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All JNKs Can Kill, but Nuclear Localization Is Critical for Neuronal Death

2008; Elsevier BV; Volume: 283; Issue: 28 Linguagem: Inglês

10.1074/jbc.m707744200

1083-351X

Benny Björkblom, Jenni C. Vainio, Vesa Hongisto, Thomas Herdegen, Michael J. Courtney, Eleanor T. Coffey,

Cytokine Signaling Pathways and Interactions

JNKs are implicated in a range of brain pathologies and receive considerable attention as potential therapeutic targets. However, JNKs also regulate physiological and homeostatic processes. An attractive hypothesis from the drug development perspective is that distinct JNK isoforms mediate "physiological" and "pathological" responses. However, this lacks experimental evaluation. Here we investigate the isoforms, subcellular pools, and c-Jun/ATF2 targets of JNK in death of central nervous system neurons following withdrawal of trophic support. We use gene knockouts, gene silencing, subcellularly targeted dominant negative constructs, and pharmacological inhibitors. Combined small interfering RNA knockdown of all JNKs 1, 2, and 3, provides substantial neuroprotection. In contrast, knockdown or knock-out of individual JNKs or two JNKs together does not protect. This explains why the evidence for JNK in neuronal death has to date been largely pharmacological. Complete knockdown of c-Jun and ATF2 using small interfering RNA also fails to protect, casting doubt on c-Jun as a critical effector of JNK in neuronal death. Nonetheless, the death requires nuclear but not cytosolic JNK activity as nuclear dominant negative inhibitors of JNK protect, whereas cytosolic inhibitors only block physiological JNK function. Thus any one of the three JNKs is capable of mediating apoptosis and inhibition of nuclear JNK is protective. JNKs are implicated in a range of brain pathologies and receive considerable attention as potential therapeutic targets. However, JNKs also regulate physiological and homeostatic processes. An attractive hypothesis from the drug development perspective is that distinct JNK isoforms mediate "physiological" and "pathological" responses. However, this lacks experimental evaluation. Here we investigate the isoforms, subcellular pools, and c-Jun/ATF2 targets of JNK in death of central nervous system neurons following withdrawal of trophic support. We use gene knockouts, gene silencing, subcellularly targeted dominant negative constructs, and pharmacological inhibitors. Combined small interfering RNA knockdown of all JNKs 1, 2, and 3, provides substantial neuroprotection. In contrast, knockdown or knock-out of individual JNKs or two JNKs together does not protect. This explains why the evidence for JNK in neuronal death has to date been largely pharmacological. Complete knockdown of c-Jun and ATF2 using small interfering RNA also fails to protect, casting doubt on c-Jun as a critical effector of JNK in neuronal death. Nonetheless, the death requires nuclear but not cytosolic JNK activity as nuclear dominant negative inhibitors of JNK protect, whereas cytosolic inhibitors only block physiological JNK function. Thus any one of the three JNKs is capable of mediating apoptosis and inhibition of nuclear JNK is protective. Application of stressful stimuli to cells leads to the activation of the stress-activated protein kinase c-Jun N-terminal kinase (JNK) 3The abbreviations used are: JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase; siRNA, small interfering RNA; GFP, green fluorescent protein; EGFP, enhanced GFP; NES, nuclear export signal; NLS, nuclear localization sequence; JBD, JNK binding domain; DIV, days in vitro; WTS, withdrawal of trophic support. 3The abbreviations used are: JNK, c-Jun NH2-terminal kinase; SAPK, stress-activated protein kinase; siRNA, small interfering RNA; GFP, green fluorescent protein; EGFP, enhanced GFP; NES, nuclear export signal; NLS, nuclear localization sequence; JBD, JNK binding domain; DIV, days in vitro; WTS, withdrawal of trophic support. pathway. Initial studies using JNK knock-out mice indicated redundant roles for JNKs 1 and 2 in survival and death during early brain development (1Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar, 2Sabapathy K. Jochum W. Hochedlinger K. Chang L. Karin M. Wagner E.F. Mech. Dev. 1999; 89: 115-124Crossref PubMed Scopus (300) Google Scholar). JNK3 deletion on the other hand reduced neurodegeneration after excitotoxin-induced seizures in adult (3Yang D.D. Kuan C.Y. Whitmarsh A.J. Rincon M. Zheng T.S. Davis R.J. Rakic P. Flavell R.A. Nature. 1997; 389: 865-870Crossref PubMed Scopus (1110) Google Scholar). Roles for JNK1 in physiological responses during neuronal development were revealed more recently (4Chang L. Jones Y. Ellisman M.H. Goldstein L.S. Karin M. Dev. Cell. 2003; 4: 521-533Abstract Full Text Full Text PDF PubMed Scopus (311) Google Scholar, 5Tararuk T. Ostman N. Li W. Bjorkblom B. Padzik A. Zdrojewska J. Hongisto V. Herdegen T. Konopka W. Courtney M.J. Coffey E.T. J. Cell Biol. 2006; 173: 265-277Crossref PubMed Scopus (146) Google Scholar, 6Björkblom B. Östman N. Hongisto V. Komarovski V. Filen J.J. Nyman T.A. Kallunki T. Courtney M.J. Coffey E.T. J. Neurosci. 2005; 25: 6350-6361Crossref PubMed Scopus (142) Google Scholar). Overall, there has been an emphasis on JNK3 as a specific target for drug development (7Manning A.M. Davis R.J. Nat. Rev. Drug Discov. 2003; 2: 554-665Crossref PubMed Scopus (528) Google Scholar, 8Bogoyevitch M.A. Boehm I. Oakley A. Ketterman A.J. Barr R.K. Biochim. Biophys. Acta. 2004; 1697: 89-101Crossref PubMed Scopus (246) Google Scholar). Although some studies have shown a contribution from JNK3 in neuronal stress responses (9Morishima Y. Gotoh Y. Zieg J. Barrett T. Takano H. Flavell R. Davis R.J. Shirasaki Y. Greenberg M.E. J. Neurosci. 2001; 21: 7551-7560Crossref PubMed Google Scholar, 10Kuan 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 (367) Google Scholar, 11Kimberly W.T. Zheng J.B. Town T. Flavell R.A. Selkoe D.J. J. Neurosci. 2005; 25: 5533-5543Crossref PubMed Scopus (94) Google Scholar), the proposal that JNK3 plays a dominant role in neuronal death (reviewed in Refs. 7Manning A.M. Davis R.J. Nat. Rev. Drug Discov. 2003; 2: 554-665Crossref PubMed Scopus (528) Google Scholar, 12Brazil M. Nat. Rev. Drug Discov. 2003; 2: 5Crossref Scopus (1) Google Scholar, 13Reed J.C. Nat. Rev. Drug Discov. 2002; 1: 111-121Crossref PubMed Scopus (595) Google Scholar, 14Johnson G.L. Nakamura K. Biochim. Biophys. Acta. 2007; 1773: 1341-1348Crossref PubMed Scopus (337) Google Scholar, 15Brecht S. Kirchhof R. Chromik A. Willesen M. Nicolaus T. Raivich G. Wessig J. Waetzig V. Goetz M. Claussen M. Pearse D. Kuan C.Y. Vaudano E. Behrens A. Wagner E. Flavell R.A. Davis R.J. Herdegen T. Eur. J. Neurosci. 2005; 21: 363-377Crossref PubMed Scopus (186) Google Scholar, and 45Resnick L. Fennell M Drug Discov. Today. 2004; 9: 932-939Crossref PubMed Scopus (105) Google Scholar) is not supported by the neuronal responsiveness of JNK3-/- mice to stress (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar), and studies suggest a possible contribution from JNK2 (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar, 17Eminel S. Klettner A. Roemer L. Herdegen T. Waetzig V. J. Biol. Chem. 2004; 279: 55385-55392Abstract Full Text Full Text PDF PubMed Scopus (94) Google Scholar, 18Keramaris E. Vanderluit J.L. Bahadori M. Mousavi K. Davis R.J. Flavell R. Slack R.S. Park D.S. J. Biol. Chem. 2005; 280: 1132-1141Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Surprisingly, however, a comparative analysis of different JNK isoforms has not been reported. A major constraint in determining JNK isoform roles in cell death has been the absence of JNK1/2/3 triple knock-out mice and consequent lack of studies with neurons depleted of JNKs 1, 2, and 3. As a result, analysis of JNK isoform involvement in neuronal death and, in most cases, even addressing whether JNK is involved at all has depended on the use of the less specific pharmacological and dominant negative approaches. These fail to unequivocally show the contributions of JNK or individual isoforms, leaving this issue unresolved. Similarly, conclusions regarding JNK substrate involvement in central nervous system neuron death responses have largely relied on correlation (19Herdegen T. Leah J.D. Brain Res. Brain Res. Rev. 1998; 28: 370-490Crossref PubMed Scopus (1155) Google Scholar). c-Jun is a major target of the JNK pathway; it contributes to the response to cellular stress and is believed to mediate apoptotic neuronal cell death (20Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (593) Google Scholar). Knock-in mice expressing c-Jun with mutations to alanine at the JNK phosphorylation sites Ser-63/73 (c-Jun-AA) exhibited the same deficiencies in the excitotoxic stress response as JNK3 knock-out mice (3Yang D.D. Kuan C.Y. Whitmarsh A.J. Rincon M. Zheng T.S. Davis R.J. Rakic P. Flavell R.A. Nature. 1997; 389: 865-870Crossref PubMed Scopus (1110) Google Scholar, 20Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (593) Google Scholar). This suggested that c-Jun and JNK3 were on the same phenotypic pathway and led to the widely held dogma that JNK3 → c-Jun signaling mediates neuronal death (reviewed in Refs. 7Manning A.M. Davis R.J. Nat. Rev. Drug Discov. 2003; 2: 554-665Crossref PubMed Scopus (528) Google Scholar, 12Brazil M. Nat. Rev. Drug Discov. 2003; 2: 5Crossref Scopus (1) Google Scholar, 13Reed J.C. Nat. Rev. Drug Discov. 2002; 1: 111-121Crossref PubMed Scopus (595) Google Scholar, 14Johnson G.L. Nakamura K. Biochim. Biophys. Acta. 2007; 1773: 1341-1348Crossref PubMed Scopus (337) Google Scholar, 15Brecht S. Kirchhof R. Chromik A. Willesen M. Nicolaus T. Raivich G. Wessig J. Waetzig V. Goetz M. Claussen M. Pearse D. Kuan C.Y. Vaudano E. Behrens A. Wagner E. Flavell R.A. Davis R.J. Herdegen T. Eur. J. Neurosci. 2005; 21: 363-377Crossref PubMed Scopus (186) Google Scholar, and 45Resnick L. Fennell M Drug Discov. Today. 2004; 9: 932-939Crossref PubMed Scopus (105) Google Scholar). In this report, a combination of methods, knock-out mice, small-molecule inhibitors, siRNAs, and compartment-targeted dominant negative inhibitors establish that JNK is required for death of cerebellar granule neurons from which trophic support was withdrawn and is used to investigate the involvement of JNK isoforms and JNK substrates. We observed that nuclear activity of JNK was required for the death, but the JNK substrates c-Jun and ATF2 were not required for the death in this system. JNK2 and JNK3 were together shown to be the main regulators of c-Jun following withdrawal of trophic support (WTS). Unexpectedly, however, they were not required for neuronal death. Only simultaneous knockdown of JNKs 1, 2, and 3 was sufficient to confer neuroprotection. This indicates that the presence of any one of the JNKs is sufficient to produce a dominant apoptotic signal. Antibodies and Reagents—Mouse anti-JNK1 (clone G151-333) and mouse anti-JNK1/2 (clone G151-666) were from Pharmingen. Rabbit anti-JNK3 (clone C05T), rabbit anti-JNK2/3 ("SAPK1a," catalogue number 06-748, characterized in Ref. 16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar), and rabbit anti pan-JNK ("SAPK1b/SAPKβ," catalogue number 06-749) were from Upstate Biotechnology (Lake Placid, NY). Mouse anti-actin was a gift from Brigitte Jockusch (Technical University of Braunschweig). Rabbit anti-ATF2 (catalogue number 9222), anti-phospho-ATF2 (Thr-69/71; catalogue number 9225), and anti-phospho-JNK (Thr-183/Tyr-185; catalogue number 9255) were from Cell Signaling Technology (Beverly, MA). Mouse anti c-Jun (clone J31920) was from BD Transduction Laboratories, and mouse anti phospho-c-Jun (Ser-63; clone KM-1) was from Santa Cruz Biotechnology (Santa Cruz, CA). Anti-rabbit Alexa-546 and anti-mouse Alexa-555 were from Molecular Probes (Eugene, OR). siRNA pools for silencing of ATF2 and JNK3 (set I) were obtained from Dharmacon (Chicago, IL). siRNA oligonucleotides for c-Jun (5′-AACAGAAAGUCAUGAACCACG-3′) and JNK1/2 (set I) (5′-GAAUGUCCUACCUUCUCUA-3′) were synthesized with a deoxythymidine deoxythymidine leader sequence by MWG-Biotech AG (Ebersberg, Germany as described previously (21Nielsen C. Thastrup J. Bøttzauw T. Jäättelä M. Kallunki T. Cancer Res. 2007; 67: 178-185Crossref PubMed Scopus (24) Google Scholar)). siRNA oligonucleotides set II for JNKs were as follows: JNK1 pool (5′-GGAAAGAACUGAUAUACAA-3′ and 5′-GAAGCAAACGUGACAACAA-3′), JNK2 pool (5′-CCGTGAACTCGTCCTCTTAAA-3′, and 5′-GTGATGGACTGGGAAGAAA-3′), and JNK3 pool (5′-GGAAAGAACUUAUCUACAA-3′ and 5′-CCAGUAACAUUGUAGUCAA-3′). Kinase inhibitors SB203580 and SP600125 were from Calbiochem and Sigma-Aldrich, respectively. GFP-NLS-SEK1kd and GFP-NES-SEK1kd were prepared, as were GFP-NLS-JBD and GFP-NES-JBD, as described previously (6Björkblom B. Östman N. Hongisto V. Komarovski V. Filen J.J. Nyman T.A. Kallunki T. Courtney M.J. Coffey E.T. J. Neurosci. 2005; 25: 6350-6361Crossref PubMed Scopus (142) Google Scholar). δMEKK1-(1174-1493) was previously described (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar). Cell Culture—Cerebellar granule neurons were prepared as described previously (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar) from postnatal day seven JNK2-/-, JNK3-/-, and JNK2-/-JNK3-/- mice where indicated. Genotyping was as described previously (1Kuan C.Y. Yang D.D. Samanta Roy D.R. Davis R.J. Rakic P. Flavell R.A. Neuron. 1999; 22: 667-676Abstract Full Text Full Text PDF PubMed Scopus (760) Google Scholar). Otherwise, neurons were prepared from FVB/N mice for JNK siRNA experiments or from Sprague-Dawley rats for ATF2/c-Jun siRNA experiments. Briefly, cells were cultured in minimal essential medium supplemented with 10% (v/v) fetal bovine serum (Hyclone, Logan, UT), 33 mm glucose, 2 mm glutamine, 50 units/ml penicillin, 50 μg/ml streptomycin, and 20 mm supplementary KCl (final 25.4 mm KCl). Cells were plated at 250,000/cm2 onto culture surfaces coated with poly-l-lysine (50 μg/ml). Culture medium was replaced after 24 h with the inclusion of 10 μm cytosine arabinofuranoside (Sigma) to reduce non-neuronal proliferation. Cells were cultured in a humidified 5% CO2 atmosphere at 37 °C. Transfections and Survival Analysis of Neurons—Cerebellar granule neurons were plated on glass coverslips (Knittel, Braunschweig, Germany) for survival assays and immunofluorescence staining, which were carried out as described previously with minor modifications (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar). Cells were transiently transfected at 5 days in vitro (DIV) with 1.0 μg of marker plasmid (pEGFP-F) together with 1.0 μg of pCMV empty vector as described previously (22Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar). siRNA oligonucleotides targeting c-Jun (200 nm), ATF2 (200 nm), JNK1/2 (100 nm), or JNK3 (100 nm) or equal amount of non targeting control siRNA were included in the DNA mixture as indicated. 48 h (c-Jun and ATF2) or 90 h (JNK1/2 and JNK3) later, culture medium was replaced with medium containing low KCl (5 mm) without serum (WTS) for 10-48 h as indicated, prior to cell fixation. Cells were fixed and DNA-stained with Hoechst 33342 (2 μg/ml) to detect pyknosis. The viability of GFP-F-expressing neurons was analyzed using a Leica DMRE microscope equipped with a Hamamatsu Orca CCD camera. Cerebellar granule neurons from JNK2-/-, JNK3-/-, and JNK2-/-JNK3-/- mice were prepared as above, and trophic support was withdrawn at 7 DIV in the presence or absence of inhibitors as indicated. A 30-min pretreatment with inhibitors was carried out prior to withdrawal of trophic support. After 24 h in low KCl and without serum, cells were fixed and nuclei were stained with Hoechst 33342. Analysis of neuronal survival was carried out blind. For analysis of compartmental JNK inhibitors, neurons were transfected at 6 DIV, and 24 h later, they were transferred to low KCl medium without serum and survival was measured as above. Immunostaining—Immunocytochemical staining was carried out as described previously (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar, 22Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar). Briefly, neurons on coverslips were fixed with 4% paraformaldehyde for 20 min at room temperature or with MeOH for 5 min at -20 °C (JNK1/2 staining) followed by permeabilization in phosphate-buffered saline/Triton X-100 (1%) for 2 min. Following washing with phosphate-buffered saline, cells were blocked with 10% serum, 0.2% Tween-20, phosphate-buffered saline for 1 h at room temperature. Incubation with primary antibodies was overnight at 4 °C using anti-c-Jun (2.5 μg/ml), anti-JNK1 (5 μg/ml), anti-JNK1/2 (5μg/ml), anti-JNK2/3 (3.3μg/ml), and anti-ATF2 (1:100) and anti-JNK3 (1:300), respectively. Immunoreactivity was detected using anti-rabbit Alexa Fluor 546 (1:500) or anti-mouse Alexa Fluor 555 (1:500). Immunoblot Analysis and Quantification—Samples were resolved on 10% SDS-PAGE and transferred by semidry transfer to nitrocellulose. Immunoblotting was carried out as described previously (22Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar). Statistical Analysis—Statistical analysis of variance was done using SPSS for Windows version 11.0.1 (SPSS Inc., Chicago, IL). Test of homogeneous sample variances was performed using Levene's test prior to one-way analysis-of-variance. Fisher least significant difference post hoc test was used in cases with more than two variable groups. Significance levels are indicated as follows, *, p < 0.05, **, p < 0.01, ***, p < 0.001. A number of studies suggest that JNK-mediated phosphorylation of c-Jun contributes to neuronal cell death (reviewed in Ref. 25Ham J. Eilers A. Whitfield J. Neame S.J. Shah B. Biochem. Pharmacol. 2000; 60: 1015-1021Crossref PubMed Scopus (212) Google Scholar), particularly in response to WTS. The majority of the evidence comes from observations that dominant negative c-Jun constructs prevent neuronal cell death upon removal of trophic support (23Eilers A. Whitfield J. Babij C. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 1713-1724Crossref PubMed Google Scholar, 24Watson A. Eilers A. Lallemand D. Kyriakis J. Rubin L.L. Ham J. J. Neurosci. 1998; 18: 751-762Crossref PubMed Google Scholar, 25Ham J. Eilers A. Whitfield J. Neame S.J. Shah B. Biochem. Pharmacol. 2000; 60: 1015-1021Crossref PubMed Scopus (212) Google Scholar, 26Whitfield J. Neame S.J. Paquet L. Bernard O. Ham J. Neuron. 2001; 29: 629-643Abstract Full Text Full Text PDF PubMed Scopus (501) Google Scholar, 27Hongisto V. Smeds N. Brecht S. Herdegen T. Courtney M.J. Coffey E.T. Mol. Cell Biol. 2003; 23: 6027-6036Crossref PubMed Scopus (129) Google Scholar, 28Besirli C.G. Wagner E.F. Johnson Jr., E.M. J. Cell Biol. 2005; 170: 401-411Crossref PubMed Scopus (60) Google Scholar). We have reported that withdrawal of trophic support from rat cerebellar granule neurons leads to selective activation of JNK2/3 isoforms in the presence of constitutive JNK1 activity (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar, 22Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar). Using 10 μm SB203580, which inhibits (at least) JNK2/3 isoforms in these cells, we found that the JNK2/3 pool contributes to c-Jun phosphorylation in neurons upon WTS (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar). However, this conflicts with the expectation that JNK3 has a specific role in neuronal c-Jun stress responses alone, largely supported by the finding that both JNK3-/- and c-Jun-AA knock-in mice possess similar resistance to kainate-induced seizure activity and the subsequent death of hippocampal neurons (3Yang D.D. Kuan C.Y. Whitmarsh A.J. Rincon M. Zheng T.S. Davis R.J. Rakic P. Flavell R.A. Nature. 1997; 389: 865-870Crossref PubMed Scopus (1110) Google Scholar, 15Brecht S. Kirchhof R. Chromik A. Willesen M. Nicolaus T. Raivich G. Wessig J. Waetzig V. Goetz M. Claussen M. Pearse D. Kuan C.Y. Vaudano E. Behrens A. Wagner E. Flavell R.A. Davis R.J. Herdegen T. Eur. J. Neurosci. 2005; 21: 363-377Crossref PubMed Scopus (186) Google Scholar, 20Behrens A. Sibilia M. Wagner E.F. Nat. Genet. 1999; 21: 326-329Crossref PubMed Scopus (593) Google Scholar). We therefore investigated which JNKs contributed to the trophic withdrawal responses using cultured cerebellar granule neurons from single and compound knock-out mice. The c-Jun Stress Response Is Strongly Suppressed in JNK2-/-JNK3-/- Neurons—Neurons were prepared from wild-type, JNK2-/-, JNK3-/-, and JNK2-/-JNK3-/- mice and treated according to the WTS paradigm. We also included in these experiments the use of small molecule JNK inhibitors that had previously been shown to influence these responses. As reported previously, phosphorylation of the JNK substrate c-Jun increased in response to WTS, and this response was blocked by 10 μm SB203580 (which inhibits JNK2/3 isoforms (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar) and 1 μm SP600125 (sufficient to inhibit JNK in neurons (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar, 29Cao J. Semenova M.M. Solovyan V.T. Han J. Coffey E.T. Courtney M.J. J. Biol. Chem. 2004; 279: 35903-35913Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar)) (Fig. 1A). Importantly, 1 μm SB203580, which is sufficient to inhibit p38α/β but not JNKs (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar, 22Coffey E.T. Hongisto V. Dickens M. Davis R.J. Courtney M.J. J. Neurosci. 2000; 20: 7602-7613Crossref PubMed Google Scholar, 29Cao J. Semenova M.M. Solovyan V.T. Han J. Coffey E.T. Courtney M.J. J. Biol. Chem. 2004; 279: 35903-35913Abstract Full Text Full Text PDF PubMed Scopus (98) Google Scholar), did not influence c-Jun phosphorylation (Fig. 1A). When the experiments were repeated with cells from JNK2-/-, JNK3-/-, or JNK2-/-JNK3-/- mice, the size of the response was somewhat diminished in JNK3-/- cultures but most strongly reduced in JNK2-/-JNK3-/- cultures, indicating that JNK2 in addition to JNK3 contributes to c-Jun phosphorylation following WTS. The total level of phospho-Ser-63 c-Jun is a somewhat complex parameter representing the specific phosphorylation of this site multiplied by the total level of c-Jun (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar). Therefore we immunoblotted the lysates with c-Jun antibody in parallel (Fig. 1B). This indicates that the total amount of c-Jun, as well as relative phosphorylation status, visualized by the retarded mobility, is the result of phosphorylation at serines 63 and 73 and threonines 91 and 93 (30Ui M. Sonobe M.H. Ito T. Murakami M. Okazaki S. Takada M. Sato T. Iba H. FEBS Lett. 1998; 429: 289-294Crossref PubMed Scopus (14) Google Scholar). To more directly compare the c-Jun mobility profiles, we plotted the film density profile against migration distance in the gel, normalizing to the peak intensity for each trace. This analysis clearly showed that following WTS, c-Jun migrates as three distinct peaks. In cells from JNK2-/-JNK3-/- mice, there was a marked reduction in retarded mobility bands when compared with JNK3-/- neurons (Fig. 1C). The c-Jun induction in the JNK2-/-JNK3-/- cultures, reduced when compared with wild type, was blocked by the pan-inhibitor of JNK, SP600125 (Fig. 1G). This indicates that the residual JNK1 is sufficient to elicit some c-Jun stress response. In summary, we have shown using knock-out cells that JNKs 2 and 3 are both required to elicit c-Jun phosphorylation in neurons, consistent with earlier results using inhibitors (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar). JNK1 Expression Is Up-regulated in the Absence of JNKs 2 and 3—We investigated the levels of JNK and P-JNK under the conditions shown in Fig. 1. Withdrawal of trophic support with or without inhibitors caused no substantial change in total JNK levels, detected with a pan JNK antibody (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar). Similarly, there was no clear change in phospho-JNK level that could not be attributed to variations in total JNK (Fig. 2A). As we noted, up-regulation of c-Jun expression in JNK2-/-JNK3-/- cells (Fig. 1, B and G), we examined whether this gene deletion may have led to an increase in JNK1. Immunoblotting with an antibody that was specific for JNK1 (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar) revealed a significant increase in JNK1 p46 and p54 isoform expression in JNK2-/- JNK3-/- cells when compared with wild type (Fig. 2, B and C). Neurons from Mice Lacking JNKs 2 and 3 Are Not Protected from WTS—An underlying assumption in the neuronal cell death field has been that c-Jun regulation by JNK can lead to cell death (reviewed in Ref. 25Ham J. Eilers A. Whitfield J. Neame S.J. Shah B. Biochem. Pharmacol. 2000; 60: 1015-1021Crossref PubMed Scopus (212) Google Scholar). This led to the subsequent expectation that inhibition of JNK-regulated c-Jun would be neuroprotective. Withdrawal of trophic support in the JNK2-/-JNK3-/- background led to a marked inhibition of c-Jun mobility relative to the wild-type background (Fig. 1). We therefore investigated the extent to which this regulation of c-Jun correlated with pyknosis induced by WTS. Once again, we included inhibitors of JNK and p38 in experiments carried out on cultures from both wild-type and knock-out backgrounds. Treatment of wild-type cells with 10 μm SB203580 provided significant protection from WTS as described previously (Fig. 3) (Ref. 16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar). SP600125 at 1 μm also provided significant protection. However, the extent of death following WTS did not differ significantly between wild-type cells and JNK2-/-, JNK3-/-, or even JNK2-/-JNK3-/- cells (Fig. 3). This was especially surprising given that the c-Jun phosphorylation shift was severely reduced in JNK2-/-JNK3-/- neurons (Fig. 1). Moreover, 10 μm SB203580, which effectively inhibits JNK2/3 activities (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar) provided a similar degree of protection in wild-type and in JNK2-/-JNK3-/- neurons. One possible explanation is that JNK1 isoforms can also contribute to neuronal death and that SB203580 also inhibits a subset of JNK1 isoforms (as reported in Ref. 31Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. Cotton M. Daub H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15434-15439Crossref PubMed Scopus (321) Google Scholar) that are up-regulated in JNK2-/-JNK3-/- neurons (Fig. 2). Indeed the JNK inhibitor SP600125 (1 μm) is also protective in neurons from JNK2-/-JNK3-/- mice. Knockdown of JNK2 and JNK3 Does Not Protect Neurons, All Three JNKs Must Be Depleted to Confer Significant Neuroprotection—The use of JNK knock-out mice did not demonstrate that JNK is required for WTS-evoked neuronal death, nor did it implicate specific isoforms, as no protection was observed either in JNK2-/-JNK3-/- (Fig. 3), neurons or in JNK1-/- neurons (data not shown). Surprisingly, SB203580 (10 μm), which inhibits JNKs 2 and 3, was neuroprotective (16Coffey E.T. Smiciene G. Hongisto V. Cao J. Brecht S. Herdegen T. Courtney M.J. J. Neurosci. 2002; 22: 4335-4345Crossref PubMed Google Scholar) (Fig. 3) even in JNK2-/-JNK3-/- neurons. One explanation would be that a JNK1 isoform is sufficient to induce death and that this isoform is sensitive to SB203580 (31Godl K. Wissing J. Kurtenbach A. Habenberger P. Blencke S. Gutbrod H. Salassidis K. Stein-Gerlach M. Missio A. Cotton M. Daub H. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 15434-15439Crossref PubMed Scopus (321) Google Scholar). Consistent with this, SP600125 also protected JNK2-/-JNK3-/- neurons (Fig. 3). Indeed, no genetic evidence for JNK requirement in WTS-induced death of central nervous system neurons has been reported, only pharmacological evidence. Therefore we addressed these issues with gene silencing. First we established the silencing efficiency of JNK siRNA. We tested antibodies for JNK isoform specificity using wild-type and knock-out cells and tissues. Using this approa