Control of Gene Transcription by Jun and Fos Proteins in the Nervous System

Transcription

1 Focus Control of Gene Transcription by Jun and Fos Proteins in the Nervous System Beneficial or Harmful Molecular Mechanisms of Neuronal Response to Noxious Stimulation? Manfred Zimmermann and Thomas Herdegen Acute and chronic stimulation of somatosensory and visceral nociceptors are followed by induction of immediate-early gene (leg) encoded transcription factors such as Jun, Fos, and Krox proteins in the central nervous system. Transection of a peripheral nerve also results in leg expression in the axotomized neurons and synaptically related central neurons. The spatial and temporal patterns in the nervous system of leg expression, as studied by immunohistochemistry, are complex and depend on the type and intensity of noxious stimulation. Immediate-early gene encoded proteins control the transcription of other target genes, a few of which have been identified. The authors hypothesize that activations of legs represent steps in the transmission of the noxious afferent input into long-lasting alterations of the neuronal cell program. Thus, stimulation induced changes in gene expression may alter the phenotype of neurons, which implies pathophysiologic modifications of neuron function. Immediate-early gene expression is meaningful beyond its present use in the neurosciences as markers of neuronal activity, because it may provide a clue to the understanding of stimulation-induced pathobiology of the nervous system that is involved in the chronicity of pain. Key words: immediate-earlygenes, neuroplasticity, pain, transcription factors, cellular oncogenes, c-fos, c-jun, nerve lesions, neuropathy, dysregulation From the Universit&t Heidelberg, II. Physiologisches Institut, Heidelberg, Germany. Reprint requests: Prof. Dr. M. Zimmermann or Dr. T. Herdegen, Universit&t Heidelberg, I1. Physiologisches Institut, Im Neuenheimer reid 326, D Heidelberg, Germany. S ome types of chronic pain and abnormal pain sensation, e.g., phantom pain, allodynia (as seen in trigeminal neuralgia or reflex sympathetic dystrophy), and most types of headache, suggest a pathophysiology that involves slow and persistent disturbances of nervous function. These disturbances are currently thought to comprise hyperexcitability of peripheral or central sensory neurons and abnormal motor, sympathetic, and neurohumoral efferent functions that in turn may increase the excitation of the nociceptive input system.14,17,2o,24,25,115 Such processes can be compared with positive feedback mechanisms known to perpetuate disturbations of a dynamic system. The persistence of the pathophysiologic states of the nervous control suggests that mechanisms at the level of gene control are involved, because these can be considered to permanently alter the functional phenotype of nerve cells. Hunt and his colleagues' discovery that a cellular oncogene, c-fos, was induced in spinal neurons of rats within 1 hour of sensory stimulation of the skin $9 formed a milestone in the neurosciences. More recent research provides evidence that at least ten genes can be induced by physiologic and pathophysiologic stimuli in the nervous system in viv042,51,112; these genes are now generally designated immediate-early genes (legs). Noxious stimuli and experimental painful conditions, such as inflammation or nerve lesions, are very powerful activators of legs. Therefore, the question naturally arised as to whether leg activation might be a clue to understanding pain-related, long-lasting changes in the nervous system. Both scientists and clinicians have APS Journal 3(1): 33-48,

2 34 FOCUS/Zimmermann and Herdegen been attracted by the idea that leg functions are part of the biological mechanisms of chronic pain. We review the general functions of legs in the nervous system and, in particular, examine their potential roles in mechanisms of pain and neuronal trauma. FUNCTIONS OF IMMEDIATE-EARLY GENES Immediate-early genes (legs) are a group of genes that become activated within a short period of time (as short as a few minutes) when an eukaryotic cell is exposed to a stimulus, such as a growth hormone. TM At present, some 100 legs have been identified by studying in vitro systems. Some of them are homologous to viral (v-) oncogenes and have therefore been termed cellular (c-) oncogenes or protooncogenes 11 such as c-fos. 18 The viral oncogene v- fos is the transforming gene of an osteosarcoma virus. Viral oncogenes are capable of inducing abnormal deregulated growth, resulting in oncogenesis. However, it is now clear that legs, which include some of the cellular oncogenes, have a more general function in gene control; they are involved in the regulation of normal growth, mitosis, and differentiation of cells (reviewed by Bravo, ~ Herschman, 54 Sheng and Greenberg99). The potential of legs is related to the proteins they encode, comprising secretory, cytosolic, and nuclear proteins. Immediate-early gene-encoded nuclear proteins regulate the expression of many other genes. They do so by controlling the transcription of these target genes. For example, leg c-fos encodes for the nuclear protein c-fos, or c-fos (capital letters indicate the protein product). The protein c-fos binds to specific regulatory nucleotide sequences in the promotor and enhancer sites of target genes. By binding to these DNA elements and by interaction with the mrna polymerase II, c-fos "trans"-activates the transcription of the target genes. Thus, c-fos acts as transcription factor; this is true also for numerous proteins encoded by other legs, such as those of the jun and krox families. The involvement of transcription factor proteins is a general operational principle in the molecular biology of gene control. The special feature of leg encoded transcription factors is that their de novo expression can be induced by external stimuli. In contrast, the constitutively expressed transcription factors such as CREB (Ca2+/cAMP response element binding protein) or SRF (serum response factor) are continually present, but they are only transiently activated by phosphorylation. 35 These two types of tran- scription factors in combination may provide an improved potential for transcription control. Inducible transcription factors are of major importance in developmental biology when growth hormones, trophic signals and secretory proteins act on cellular surface receptors and induce leg expression (reviewed by He and Rosenfeld39). Expression of legs alters the morphology and function of cells and underlies the complex interplay of growth, mitosis, and morphological differentiation in both the developing and the maturing organism. However, transcription control by legs is not confined to growth and differentiation, but precedes further modifications of cell function induced by exogenous and endogenous stimuli. Functional plasticity is a particular property of nerve cells, and here, too, the induction of legs is the first step of gene expression. In vitro studies on the activation, by external stimuli, of leg c-fos in cultures of fibroblasts or the pheochromocytoma cell line PC12 have been provided a basic understanding of the induction of legs (Fig. 1 ).s,9,el For example, the cellular surface receptor for a growth hormone activates second messengers such as Ca 2+ or camp, which in turn induce phosphorylation of the constitutively expressed transcription factor CREB by activation of specific protein kinases, such as PKA or PKG. 4'33'35'10 Phosphorylated CREB then interacts with the DNA consensus sequence CRE (Ca2+/cAMP response element), a DNA regulatory consensus sequence in the promotor region of legs such as c-jun, junb, c-fos, and krox-24. Binding of CREB results in the initiation of transcription of these legs with consequent translation into the proteins c-jun, JunB, c-fos, and Krox-24. The transcription promoting capability of c-fos becomes significant only after association with the nuclear protein c-jun or its related proteins JunB and JunD. 36,8,92,95 The c-jun protein is encoded by another cellular oncogene and leg, c-jun. 2'94'96'1 8 The proteins c-fos and c-jun form a heterodimer by binding to each other at a series of leucine sites named the leucine zipper. 32,36,62,83 The Fos/Jun containing transcription complex, called AP-1 (activator protein- 1) complex, binds to specific consensus sequences of other genes to induce transcription of these downstream target genes. 92 Because many genes contain AP-1 sites, Fos and Jun proteins form a superordinate master switch that controls transcription of several other target genes subsequent to surface stimulation of the cell. These mechanisms are also at work in the nervous system.

3 FOCUS/Zimmermann and Herdegen 35 Hormones~ SRF ~ PKC fos-genes Transrnitters--~-PKA CREB ~ jun- Genes =- ~'~F~OS~c- FOS \ -I,J ros B ~Fro- 1 2 los- Genes CREB-(E~),_,_~ II --JUN B _V<( J ~ w ~JUN D r"l jun - Genes AP-1 fe_...~ct Ef o r ~ Protein Effedor- Gene Cell stimulus Expression of DNA binding of heterodimeric transcription factors transcription factors Figure 1. Sequence of molecular genetic events between transmembrane stimulation and expression of effector genes: stimulation-transcription coupling. Cell stimulus: constitutively expressed transcription factors such as CREB or SRF, or both, are activated by phosphorylation via protein kinases C or A (PKC, PKA). Expression of transcription factors: phosphorylated SRF and CREB (SRF-P, CREB-P) bind with high affinity to specific regulatory DNA sequences in the promotors of immediate-early genes such as fos and jun genes. Transcribed fos and jun mrna are translated into the corresponding Fos and Jun proteins in the ribosomes (R). DNA binding of heterodimeric transcription factors: the Jun and Fos proteins form dimers designated as AP-1 complexes that bind to specific regulatory DNA sites in the promotors of other downstream genes, resulting in transcription of these (target or effector) genes. Variable Composition of Jun- and Fos-containing Complexes Differ in Their Transcription Properties Jun and Fos proteins differ in their binding affinities to the regulatory DNA consensus sequences and in their activation potential. 81,92 Moreover, at least some of the transcription factor proteins can act as suppressors of gene expression, as has been reported for FosB. ee,z Thus, the late and persistent appearance of FosB following somatosensory stimulation and under pathophysiological conditions 29,3,42, 4s.52,53 might contribute to the termination of gene transcription controlled by leg. The JunB can negatively modify the transcription operations of c- Jun. ~9'98 Different second messenger pathways converge on leg proteins and modulate their transcription activities, e.g., phosphorylation of c-jun protein can increase or decrease its transcriptional potency. 91 IMMEDIATE-EARLY GENES IN THE NERVOUS SYSTEM Immediate-early gene-encoded proteins such as c- Jun, JunD, and Krox-24 are present in the nervous system in the absence of any intentional stimuli. 41,44, ~3 Because of their known general function in the control of growth, they were originally associated with developmental growth and differentiation of the nervous system, ssj~ More recently it became evident that legs are rapidly activated by a variety of hormonal and neuronal (e.g., synaptic) stimuli under normal physiological and pathophysiological conditions in adult nerve cells. The processes of leg expression with subsequent transcription of target genes following physiological stimuli are now subsumed under the concept of stimulation-transcription coupling. 1'5,16,38,78,99 According to this concept, a main function of the leg-encoded transcription factor proteins is the control of gene transcription as previously described.

4 36 FOCUS/Zimmermann and Herdegen Research into the possible target genes in the nervous system is still at an elementary stage. The increase in nerve growth factor mrna induced by neuronal lesions is mediated by c-fos, 4 and Fos and Jun proteins are involved in the regulation of proenkephalin and prodynorphin expression. 82,1 3 In relation to pain mechanisms, an experimental inflammation in the rat's paw induced fast rises in c-fos mrna, coinciding with modest increases in preproenkephalin mrna, and followed by marked, prolonged increases in preprodynorphin mrna in spinal dorsal horn neurons. 23 These results corroborate the hypothesis, that a set of legs are first activated by the stimulus in neurons, 42,83,~2 which in turn induce transcription of the genes corresponding to the observed changes in neuropeptide synthesis. 93 Immunohistochemical visualization of Jun (c-jun, JunB, JunD), Fos (c-fos, FosB, Fra-1, Fra-2), and Krox (Krox-20, Krox-24) proteins has been used to demonstrate induction of the corresponding genes in the nervous system in vivo. Thus, c-fos was increasingly used for metabolic mapping at the cellular level, in order to explore the topographical distribution of nervous system activity, 97 for instance, during epileptic seizures. 77 Other studies have attempted to associate the induction of c-fos with long-lasting enhancements of synaptic transmission like those involved in long-term potentiation (LTP) and kindling in the hippocampus.2~,~ ~,~2 A differential spatiotemporal pattern of c-jun, c-fos, and krox-24, but not c- myc, was established after a kindling discharge in the hippocampus. ~ 2 In single hippocampal CA1 neurons, an increase of krox-24 mrna (synonymous for zif/268, NGFI-A, Egr-1, and Tis 8) was associated with subsequent increases of protein kinase II mrna and GABA-A receptor mrna, and these LTP-induced changes could be prevented by blocking NMDA receptors. 72 Long-term potentiation has been considered a cellular model for learning, and legs have therefore generally been associated with mechanisms of memory. 3,34,55 The activation of legs has recently been studied and recorded in experimental learning protocols in animals: in chickens exposed for the first time to a discrimination task after hatching, c-jun, but not c-fos, mrna was enhanced in forebrain areas. 3 EVIDENCE FOR PAIN-RELATED, LONG- LASTING MODIFICATIONS OF NERVOUS FUNCTION Clinical evidence suggests that an engram of pain may be formed in the central nervous system (CNS), consisting of a focus of hyperexcitable neurons that generate abnormal discharges. A well known example is phantom pain, i.e., pain that has eventually become independent of the original peripheral nerve pathology. Referred pain and allodynia may be other cases of central hyperexcitability where nonnoxious sensory input is sufficient to trigger a discharge of hyperexcitable neurons. Other mechanisms of neural pathophysiology are inadequate motor, sympathetic, or hormonal responses of the nervous system that may result in dysfunction of the target organs under efferent neural control, e.g., postural muscles or intracranial vessels. Animal experiments have revealed that long-lasting modifications occur in the CNS in response to a noxious peripheral event or to the lesion of a peripheral nerve. For example, the threshold of the flexor reflex in rats was decreased for weeks in both hindlimbs after a transient burn trauma had been inflicted on one leg. 113 Experimental inflammation in the hindlimb is paralleled by slow biochemical and cellular processes in the spinal cord that may contribute to hyperalgesia and, in addition, to nociceptor sensitization. For example, tachykinin receptor mrna and nitric oxide synthase (NOS) are increased in the spinal dorsal horn after subcutaneous injection of formalin. 52,8~ In the spinal cords of rats with an experimental arthritis, gene expression of prodynorphin mrna and its product dynorphin is increased, $6,7~,93 opioid receptor binding is modified, 7 and release of amino acids and prostaglandin E2 by C-fiber stimulation is much enhanced. 1 4 Spinal neurons develop abnormal hyperactivity after transection of dorsal roots in rats. 71 Animals show behavioral signs of pain and hyperalgesia after transection or compression of a major limb nerve, e,1 9 The patterns of sympathetic reflexes to skin and muscle are persistently changed after transection of a peripheral nerve, an observation that has been associated with sympathetic reflex dystrophy. 8 Gene expression of prodynorphin and dynorphin in spinal dorsal horn neurons is dramatically increased after a peripheral nerve or spinal cord lesion. 9 INDUCTION OF IMMEDIATE-EARLY GENE EXPRESSION FORMS A BRIDGE BETWEEN NOXIOUS STIMULATION AND LASTING ALTERATIONS OF GENE EXPRESSION From the available body of evidence, it is conceivable that virtually every long-term change in nervous system function implies modified gene expression in nerve cells. At the DNA level, processes of transcription control can be assessed by studying the expression of legs, the master switches of the transcription

5 FOCUS/Zimmermann and Herdegen Figure 2. Locations of c-fos immunoreactive cell nuclei (per 50 m section) in the brain 2 hours following injection of 5% formalin into one hindpaw of awake rats. Each dot represents one immunopositive nucleus. Note that the distribution of c-fos-ir is symmetrical, i.e., does not show preference for the ipsi- or contralateral side of formalin injection. The numbers indicate the distances of frontal brain sections with reference to Bregma. Abbreviations are according to Paxinos and Watson: The rat brain in stereotaxic coordinates, machinery. TM Hunt and his colleagues 57 were the first to show by immunocytochemistry that expression of c-fos protein occurs in the nuclei of dorsal horn neurons after noxious stimulation. This finding has repeatedly been confirmed. 12,74,88 More recently, it was shown that nociceptive input to spinal neurons also results in the expression of other legs and their protein products, such as Krox-24, c-jun, JunB, JunD, and FosB. 41'42'44'45'52'1 7'112 The Fos and Jun proteins induced by noxious cutaneous stimulation have also been found in the brain.12,13,41,46,88 The distribution pattern of these immunoreactivities (IR) in the CNS was only partially homologous to what is generally considered the central pain system (Fig. 2). However, areas with definite neuronal excitation by acute somatosensory noxious stimulation, such as the cerebellum and the hippocampus, do not express legs. 13'46,86 After noxious stimulation, legs are also visible in nuclei of the limbic system and hypothalamic areas, whereas they are absent in the ventrobasal complex of thalamus and in the dorsal column nuclei. It is conspicuous that basal leg expression can be seen in many areas that show elevated levels of leg expression following noxious peripheral stimulation. 13,41,4e,86 Thus, leg encoded proteins are not just markers for neuronal activity, they specifically indicate that processes have been activated at the nuclear level in selected neuronal populations. Therefore, leg immunohistochemistry is a valuable new tool to map neuronal functions that involve activation of transcription processes following sensory stimulation or pathological activation of the nervous system. Mechanisms of Neuronal Immediate-Early Gene Activation In addition to these mapping studies, research has been aimed at revealing the mechanisms of leg induction in the nervous system. Activation of legs in nerve cells depends on the type of stimulation acting

6 38 FOCUS/Zimmermann and Herdegen on the cell. Thus, the rapid increase and the suppression of jun, fos, and krox-24 mrna in brain neurons depend on the activation of synaptic NMDA receptors. 1~'22,3 Conversely, c-fos induction in the spinal cord following noxious stimulation is prevented by opiates and adrenoceptor agonists, but not by NMDA receptor antagonists. 86,88,1 5,~ 6 Several findings suggest that electrical stimulation per se is not a sufficient condition for leg induction: Primary sensory neurons of the somatosensory system, i.e., neurons in the dorsal root ganglia (DRG), do not express leg encoded proteins after their direct electrical or adequate sensory nerve stimulation. 42 KCI induced cortical spreading depression only weakly affects c-jun and JunD expression in the cortex. 53 KCI induced membrane depolarization is not followed by c-jun mrna induction in fibroblasts, s Electrical stimulation of sciatic nerve fibers at A-fiber intensity does not evoke leg expression in spinal neurons. 42,57,75 In addition to excitatory amino acids, leg expression might be mediated by neuropeptides, 67 nitric oxide (NO), and molecular genetic mechanisms such as phosphorylation of CREB. The neuropeptides substance P and calcitonin gene-related peptide (CGRP) are transmitters predominantly of nonmyelinated peripheral nerve fibers, 6s and their release is a consequence of C-fiber stimulation. 26 Previously it was demonstrated that neurons labeled by c-fos following application of mustard oil receive input from peptidergic primary afferent fibers containing substance P, serotonin, and enkephalin. 89 The gas molecule NO has became a focus of attention in neurobiology because it represents a novel class of neurotransmitters ~'76 and evokes lasting alterations in the function of neurons. 73 Nitric oxide and its catalyzing enzyme NOS are most likely involved in a neuronal network contributing to leg expression, as suggested by the following findings: The induction of c-fos in spinal neurons by noxious peripheral stimulation can be prevented by inhibition of NO synthesis. 7 Double labeling for nicotinamide-adenine dinucleotide phosphate (NADPH) diaphorase (indicating the presence of NOS) and Jun, Fos, and Krox proteins has revealed that many neurons labeled by leg-encoded proteins fol- Figure 3. Histological section of lumbar spinal cord of a rat killed 2 hours following injection of 5% formalin into one hindpaw. Double-labeling of c-fos-ir and NADPH-diaphorase reaction in the ipsilateral dorsal horn is shown. The numbers mark NADPH-diaphorase labeled neurons with extended axons forming a dense network between superficial and deep dorsal horn, ipsilateral to the formalin injected paw. The arrows mark c-fos labeled nuclei of neurons that are in close proximity to NADPH-diaphorase labeled fibers. lowing application of formalin are in close proximity to fibers labeled by NADPH diaphorase (Fig. 3). 52 Subcutaneous formalin increases NOS expression in the superficial dorsal horn. The superficial dorsal horn shows the highest number of neurons labeled by leg encoded transcription factors, and some of these neurons are double labeled by NOS. ~2 Thus, induction of NOS and legs may be interrelated, and this could explain the hyperalgesic effects of NOy according to the following cascade of events: the peripheral noxious stimulation might evoke release of neuropeptides and NO from primary afferents, resulting in induction of leg, which in turn results in the upregulation of NOS expression with concomitant hyperalgesia via NO mediated hyperexcitation of spinal neurons.

7 FOCUS/Zimmermann and Herdegen 39 Induction of leg also depends on the specific regulation mechanism of the individual leg promotors. Recently, it was reported that the induction of c-fos by NO is much enhanced if Ca 2+ ions are present and that this effect is mediated via PKA-dependent activation of the transcription factor CREB. 87 Under the same conditions, induction of c-jun is much less, which supports the hypothesis of differential fine tuning of leg expression in a neuronal system. Phosphorylation of CREB is presumably an important step in the activation of leg following transsynaptic stimulation of neurons 33 which are dominated by an intense c-fos and JunB expression. 29,31,42,45,53 In contrast, the transcription operation, during the cell body response of axotomized neurons, is dominated by c-jun and JunD, and this selective induction might be due to the absence of phosphorylation of CREB. PATTERNS OF IMMEDIATE-EARLY GENE EXPRESSION IN THE NERVOUS SYSTEM FOLLOWING NOXIOUS STIMULI In collaboration with several research groups, we studied the complex immunohistochemical patterns and time courses involved in the activation of several legs after acute and chronic noxious stimulations. Following repeated application of noxious heat or injection of formalin into one hindpaw, we observed individual spatiotemporal patterns of leg expression in anesthetized (noxious heat) and awake (formalin) rats45'52'86'l 6: within 2 hours, a maximal expression of c-jun, JunB, c-fos, and Krox-24 proteins was visible in nuclei per 50 #m section of the ipsilateral dorsal horn (Fig. 4). These IR declined to base levels after 10 hours. The JunD and FosB reached their maximal expression between 5 and 10 hours and were still present after 24 hours. The numbers of neuronal nuclei labeled for c-fos, JunD, and Krox-24 were fairly equally distributed between superficial and deep dorsal horn, whereas c- Jun and FosB were predominantly expressed in the superficial layers. Thus, varying compositions are possible of AP-1 complexes from the available transcription factor proteins, depending on the specific temporospatial expression of the individual leg proteins: within the first 2 hours, dimers containing c- Jun, JunB, and c-fos can be formed in the superficial layers and JunB:c-Fos dimers in the deep layers, whereas between 10 and 24 hours, AP-1 complexes may consist of FosB:JunD dimers. It is noteworthy that c-jun and FosB, which have the highest DNA = a) _.] 100" 75._= 50 E C -.J i i / [] JUN B [] JUN D [] FOS B E 0 x =- :> E O- r-._~ E 50-..J J hours Figure 4. Temporal and spatial patterns of legs in the rat spinal cord following noxious skin heating. Under anesthesia (halothane) one hindpaw was repeatedly immersed in hot water (52 C, 10 times for 20 seconds each, at 90-second intervals). Animals were sacrificed 24 hours after the end of stimulation, and the lumbar spinal cord was processed for immunohistochemistry of JunB, JunD, and FosB. The mean numbers of labeled neurons (_+SD) are given per 25 m slice. The means were calculated from 15 slices (5 slices each, from 3 rats). The upper part gives the number of labeled neurons in ipsilateral laminae I-III, the lower part in laminae IV-VII and X (Herdegen et al.4~).

8 40 FOCUS/Zimmermann and Herdegen binding affinity and transcription activity, 9s are expressed in a fairly low number of neurons compared to c-fos or JunB. This suggests a meaningful neural control of transcription factors. Similarly, lasting differential expression of leg-encoded proteins with putatively specific formation of AP-1 complexes could also be observed following chronic somatic inflammation. 63 Repetitive electrical stimulation of the sciatic nerve was performed for 10 minutes. 42 Stimulation of large A-fibers alone did not result in the expression of leg-encoded proteins beyond base expression in spinal dorsal horn neurons in the area of termination of sciatic nerve fibers. However, when stimulation (at 5 Hz) included A.~- and C-fibers, many neurons of the ipsilateral dorsal horn showed nuclear labeling for these six proteins, with an onset of detection at about.5 hour after the start of stimulation and a maximum of labeled neurons between 1 and 4 hours. The expression declined to base levels between 8 and 16 hours, except for JunD and FosB, which were still expressed after 32 hours. After 4 hours, the six proteins were also seen in some neurons of the contralateral dorsal horn. The leg expression patterns following electrical stimulation at C-fiber strength of sciatic nerve were fairly congruent with those following stimulation of hindlimb nociceptors by noxious heating or formalin injection. In the brain, the leg-encoded proteins studied showed stimulation-dependent increases in the numbers of labeled neurons mostly bilaterally in numerous brain nuclei, such as the lateral reticular formation of medulla, locus coeruleus, dorsal raphe and pontine nuclei, periaqueductal gray, parabrachial nucleus, supramammillary nucleus, midline nuclei of thalamus and hypothalamus, amygdala, and lateral habenular nucleus. Again, c-fos, JunD, and Krox-24 showed the greatest numbers of labeled neurons, whereas c-jun, JunB, and FosB were absent in some areas with induced c-fos and JunD labeling. Thus, similar to the spinal cord, different AP-1 compositions are formed in individual brain areas resulting in differential transcriptional operations. Immediateearly genes were absent or not altered in nucleus gracilis and nucleus cuneatus, ventrobasal complex of thalamus, and the cerebellum. Thus, differential spatiotemporal patterns of stimulation-induced leg expression suggest that the legs do not just reflect neuronal activity. Rather, leg expression may indicate specific neuronal populations, which alters their neuronal program and their de novo protein synthesis, respectively. Provided that changes in protein synthesis are a major attribute of plasticity, legs can be used to visualize those neurons reacting with plasticity to somatosensory stimulation. Recruitment of Immediate-Early Genelabeled Neurons Following Persistent Noxious Stimulation or Increase of Stimulation Intensity, or Both Expression of leg in neurons is not a yes/no answer, but likely depends on the intensity of transynaptic activation and other facilitatory or inhibitory conditions. Thus, spinal neurons of deep dorsal horn express legs in NADPH labeled neurons only following repeated noxious stimulation, not after a single stimulus, s2 which indicates that temporal facilitation is required to initiate transcription operations in specific neurons. Following auditory stimulation at low intensity, numerous neurons express c-fos in the auditory cortex, but not neurons that contain parvalbumin, a calcium binding protein. In these neurons, c-fos became visible only after increasing the intensity of stimulation. 116 Electrical stimulation of the proximal stump of the sciatic nerve 20 days after nerve transection was highly effective in inducing expression of c-fos in neurons of spinal lamina III and nucleus gracilis. In these areas, no leg expression was seen following electrical stimulation of the intact sciatic nerve. 7s ACTIVATION OF IMMEDIATE-EARLY GENES BY NERVE LESIONS Another case of a differential pattern of leg expression was obtained after transection and ligation of one sciatic nerve of the rat, which is part of a great variety of reactive changes in the primary afferent neuron, its peripheral target organs, and the second order spinal neurons (Fig. 6). In this experiment, we studied the leg-encoded proteins in the lumbar spinal cord and in the related DRG. 42'68 After the nerve transection, we found an early and transient induction of c-fos, FosB, c-jun, JunB, JunD, and Krox-24 in the ipsilateral dorsal horn neurons of those segments related to the ipsilateral hindlimb, predominantly in the superficial laminae. We found a pattern of expression of the various legs in the dorsal horn, similar to electrical sciatic nerve stimulation or noxious skin stimulation. The numbers of leg protein labeled neurons reached a maximum a few hours after nerve transection and then declined between 24 and 48 hours. These expression patterns are most likely due to transynaptic impulse dis-

9 FOCUS/Zimmermann and Herdegen % of labelled neurons Control 15h 24h 48h 10d 30d-- 150d Time after transection c-jun ~ Galanin ~ Galanin and c-jun Figure 5. Expression of c-jun and galanin in neurons of ipsilateral L5 dorsal root ganglion following transection of the sciatic nerve in the rat. Transection and ligation of the sciatic nerve was performed under anesthesia (pentobarbital). A few hours to 150 days later, the animals were reanesthetized and perfused for histological analysis. Dorsal root ganglia were dissected and processed for immunohistochemistry for c-jun and galanin. The open and hatched columns give the numbers of neurons labeled for c-jun and galanin, respectively, relative to the total number of neurons. The black columns give the percentage of neurons labeled for both galanin and c-jun. Each column is the mean of 3 sections each, from 3 rats, selected at random (Herdegen et al.~ ). charges peri- and postoperatively evoked by surgery in the leg and sciatic nerve transection. Treatment of the wound and nerve with lidocaine prevented this leg expression. In our nerve transection experiments, the most important difference to our findings with afferent noxious stimulation was the delayed and selective expression of c-jun and JunD, starting between 10 and 24 hours in the axotomized neurons of the DRG and sciatic motoneurons. 47,5,68 The c-jun expression persisted for days at a maximal level and was still suprabasal in small diameter DRG neurons after 15 months. The selective expression of c-jun and JunD in the absence of JunB, c-fos, and FosB is a general finding following axotomy of peripheral and central neuro ns.43,48,49,59,60,69 Double labeling with the tracers fast blue or horseradish peroxidase-labeled gold transported retrogradely from the site of axotomy, showed that c-jun protein expression was confined to the neurons that had undergone axotomy. 68 Most probably the c-jun response to the nerve lesion is mediated by signals transmitted along retrograde axons, as blocking axonal transport of the intact nerve with topical colchicine had a similar result, s8 It would be of utmost importance to see which of these genetic responses can be associated with the persistent pain that can occur following nerve lesion in man and animals, e.g., by correlation analysis of leg expression and pain behavior in individual animals after a nerve lesion. In order to explore the functional significance of leg expression following nerve transection, we investigated coexpression of the neuropeptides galanin and CGRP and of NOS in axotomized neurons. We found that many DRG neurons labeled for c-jun after sciatic nerve transection also showed increased galanin IR (Fig. 5) which is very low in untreated DRGs. s The time course of the increased galanin expression was nearly parallel to that of c-jun, but it was clearly delayed by 5-10 hours. These findings are in accordance with the hypothesis that expression of c-jun and galanin is functionally related and that the transcription of the galanin gene might be

10 42 FOOUS/Zimmermann and Herdegen Dorsal root ganglion SP ~ Spinal CGRP~ c-jun f neuron GAL f JUN D f VlP st E., NOS Neuroma Figure 6. Changes in gene expressions following sciatic nerve transection: schematic diagram of functional implications. Transected axons start to regenerate by forming axonal sprouts. In the cell body of the dorsal root, ganglion neurons c- Jun and JunD proteins are expressed in the nucleus. Contents of substance P (SP) and calcitonin gene-related peptide (CGRP) decrease over weeks, whereas galanin (GAL), vasoactive intestinal peptide (VIP), and nitric oxide synthase (NOS) are upregulated for weeks and months. In dorsal horn neurons, c-fos, c-jun, and Krox-24 are transynaptically induced by nerve transection, followed by upregulations of dynorphin (DYN), enkephalin (ENK), neurotensin (NT), and NOS. Colocalisations, covariances, and temporal order of expressions suggest that the leg encoded proteins act as transcription factors for the changes in biosynthesis of neuropeptides and NOS. controlled by c-jun. Galanin has been shown to have inhibitory effects in the spinal cord, and therefore it is conceivable that the increase in galanin content in afferent fibers could contribute to a mechanism counteracting the hyperexcitability of spinal neurons following peripheral nerve lesion. 114 Spinal motoneurons with axons projecting into the sciatic nerve showed an increase in CGRP expression after nerve transection.5 As with galanin in DRG neurons there was a high incidence of colocalization with c-jun. The c-jun and CGRP also had a parallel time course, and again c-jun expression preceded CGRP by about 5 hours. This suggests that c-jun regulates transcription of the CGRP gene. The CGRP regulates the synthesis of muscle acetylcholine receptor 84 and substantially contributes to the functional reestablishment of the neuron-muscle axis after nerve regeneration. Similarly, we found coexpression of NOS, the enzyme controlling synthesis of the novel messenger substance NO, in many DRG neurons labeled for c- Jun after nerve transectiony In this case, however, c-jun IR preceded NOS IR by 5 days, whereas the subsequent time courses of c-jun IR and NOS IR were similar for over 100 days. Here again, it is conceivable that c-jun is involved in the control of NOS gene expression, where the long time lag might indicate a more complex sequence of events being interposed between c-jun and NOS transcriptions. The upregulation of NOS in the afferent neurons, with consequent increase in NO availability, could be a mechanism to compensate the decreases in substance P and CGRP contents in afferent neurons after nerve transection: both these peptides and NO have similar neurotransmitter effects in the periphery (neurogenic inflammation) and in the spinal dorsal horn (excitatory neurotransmission or neuromodulation). CONCLUSIONS The observation that nervous activity can alter gene transcription at first seemed paradoxical, as adult nerve cells do not undergo mitosis and normally do

11 FOCUS/Zimmermann and Herdegen 43 not show signs of growth. Why, then, should the nuclear transcription machinery and other processes related to nervous system development be activated by physiological nerve cell stimulation? Immediate-early genes might be an essential part of the mechanisms that enable nerve cells to modify their working range at the molecular level in response to the requirements of changing conditions. This would best correspond to the dominant principle of the nervous system, i.e., to enable the organism to continuously adapt to the external world. Many nervous system adaptations are related to learning, and learning is mostly a long-term modification that might last for the entire life span. Hence, learning and memory research, since Hyden, has had a great interest in assessing what happens at the level of gene transcription, although the changes that may occur in single neurons during a learning experiment might be below the detection level of currently available methods. It is impressive to see that noxious stimulation has particularly strong effects at the transcription level of nerve cells, as indicated by the powerful induction of legs by noxious stimuli and pain conditions. We do not yet know precisely what this long-lasting activation of legs means in relation to nociception and pain. However, it reflects profound regulatory responses in the functional molecular repertoire of the nerve cells, which might include modifications of enzymes, transmitter synthesis, and synaptic and hormonal receptor configurations (Fig. 7). A few of the relationships between an leg and some of the target genes, whose transcription it controls, have been well established by direct molecular biological analysis, such as transcription control of nerve growth factor (NGF) expression by AP-1 proteins in glial cells following neuronal lesions. Other presumed relationships between leg and potential target genes seem likely, as has been concluded, for instance, from the covariance of legs and neuropeptides in immunohistochemical or RNA analysis. Some of these have been reviewed in this article. Excitatory inputs re ~ C fibre ( ~ Neuropeptides _~'~ i / A K,oX~/ Inhibitory inputs Figure 7. Schematic diagram of stimulation-transcription coupling in nerve cells. Stimulation of a neuron by excitatory inputs can induce, via second messengers, activation of immediate-early genes (legs, such as fos, jun, krox-24) in the cell nucleus. The protein products of these legs control the expression of other genes relevant for the synthesis of constituents of neuronal function, e.g., neurotransmitters, synaptic proteins, channel proteins, and enzymes. Nociceptive inputs via neuropeptides released from C-fibers, excitatory.amino acids (EAAs), or signals transmitted by axonal transport can activate the transcription-controlling cascade of events shown. Hypothetically, noxious stimuli can result in changes in neuronal excitability by modifying the gain of excitatory transmission or the efficacy of inhibitory systems. Diagram based on results and ideas by several authors as discussed in the text (ZimmermannllS).

12 44 FOCUS/Zimmermann and Herdegen Can Transcription Control Result in Neuronal Dysfunction and Chronic Pain? The view we presented emphasizes the basically beneficial functions of transcriptional control by external stimuli mediated by legs. However, clinical and experimental evidence overwhelmingly shows that any somatic trauma, lesion to the peripheral or central nervous system, or transient pain conditions can result in chronic pain and chronic nervous system disease, obviously due to lasting changes in the nervous system that are also based on gene expression controlled by legs. Some far-reaching regulatory responses of the nervous system that imply mechanisms of gene control may carry the risk of having long-lasting detrimental consequences. It seems to be a general principle in the nervous system that when a regulatory response exceeds a certain range, it can result in disturbance. Thus, negative feedback aimed at stabilizing the system under challenge is then changed to positive feedback that detracts from stability. To make these matters more concrete, in terms of nervous function, we hypothesize that most of the changes related to chronic pain result from deregulated gene expression under conditions when legs are at work. Specifically, according to our hypothesis, inappropriate and dysfunctional neurotransmitters and synaptic receptor proteins may have formed, as indicated in Figure 7. We do not know what determines the outcome of leg induction by transient trauma or noxious stimulation. The same c-fos/c-jun transcription complex can activate hundreds or even thousands of target genes, and the number of legs, and other transcription controlling molecules, currently known is too small to provide specificity of control in relation to the subsequent processes. However, we do not know what happens beyond the few transcription factors we have been able to observe so far, and it might well be that what we can see today is a mere fraction of what will be discovered in our nervous system in the future. Future research in this field will have to examine in detail the molecular genetic processes in our brain. However, many questions aimed at manipulating the system and its therapeutic implications are being posed, and we are confident that there will be intensive research into therapeutic possibilities at the level of transcription control, as the research methods used in the field suggest the possibility of highly selective approaches. Thus, if we consider that some kinds of chronic pain are diseases of acquired transcription failures, the therapeutic and preventive strategies to be followed might not be too different from those now being developed for other diseases, which are due to inherited or acquired gene defects or transcription deficiencies. References 1. Alrnendral JM, Sommer D, MacDonald-Bravo H et al: Complexity of the early genetic response to growth factors in mouse fibroblasts. Mol Cell Biol 8: , Angel P, Allegretto EA, Okino ST et al: Oncogene jun encodes a sequence-specific trans-activator similar to AP-I. Nature 332: , Anokhin KV, Rose SPR: Learning-induced increase of immediate early gene messenger RNA in the chick forebrain. Eur J Neurosci 3: , Bading H, Ginty DD, Greenberg ME: Regulation of gene expression in hippocampal neurons by distinct calcium signaling pathways. Science 260: , Bartel DP, Sheng M, Lau LF, Greenberg ME: Growth factors and membrane depolarization activate distinct programs of early response gene expression: dissociation of fos and jun induction. Genes Dev 3: , Bennett GH, Xie Y-K: A peripheral mononeuropathy in rat that produces disorders of pain sensation like those seen in man. Pain 33:87-107, Besse D, WeiI-Fugazza J, Lombard MC, Butler SH, Besson JM: Monoarthritis induces complex changes in mu-, delta- and kappa-opioid binding sites in the superficial dorsal horn of the rat spinal cord. Eur J Pharm 223: , Blumberg H, J&nig W: Changes of reflexes in vasoconstrictor neurons supplying the cat hindlimb following chronic nerve lesions: a model for studying mechanisms of reflex sympathetic dystrophy. J Auton Nerv Syst 7: , Bravo R, MacDonald-Bravo H, M~ller R, HL~bsch D, Almendral JM: Bombesin induces c-fos and c-myc expression in quiescent swiss 3T3 cells. Exp Cell Res 170: , Bravo R: Growth factor inducible genes in fibroblasts. pp Herschman A (ed): Growth factors, differentiation factors and cytokines. Springer-Verlag, Heidelberg, Bredt DS, Snyder SH: Nitric oxide, a novel neuronal messenger. Neuron 8:3-11, Bullit E: Induction of c-fos-like protein in the lumbar spinal cord and thalamus of the rat following peripheral stimulation. Brain Res 493: , Bullit E: Expression of c-fos like protein as a marker for neuronal activity following noxious stimulation in the rat. J Comp Neurol 296: , Casey KL (ed): Pain and central nervous system dis-

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