c-Jun N-terminal Kinase 3 Deficiency Protects Neurons from Axotomy-induced Death in Vivo through Mechanisms Independent of c-Jun Phosphorylation
2004; Elsevier BV; Volume: 280; Issue: 2 Linguagem: Inglês
10.1074/jbc.m410127200
ISSN1083-351X
AutoresElizabeth Keramaris, Jacqueline L. Vanderluit, Mohammad Hadi Bahadori, Kambiz Mousavi, Roger J. Davis, Richard A. Flavell, Ruth S. Slack, David S. Park,
Tópico(s)Neuroscience and Neuropharmacology Research
ResumoBoth the transcription factor c-Jun and the c-Jun N-terminal kinases (JNKs) have been associated with neuronal loss in several death paradigms. JNK are key regulators of c-Jun and a common accepted model has been that JNKs mediate neuronal death through modulation of c-Jun activation. In the present study, we examined whether JNK2 and -3 (JNK members most associated with neuronal loss) deficiency can rescue neuronal loss caused by facial and sciatic nerve axotomy in the neonate in vivo. JNK2, JNK3, and JNK2/3 double-deficient neurons displayed significantly less death in the facial nerves of the CNS when compared with controls. JNK2 and JNK2/3 double-deficient animals also showed reduced c-Jun phosphorylation and induction following axotomy, consistent with the model that JNK acts to regulate death by activating c-Jun. Of significance, however, protection of facial nerves in JNK3-deficient animals was not accompanied by reduction in c-Jun activation. These results suggest that JNKs can mediate death independently of c-Jun. Importantly, the lack of correlation between JNK3 deficiency and c-Jun induction was not universal. In a sciatic axotomy model of neuronal injury in the neonate, death of DRG neurons was also reduced by JNK3 deficiency. However, in this case, c-Jun activation was also eliminated. Both the transcription factor c-Jun and the c-Jun N-terminal kinases (JNKs) have been associated with neuronal loss in several death paradigms. JNK are key regulators of c-Jun and a common accepted model has been that JNKs mediate neuronal death through modulation of c-Jun activation. In the present study, we examined whether JNK2 and -3 (JNK members most associated with neuronal loss) deficiency can rescue neuronal loss caused by facial and sciatic nerve axotomy in the neonate in vivo. JNK2, JNK3, and JNK2/3 double-deficient neurons displayed significantly less death in the facial nerves of the CNS when compared with controls. JNK2 and JNK2/3 double-deficient animals also showed reduced c-Jun phosphorylation and induction following axotomy, consistent with the model that JNK acts to regulate death by activating c-Jun. Of significance, however, protection of facial nerves in JNK3-deficient animals was not accompanied by reduction in c-Jun activation. These results suggest that JNKs can mediate death independently of c-Jun. Importantly, the lack of correlation between JNK3 deficiency and c-Jun induction was not universal. In a sciatic axotomy model of neuronal injury in the neonate, death of DRG neurons was also reduced by JNK3 deficiency. However, in this case, c-Jun activation was also eliminated. Neuronal apoptosis is regulated by a complex series of molecular events, which in many cases culminate in activation of the conserved mitochondrial pathway of death characterized by the activation of the apoptosome. 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Flavell R.A. Davis R.J. Rakic P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15184-15189Crossref PubMed Scopus (374) Google Scholar). JNK3 is also critical for c-Jun phosphorylation and dopaminergic loss in a 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine model of Parkinson's disease (11Hunot S. Vila M. Teismann P. Davis R.J. Hirsch E.C. Przedborski S. Rakic P. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 101: 665-670Crossref Scopus (363) Google Scholar). However, in this case, JNK2 also appears to mediate neuronal loss (11Hunot S. Vila M. Teismann P. Davis R.J. Hirsch E.C. Przedborski S. Rakic P. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 101: 665-670Crossref Scopus (363) Google Scholar). The crucial role for c-Jun as a downstream target of JNK-mediated neuronal death is supported by the observations that in cases where JNK deficiency inhibits death, c-Jun phosphorylation is also reduced. In these paradigms, c-Jun has been shown to be a required factor in death signaling. 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Neurosci. 1998; 18: 5124-5135Crossref PubMed Google Scholar) and it has been suggested that c-Jun may in fact participate in regenerative processes (31Herdegen T. Waetzig V. Oncogene. 2001; 20: 2424-2437Crossref PubMed Scopus (179) Google Scholar, 32Raivich G. Bohatschek M. Da Costa C. Iwata O. Galiano M. Hristova M. Nateri A.S. Makwana M. Riera-Sans L. Wolfer D.P. Lipp H.P. Aguzzi A. Wagner E.F. Behrens A. Neuron. 2004; 43: 57-67Abstract Full Text Full Text PDF PubMed Scopus (374) Google Scholar). In the present study, we examined the importance of the JNK/c-Jun pathway in the context of two neonatal models (sciatic and facial) of axotomy. Here we demonstrate that c-Jun activation is not necessarily the main component of JNK-mediated death. We show that while JNK3 deficiency inhibits c-Jun activation and protects peripheral sensory neurons from death, the protection afforded by JNK3 deficiency in facial motor neurons is not accompanied by reduced c-Jun phosphorylation. Accordingly, we suggest that, at least in certain neuronal contexts, JNK-mediated death signals act through other c-Jun-independent signals. Facial Nerve Axotomy—Axotomy of the right facial nerve was performed on postnatal day 1 mouse pups. Briefly, pups (C57/Bl background) were anesthetized with a gas mixture of 2–5% isofluorane and O2 at a flow rate of 1 liter/min, a 1-cm incision (medial to lateral) was made caudal to the right ear. The musculature was separated to expose the facial nerve. The branches of the right facial nerve were transected as they exited the stylomastoid foramen. The musculature was closed and the skin was sutured together with Prolene 6–0 suture (Johnson & Johnson). Mice were sacrificed 24 h or 4 days following axotomy. For quantitation of survival, every fifth section through the facial nucleus was evaluated by cresyl violet staining and the total number of live motor neurons was evaluated both ispialteral and contralateral to the axotomy as previously described (33Vanderluit J.L. McPhail L.T. Fernandes K.J. McBride C.B. Huguenot C. Roy S. Robertson G.S. Nicholson D.W. Tetzlaff W. Eur. J. Neurosci. 2000; 12: 3469-3480Crossref PubMed Scopus (51) Google Scholar). Sciatic Nerve Axotomy—Neonatal pups from mice (JNK knockout and littermate controls; C57/Bl background see below) were utilized for these studies. Sciatic nerve axotomy was performed on postnatal day 1 pups as previously described (34Sun W. Oppenheim R.W. Mol. Cell. Neurosci. 2003; 24: 875-886Crossref PubMed Scopus (56) Google Scholar). Briefly, the animals were anesthetized by a gas mixture of 2–5% isofluorane and O2 at a flow rate of 1 liter/min, an incision was made in the right leg parallel to the femur. The nerve was transected at mid-thigh level. The pups were returned to their mothers after suturing. At the indicated times, the animals were sacrificed by transcardial perfusion as described above. Right and left L4-L6 dorsal root ganglia (DRGs) with attached vertebra were dissected and fixed by immersion in Bouins fixative (34Sun W. Oppenheim R.W. Mol. Cell. Neurosci. 2003; 24: 875-886Crossref PubMed Scopus (56) Google Scholar). Tissue was then embedded in paraffin and 5-μm serial sections were cut using a rotary microtome and stained with hematoxylin and eosin as previously described (35Wang F. Corbett D. Osuga H. Osuga S. Ikeda J.E. Slack R.S. Hogan M.J. Hakim A.M. Park D.S. J. Cereb. Blood Flow Metab. 2002; 22: 171-182Crossref PubMed Scopus (99) Google Scholar). To obtain neuronal counts, neurons showing a nucleolus were counted in every fifth section of the L5 ganglion both ipsilateral and contralateral to the axotomy. Alternatively, spinal cord containing L4-L6 DRGs were removed after perfusion and postfixed in 4% PFA in 0.1 m phosphate buffer overnight at 4 °C as previously described (36Crocker S.J. Smith P.D. Jackson-Lewis V. Lamba W.R. Hayley S.P. Grimm E. Callaghan S.M. Slack R.S. Melloni E. Przedborski S. Robertson G.S. Anisman H. Merali Z. Park D.S. J. Neurosci. 2003; 23: 4081-4091Crossref PubMed Google Scholar). The tissue was cryoprotected in 10% sucrose solution in 0.1 m phosphate buffer as previously described (36Crocker S.J. Smith P.D. Jackson-Lewis V. Lamba W.R. Hayley S.P. Grimm E. Callaghan S.M. Slack R.S. Melloni E. Przedborski S. Robertson G.S. Anisman H. Merali Z. Park D.S. J. Neurosci. 2003; 23: 4081-4091Crossref PubMed Google Scholar). 14-μm cryostat sections were obtained for immunofluorescent analyses (see below). Knockout Mice—For knockout studies, JNK3 and JNK2 knockouts were obtained by heterozygous or heterozygous/homozygous knockout pairings. Newborn pups were axotomized as described above. Immediately prior to perfusion and fixation, tail clips were obtained for genotyping. JNK3 knockout embryos were genotyped using CCTGCTTCTCAGAAACACCCTTC (MJ3B3), CGTAATCTTGTCCACAGAAATCCCATAC (MJ3F3) and CTCCAGACTGCCTTGGGAAAA (PGKP1) primers in one PCR reaction under following PCR conditions: 95 °C, 5 min (1 cycle); 95 °C, 1 min; 58 °C, 1 min (–1.0 °C/cycle); 72 °C, 1 min (10 cycle); 95 °C, 1 min; 48 °C, 1 min; 72 °C, 1 min (20 cycle); 72 °C, 5min. JNK2 were genotyped under identical conditions. However GTTAGACAATCCCAGAGGTTGTGTG (MJ2F5), CCAGCTCATTCCTCCACTCATG (PGKT1), and GGAGCCCGATAGTATCGAGTTACC (MJ2B3) primers were utilized. Immunofluorescence—Fixed sections containing L5 DRGs or the facial motor nucleus were incubated with Ser63 c-Jun, Ser73 c-Jun, c-Jun, p53, phospho-Ser15 p53, Elk-1, phospho-Ser383 Elk-1, ATF2, phospho-Thr69/71 ATF-2 (1:200; Cell Signaling), phospho-Ser65 BIM (1:200; Upstate) antibodies overnight at 4 °C in 0.01 m phosphate-buffered saline, pH 7.4 containing 0.3% Triton X-100. After 3 washes with phosphate-buffered saline, sections were then incubated with CY3-conjugated secondary antibody (Jackson 1:200) for 3 h at room temperature. Sections were then incubated with Hoechst 33258 to visualize nuclei as previously described (37Keramaris E. Stefanis L. Maclaurin J. Harada N. Takaku K. Ishikawa T. Taketo M.M. Robertson G.S. Nicholson D.W. Slack R.S. Park D.S. Mol. Cell Neurosci. 2000; 15: 368-379Crossref PubMed Scopus (85) Google Scholar). Quantitation of phosphorylated c-Jun or c-Jun positive neurons was assessed by counting the number of CY3-positive neurons in at least two separate midsections of each nuclei. To evaluate the intensity of staining, a random field for each nucleus was chosen. Densitometry was performed by subtracting background signal from each positive staining neuron in that field using a computer-based image analysis system (Northern Eclipse, Empix Imaging). For each nucleus, the densitometry values were averaged. This was performed for nuclei both ipsilateral and contralateral to the axotomy. Each data point is the representation of at least three different animals and is presented as mean ± S.E. JNK3 Modulates Death of Facial Motor Neurons—Axotomy of the facial nerve in neonates results in death of neurons of the facial motor nuclei. Previous work has shown that this results in apoptotic death, which is associated with caspase induction (33Vanderluit J.L. McPhail L.T. Fernandes K.J. McBride C.B. Huguenot C. Roy S. Robertson G.S. Nicholson D.W. Tetzlaff W. Eur. J. Neurosci. 2000; 12: 3469-3480Crossref PubMed Scopus (51) Google Scholar, 38Rossiter J.P. Riopelle R.J. Bisby M.A. Exp. Neurol. 1996; 138: 33-44Crossref PubMed Scopus (107) Google Scholar). Utilizing this model, we first asked whether JNKs might be required for death. Because, JNK2 and -3 are most associated with death of neurons, we focused on these two JNK family members. Axotomy was performed on JNK3 knockout mice or littermate controls and the number of surviving neurons in the facial motor nucleus was examined 4 days following axotomy. As shown in Fig. 1B, axotomy resulted in survival of ∼13% of facial motor neurons in littermate control mice 4 days following axotomy. In contrast, there was almost 3-fold the number of surviving neurons (37% survival) in JNK3-deficient mice under the same conditions (Fig. 1D). JNK2-deficient nuclei were slightly more resistant to death than JNK3-deficient neurons (43% survival). Interestingly, while JNK2/3 double-deficient mice showed the best protection (52% survival), the protection was not synergistic or even additive in nature. In the uninjured control nucleus, healthy neurons displayed centrally located nucleus and dark nissl staining of the cytoplasm. In contrast, dying neurons of control animals showed pyknotic nuclei and loss of Nissl staining. This was partially reversed in JNK3-, JNK2-, and JNK2/3-deficient animals (Fig. 1). Taken together, these data indicate that both JNK3 and JNK2 play roles in neonatal facial axotomy induced death of motor neurons. JNK3 Modulation of Motor Neuron Death Occurs Independently of c-Jun Phosphorylation—We next determined whether JNK2 or JNK3 deficiency mediated protection is accompanied by reduced c-Jun phosphorylation and activation as reported in other neuronal death paradigms (for example, Refs. 9Kuan C.Y. Whitmarsh A.J. Yang D.D. Liao G. Schloemer A.J. Dong C. Bao J. Banasiak K.J. Haddad G.G. Flavell R.A. Davis R.J. Rakic P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15184-15189Crossref PubMed Scopus (374) Google Scholar and 11Hunot S. Vila M. Teismann P. Davis R.J. Hirsch E.C. Przedborski S. Rakic P. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 101: 665-670Crossref Scopus (363) Google Scholar)). We first examined c-Jun phosphorylation at Ser63 using a phospho-Ser63-specific antibody. An increase in the number of Ser63 c-Jun-positive neurons was detected 24 h following axotomy in the ipsilateral axotomized side when compared with non-axotomized contralateral nuclei in wild-type mice (Fig. 2). This increase was not apparent 4 days following axotomy as expected due to the death of axotomized facial neurons at this time (see Fig. 1). Interestingly, the basal level of phospho c-Jun positive neurons in the contralateral side was slightly elevated at 24 h in comparison to 4 days. This is likely due to indirect effects of the axotomy on the contralateral control nucleus. Consistent with previous reports (9Kuan C.Y. Whitmarsh A.J. Yang D.D. Liao G. Schloemer A.J. Dong C. Bao J. Banasiak K.J. Haddad G.G. Flavell R.A. Davis R.J. Rakic P. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15184-15189Crossref PubMed Scopus (374) Google Scholar, 11Hunot S. Vila M. Teismann P. Davis R.J. Hirsch E.C. Przedborski S. Rakic P. Flavell R.A. Proc. Natl. Acad. Sci. U. S. A. 2003; 101: 665-670Crossref Scopus (363) Google Scholar), JNK2 deficiency mediated neuroprotection was accompanied by reduced number of phospho c-Jun-positive neurons (Fig. 2, A and B). The number of phospho-c-Jun-positive neurons was elevated in JNK2-deficient mice at 4 days in comparison to wild-type mice as one would expect from an increased number of surviving neurons. It was also elevated in comparison to the contralateral side at 4 days, suggesting that Ser63 c-Jun phosphorylation is sustained, albeit at lower levels than at 24 h. JNK2/3 double-deficient mice showed further reduced Ser63 c-Jun phosphorylation in comparison to JNK2 alone (Fig. 2, A and B), reflecting the protection observed (Fig. 1). In this case, the number of Ser63 c-Jun-positive neurons was reduced down to levels observed on the contralateral side at 24 h, suggesting that a combination of JNK2 and -3 are important for the c-Jun phosphorylation increase observed following axotomy. Surprisingly, however, JNK3 deficiency alone failed to reduce the number of Ser63 c-Jun-positive neurons at 24 h. There was no significant difference between JNK3-deficient and wild-type mice in Ser63 phosphorylation at 24 h. This finding contrasts to that of JNK2 or JNK2/3 double-deficient neurons. While the above observations indicated that JNK3 deficiency alone did not affect the total number of Ser63-positive neurons, we examined whether JNK3 deficiency might affect the overall intensity (per neuron) of c-Jun phosphorylation. To examine this, we performed densitometric analyses of Ser63-positive neurons in the different animal groups (Fig. 2C). In wild-type animals, the average intensity of Ser63 staining was elevated following axotomy at 24 h. While this intensity was reduced in JNK2- or JNK2/3-deficient nuclei following axotomy at 24 h, JNK3-deficient nuclei showed no such reduction. Similarly, total Ser63 phosphorylation activity, as defined by the number of positive neurons multiplied by average intensity of each neuron, was also not affected in JNK3-deficient facial nuclei (Fig. 2D) when compared with wild-type mice (24 h). In contrast, JNK2 or JNK2/3 deficiency significantly reduced total Ser63 c-Jun phosphorylation activity. At 4 days, the intensity of Ser63 staining was still sustained when compared with the contralateral side. However, there was no difference between any of the groups. c-Jun, depending upon the context, may be differentially phosphorylated at the two major phosphorylation sites of c-Jun. For example, following medial forebrain axotomy in the adult rat, Ser73 phosphorylation and not Ser63 phosphorylation is observed (7Crocker S.J. Lamba W.R. Smith P.D. Callaghan S.M. Slack R.S. Anisman H. Park D.S. Proc. Natl. Acad. Sci. U. S. A. 2001; 98: 13385-13390Crossref PubMed Scopus (80) Google Scholar). Accordingly, we also evaluated phosphorylation of c-Jun at Ser73. Similar to that observed with Ser63 phosphoepitope-specific antibody, we observed an increase in levels of Ser73 c-Jun phosphorylation in axotomized control mice, which did not differ from that observed with JNK3-knockout mice (Fig. 3). Like with Ser63, Ser73 phosphorylation was negatively impacted by JNK2 or JNK2/3 deficiency. Finally, we determined whether c-Jun levels by itself may be regulated by JNK deficiency. As shown in Fig. 4, there were relatively high basal levels of c-Jun in the contralateral nuclei at both 24 and 4 days and a modest increase in numbers (148 ipsi versus 104 contra) and intensity (109 ipsi versus 74 contra) of c-Jun at 24 h. Similar to that observed with Ser63 or Ser73 c-Jun phosphorylation, this increase was not affected by JNK3 deficiency while JNK2 or JNK2/3 double-deficient mice showed reduced c-Jun levels. Taken together, these data indicate that JNK3-mediated protection is not correlated with a reduction in c-Jun activation. Most importantly, these data indicate that c-Jun activation per se is not the only JNK-mediated signal required for facial axotomy-induced death. Because protection afforded by JNK3 deficiency was not accompanied by reduced c-Jun phosphorylation following facial axotomy, we asked whether other potential JNK targets may be modulated and required for neuronal death. We examined additional select transcription factors, ATF2 and Elk1, which are also regulated by the JNKs (39Cesari F. Brecht S. Vintersten K. Vuong L.G. Hofmann M. Klingel K. Schnorr J. Arsenian S. Schild H. Herdegan T. Wiebel F. Norhheim A. Mol. Cell. Biol. 2004; 24: 294-305Crossref PubMed Scopus (77) Google Scholar, 40Martin-Villalba A. Winter C. Brecht S. Buschmann T. Zimmermann M. Herdegen T. Mol. Brain Res. 1998; 62: 158-166Crossref PubMed Scopus (47) Google Scholar). However, we could not detect any increased phosphorylation or induction of these factors following axotomy 24 h following axotomy when c-Jun activation was pronounced (data not shown). Evaluations of potential induction in these cases were performed by immunofluorescent analyses utilizing ATF2 and ELK1 antibodies or phosphospecific antibodies recognizing activated ATF2 and Elk1. In fact, the phospho-ATF2 and phospho-Elk1 signals decreased 24 h following axotomy on the ipsilateral side consistent with what has previously been reported in other neuronal stress situations
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