4008_001 - The Journal of Neuroscience, January 1, 1998,...

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Unformatted text preview: The Journal of Neuroscience, January 1, 1998, 18(1i:104—l 11 Motoneuron Apoptosis ls Blocked by CEP-1347 (KT 7515), a Novel Inhibitor of the JNK Signaling Pathway Anna C. Maroney,1 Marcie A. Glicksman,1 Alie N. Basma,1 Kevin M. Walton,1 Ernest Knight Jr,‘ Carol A. Murphy,1 Becky A. Bartlett,1 James P. Finn,1 Thelma Angeles,1 Yuzuru Matsucia,2 Nicola T. Neil,1 and Craig A. Dionne1 1Cepnaion incorporated, West Chester, Pennsylvania 19880, and 2Kyovva-Hal/«kc Kogyo Company, Limited, Machida—sni, Tokyo 194, Japan Neurons undergoing apoptosis can be rescued by trophic tac- tcrs that simultaneously increase the activity of extracellular signaturegulated kinase {ERK) and decrease c-dun N-terminal kinase (JNK) and p38. We identified a molecule, CEP—1347 (KT7515), that rescues motoneurons undergoing apoptosis and investigated its effect on ERK1 and JNKt activity. Cultured rat embryonic motoneurons, in the absence of trophic factor. be- gan to die 24—48 hr afterr plating. During the first 24 hr ERKl activity was unchanged, whereas JNKt activity increased four— fold. CEP-is47 comptetely rescued motoneurons for at least 72 hr with an E050 of 20 t 2 nu. CEP—i347 did not alter ERKl activity but rapidly inhibited JNKl activation. The IO.30 of (DEFw 1347 for JNK1 activation was the same as the E050 for me- toneuron survival. Inhibition of JNK? activation by CEP-t347 was not selective to mctcneurcns. CEP—1347 aiso inhibited During development, populations of neuronal embryonic cells in viva undergo a predetermined process of programmed cell death (PCD) {for review, see Oppenheiin, I991). in particular, between embryonic days Md and l8 “60% ofral spinal cord motoneurons undergo a form of PC!) inorplmiogically identified as apoptosis. Cultures of motoncurons isolated from 1314.5 rat spinal cord also apoptose in viii-o (Cornelia et a]., i994; Milligan et al., l994). Cell death in this relatively pure population of neurons can be partially prevented by the addition of growth factors such as brain-derived neurotrophic factor and insulin-like growth factor-1 (Henderson et al., 1993; Hughes et al., 1993). Because the environmental cues leading to in vivo apoptosis of motoneurons are not well underw stood, cultures enriched for motoneurons provide a useful, rela— tively homogeneous model for identifying neuronal survival agents and examining the biochemical events that govern survival. Growth factors may mediate neuronal survival by regulating signaling cascades downstream of the small G'I‘I> binding proteins ras, rac, and cdc42 (for review, see Denhardt, 1990). Activation of the smail G'I‘P binding proteins leads to modulation of serinei thrconinc kinases in the mitogenactivated protein kinasc Received June It, l997; revised Oct. (1. 1997: accepted Oct. 15. NW. We thank J, Silvio Gutkind for providing the MEEKK and JNK vectors. Anne (‘auioritttn for helpful review of this manuscript. and Judy Richardson for manu- script preparation. Correspondence sliuuid be atldrc scd to Dr. Anna Coco Maroney, Cephalon incorporated, l-iS Brandywinc Park 1y, West Chester, PA 19380. Dr. Glicksman's present address: DuPont Merck, Experimental Station, Route Hi and licnry (Slay Road, Wilmington. DE I‘JSSU. Dr. Murphy‘s present address: institute of 'l'oxicology, Schoi‘enstrassc lo, Schw- crzcnhach Ci'l-h’hlB, Switzerland. Copyright 53.: 199'} Society for Neuroscience {Bill-(1474597:ltitlitli-titiSllSilililJ JN K1 activity in Cos? cells under conditions of ultravloiet irra~ diation, osmotic shock, and inhibition of glycosylation. lnhibi» tion by CEP-1347 of the JNKl signaling pathway appeared to be seiective, because CEP~1347 did not inhibit p38-reguiated mitegen-activated protein kinase—activated protein kinase—2 (MAPKAPQ) activity in Cos? cells subjected to osmotic shock. The direct molecular target of (DEF-1347 was not JNKl, be— cause CEPu1347 did not inhibit JNK? activity in Cos? cells cotransfected with MEKKl and JNKl (DNA constructs. This is the first demonstration of a small organic molecule that pro- motes motoneuren survival and that simultaneously inhibits the JNKt signaling cascade. Key words: mofoneurons; indolocarbazole; CEP-134 7,‘ suru vival; apoptosis,‘ c-Jun N-termr'nai kinase; mitogen-activated protein kinase; p38 (MAPK) family. Specifically, activation of ras leads to phosphor- ylation and activation of extracellular receptor-activatcd kinasc (ERK), which has been linked biologically to growth and differ“ entiation processes, whereas stimulation of rac/ede42 leads to an increase in the activity of JNK and p38, a response that is associated with stress and apoptosis. In neuronally differentiated l’Cl2 cells, withdrawal of NGF causes apoptosis that is preceded by a decrease in lZiRK activity and an increase in JNK/p38 activity (Xi-a ct al., 1995). These results suggest that ERK and JN KipSli are tightly coupled in an opposing reiationship to each other. l-lowever, more recent studies indicate that inhibition of E‘LRK activation {ailed to block NGF-depcndent survival of supe« rior cervical ganglion (SCG) neurons (Creedon et al., 1990; Virdec et al., 1996). Furthermore, insulin promotes survival of fetal chick forebrain neurons concomitant with inhibition of p38 in the absence of an effect on ERK and JNK activity (l-leiden- reich and Kummcr, 1996}. Clearly, the activity of several MAPK members is altered during apoptosis, but at present it is uncertain which, if any, of these activities is necessary and/or sufficient for neuronal apoptosis. In an effort to identify small molecules that promote neuronal survival, we selected (SEEP—1347, also known as K'IVSIS, for its ability to induce choline acctyl ti'ansfcrasc (Ch/\T) activity in cultures prepared from embryonic spinal cord and basal forebrain tissue (Kaneko et al., 1997). Clip-1347 is a semisynthetic deriv- ative of the fermentation product K-252et, an indolocarlulzole that promotes neuronal survivalin chick dorsal root ganglion cultures {Borasim 1998) and ChA'I‘ activity in cultures of rat embryonic spinal cord (Cilicl-tsman et al., 1993), basal forebrain, and striatal Maroney et at. o CEP»134? Inhibits Motoneuron Apoptosts and JNKt Activity neurons (Glicksman ct al., 1995); it also protects hippocampal, septal, and cortical cultures against glucose deprivation-induced death (Cheng et at, 1994). CIlEP-‘l347 effectively protects ino- toneurons in several in viva models of PCD such as the postnatal rat motoneurons of the spinal nucleus of the bulbocavernosus, adult rat hypoglossal axotomy, and chick lumbar motoneurons in ova (M. Glicksman, unpublished observation). To elucidate the mechanism by which CEP-1347 promotes survival of motoneuv runs in viva, we examined the survival activity and determined the effect of UHF—1347 on members of the MAPK family in cultures enriched for embryonic motoneurons. MATERlALS AND METHODS tlt’urcriuir. CEP—l347. also known as KT75l5. is a scmisynthctic deriva- tive of 1025221 provided by [(yowa-l-Iakko Kogyo (Tokyo, Japan) (Kaneko ct al., l997). (SI-IP-lfltl'ii was dissolved in cell culture grade dimethylsttlfoxide (DMSO) attd stored in the dark at 4GC. All dilutions of (YEP-1347 were made in DMEM containing 1% bovine serum albumin. c-Jun Nvterminal kinase 1 (JNKI) antibody (catalog #sc~474AG) was purchased from Santa Cruz Biotechnology (Santa Cruz, CA). ERKJ antibody (catalog #06482), mitogen-activated protein kinase--activated protein kinasc 2 (MAI’KAI’Z) antibody (catalog #66634), and MAP— KAPL’ peptide substrate (catalog #lE-Ecttl) were pttrchascd from Upstate Biotechnology (Lake Placid, NY). HA antibody was purchased from Babco (Richmond, CA). Al’-i (c-fim) substrate was purchased from Promega (Madison. WI). Myclin basic protein substrate, I'loechst dye. and tunicamycin were purchased from Sigma (St. Louis, MO). Sl32ll3580 was Customrsyntltesized by RIT International 'I‘echnology {Snellville. GA). [y-“PJATP (6000 Ci/mmol) was purchased from Amersham {Ar- lington Heights, 11.). Rat spinal cord t'ttiriires enriched for motoncm'ons. Spinal cords were dissected frotn Sprague Dawley rat fetuses (Charles River Laboratories, Wilmington, MA) of embryonic age ([3) 14.5"]5. Cells from only the ventral portion of the spinal cord were dissociated and further enriched for motoneurons by centrifugation on a 6.5% step metrizamide gradient, as described previously (Inlenderson et al., 1993), and were analyzed for purity by staining with low-ailinity neurotrophin receptor antibody (IgG— I92. Boehringcr Mannheim, Indianapolis, EN) (data not shown). Cells were seeded onto 96-well plates previously coated with poly-t.—ornithine. and laminin {.‘i lug/ml each) at a density of 6 X 10“ cellslcm'w' in chemically defined serumvfrec N2 medium (Bottenstcin and Sum. 1979). To separate attachment from stIrVival effects. we added (L‘El’kl347 to the cultures after an initial attachment period of l~3 hr. Neuronal survival was assessed after 4 d by using calccin AM (Molecular Probes. Eugene, OR) in a ituorimetric viability assay (Bozyczko—Coyne ct al., 1993}. Microscopic counts of neurons correlated directly with relative fluores- cence values. In brief. culture medium was diluted serially in Dl’ISS (Dulbecco‘s PBS). and a final concentration of (i you calcein AM stock was added to each 9bvwe|l plate. The plates were incubated for 30 min at 37%;, followed by serial dilution washes in DPBS. The fluorescent signal was read with a plate-reading iiuorimeter from Millipore (Cytoltuor 2350; Bedfot‘d. MA) at an excitation equal to 485 nm and an emission equal to 538 nm. For each plate, mean background derived from wells receiving calcein AM, bttt containing no cells. was subtracted from all values. Linearity of the fluorescence signal was verified for the concen- tration and incubation time for the range of cell densities in these experiments. For assessing apoptotic nuclei. we plated enriched tnotoncuron cub turcs onto eight~chamber slides (Lab-Tek, Nunc. Naperville, IL); they were fixed with 4% paraformaldehyde for 20 min. rinsed with DPBS. and then stained with l ,ugi'ml l-locchst dye for 15 min. After staining. cells were rinsed again and coverslipped, using the aqueous mountant li'luoro- mount (Vector I..aboratories. Burlingame. CA). and then they were. examined and photographed with a Nikon Diaphot microscope (Garden City, NY). (.‘os7t'ct'i culture. Green monkey kidney (3057 cells were obtained from American Type Culture Collection (CRL lofil: Roekville, MD) and ‘ maintained in DM FVI containing 10% bovine serum, 2 inst glutamine. ] m.\-t pyruvate. and 50 U/ml penicillint/streptomycin at 37°C in “1% (303/ 9t I" air atmosphere. (Tbs? cells were detached for pa. aging by adding . .. lrypsin. In vitro kittirsc activity. The inhibitory activities of various concentra» tions of K2522] and CEP~1347 were measured in kinasc assays. Partially J. Neurosci.. January 1, 1998. 18(1):104—111 105 purified protein kinase C (PKC) was prepared from rat brain. and the holoenzyme of cyclic adenosinc monophospbate~dependcnt protein ki~ nase type I (I’KA) was partially purified front rabbit skeletal muscle as described (Kase et al.. 1987). I’hosphoinositol serine— and Ca” - dependent PKC activities were assayed under the conditions described, using 280 ,ugi’ml histone l'i-l and 5 pstl'ywfll’]A'I‘P (Kase et al.. l987). The activity of PKA was assayed in the presence of 100 pg/ml histone II-AS and 10 MM Ey-‘nl’lA'I‘P by the methods in Kase et al. (1987). Myosin light chain kinasc (MLCK) was purified from chicken gizzard and assayed as previously described by I-lPLC analysis (Nakanishi ct al., 1991). Pliosphatidylinositolii kinase (PI3K) was partially pnritied from calf thymus and assayed as described in Yano et ill. U993). The tyrosine kittase activity of the cytoplasmic domain of human recombinant trkA was assessed in an ELISA assay (Angeles or al., 199(1). The IC50 values reported in Table l were calculated front plots of the percentage of inhibition versus log“, concentration of the compound. 1mmtmoprccipimrion rim! kinast’ assay from tt'imt'e cells. Purified ino- toneurons were plated at a density of 6 X 10‘1 cellsiem3 in 16-min- diameter wells. Cells were allowed to attach for 2 hr before treatment. Cells were treated with either 0.01259"? DMSO or 500 nM CIIP—l347 for the indicated time points in defined N2 medium. Then cells were rinsed with ice-cold PBS, followed by lysis in 0.4 ml of'l‘riton buffer (1% Triton X—ltltl. 50 not sodium chloride. 10 mat Tris, pH 7.6, 0.1% bovine serum albumin. 30 pct-t sodium pyrophospltate. 5t] mat sodium fluoride, 20 pgiml aprotinin. 1 mM phenylmethylsulfonylfluoridc, 5 tag/ml leupeptin. and 1 mar sodium vanadate). Immunoprecipitation and kinase assay were per- formed as previously described (Maroncy et al., 1995). Lysatc from motoneuron cultures was normalized to cell number. and lysate from Cos"! cells was normalized to protein concentration. For JNKJ and ERKI iinmunoprecipitations. antibodies were used as recommended by the providers. Immunoprecipitatcs were rinsed three times with Triton buffer, followed by a final wash in kinase buffer (20 11131 H IEl’lES. pH 7.4, 20 111M MgClg. 2 mM dithiothreitol, and (l.| um sodium vanadatc}. Reactions were incubated in hinasc butler containing 1 ptM ATP and 5 |uCi [y-“f’lA’I‘P and substrate {20 .ngl‘SilI'l‘lplC of niyelin basic protein for II:'.RI(I or 1 pig/sample of AP-l for JNKI) for 15 min at 30°C. The kinase reaction was stopped by the addition of sample buffer. Samples were heated to 80°C for 5 min and loaded onto polyacrylamide gels. Qttitltllr tation of results was performed on a Molecular Dynamics Pltospltorltn- tiger (Sunnyvale. CA). For the M AI’KAPZ assay. Cos? cells were grown to conthtency in a (10 mm dish. Cells were preincubatcd in serum-free medium containing either ll.0]?.5‘i’i.- DMSO or 500 its-t CEI’v1347 for l hr. followed by treatment with 500 not sorbitol for l hr. Then cells were rinsed with ice-cold PBS attd lysed in Triton bullet". lysate was normalized to protein concentration. Lysate was immunoprecipitated with the MAPKAPZ antibody and assayed for kinasc activity with 0.l25 inst MAPKAPZ peptide substrate, as described by the provider. The kinase reaction was stopped with 75 mM phosphoric acid, loaded onto phosphoccllulosc tilters (Pierce, Rockford. IL), and washed with 75 mm phosphoric acid, followed by elution with ]N NaOl'l. Radioactivity front the eiuate was counted in a Beckman [.8380] (f-‘ullcrton, CA). .S'n'css-iittiitced' INK! rtciit'iry in Car? cell's. Cos7 cells were grown to contiuency in (all mm plates. The cells were preincubated with 011W- DMSO or 500 IN (YEP-I347 for 1 hr, followed by the following stresses: 500 mm sorbitol for ] hr; exposure to ultraviolet irradiation from the U V2400 Stratolinker (Stratagene. La .Iolla, CA} for 5 min. followed by 1 hr incubation at 37°C; and 50 peg/It‘ll tunicamycin for 5 hr. The cells were rinsed with cold PBS. lysed with Triton butler, immunoprecipitated with the JNKl antibody. and assayed for kinasc activity as described above. Ot-‘cttfxttmssion Of.‘l'tifogt‘h"t’tc'lit’ai’t’d kitttise kittirsc Mouse 1' (AKHEKKI) and JNK.’ in (7057 cells. (.‘os'i cells were plated to 86% conilttcncy and transfected with 2 ,ug each of cDNA constructs, using l,,ipofectatnine as recom mended by the provider (Life "Technologies. Gaithersbtu‘g. MD). A truncated CDNA of mouse MIEKKI (corresponding to amino acids 8174493 of full—length rat MIEKKI) and full-length human JNKl. kindly provided by .l. Silvio thtkind (National Institutes of Health. Bethesda. MD). were subcloncd ittto the pCDNA3 vector (lnvitrogen, San Diego. CA). The JNK construct contained a hemagglutinin tag. After a 48 hr transl‘cction. the cells were treated with DMSO or Silt) n.\41 (iflEi’-l347 for 2 hr. followed by immtinoprecipitation with the HA antiv body. lmatunoprecipitates were normalized for protein anti assayed for kinasc activity in the presence of c-jmi substrate as described above. 105 J. Neurosci. January 1. 1998, 18(1):‘lOd--1 11 Figure I. $tructure of CUP-1347. (1331347 was synthesized by deriviti- zation of K-252a, an indolocarlntzole isolated from culture brotlts of Ner'ru'tt'iosis. RESULTS Low nanomolar concentrations of K—252a, a natural product indoloearbazole of the bacterium Non-(mitosis species, induced ChA’l‘ activity in spinal cord cultures (Glicksman et al., 1993). The ChAT enzyme catalyzes the synthesis of the neurotransmit- ter, acetylcholine. and serves as a marker for motoncurons in spinal cord cultures (Phelps ct al., 1988, 1990}. This neurotrophie activity was optimized by examining novel K~252a analogs in embryonic rat spinal cord cultures and measuring ChAT activity. As reported elsewhere, an ethyithiomethyl analog of K-252a} (HEP—1347 (for structure, Fig. 1). exhibited greater efiieaey (250% of control) and potency (lfiCsn : 5t) 11M) than K-252a in spinal cord ChAT assays (Kanelto ct al., 1997). The neurotrophic properties of CEP- 1347 were not attributable to the inhibition of several known target ltinascs of K-252a. ln contrast to the inhibition of several serine/tln'eonlne kinases by K-252a in the nanomolar range, the ICSU values of ("JEEP-B47 for PKC, l’KA, MLCK, and E’I3K were all >10 ps4 (fable 1). K-252a inhibited trk tyrosine kinase activity with an iCSU of 2.5 nM, whereas the [CSOofCliILIIldT’ was >3 psi (Table i). These results demonstrated a broad separation of neurotrophic activity from inhibition of several known target kinases of K-252a. To examine whether the increased ChAT activity in spinal cord cultures was attributable, at least in part, to inotoneuron survival, we assessed the activity of Cliit’~l347 in cultures of enriched motonenrons. Cell viability in untreated cultures decreased by 35% after 48 hr and by 65% after 72 hr. An inhibitor of p38, SB203580, did not rescue tnotoneurons from cell death (Fig. 2) Figure 2. Time course of tnotoneuron death in the absence or presence of (HEP-1347. (‘clls were plated at a den sity of a X it)" cells/cm2 in chemically defined N2 medium. After 2 hr to allow 100 for attachment, cells were incubated with (Hitlofr‘i- DMSO (control). 250 nM (TILSPAlBLlT. or Silt) 11M 38203580 and monitored for cell viability over 3 (I. Cell viability was measured by using the calcein AM assay as described in Mate rials and Methods. Experimental data represent the mean :1: SI), n =---' 4; DMSO control data represent the mean f: SD." =-'- l2. Three independent experiments were performed; data pre- sented are from one representative cs- 0 pcrimcnt. “p < 0.05. by Dunnctt‘s I statistics. significantly dill'crent front control cultures. 120 80 60 {n of T=tJ) 40 Cell Via ‘li 20 Maroney et at. o (HEP-1347' Inhibits Motoneuron Apoptosis and JNKi Activity Table 1. Comparison of in virro kinasc inhibitory activity between K- 252a and HEP—1347 ICSU (Pew) Kinase K-252a CEPw l347 PKC 0.028 lti.3 [’KA H.072 >10 MLCK 0.19 >ltl PISK 2.0 >10 TRK 0.002 >l {Cuenda et al., 1995). In contrast. viability in the presence of CEP~1347 did not differ substantially from cultures assayed at initial plating (Fig. 2). The morphology of motoneuron cultures was assessed in the presence of CEI’~}347. In contrast to control cultures that rapidly underwent neurite retraction and cellular fragmentation, those cells treated with (TSP—1347 displayed a flattened cell body mor- phology with extensive neuritic processes for at least 5 d (Fig. 30,15). To determine whether cells in control cultures were dying by apoptosis, we examined chromatin condensation by staining the DNA with fluorescent l-{oechst dye. By 48 hr a significant proportion of untreated ntotoneurons exhibited clear hallmarks of chromatin condensation, consistent with a previous report that cultures of enriched tnotoneurons in the absence of neurotrophic factors die in an apoptotic manner (Fig. (Cornelia et al., 1994; Milligan et al., 1994). In contrast. (IE31’4347—trcatcd cultures exhibited diffuse nuclear staining, consistent with the survival activity detected by the calcein assay (Fig. 30’). Changes in the activities of members in the MAPK family have been implicated in neuronal survival and apoptosis {Xia et al., [995). We examined whether neuronal survival induced by (781’- 1347 was accompanied by changes in the activities of lERl-(l and JNKI. The basal level of l-ERKl activity (lid not change over time in ttntreated cultures, and 506 NM Clip—i347 had no significant eifect on ERKi activity (Fig. 4/1}. These data demonstrated that Clip-1347 promoted survival in the absence of a change in Ii-ZRKI activity. In contrast, JNKI activity in untreated cultures in- creased approxintz-ttely fourfold within 24 hr after plating. As early as 15 min after the addition of Slit] HM (313134347, JNKI activity sharply decreased to ~5(l% of control levels and contin— ued to decrease for the next 24 hr (Fig. 413). H control 500 nM DEF-1347 E 5 pM 88203580 m to uM 88203580 Time (hr) Maroney et al. - CEP-1347 inhibits Motoneuron Apoptosis and JNKt Activity CONTROL J. Neurosci., January 1. 1998, 18(1):104-t‘l1 107 250 nM CEP1347 Figure 3. Apoptosis of enriched E145 motoneurons in the absence or presence of (51317-1347. Cells were plated at a density of (1 >1 l0" cells/cm] in chemically dclined N2 medium. After 2 hr to allow for attaclnncnt. control cells were incubated with 6.006% DMSO control (a, c) or 25.0 mt 0313-1347 (I), (i) for 5 d. followed by fixation and photography with Hellman modulating contrast optics (rt. 1)}, or for 2 (I. followed by staining with Hocchst dye (c, (I) to detect condensed chromatin. To resolve whether inhibition of JN K1 activity by (31313-1347 correlated with tnotoneuron survival, we compared the ICSU for JNK] activity with the [-1150 for sttrvival by (HEP-1347. Cultures enriched for motoneurons were grown in tbe pt'eSence of increas- ing concentrations of USP-1347, and .IN K1 activity and cell survival were determined. The 1C5” for JNK] activity measured at 22 hr was 2i 1: 2 mt, whereas the ECfm for cell survival measured at 5d was 20 :i: 2 1th (Fig. 5). These results suggested that cell survival and inhibition of JNKI activity by (KEEP-1347 were integrally linked processes. To determine whether the observed decrease in JNK] activa~ tion was secondary to effects on neuronal survival or was an intrinsic property of (HEP-1347, we examined the etl'ect of (Eli-JP» 1347' on JN K1 activation in Cos7 cells exposed to various exter— nal stresses. JNKI activity increased after treatment with irradL ation. sorbitol, and tunieamyein, censistent with previous studies (Derijard et al., E994; Kyriakis et al., 1994; Rosette and Karin, 1996; Zanke et al.. 199(3) (Table 2a). CEP-l347 prevented the increase in JN K1 activity to a significant degree under all three stress conditions. 'I‘herei‘ore. inhibition of JNK'] activation by (HEP-1347 was not neuronal or stimulinspcciiic. In addition, inhi— bition oi JNKI activation was not a Consermcnce of neuronal survival but appears to be an intrinsic property of (iTIE-Il’u1347. Because p38 also has been implicated in neuronal apoptosis (Xia et al., 1995.), it was of interest to determine whether C13?- 1347 inhibited the p38 signaling pathway. MAPKAPZ is a sul - 108 J. Neurosci,. January 1, 199B, 18(1):104w111 A. 250 g —I—Control g 200 + 500 nM CEP1347 3:" .. :2 3, 150 '2" as .E *- L- 100 s s m g 50 O .c a. "” 0 Time {hr} B. 500 +Control E. —*- 500 nM CEP1347 ': 400 ,3: '3 a 300 33 D? < E - h- 200 § .2 ...., a. g 100 .1: E. 0 o 10 20 30 Time {hr} Figure 4. ERKI and JNK] activity in the absence or presence of C15. 71347. Cultures of enriched i 4.5 ntoloneurons were treated with 0.0 1% DMSO control or Silt) nM C, 1.111347 for various times, as indicated. Ceiis were lysed in 1"}?- Triton butter. and the iysate was imanumprccipiv tated with the ERR} (/1) orlN K] (8) antibody. The immunoprecipitatcs were assayed for itinase activity hy using myeiin basic protein or c-lun, respectively, as substrates. Ii-Isperiments were performed at least two times. and results from representative experiments are shown. Points represent the average of duplicate sampies; error bars indicate the SEM. stratc of p38 and reilects p38 activation {Rouse ct at, 1994). Attempts to measure p38 activity in motoneurons hy assaying the activity of MAPKAPZ were unsuccessfui, probably because of a lack of detection sensitivity in the low-density motoneuron cul’ tures. We therefore tested the effect of CEP~1347 on MAPKAPZ activity in osmoticaliy stressed Cos? celis, a treatment that has been shown previously to activate p38 (Raingcaud et a]., 1995). (YEP-1347 had no effect on MAPKAP2 activity, whereas a p.38 inhibitor, 813203580, completely blocked the stress-induced p38 activity (Tahle 2h) (Cuenda et al., 1995). These data suggested that (HEP-1347 did not inhibit p38 directly or inhibit the upstream regutators of the osmotic shock-induced MAPKAP2 activity. To determine whether JN K1 was a direct molecular target of (HEP-i347 in Cos? celis, we overexpressed eDNA coastructs of Maroney et al. - (DEF-1347' Inhibits Motoneuron Apoptosis and JNKt Activity HAwJNKl alone or with an upstream activator of JN K], MEKKI (i..angc-Cartcr et al., 1993; Minden et 211., i994), and examined JN Kl activity in the absence or presence of CEP—1347. After a 48 hr transfection, the JNKI activity in (3057 cc-ils co- transfccted with varying amounts of MEKK and HA—lNKi constructs was ~threc£old to Stl-foid above the JNKI activity in cells transfected with i--lA—-JN K1 atone (Fig. (i). CHEF-1347 (lid not prevent JN Kl activity under any of the conditions tested. These results indicated that .lN K] was not the direct molecuiar target of CEP-1347 and that the molecular target of CEP»1347 was either upstream of MEKKJ or independent of an MEKKl—aclivaled 1N K1 pathway. DISCUSSION We have shown that CEP-1347 rescues motoncurons from apo- ptotic death in via-o {see Fig. 2) and that survival correlates with the inhibition rileKI activation {see Figs. 4, 5). An endogenous substrate ole K1 is the nuclear transcription factor c-jtm (Hibi et al., 1993; Dcrijard ct al., 1994; Kyriakis ct al., 1994), and (313317“ [347 suppressed r-jmt mRNA in motoneurons at 24 hr after treatment (M. Glichsman, unpublished data). This is consistent with reports demonstrating that a dominant negative mutant of c~fun or a ("jun neutralizing antibody blocks apoptosis induced by neurotrophin withdrawal in SCG neurons and in PC12 cells (liisttts et al., 1994; Ham et a]., 1.995; Xia et al., 1995). Further- more, a dominant negative mutant of MEKKI, which is one upstream regulator 0le K activation, blocked apoptosis in NGF- withdrawn ncuronally differentiated PCIZ coils (Xia ct al., 2995). These data provide convincing evidence to impiicate the JNK signaiing cascade in neuronal models of cell death. However. the sufficiency of .iNK and cr—jtm activation to promote cell death is questionabic. 1n SCG neurons undergoing protongcd NGF depri- vation, suppression of elevated .iN K activity by the readdition of NGF is insuilicicnt to rescue all of the cclis (Virdce et ah. 1997). Furthermore, c—jtm is eievated in choiinergic neurons after for- nix—fitnbria transection, and these cclis do not die (Butterworth and Dragunow, 1996). These reports suggest that, in addition to .lN K/c-jtm activation, other signaling events may be invoived in committing neurons to a death pathway. A rote for INK in the induction of apoptosis also has been examined in non-neuronai systems. Inhibiting JNK or kinascs upstream 0le K protects different eelt types from death induced by a variety of stimuli such as camptothecin, thermal shock, cis-piatintnn, and ceramide (Verhcij et al., 1996; Zanke ct al., i996; lehijo et al., 1997'; Scimiya ct 3%., 1997). However, inactiva- tion of the JNK signaling cascade does not protect against all types ofstrcss—induccd death. For example, high doses of arabino- furanosyicytosine lead to apoptosis in monocytic ieukemia U937 cells, and this death is not blocked with a dominant negative ntutant of c-Jun (Grant et al., 1996}. Also, developmental PCD occurs in c-jmt null embryos. suggesting that (jun is not essential for apoptosis to occur in a variety of tissues (Rolfler-Tarlov et al., 1996); in one study CD95- and CD3~mediatcd apoptosis was exacerbated in MEKfii nnli immature thymocytes (Nishina Cl al., 1997). li‘urthermore, the addition of tumor necrosis factor~a leads to apoptosis in many ccil types and activates the .iNK pathway; however, the role of JN K activation in these apoptotic models is controversial (Gardner and Johnson, 1996; Lin ct al., 1996; Vet'— heij et al.. 1996; Natoli ct ah. i997). Certainly, muitiple pathways leading to coil death exist and may have diii‘crc-nt dependencies on the MAPKs for ftmctionai outcome, subject to the death stimulus and cclluiar environment. Marortey et at, - CEP-134?‘ Inhibits Motonettron Apoptosts and JNK1 Activity JNK1 Activity (Phosphorimager Units} O 1 ‘10 100 [CEP1347] (th Dominant negative forms of various components of the JN K! p38 signaling pathway interfere with death, whereas constitutively active forms of components in the IESRK signaling pathway pro- mote survival after NGF withdrawal from neuronally differenti— ated PClz cells (Xia et al., i995). These resuits suggest that an opposing balance between the IERK and stress-activated sinuses may be crucial for determining whether a coil survives or dies. l'iowever, NGF promotes survival in SCG in the presence of the MEKI inhibitor, P1398059, which blocks ERR activation (Creedon et al., 1996; Virdec and Tolkovsity, 1996). li‘urthermorc, sustained activation of ERK is insufficient to protnote survival in hippo ‘ampal pyramidal neurons (Marsh and Palfrey, 1996). Be- cause UHF-1347 promotes survival in the absence ofany effect on IERKI activity, results reported here support evidence that ERK activation is not necessary or essential for neuronal survival. Although we could not measure MAPKAPZ activity in the mo- toneuron model= (YEP-i347 does not inhibit MAPKAPZ activity in sorhitol-treated Cos? cells (Tabie 2}. Furthermore, the p38 inhibitor, 313203580, inhibits MAPKAPZ activity in Cos? ccils (Table 2; Cuenda et al.. 1995) but does not promote motonenron survival (see Fig. 2). These data suggest that inhibition of p38 is not sttiiicient for motoncut'on survival. Taken together, we con— Tablc 2. Activity in C03? cclis .lNiit activity in stressed-induced Cos? cells Treatment Controi (711111347 Untreated 1.11 Lil irradiation 5.3 2.4 Sorbitol 7.8 2.4 'l'tmicamyciu Lo (1.9 MAPKAI’Z activity in osmotic shocked (Tos? cclls Treatment Controi ($511334? $820358“ Untreated LU 1.4 U!) Sorhitot 8.] 7.6 1.0 Celts w 'c grown to conlluency and pretreated with DMSO. 5th mt Ciil’JBH. or It) [1,.“ 515203580 for | In before treatment with UV irradiation (:3 min in Stra- l0li|1i\‘tli‘. t'oiiou-cd by i hr incubation at 37°C). sot'hitol {Still not sorbitoi for i hr}. or lttnicamycin (5t) tittr‘mi for 5 hr). Lysatcs were collected. normalized for protein. and iunnintoprceipitatul with the JNK1 or MAPKAFZ antibody, as described in Ma- terials and Methods. .spcritncnts were performed at least ttto times, and results from a representative experiment are shown. Results are expressed as the fold increase relative to untreated couti‘oi. J. Neurosci.. January 1, 1998, 18(1):t04—111 109 figure 5. Dose—response of inhibition of iNKl ac- tivity and ceii survival by (HEP-33:17. Cultures of en- riched 1314.5 motonettt‘ons \vct‘c platted and allowed to adhere 2.5 hr before the addition of the indicated concentrations of CEIH347. For 1N K1 activity. cells were collected 22 hr after the addition of compound and assayed for kinase activity as described in Figure 4; ceil viability was determined by calcein AM assay after 5 d in culture. The percentage of cell viability is relative to untreated controls. which is equivalent to ttitm. Points represent the average of duplicate sanr pics; the error bars indicate the $13M. (tenuoo .40 %) MEIMPIA 1190 elude that the activity of the MAPK members is not tightly coupled in an opposing relationship during inotoueuronal surviv- al/death processes. The enhancement of motoncuronai survival by CEPJM? in WHY) is comparable to that elicited by optimal concentrations of protein growth factors (Arakawa ct al.. 1990; Henderson et at, 1993, 1994; Hughes et al., i993). CEI’-1347 does not exhibit selectivity for motoneurons but is also neurotrophic for neurons dissociated from other regions of the vertebrate embryo, for example, spinal cord and basal forcbrain (Kancko et al., 1997), dorsai root gangiia, striatum, basal forebrain, and entorhinal cortex (M. Glici-zsinan, unpublished data). Growth factors, such as BDN F and iGF—l, rescue motoneurons from trophic dcprivatitm-induccd cell death. in our hands the effect of BUN i" on tnotoneuron survivai was highly dependent on cell density. Under extremely stringent conditions (200 celisfcm BDNF, as well as (HEP-134?, rescued motoneurons. This low- density plating prohibited biochemical attaiysis of signaling path- ways. l-lowevcr, at higher plating density, as presented in this manuscript, BDN I: activated ERKI but did not promote survival nor inhibit the rise in JNK1 activity (data not shown). Thus, BDNiT-indttced IEiRKi activation was not sufficient for motoneuw ron survival in these cuiturcs. The mechanism by which growth factors promote survival of neurons is unclear and perhaps may depend on the activation of other proteins such as phosphatidylinositol-3 itinase and Altt, which ultimately may lead to a decrease in JNK activity (Yao and Cooper, 1995; Dttdek et a1., 1997). Inhibition of JNK1 activation appears to be an intrinsic prop- erty of (STEP-1347, because the activation of JNK1 by multipie stress stimuli also was blocked by (3i231’~134? in Cos? celis (Tabie 2). Because dill‘ercnt stimuli can activate JNK via distinct path- ways, these data suggest that ($111134? is acting at a site at or proximal to tiNK itself. Directly upstream of JNK is MEK4, which can be phosphorylated by a number of kinases, one of which is MIZEK K1 (Langcfiartcr et ai., 1993; Mindcn c-t al.. 1994; Dcrijard ct al., 1995; Lin ct al., 1995}. "[‘ransfcction data reported here suggest that JNK1 is not the direct target of (YEP-{347 and that the molecular target is either upstream or indepent‘le‘r‘tt of MEKKl. Kinases of the germinai center and tnultiplc iineage kinasc families activate 3N K independently of M EKKt and are also potcntitll targets for (HEP-1347 action (for review, see hanger et al., 199?). As has been shown in Table i, (HEP-134? does not 110 J. Neurosci., January 1, 1998, 18(1):104—111 7U 60 50 —~ 40 's E 2° 'fi 2 < i=3 ._ x 2 :2 o 10 " E O - m MEKK O 0.1 D HA-JNK 0.2 0.2 1 Figure 0. Maroney et al. - (REP—1347 inhibits Motoneuron Apoptosis and JNKt Activity control CEPnt 347 0.05 1 1 1 JNKI activit t in Cos7 cells overcx )rcssina I’lA-JNK] alone or with M IEKKI. Cos? cells were 'rown to 80% confluent: ' and transfeeted with It 1 e E } 1'1A-tttgged JN [(1 alone or with MEKKI at various amounts of cDNA. as indicated. After a 48 hr period the cells were incubated with (1.01% DMSO or Silt) nM CEP-i347 for 2 hr. foilowed by lysis in 1% Triton butler. Lysztte was normalized to protein and intmunoprecipitated with HA antibody. The immunoprecipitates were assayed for kinase activity with the crjmr substrate. Experiments were performed at ieast two times. and t'esuits from representative experiments are shown. Activity is expressed by fold increase relative to untreated HAuJNK-transfeetcd ceiis. Columns represent the average of duplicate samples; the error bars indicate the SEM. display the broad kinase inhibitory activities of K-252a. Although (HELP-1347 appears to be more selective than K-252a, it also tnay have multipie ceilular targets. The recent discovery of stress-activated signaling pathways in dying neurons broadens the concept of ne-tu'otropitism. which eiassically has been defined by activation of the ras signaling cascade by NGF, resulting in the attenuation ofdeath (Borasio et 211.. 1989; Nobes and ’I‘olkovsky, 1995; Weng et al., 1996). We have demonstrated here that a nonpoiypeptide organic molecule can mwmmwimmomdduMithmtmeaQWMmutna MAPK pathway. Importantly, CEPJ347wtnediated motoneuron survival correlates with the inhibition of the JN K signaiing cascade. These observations suggest that intervention in the JNK signaling eas- eade may ofl‘er opportunities for the development of therapeutic agents for nettrodegenerative disease. REFERENCES Angeles TS. Stetller C. Bartlett BA. Hudkins RL, Stephens RM. Kaptan DR, Dionne CA (1996) Enzymeilinked imtmmosorbeut assay for tritA tyrosine kinase activity. Attai Biochcm 23631945. 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