabolism that might be associated with the ODC increase are probably not enhanced. This view is supported by the observation of Langford [quoted by Austin, (2)] who studied the RNA changes after axotomy in the nodose ganglion of the rat. A second crush applied 3 or 7 days alter the first did not stimulate a second rise in uridine incorporation into RNA. This fits with our recent results, that in the facial nucleus the neuronal hexose monophosphate shunt enzymes are not further activated by a preceding axotomy (38). Nevertheless, the conditioning lesion reduces the period of functional recovery by 10%
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in the facial nerve (38). Therefore, if no further stimulation of the neuronal metabolism itself takes place, this conditioning lesion effect would be due to a shortening of the initial delay, i.e., the time before axonal outgrowth begins. Another possibility which must be considered is that stimulator-y factors in the distal nerve stump, which is undergoing Wallerian degeneration, enhance the fiber outgrowth velocity, as was recently shown for the rat sciatic nerve (3). REFERENCES 1. ANDO, M., M. MIWA, K. KATO, AND Y. NAGATA. 1984. Effects of denervation and axotomy on nervous system-specific protein, omithine decarboxylase, and other enzyme activities in the superior cervical sympathetic ganglion of the rat. J. Neurocbem. 42: 94100. 2. AUSTIN, L. 1985. Molecular aspects of nerve regeneration. Pages 1-29 in A. LAJTHA, Ed., Alterations of Metabolites in the Nervous System. Handbook of Neurochemistry, 2nd ed., Vol. 9. Plenum, New York. 3. BISBY, M. A., AND B. POLLOCK. 1983. Increased regeneration rate in peripheral nerve axons following double lesions: enhancement of the conditioning lesion phenomenon. J. Neurobiol. 14: 467-47 1. 4. CANELLAKIS, E. S., D. VICE&MADORE, D. A. KYRIAKIDIS, AND J. S. HELLER. 1979. The regulation and function of omithine decarboxylase and of the polyamines. Pages 155202 in B. L. HORECKER, AND E. R. STADTMAN, Eds., Current Topics in Cellular Regulation, Vol. 15. Academic Press. New York. 5. COUSIN, M. A., D. LANDO, AND M. MOGUILEWSKY. 1982. Omithine decarboxylase induction by ghtcocorticoids in brain and liver of adrenalectomized rats. J. Neurochem. 38: 1296-1304.
DIENEL, G. A., AND N. F. CRUZ. 1984. Induction of brain omithine decarboxylase during recovery from metabolic, mechanical, thermal or chemical injury. J. Neurochem. 42: 1053-1061. 7. FONG, W. F., J. S. HELLER, AND E. S. CANELLAKIS. 1976. The appearance of an omithine decarboxylase inhibitory protein upon the addition of putrescine to cell cultures. Biochim. Biophys. Acta 428: 456-465. 8. GILAD, G. M., AND V. H. GUD. 198 1. Cytochemical localization of omithine decarboxylase with rhodamine or biotin-labeled cuditluoromethylomithine. An example for the use of labeled irreversible enzyme inhibitors as cytochemical markers. J. Histochem. Cytochem.
6.
29: 687-692.
GILAD, G. M., AND V. H. GILAD. 1983. Early rapid and transient increase in omithine decarboxylase activity within sympathetic neurons after axonal injury. Exp. Neural. 81: 158-166. 10. GRAFSTEIN, B., AND I. G. MCQJARRIE. 1978. The role of the nerve cell body in axonal regeneration. Pages 155-195 in C. W. COTMAN, Ed., Neuronal Plasticity. Raven Press, New York. 11. GREENE, L. A., AND J. C. M&&IRE. 1978. Induction of omithine decarboxylase by nerve growth factor dissociated from effects on survival and neurite outgrowth. Nature 276: 191-193. 9.
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12. HALL,M. E. 1982.Changes in synthesis of specificproteinsin axotomixeddorsalroot gangliaExp. Neural. 76: 83-93. 13. HULLER, J. S.,W. F. FONO,ANDE. S.CANELLAKIS. 1976.Inductionof a proteininhibitor to omithinedecarboxylase by the endproductsof its reaction.Proc.Narf.Acad. Sci. USA73: 1858-1862. 14. HIETALA, 0. A. 1983.Detectionof omithinedecarboxylase antixymein mousebrain. J. Neurochem. 40: 1174-1177. 15. KLEIHUES, P., K.-A. HOSSMANN, A. E. PEGG,K. KOBAYASHI, ANDV. ZIMMERMANN. 1975.Resuscitation of the monkeybrain after one hour complete&hernia. III. Indicationsof metabolicrecovery.Brain Res. 95: 61-73. 16. KOSHAKA, S.,M. SCHWARTZ, AND B. W. AGRANOFP. 1981.Increased activity of omithine decarboxylase in goldfishfollowingopticnervecrush.Dev.BrainRex 1: 391-401. 17. KREUTZBERG, G. W. 1982.Acuteneuralreactionto injury. Pages 57-69in J. G. NICHOIS, Ed., Repair and Regeneration of the Nervous System. Dahlem Workshop Reports-Life Sciences Research Report, Vol. 24.Springer,Berlin. 18. LANDRETH, G. E., ANDB. W. AGRANO~.1979.Explantcultureof adultgoldfishretina: a modelfor thestudyof CNS regeneration. Brain Res. 161:39-53.
19. LEWIS,M. E., J. LAKSHMANAN, K. NAGAIAH,P. C. MACDONNELL, ANDG. GUROFF. 1978.Nervegrowthfactor increases activity of omithinedecarboxylase in rat brain. Proc. Natl. Acad. Sci. USA 75: 1021-1023. 20. LOWRY,0. H., N. J. ROSEBROUGH, A. L. FARR,ANDR. J. RANDALL.1951. Protein measurement with the Folia phenolreagent.J. Biol. Chem. 193: 265-275. 21. MACDONNEU,P. C., K. NAGAIAH,J. LAKSHMANAN, ANDG. GUROFF.1977.Nerve growthfactor increases activity of omithinedecarboxylase in superiorcervicalganglia of youngrats. Proc. Nad Acad. Sci. USA 74: 4681-4684. 22. MAUDSLEY, D. V. 1979.Regulation of polyaminebiosynthesis. Biochem. Pharmaco[. 28: 153-161. 23. MCQUARRIE, I. G., B. GRAFSTEIN, AND M. D. GERSHON. 1977.Axonalregeneration in the rat sciaticnerve:effectof a conditioninglesionandof dbcAMP.Brain Res. 132: 443-453. 24. MCQUARRIE, I. G., ANDB. GRAFTXEIN. 1982.Proteinsynthesis andaxonaltransportin goldfishretinalganglioncellsduringregeneration accelerated by a conditioninglesion. Brain Res. 251:25-37. 25. MITCHELL, J. L. A. 1981.Post-translational controlsof omitbinedecarboxylsse activity. Pages15-26in C. M. CALDARERA, V. ZAPPIA,ANDU. BACHRACH, Eds., Advances in Polyamine Research, Vol. 3. RavenPress, NewYork. 26. OBLINGER, M. M., ANDR. J. LASEK.1984.A conditioninglesionof the peripheral axons of dorsalroot ganglioncellsaccelerates regeneration of only their peripheralaxons.J. Neurosci. 4: 1736-1744. 27. PEGG,A. E., ANDH. G. WILLIAMS-ASHMAN. 1968.Biosynthesis of putrescinein the prostateglandof the rat. Biuchem. J. 108: 533-539. 28. ROGER, L. J., ANDR. E. FELLOWS. 1980.Stimulationof omithinedecarboxylase activity by insulinin developing rat brain.Endocrinology 106: 619-625. 29. RUSSELL, D. H. 1981.Omithine decarboxylase: transcriptionalinductionby trophic hormones via a CAMPandCAMP-dependent proteinkinasepathway.Pages 109-125in D. R. MORRIS, ANDL. R. MARTON, Eds., Polyamines in Biology and Medicine, The Biochemistry of Disease, Vol. 8. Dekker,NewYorQBasel. 30. RUSSELL, D. H. 1983.Microinjectionof purifiedomithinedecarboxylase into Xenopw
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oocytes selectively stimulates rihosomal RNA synthesis. Proc. NatI. Acad. Sri. USA 80: 1318-1321. 31.
RUSSELL, D. H., V. J. MEDINA, AND S. H. SNYDER. 1970. The dynamics of synthesis and degradation of polyamines in normal and regenerating rat liver and brain. J. Biol. Chem.
245: 6732-6738.
32. SCHWARTZ, M., S. KOHSAKA, AND B. W. AGRANOFF. 1981. Omithine decarhoxylase activity in retinal explants of goldfish undergoing optic nerve regeneration. Dev. Brain Rex
1: 403-413.
33. SEILER, N. 198 1. Turnover of polyamines. Pages 169-180 in D. R. MORRIS, AND L. R. MARTON, Eds., Polyarnines in Biology and Medicine. The Biochemistry of Disease, Vol. 8. Dekker, New York/Basel. 34. SEILER, N., AND U. LAMBERTY. 1975. Interrelations between polyamines and nucleic acids: changes of polyamine and nucleic acid concentrations in the developing rat brain. J. Neurochem.
24: 5-13.
35. SHASKAN, E. G., AND S. H. SNYDER. 1973. Polyamine turnover in different regions of rat brain. J. Neurochem. 20: 1453-1460. 36. SKENE, J. H. P., AND M. WILLARD. 1981. Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells. J. Cell Biol. 89: 86-95. 37. TABOR, C. W., AND H. TABOR. 1976. 1,4-Diaminobutane (putrescine), spermidine, and spermine. Pages 288-305 in E. S. SNELL, P. D. BOYER, A. RICHARDSON, AND C. C. MEISTER, Eds., Annual Review of Biochemistry, Vol. 45. Annual Reviews Inc., Palo Alto. 38. TETZLAFF, W., AND G. W. KREUTZBERG. 1984. Enzyme changes in the rat facial nucleus following a conditioning lesion. Exp. Neural. 85: 547-564. 39, TORVIK, A., AND A. HEDING. 1969. Effect of Actinomycin D on retrograde nerve cell reaction. Acta Neuropathol. (Berlin) 14: 62-7 1.
NEUROLOGY
Ornithine
89,679-688
(1985)
Decarboxylase in Motoneurons during Regeneration
WOLFRAM TETZLAFF AND GEORG
W. KREUTZBERG’
Max-Planck-Institute for Psychiatry, Am Klopferspitz 18a. 8033 Planegg-Martinsried, Federal Republic of Germany Received April 29, 1985 The activity of omithine decarboxylase, the rate-limiting enzyme in polyamine synthesis, was assayed in the isolated facial nucleus of the rat at various times after axotomy of the facial nerve. In addition, it was measured 24 h after the second of a series of two lesions (conditioning lesion design) with various times between the first and second operations. Ornithine decarboxylase activity was found to increase 8 h atIer nerve transection and was maximum after 24 h (300% of control). Thereafter the activity declined to subnormal levels where it remained for several weeks. Omithine decarboxylase activity did not increase again when a second axotomy was made 2 weeks after the first lesion. However, omithine decarboxylase did respond to the second axotomy if it was carried out 3 weeks after the first lesion. Histochemical localization of omithine decarboxylase demonstrated that the increase in enzyme activity was mainly confined to the perikarya of the motoneurons. These data suggest that this enzyme is somehow involved in triggering the “regeneration program” and clearly indicate that at least some aspects of the neuronal response to axotomy are not further stimulated by a conditioning lesion. o 1985~csdemic PIUS,IIN.
INTRODUCTION
The interruption of the continuity of peripheral nerve axons leads to a number of morphologic, metabolic, and physiologic changes in the nerve cell body known as chromatolysis or “axonal reaction” [reviewed in (10, 17)]. Among the earliest changes observed so far, an increase in omithi* decarboxylase (ODC) activity has been reported in the superior cervical ganglion of the rat (1, 9) and in the goldfish retina (16). Omithine Abbreviations: ODC-omithine decarboxylase; a-DPMO-a-ditluoromethylomithine. ’ The authors thank Dr. M. Reddington for critical reading of the manuscript. Please address correspondence to Professor Kreutzberg.
679 00144886/85
$3.00
Copyright 8 1985 by Academic Ress Inc. All riglm of npmdwlion in any form reserved
680
TETZLAFF
AND
KREUTZBERG
decarboxylase catalyzes the conversion of omithine to the diamine, putrescine (27), which is the rate-limiting step in the synthesis of the polyamines, spermidine and spermine [reviewed in (4, 22)]. High levels of ODC activity and polyamines are found in embryonic, proliferating, and regenerating tissues [for review see (4, 22, 3 1, 34)]. They are presumed to be involved in gene expression by regulating DNA replication and RNA synthesis (4, 37). This function could be of interest in the switching of the nerve cell from its normal function to the metabolic program typical of regeneration. In the present study we investigated the activity of ODC for the first time in regenerating motoneurones. We also applied the conditioning lesion paradigm and studied the response of this enzyme to a repeated lesion. MATERIALS
AND
METHODS
Male Sprague-Dawley rats weighing 230 to 280 g were kept in a 12 h light:12 h dark cycle and fed a standard diet and water ad libitum. Surgery. Design A. Under deep ether anesthesia the left facial nerve was transected at the stylomastoid foramen and the right facial nerve was shamoperated, i.e., exposed but not mobilized. The rats were killed 3, 5, 8, or 24 h, 2, 3, 7, 12, 14, or 20 days after the operation. Design B. The left facial nerve was crushed 3 mm distal to the stylomastoid foramen and the right facial nerve was sham-operated. In a second operation, both the right and the left facial nerve were transected. The interval between the first and the second operations was 14, 20, or 30 days. The rats were killed 24 h after the second operation. Biochemical assays. The isolated facial nuclei from six rats were pooled at each timepoint; the 24-h experiment was conducted twice. A control group of three untreated rats, i.e., six combined left and right facial nuclei, was included in each assay series. The brain stems were frozen and the facial nuclei were punched out with a blunt canula of 1.1 mm inner diameter. Omithine decarboxylase (E.C. 4.1.1.17) activity was assayed in supernatants (13.000 g/10 min) of the homogenated facial nuclei using L(1-14C)-labeled omithine and counting the cleaved radioactive CO;! after 1 h or 2 h of incubation (27). The assay medium contained 5 mMdithiothreito1, 0.3 mM pyridoxal phosphate, 0.1 mM nonradioactive L-omithine in 50 mM Tris/HCl, pH 7.2. To start the assay 0.25 &i L-[l-‘4C]omithine (56 mCi/mmol) was added to 1 ml of the medium/supematant. The reaction was stopped with 0.5 ml 40% trichloroacetic acid and 2 h later the KOH-moistened (200 ~1, 5 N) filters were removed from the center of the rubber-stoppered vials and counted in Supersolve (Zinsser). Low ODC activity made it necessary to pool six facial nuclei for each duplicate measurement. The ODC activities were related to the protein content of the homogenates assayed according to the method of Lowry et al. (20). The
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activities were expressedas a percentage of the unoperated control group which typically displayed activities of ca. 536 + 34 dpm/mg protein/h. Histochemistry For histochemical localization of ODC activity, unfixed cryostat sectionsof the brain stem (20 pm) were used 24 h after facial nerve transection. The unlixed sections were incubated with biotinylated (rdifluoromethylornithine (cy-DFMO), an irreversible inhibitor of ODC, according to the technique of Gilad and Gilad (8). The ODC-DFMO-complex was visualized with avidin-coupled horseradish peroxidase (avidin-HRP) that was processedwith diaminobenzidine. Chemicals. If not mentioned otherwise, all chemicals were purchased from Sigma except avidin-HRP (Vector-Chemicals), L( 1-‘4C)-omithine (Amersham), and Supersolve (Zinsser). Biotinylated cr-DFMO was a gift from Dr. G. Gilad (Weizmann Institute, Rehovot). RESULTS There was no obvious difference in ODC activity between the control facial nuclei, i.e., pooled left and right sidesfrom three unoperated animals, and those contralateral to the operated sides (design A) on days 1, 7, and 12. The unoperated contralateral values of days 2 and 3 were higher than in the control groups (122 and 146% of control values, respectively). In the axotomized facial nuclei the ODC activity started to increase 5 h after axotomy and was twice the control values after 8 h (Fig. 1). The maximum was found after 24 h when the activity was about 300% of controls. On the 2nd and 3rd postoperative days,the ODC activity declined and was at subnormal levels 1 week after operation at which it remained for several weeks.
IQ”,
I;’ days
. 2bd
post OP
FIG. 1. BiochemicaUy measured omithine decarboxylase (ODC) activity in the facial nucleus of the rat at various time intervals after facial nerve transection. The ODC activities are expressed as a percentage of a control group of normal rats (unoperated). Bach symbol represents duplicate measurements of six pooled facial nuclei. The time scale (abscissa) is interrupted.
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To investigate how this ODC activity reacts to a second axotomy, experiments were conducted according to design B (see Materials and Methods). The ODC activity was measured 24 .h after the second operation as this was the timepoint of maximal activity after single axotomy. When the second axotomy was carried out 14 days after the first, no reincrease of the ODC activity was detectable when measured 24 h later. By that time most axons had already reached the peripheral targets again, as indicated by the reonset of whisker movement on day 12. However, axonal maturation was not yet accomplished. Twenty or thirty days after the first axotomy the facial nucleus responded to a second axotomy by a second increase of ODC activity. This second increase was somewhat smaller compared with the first increase: after an operation interval of 30 days, this second increase was about 250% of control whereas the first increase after a single lesion was about 300% of the unoperated control group (Fig. 2). Histochemistry was carried out 24 h after axotomy in order to localize the increasing ODC activity. The activity was rather low in the facial nucleus and the biochemically measured differences were not as pronounced in histochemistry using an irreversibly binding enzyme inhibitor (a-DFMO) for enzyme visualization. The increasing ODC activity was localized in the perikarya of the facial motoneurons (Fig. 3a). There was also some activity in the neuropil whereas the bundles of nerve fibers that cross the facial nuclei appeared blank. The contralateral facial nuclei displayed a positive neuropil but the perikarya were not visible as they were on the axotomized side (Fig. 3b). Control i’ncubations also displayed a slightly positive neuropil, which was less intense than in the unoperated contralateral sides. This indicated that the increasing
1 all data
24h
POI~ 2nd
OP
OP-Interval
FIG. 2. ODC activity in the facial nucleus of the rat 24 h after a second axotomy (CL). The interval between the first (conditioning) and the second (testing) lesion was varied (OP-interval). The ODC increase 24 h after single lesion (SL) is indicated at the left margin of the diagram. Each bar represents a group of six pooled facial nuclei, expressed as a percentage of a control group.
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FIG. 3. Histochemistry of ODC activity in normal (3b) and in axotomized facial nucleus 24 h after nerve transection (3a). Unfixed cryostat sections were incubated with biotinylated (Ydilluoromethylomithine, which binds irreversibly to ODC. This was coupled to avidin-HRP that was visualized with diaminobenzidine.
ODC activity is localized mainly motoneurons.
in the perikarya of the axotomized
facial
DISCUSSION The main finding of the present study is an early and transient increase in ODC activity in the axotomized motoneurons, which did not increase
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again if the neurons were axotomized for a second time during the regeneration period according to the conditioning lesion paradigm. The increase of ODC activity in the axotomized facial nucleus is the earliest sign known of the neuronal response to axotomy in a motoneuron system. An increase in ODC activity, which represents the rate-limiting step in polyamine synthesis, can be found in various tissues and tissue cultures [reviewed in (4, 22)] including the nervous system (5, 6, 15, 19, 21, 28) as a response to a variety of stimuli such as hormones (5, 28), growth factors (11, 19, 21), &hernia (6, 15), and tissue damage (6). At present it is not possible to distinguish whether a de nova synthesis or a posttranslational regulation mechanism is involved in this ODC activation of the facial motoneurons. Both mechanisms have been discussed in a variety of experiments (22, 25). Generally the latency of the ODC increase takes 2 to 3 h. It is mediated by CAMP (29) and in some systems it has been shown to be sensitive to actinomycin D or cycloheximide [reviewed in (22, 29)]. This is of interest with respect to the observation that actinomycin D inhibits the chromatolysis of mouse facial motoneurons when applied within the first 9 h after axotomy; later applications (15 h) are no longer effective (39). These experiments indicate that at least some RNA species must be synthesized within a few hours after injury before the nerve cell body can respond to axotomy. As is known from other systems, polyamines can be involved in the regulation of DNA and RNA synthesis (4, 34, 37) and the ODC molecule itself can stimulate RNA polymerase I (30). Thus it is possible that the increase in ODC activity in the axotomized motoneurons is somehow linked to these early changes in RNA metabolism [see Austin, for recent review (2)]. These changes give rise to the formation of a group of polypeptides and proteins that are either specific for regeneration or have a highly stimulated turnover during regeneration (12, 36). Preliminary studies indicate that such specific changes in the protein pattern also occur in the facial nucleus of the rat (in preparation). Whether or not the increase in ODC activity in the axotomized motoneurons is a prerequisite for these changes in RNA and protein metabolism and thus for the initiation of the regeneration program can be only speculated on at present. The results of the conditioning lesion experiment support the view that the ODC increase may be involved in the initiation of the regeneration program. The ODC activity of the facial nucleus did not increase again when a second axotomy was performed 14 days after nerve crush. At this time the neuronal metabolism is still in the regeneration mode and a second initiation of this program, which includes an ODC activation, probably does not take place. It might therefore be expected that a second
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increase of OLX would occur only after nerve regeneration is accomplished and the neurons are again at normal metabolism. This would agree with our observation of triggering an ODC increase only 20 days after the first lesion. We assume that the second lesion is signaled to the nerve cell body because the facial nucleus reacts by further decreasing acetylcholinesterase activity and by increasing 5’ nucleotidase activity (38). However, a simple amplification of the original response to axotomy does not take place. The signal after a second axotomy could be different or differently processed. Also, after a second axotomy the signal may arrive at the cell body while the cell is in a different metabolic state. This different state could mean that the pool of polyamines formed during the early transient increase in ODC activity still persists and causes a negative feed-back inhibition of ODC activity. Polyamines can have half-lives of several days (3 1, 33, 35) and can induce the formation of a complexing protein that inactivates ODC activity (7, 13, 14). In contrast to the facial nucleus, there is a second increase of ODC activity in the goldfish visual system (32). This second ODC increase is even enhanced by 60% in those retinal explants whose optic nerves had been crushed 2 weeks previously. This is correlated with a stimulated fiber outgrowth from precrushed retinae (18, 32) that can be inhibited by CXDPMO, an irreversible ODC inhibitor (32). In vivo, the response to a second axotomy is more pronounced in the goldfish visual system than in the facial nerve. In the goldfish the velocity and amount of axonal transport are stimulated by about 50% and axonal regeneration is accelerated by more than 90% (23). This contrasts to the faint stimulation of regeneration velocity (23 to 30%) by a conditioning lesion in the rat, as was measured in the sciatic nerve (3, 24, 26). Thus, in the goldfish the enhanced ODC response after a preceding crush of the optic nerve seems to be related to a stimulation of the regenerative response of the retinal ganglion cells. The above considerations prompt us to speculate whether the missing increase of ODC in the facial nucleus reflects a lack of further stimulation of the “regenerative response” of the facial motoneurons aher a conditioning lesion. At least the early changes in RNA metabolism that might be associated with the ODC increase are probably not enhanced. This view is supported by the observation of Langford [quoted by Austin, (2)] who studied the RNA changes after axotomy in the nodose ganglion of the rat. A second crush applied 3 or 7 days alter the first did not stimulate a second rise in uridine incorporation into RNA. This fits with our recent results, that in the facial nucleus the neuronal hexose monophosphate shunt enzymes are not further activated by a preceding axotomy (38). Nevertheless, the conditioning lesion reduces the period of functional recovery by 10%
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in the facial nerve (38). Therefore, if no further stimulation of the neuronal metabolism itself takes place, this conditioning lesion effect would be due to a shortening of the initial delay, i.e., the time before axonal outgrowth begins. Another possibility which must be considered is that stimulator-y factors in the distal nerve stump, which is undergoing Wallerian degeneration, enhance the fiber outgrowth velocity, as was recently shown for the rat sciatic nerve (3). REFERENCES 1. ANDO, M., M. MIWA, K. KATO, AND Y. NAGATA. 1984. Effects of denervation and axotomy on nervous system-specific protein, omithine decarboxylase, and other enzyme activities in the superior cervical sympathetic ganglion of the rat. J. Neurocbem. 42: 94100. 2. AUSTIN, L. 1985. Molecular aspects of nerve regeneration. Pages 1-29 in A. LAJTHA, Ed., Alterations of Metabolites in the Nervous System. Handbook of Neurochemistry, 2nd ed., Vol. 9. Plenum, New York. 3. BISBY, M. A., AND B. POLLOCK. 1983. Increased regeneration rate in peripheral nerve axons following double lesions: enhancement of the conditioning lesion phenomenon. J. Neurobiol. 14: 467-47 1. 4. CANELLAKIS, E. S., D. VICE&MADORE, D. A. KYRIAKIDIS, AND J. S. HELLER. 1979. The regulation and function of omithine decarboxylase and of the polyamines. Pages 155202 in B. L. HORECKER, AND E. R. STADTMAN, Eds., Current Topics in Cellular Regulation, Vol. 15. Academic Press. New York. 5. COUSIN, M. A., D. LANDO, AND M. MOGUILEWSKY. 1982. Omithine decarboxylase induction by ghtcocorticoids in brain and liver of adrenalectomized rats. J. Neurochem. 38: 1296-1304.
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12. HALL,M. E. 1982.Changes in synthesis of specificproteinsin axotomixeddorsalroot gangliaExp. Neural. 76: 83-93. 13. HULLER, J. S.,W. F. FONO,ANDE. S.CANELLAKIS. 1976.Inductionof a proteininhibitor to omithinedecarboxylase by the endproductsof its reaction.Proc.Narf.Acad. Sci. USA73: 1858-1862. 14. HIETALA, 0. A. 1983.Detectionof omithinedecarboxylase antixymein mousebrain. J. Neurochem. 40: 1174-1177. 15. KLEIHUES, P., K.-A. HOSSMANN, A. E. PEGG,K. KOBAYASHI, ANDV. ZIMMERMANN. 1975.Resuscitation of the monkeybrain after one hour complete&hernia. III. Indicationsof metabolicrecovery.Brain Res. 95: 61-73. 16. KOSHAKA, S.,M. SCHWARTZ, AND B. W. AGRANOFP. 1981.Increased activity of omithine decarboxylase in goldfishfollowingopticnervecrush.Dev.BrainRex 1: 391-401. 17. KREUTZBERG, G. W. 1982.Acuteneuralreactionto injury. Pages 57-69in J. G. NICHOIS, Ed., Repair and Regeneration of the Nervous System. Dahlem Workshop Reports-Life Sciences Research Report, Vol. 24.Springer,Berlin. 18. LANDRETH, G. E., ANDB. W. AGRANO~.1979.Explantcultureof adultgoldfishretina: a modelfor thestudyof CNS regeneration. Brain Res. 161:39-53.
19. LEWIS,M. E., J. LAKSHMANAN, K. NAGAIAH,P. C. MACDONNELL, ANDG. GUROFF. 1978.Nervegrowthfactor increases activity of omithinedecarboxylase in rat brain. Proc. Natl. Acad. Sci. USA 75: 1021-1023. 20. LOWRY,0. H., N. J. ROSEBROUGH, A. L. FARR,ANDR. J. RANDALL.1951. Protein measurement with the Folia phenolreagent.J. Biol. Chem. 193: 265-275. 21. MACDONNEU,P. C., K. NAGAIAH,J. LAKSHMANAN, ANDG. GUROFF.1977.Nerve growthfactor increases activity of omithinedecarboxylase in superiorcervicalganglia of youngrats. Proc. Nad Acad. Sci. USA 74: 4681-4684. 22. MAUDSLEY, D. V. 1979.Regulation of polyaminebiosynthesis. Biochem. Pharmaco[. 28: 153-161. 23. MCQUARRIE, I. G., B. GRAFSTEIN, AND M. D. GERSHON. 1977.Axonalregeneration in the rat sciaticnerve:effectof a conditioninglesionandof dbcAMP.Brain Res. 132: 443-453. 24. MCQUARRIE, I. G., ANDB. GRAFTXEIN. 1982.Proteinsynthesis andaxonaltransportin goldfishretinalganglioncellsduringregeneration accelerated by a conditioninglesion. Brain Res. 251:25-37. 25. MITCHELL, J. L. A. 1981.Post-translational controlsof omitbinedecarboxylsse activity. Pages15-26in C. M. CALDARERA, V. ZAPPIA,ANDU. BACHRACH, Eds., Advances in Polyamine Research, Vol. 3. RavenPress, NewYork. 26. OBLINGER, M. M., ANDR. J. LASEK.1984.A conditioninglesionof the peripheral axons of dorsalroot ganglioncellsaccelerates regeneration of only their peripheralaxons.J. Neurosci. 4: 1736-1744. 27. PEGG,A. E., ANDH. G. WILLIAMS-ASHMAN. 1968.Biosynthesis of putrescinein the prostateglandof the rat. Biuchem. J. 108: 533-539. 28. ROGER, L. J., ANDR. E. FELLOWS. 1980.Stimulationof omithinedecarboxylase activity by insulinin developing rat brain.Endocrinology 106: 619-625. 29. RUSSELL, D. H. 1981.Omithine decarboxylase: transcriptionalinductionby trophic hormones via a CAMPandCAMP-dependent proteinkinasepathway.Pages 109-125in D. R. MORRIS, ANDL. R. MARTON, Eds., Polyamines in Biology and Medicine, The Biochemistry of Disease, Vol. 8. Dekker,NewYorQBasel. 30. RUSSELL, D. H. 1983.Microinjectionof purifiedomithinedecarboxylase into Xenopw
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32. SCHWARTZ, M., S. KOHSAKA, AND B. W. AGRANOFF. 1981. Omithine decarhoxylase activity in retinal explants of goldfish undergoing optic nerve regeneration. Dev. Brain Rex
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33. SEILER, N. 198 1. Turnover of polyamines. Pages 169-180 in D. R. MORRIS, AND L. R. MARTON, Eds., Polyarnines in Biology and Medicine. The Biochemistry of Disease, Vol. 8. Dekker, New York/Basel. 34. SEILER, N., AND U. LAMBERTY. 1975. Interrelations between polyamines and nucleic acids: changes of polyamine and nucleic acid concentrations in the developing rat brain. J. Neurochem.
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35. SHASKAN, E. G., AND S. H. SNYDER. 1973. Polyamine turnover in different regions of rat brain. J. Neurochem. 20: 1453-1460. 36. SKENE, J. H. P., AND M. WILLARD. 1981. Changes in axonally transported proteins during axon regeneration in toad retinal ganglion cells. J. Cell Biol. 89: 86-95. 37. TABOR, C. W., AND H. TABOR. 1976. 1,4-Diaminobutane (putrescine), spermidine, and spermine. Pages 288-305 in E. S. SNELL, P. D. BOYER, A. RICHARDSON, AND C. C. MEISTER, Eds., Annual Review of Biochemistry, Vol. 45. Annual Reviews Inc., Palo Alto. 38. TETZLAFF, W., AND G. W. KREUTZBERG. 1984. Enzyme changes in the rat facial nucleus following a conditioning lesion. Exp. Neural. 85: 547-564. 39, TORVIK, A., AND A. HEDING. 1969. Effect of Actinomycin D on retrograde nerve cell reaction. Acta Neuropathol. (Berlin) 14: 62-7 1.