Functional Repression of cAMP Response Element in 6-Hydroxydopamine-treated Neuronal Cells
2006; Elsevier BV; Volume: 281; Issue: 26 Linguagem: Inglês
10.1074/jbc.m602632200
ISSN1083-351X
AutoresElisabeth Mole Chalovich, Jian-hui Zhu, John Caltagarone, Robert Bowser, Charleen T. Chu,
Tópico(s)Nerve injury and regeneration
ResumoImpaired survival signaling may represent a central mechanism in neurodegeneration. 6-Hydroxydopamine (6-OHDA) is an oxidative neurotoxin used to injure catecholaminergic cells of the central and peripheral nervous systems. Although 6-OHDA elicits phosphorylation of several kinases, downstream transcriptional effects that influence neuronal cell death are less defined. The cAMP response element (CRE) is present in the promoter sequences of several important neuronal survival factors. Treatment of catecholaminergic neuronal cell lines (B65 and SH-SY5Y) with 6-OHDA resulted in repression of basal CRE transactivation. Message levels of CRE-driven genes such as brain-derived neurotrophic factor and the survival factor Bcl-2 were decreased in 6-OHDA-treated cells, but message levels of genes lacking CRE sequences were not affected. Repression of CRE could be reversed by delayed treatment with cAMP several hours after initiation of 6-OHDA injury. Furthermore, restoration of CRE-driven transcription was associated with significant neuroprotection. In contrast to observations in other model systems, the mechanism of CRE repression did not involve decreased phosphorylation of its binding protein CREB. Instead, total CREB and phospho-CREB (pCREB) were increased in the cytoplasm and decreased in the nucleus of 6-OHDA-treated cells. 6-OHDA also decreased nuclear pCREB in dopaminergic neurons of primary mouse midbrain cultures. Co-treatment with cAMP promoted/restored nuclear localization of pCREB in both immortalized and primary culture systems. Increased cytoplasmic pCREB was observed in degenerating human Parkinson/Lewy body disease substantia nigra neurons but not in age-matched controls. Notably, cytoplasmic accumulation of activated upstream CREB kinases has been observed previously in both 6-OHDA-treated cells and degenerating human neurons, supporting a potential role for impaired nuclear import of phosphorylated signaling proteins. Impaired survival signaling may represent a central mechanism in neurodegeneration. 6-Hydroxydopamine (6-OHDA) is an oxidative neurotoxin used to injure catecholaminergic cells of the central and peripheral nervous systems. Although 6-OHDA elicits phosphorylation of several kinases, downstream transcriptional effects that influence neuronal cell death are less defined. The cAMP response element (CRE) is present in the promoter sequences of several important neuronal survival factors. Treatment of catecholaminergic neuronal cell lines (B65 and SH-SY5Y) with 6-OHDA resulted in repression of basal CRE transactivation. Message levels of CRE-driven genes such as brain-derived neurotrophic factor and the survival factor Bcl-2 were decreased in 6-OHDA-treated cells, but message levels of genes lacking CRE sequences were not affected. Repression of CRE could be reversed by delayed treatment with cAMP several hours after initiation of 6-OHDA injury. Furthermore, restoration of CRE-driven transcription was associated with significant neuroprotection. In contrast to observations in other model systems, the mechanism of CRE repression did not involve decreased phosphorylation of its binding protein CREB. Instead, total CREB and phospho-CREB (pCREB) were increased in the cytoplasm and decreased in the nucleus of 6-OHDA-treated cells. 6-OHDA also decreased nuclear pCREB in dopaminergic neurons of primary mouse midbrain cultures. Co-treatment with cAMP promoted/restored nuclear localization of pCREB in both immortalized and primary culture systems. Increased cytoplasmic pCREB was observed in degenerating human Parkinson/Lewy body disease substantia nigra neurons but not in age-matched controls. Notably, cytoplasmic accumulation of activated upstream CREB kinases has been observed previously in both 6-OHDA-treated cells and degenerating human neurons, supporting a potential role for impaired nuclear import of phosphorylated signaling proteins. Oxidative stress has been implicated in aging and a variety of pathological processes. In particular, oxidative mechanisms have been strongly linked to the pathogenesis of age-related neurodegenerative diseases (1Barnham K.J. Masters C.L. Bush A.I. Nat. Rev. Drug. Discov. 2004; 3: 205-214Crossref PubMed Scopus (2639) Google Scholar). Neurons may be particularly susceptible to oxidants because of high metabolic activity and relatively low levels of endogenous antioxidants in the brain. Indeed, markers for lipid peroxidation and protein nitration are increased in affected brain areas in Alzheimer disease, Parkinson disease, and amyotrophic lateral sclerosis (2Beal M.F. Ann. Neurol. 1995; 38: 357-366Crossref PubMed Scopus (1246) Google Scholar, 3Tyurin V.A. Tyurina Y.Y. Borisenko G.G. Sokolova T.V. Ritov V.B. Quinn P.J. Rose M. Kochanek P. Graham S.H. Kagan V.E. J. Neurochem. 2000; 75: 2178-2189Crossref PubMed Scopus (196) Google Scholar, 4Souza J.M. Giasson B.I. Chen Q. Lee V.M. Ischiropoulos H. J. Biol. Chem. 2000; 275: 18344-18349Abstract Full Text Full Text PDF PubMed Scopus (489) Google Scholar), and antioxidants provide protection in their respective animal and culture models (5Cassarino D.S. Fall C.P. Swerdlow R.H. Smith T.S. Halvorsen E.M. Miller S.W. Parks J.P. Parker Jr., W.D. Bennett J.P. Jr. Biochim. Biophys. Acta. 1997; 1362: 77-86Crossref PubMed Scopus (236) Google Scholar, 6Wu D.C. Teismann P. Tieu K. Vila M. Jackson-Lewis V. Ischiropoulos H. Przedborski S. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 6145-6150Crossref PubMed Scopus (529) Google Scholar, 7Callio J. Oury T.D. Chu C.T. J. Biol. Chem. 2005; 280: 18536-18542Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 8Yang F. Lim G.P. Begum A.N. Ubeda O.J. Simmons M.R. Ambegaokar S.S. Chen P.P. Kayed R. Glabe C.G. Frautschy S.A. Cole G.M. J. Biol. Chem. 2005; 280: 5892-5901Abstract Full Text Full Text PDF PubMed Scopus (1911) Google Scholar, 9Wu A.S. Kiaei M. Aguirre N. Crow J.P. Calingasan N.Y. Browne S.E. Beal M.F. J. Neurochem. 2003; 85: 142-150Crossref PubMed Scopus (79) Google Scholar). However, the mechanism(s) by which reactive oxygen species influence cell survival and death decisions are incompletely defined (10Ellerby L.M. Bredesen D.E. Methods Enzymol. 2000; 322: 413-421Crossref PubMed Google Scholar, 11Barlow C.A. Shukla A. Mossman B.T. Lounsbury K.M. Am. J. Respir. Cell Mol. Biol. 2006; 34: 7-14Crossref PubMed Scopus (24) Google Scholar). Given the progressive nature of neurodegenerative diseases, a better understanding of mechanisms that could impair adaptive responses to injury would be particularly pertinent. 6-Hydroxydopamine (6-OHDA) 2The abbreviations used are: 6-OHDA, 6-hydroxydopamine; BDNF, brain-derived neurotrophic factor; CRE, cAMP response element; CREB, CRE-binding protein; CBP, CREB-binding protein; ERK, extracellular signal-regulated protein kinase; RSK, ribosomal S-6 kinase; pCREB, activated CREB phosphorylated at Ser-133; pCRE-luc, reporter plasmid in which luciferase expression is regulated by CRE sequences; pERK, activated ERK phosphorylated at Thr-202/Tyr-204; PKA, protein kinase A; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; TH, tyrosine hydroxylase; RT, reverse transcription; Z, benzyloxycarbonyl; AFC, N-acetyl-S-farnesyl-l-cysteine; EMSA, electrophoretic mobility shift assay; ANOVA, analysis of variance; Bt2cAMP, dibutyryl cAMP. 2The abbreviations used are: 6-OHDA, 6-hydroxydopamine; BDNF, brain-derived neurotrophic factor; CRE, cAMP response element; CREB, CRE-binding protein; CBP, CREB-binding protein; ERK, extracellular signal-regulated protein kinase; RSK, ribosomal S-6 kinase; pCREB, activated CREB phosphorylated at Ser-133; pCRE-luc, reporter plasmid in which luciferase expression is regulated by CRE sequences; pERK, activated ERK phosphorylated at Thr-202/Tyr-204; PKA, protein kinase A; PBS, phosphate-buffered saline; LDH, lactate dehydrogenase; CHAPS, 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonic acid; PIPES, 1,4-piperazinediethanesulfonic acid; TH, tyrosine hydroxylase; RT, reverse transcription; Z, benzyloxycarbonyl; AFC, N-acetyl-S-farnesyl-l-cysteine; EMSA, electrophoretic mobility shift assay; ANOVA, analysis of variance; Bt2cAMP, dibutyryl cAMP. is a redox active analog of catecholamine neurotransmitters, which has been used to lesion the nigro-striatal system that degenerates in Parkinson and related diseases, to ablate the sympathetic nervous system, and as a chemotherapeutic agent for catecholaminergic neoplasms (12Schor N.F. Kagan V.E. Liang Y. Yan C. Tyurina Y. Tyurin V. Nylander K.D. Biochemistry (Mosc.). 2004; 69: 38-44Crossref PubMed Scopus (10) Google Scholar, 13Przedborski S. Ischiropoulos H. Antioxid. Redox. Signal. 2005; 7: 685-693Crossref PubMed Scopus (169) Google Scholar). Spontaneous autoxidation of 6-OHDA results in production of reactive oxygen species such as hydrogen peroxide, superoxide and hydroxyl radical (14Cohen G. Heikkila R.E. JBC. 1974; 249: 2447-2452Abstract Full Text PDF PubMed Google Scholar). 6-OHDA treatment elicits activation of several kinases including extracellular signal regulated protein kinases (ERK) (15Kulich S.M. Chu C.T. J. Neurochem. 2001; 77: 1058-1066Crossref PubMed Scopus (169) Google Scholar, 16Horbinski C. Chu C.T. Free Radic. Biol. Med. 2005; 38: 2-11Crossref PubMed Scopus (208) Google Scholar), glycogen synthase kinase-3β (17Chen G. Bower K.A. Ma C. Fang S. Thiele C.J. Luo J. FASEB J. 2004; 18: 1162-1164Crossref PubMed Scopus (180) Google Scholar), and stress-activated kinases (18Choi W.S. Eom D.S. Han B.S. Kim W.K. Han B.H. Choi E.J. Oh T.H. Markelonis G.J. Cho J.W. Oh Y.J. J. Biol. Chem. 2004; 279: 20451-20460Abstract Full Text Full Text PDF PubMed Scopus (179) Google Scholar, 19Eminel 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). Both in vivo and in vitro studies implicate intracellular oxidative stress in 6-OHDA neurotoxicity (7Callio J. Oury T.D. Chu C.T. J. Biol. Chem. 2005; 280: 18536-18542Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 20Asanuma M. Hirata H. Cadet J.L. Neuroscience. 1998; 85: 907-917Crossref PubMed Scopus (78) Google Scholar, 21Barkats M. Millecamps S. Bilang-Bleuel A. Mallet J. J. Neurochem. 2002; 82: 101-109Crossref PubMed Scopus (31) Google Scholar), and only antioxidants that can affect intracellular reactive oxygen species act to inhibit 6-OHDA-mediated ERK activation (22Kulich S.M. Chu C.T. J. Biosci. 2003; 28: 83-89Crossref PubMed Scopus (51) Google Scholar). Although recent gene profiling studies indicate increases in both death-associated and neuroprotective genes (23Ryu E.J. Angelastro J.M. Greene L.A. Neurobiol. Dis. 2005; 18: 54-74Crossref PubMed Scopus (71) Google Scholar), transcriptional mechanisms by which 6-OHDA modulates cell survival/death decisions remain incompletely defined. Cyclic AMP response element-binding protein (CREB) is a transcription factor that plays an important role in neuronal survival, in part by controlling the transcription of neuroprotective genes (24Finkbeiner S. Neuron. 2000; 25: 11-14Abstract Full Text Full Text PDF PubMed Scopus (410) Google Scholar). The promoter regions of the genes for brain-derived neurotrophic factor (BDNF) and the pro-survival protein Bcl-2 contain cAMP response elements (CREs) (25Mayr B. Montminy M. Nat. Rev. Mol. Cell Biol. 2001; 2: 599-609Crossref PubMed Scopus (2019) Google Scholar). Studies show that cAMP, a potent activator of CREB, acts as a trophic or protective signal for several populations of catecholaminergic neurons (Reviewed in Refs. 26Goldberg J.L. Barres B.A. Annu. Rev. Neurosci. 2000; 23: 579-612Crossref PubMed Scopus (300) Google Scholar and 27Hulley P. Hartikka J. Lubbert H. J. Neural Transm. Suppl. 1995; 46: 217-228PubMed Google Scholar). Additionally, cAMP has been shown to enhance the protective effects of noradrenaline (28Troadec J.D. Marien M. Mourlevat S. Debeir T. Ruberg M. Colpaert F. Michel P.P. Mol. Pharmacol. 2002; 62: 1043-1052Crossref PubMed Scopus (71) Google Scholar) and glial cell line derived neurotrophic factor (29Engele J. Franke B. Cell Tissue Res. 1996; 286: 235-240Crossref PubMed Scopus (44) Google Scholar), further emphasizing the important role that the CREB pathway plays in controlling neuronal fate. Taken together, these results suggest that a loss of CREB function could contribute to neuronal dysfunction. The potential role of the CREB pathway in neuronal cell responses to 6-hydroxydopamine was investigated in this study. The results indicate that suppression of CRE transactivation contributes to 6-OHDA-induced toxicity and that this loss of function is not due to decreased phosphorylation of CREB. Instead, phospho-CREB accumulates in the cytoplasm and is decreased in the nucleus of 6-OHDA-treated cells, and CRE-controlled genes such as BCL-2 and BDNF are down-regulated. Reversal of CRE repression by cAMP treatment confers significant protection from 6-OHDA toxicity, even when the cAMP is added 4 h after initiation of injury. These results demonstrate a transcriptional mechanism for oxidative stress-induced neurotoxicity with potential relevance to neurodegenerative mechanisms. Cell Culture—B65 cells (ECACC 85042305) were the gift of Dr. David Schubert of the Salk Institute, La Jolla, CA. SH-SY5Y cells were purchased from ATCC (Manassas, VA). Cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal calf serum, 2 mm l-glutamine, and 10 mm HEPES. Low serum medium consisted of Dulbecco's modified Eagle's medium supplemented with 0.5% heat-inactivated fetal calf serum, 2 mm l-glutamine, and 10 mm HEPES. Cell culture reagents were purchased from BioWhittaker (Walkersville, MD) unless otherwise indicated. Cells were plated at a density of 3 × 105 cells/well for 6-well plates, 1 × 104 cells/well in 96-well plates, or 1 × 106 cells/well in 100-mm dishes. For all experiments, cells were cultured in a humidified incubator with 5% CO2 at 37 °C. Primary Midbrain Cultures—Primary midbrain neuronal cultures were derived from 15-day C57BL/6 mouse embryos as described previously (30Chu C.T. Zhu J.H. Cao G. Signore A. Wang S. Chen J. J. Neurochem. 2005; 94: 1685-1695Crossref PubMed Scopus (74) Google Scholar). Glial proliferation was inhibited using cytosine arabinoside (2 μm), and cultures were treated at 7 days in vitro with 50 μm 6-OHDA, which kills 50–65% of midbrain TH+ neurons (31Guo X. Dawson V.L. Dawson T.M. Eur. J. Neurosci. 2001; 13: 1683-1693Crossref PubMed Scopus (92) Google Scholar). After 3 h, the chamber slides were processed for immunofluorescence as described below. Transcription Assays—The CRE reporter vector from the Mercury Pathway Profiling System (Clontech, Palo Alto, CA) was cloned and purified using endotoxin-free Maxi-Prep kits (Qiagen, Valencia, CA). Cells were plated in 6-well plates and grown overnight. For each well, 1 μg of plasmid DNA was prepared in 500 μl of Opti-MEM medium, combined with 10 μl of Lipofectamine 2000 (Invitrogen, Carlsbad, CA) in 500 μl of Opti-MEM, and incubated for 20 min before being applied to plated cells. After 8 h, the cells were switched to low serum medium for 24 h prior to the experiment. For calcium phosphate transfections using the CalPhos mammalian transfection kit (Clontech), 1 μg of plasmid DNA was combined with water and calcium phosphate reagent, allowed to incubate for 20 min at room temperature, and then added dropwise to cells. Transfections were allowed to proceed overnight before switching to low serum medium for 24 h prior to use. Parallel experiments were performed using either Lipofectamine 2000 or calcium phosphate with similar results. Luciferase Assay—Cells were assayed for CRE reporter activity using a luciferase reporter assay kit (Clontech). Briefly, cells were washed with phosphate-buffered saline (PBS) (without calcium and magnesium) and then lysed with gentle agitation for 20 min. After centrifugation, supernatants were assayed for luciferase activity within 20 min or frozen at –70 °C and assayed within 3 weeks. Equal volumes of lysate and luciferin substrate were combined (200 μl final volume) followed by a 20 s measurement of emitted light using a Lumat LB 9507 tube luminometer (Berthold Technologies, Oak Ridge, TN). Luciferase, expressed in response to CRE transactivation, catalyzes the oxidation of luciferin, resulting in photon production. Light output is directly proportional to the amount of luciferase, and thus transcriptional activity that is driven by CRE in the cells. Toxicity Assays—For each assay, 6-OHDA (Sigma) was prepared in ice cold 0.5% (w/v) ascorbate or sterile water immediately before use. Equivalent results were obtained with either vehicle system. Dibutyryl cyclic AMP (Bt2cAMP; Sigma) was prepared in sterile water. Toxicity was measured using a lactate dehydrogenase (LDH) release assay (Sigma 340-UV LDH detection system). Briefly, cells were grown in 96-well plate format and exposed to the LD50 concentration of 6-OHDA (500 μm for B65 cells; 150 μm for SH-SY5Y cells) or to vehicle for 18–20 h. Cells were pre- or post-treated with Bt2cAMP (250 μm) as indicated in the figure legends. The level of LDH released into the culture medium was expressed as percent of total LDH in the wells, as described previously (15Kulich S.M. Chu C.T. J. Neurochem. 2001; 77: 1058-1066Crossref PubMed Scopus (169) Google Scholar). The fluorogenic substrate Z-DEVD-AFC (Calbiochem) was used to measure caspase-3 activity. Lysate (15 μg of protein) was prepared in 200 μl of reaction buffer (0.1% CHAPS buffer containing 20 mm PIPES, 100 mm NaCl, 10 mm dithiothreitol, 1 mm EDTA, 10% sucrose) and mixed with 20 μm Z-DEVD-AFC. The fluorescence of the cleavage product was measured kinetically at 37 °C in a microplate spectrofluorometer (Molecular Devices; excitation wavelength 400 nm, emission 505 nm). Proteolytic activity was expressed as relative fluorescence units normalized to control cultures. Immunoblotting—Cell lysates were obtained as described previously (15Kulich S.M. Chu C.T. J. Neurochem. 2001; 77: 1058-1066Crossref PubMed Scopus (169) Google Scholar) using a Triton X-100-based lysis buffer in the presence of protease and phosphatase inhibitors. For separation of nuclear and cytoplasmic protein fractions, the NePUR Kit (Pierce) was used as directed by the manufacturer. Protein concentration was determined using the Coomassie Plus protein assay (Pierce). Purity of the fractions was assessed by immunoblotting for cytoplasmic and nuclear markers. Equal amounts of protein were electrophoresed through 5–15% SDS-polyacrylamide gels, transferred to Immobilon-P membranes (Millipore, Bedford, MA), and blocked in 5% nonfat dry milk in PBS-T (20 mm potassium phosphate, 150 mm potassium chloride, and 3% (w/v) Tween 20, pH 7.4) for 1–2 h at room temperature. Blots were probed overnight at 4 °C with the following antibodies and dilutions: total CREB, 1:1000 (Cell Signaling Technology, Danvers, MA); phospho-CREB, 1:1000 (Cell Signaling Technology); β-actin, 1:10,000 (Sigma); lamin A/C, 1:1000 (Cell Signaling Technology). CREB control extracts (Cell Signaling Technology) were used as positive control. After washing, blots were developed using a horseradish peroxidase-conjugated IgG secondary antibody and a chemiluminescence detection kit (Amersham Biosciences). Blots were stripped and reprobed as indicated in the figure legends. After use, blots were stained with Coomassie Blue to confirm equal protein loading and transfer. Isolation of RNA and RT-PCR—Total RNA was isolated from treated B65 cells using Qiagen RNeasy kits (Qiagen). RNA was quantified by spectrophotometry, and 1 μg of RNA was used for each PCR reaction. Primers were designed using the on-line Primer 3 software (68Rozen S. Skaletsky H.J. Bioinformatics Methods and Protocols: Methods in Molecular Biology. 2000; (Humana Press, Totowa, NJ): 365-386Google Scholar) unless otherwise stated. The sequences of forward and reverse primers were: 5′-ATACCTGGGCCCAAGTG-3′ and 5′-TGATTTGACCATTTGCCTGA-3′ for Bcl-2; 5′-GTGACAGTATTAGCGAGTGGG-3′ and 5′-GGGTAGTTCGGCATTGC-3′ for BDNF (69Tokuyama W. Hashimoto T. Li Y.X. Okuno H. Miyashita Y. Brain Res. Mol. Brain Res. 1998; 62: 206-215Crossref PubMed Scopus (19) Google Scholar); and 5′TGTTTGAGACCTTCAACACC-3′ and 5′-TAGGAGCCAGGGCAGTAATC-3′ for β-actin. Samples were amplified in a PTC-100 programmable thermal controller (Bio-Rad), using the GeneAmp EZ rTth RNA PCR kit (PerkinElmer Life Sciences/Roche Applied Science) following the manufacturer's instructions. Reaction products were electrophoresed through 1% agarose gels and stained with ethidium bromide. Quantitative RT-PCR was performed using a LightCycler instrument (Roche Applied Science) and the LightCycler-RNA amplification kit SYBR Green I (Roche Applied Science) to measure RNA in a one-step RT-PCR reaction in real time (32Oury T.D. Schaefer L.M. Fattman C.L. Choi A. Weck K.E. Watkins S.C. Am. J. Physiol. 2002; 283: L777-L784PubMed Google Scholar). Melting curves and gel electrophoresis of the products were used to ensure specificity of amplification products. Electrophoretic Mobility Shift Assay (EMSA)—EMSA was performed as described on double-stranded oligonucleotides by hybridizing complementary denatured single-stranded oligonucleotides with 10× annealing buffer (200 mm Tris-HCl, pH 8.0, 100 mm MgCl2, 500 mm NaCl) (33Chandran U.R. Attardi B. Friedman R. Zheng Z. Roberts J.L. DeFranco D.B. J. Biol. Chem. 1996; 271: 20412-20420Abstract Full Text Full Text PDF PubMed Scopus (67) Google Scholar, 34Jordan-Sciutto K.L. Dragich J.M. Rhodes J.L. Bowser R. J. Biol. Chem. 1999; 274: 35262-35268Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). A palindromic CRE promoter sequence (5′-AGAGATTGCCTGACGTCAGAGAGC-3′) (35Zhang B. Liu S. Perpetua M.D. Walker W.H. Harbrecht B.G. Hepatology. 2004; 39: 1343-1352Crossref PubMed Scopus (18) Google Scholar) was used for the probe. The CRE probe (32P end-labeled; 20,000 cpm/lane) was incubated with 30 μg of nuclear protein extract in 5× binding buffer (50 mm Tris-HCl, pH 8.0, 62.5% glycerol, 2.5 mm EDTA, 750 mm KCl) in a 30 μl total reaction volume for 15–20 min at 25 °C. Specificity was assessed by competition with unlabeled CRE probe. Poly(dI-dC) (15 ng) was used as a nonspecific competitor. For supershift assays, a mixture of antibodies to CREB and pCREB (Cell Signaling Technology; 4 μl each) was added 15 min prior to the addition of labeled CRE probe. Protein-DNA complexes were resolved on 1× Tris borate-EDTA/10% polyacrylamide gels and detected by autoradiography. Immunofluorescence—Cells were plated on glass coverslips in 12-well plates at a density of 1.5 × 105 cells/well, treated with 6-OHDA or Bt2cAMP as indicated in the figure legends, and then washed with PBS and fixed in ice-cold 4% paraformaldehyde for 15 min (30Chu C.T. Zhu J.H. Cao G. Signore A. Wang S. Chen J. J. Neurochem. 2005; 94: 1685-1695Crossref PubMed Scopus (74) Google Scholar). Cells were permeabilized with 0.1% Triton X-100/PBS and then blocked in 5% normal donkey serum. To visualize phosphorylated CREB, a pCREB monoclonal antibody (1:1000, overnight at 4 °C, Cell Signaling Technology) was used. Coverslips were then washed with PBS and incubated with fluorescent secondary antibodies (1:200, Jackson ImmunoResearch Laboratories) for 1 h at room temperature. Nuclei were counter-stained with 4,6-diamidino-2-phenylindole (DAPI, Molecular Probes). Coverslips were mounted in gelvatol, and cells were visualized and photographed using a Nikon Eclipse II microscope. Paraffin-embedded human midbrain sections, from a previously characterized set of parkinsonian and control brains (7Callio J. Oury T.D. Chu C.T. J. Biol. Chem. 2005; 280: 18536-18542Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar, 36Zhu J.-H. Kulich S.M. Oury T.D. Chu C.T. Am. J. Pathol. 2002; 161: 2087-2098Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar, 37Zhu J.-H. Guo F. Shelburne J. Watkins S. Chu C.T. Brain Pathol. 2003; 13: 473-481Crossref PubMed Scopus (204) Google Scholar), were stained for pCREB (36Zhu J.-H. Kulich S.M. Oury T.D. Chu C.T. Am. J. Pathol. 2002; 161: 2087-2098Abstract Full Text Full Text PDF PubMed Scopus (133) Google Scholar). The red 3-amino, 9-ethyl-carbazole (AEC) chromogen was used with hematoxylin counterstain. Equivalent results were obtained using avidin-biotin or tyramide amplification. 6-OHDA Represses the CRE Promoter—The luciferase reporter transcription assay was used to monitor induction of the CRE in B65 cells treated with medium, vehicle, or 6-OHDA. Luciferase activity from six independent experiments were normalized to the untreated control of each experiment and averaged (Fig. 1A). There was a significant repression in the basal activity of the CRE promoter after 6-OHDA treatment. To verify that the observed repression was not due to cell death, several measures of cell viability and the transcription of control luciferase constructs lacking CRE elements (Fig. 1B) were examined. At the time points used for transcriptional assays (30 min to 3 h of 6-OHDA exposure) no morphological changes of cell death are apparent (Fig. 1C). Moreover, there is no evidence of LDH release until 8 h after 6-OHDA exposure (Fig. 1D). Control experiments were conducted using the pTAL plasmid, which lacks CRE sequences but is otherwise identical to the TATA-like promoter-containing CRE reporter vector. 6-OHDA treatment had no effect on pTAL-driven luciferase expression (Fig. 1B), indicating that 6-OHDA does not cause a general disruption in transcriptional responses. Therefore it is likely that the observed transcriptional repression is due to disrupted CRE transactivation and not to general defects in transcription or cell viability. Disruption in CREB Function Contributes to 6-OHDA-induced Cell Death—Next, the potential ability of cAMP treatment to reverse the repressive effects of 6-OHDA on the CRE promoter was examined. Cells were treated with a cell-permeable form of cAMP 10 min prior to exposure to 6-OHDA. The results demonstrate that Bt2cAMP pretreatment prevented the 6-OHDA-induced repression of CRE activity (Fig. 2A). Treatment of B65 cells with Bt2cAMP in the presence of 6-OHDA not only restored CRE activity but also resulted in CRE activity levels that were higher than those elicited by treatment with the same concentration of Bt2cAMP alone. Experiments were conducted to determine whether Bt2cAMP treatment during 6-OHDA exposure would also confer protection against 6-OHDA-mediated toxicity. Indeed, a 10-min pretreatment with Bt2cAMP resulted in significantly decreased cell injury at 18 h by both morphological examination and LDH release assay (Fig. 2, B and C). Because the addition of Bt2cAMP reversed the 6-OHDA-induced repression while conferring protection, these data suggest that disruption in CRE function contributes to 6-OHDA-induced cell death. Delayed Administration of cAMP Reverses CRE Repression and Confers Protection—This effect was further characterized by determining whether delayed treatment with Bt2cAMP following initiation of 6-OHDA toxicity would still effectively reverse 6-OHDA-mediated CRE repression. We exposed the CRE-transfected B65 cells to Bt2cAMP at different intervals after 6-OHDA exposure. Results showed that delayed addition of Bt2cAMP caused a reversal of 6-OHDA-induced repression (Fig. 3A). Moreover, delayed Bt2cAMP treatments also resulted in significant protection against toxicity even when administered up to 4 h after initiation of 6-OHDA treatment (Fig. 3B), further supporting the interpretation that perturbations to the CREB signaling pathway contributes to 6-OHDA toxicity. 6-OHDA Reduces Expression of CRE-regulated Genes—To confirm that 6-OHDA-induced repression of CRE transactivation was associated with functional effects on endogenous genes, we examined the expression of downstream CRE-regulated genes (25Mayr B. Montminy M. Nat. Rev. Mol. Cell Biol. 2001; 2: 599-609Crossref PubMed Scopus (2019) Google Scholar). BDNF and Bcl-2 were selected because of their well documented roles in neuronal growth and survival as well as prior studies showing that both CREB-induced genes can protect from 6-OHDA toxicity (38Klein R.L. Lewis M.H. Muzyczka N. Meyer E.M. Brain Res. 1999; 847: 314-320Crossref PubMed Scopus (118) Google Scholar, 39Jordan J. Galindo M.F. Tornero D. Gonzalez-Garcia C. Cena V. J. Neurochem. 2004; 89: 124-133Crossref PubMed Scopus (66) Google Scholar). Quantitative RT-PCR showed decreased BCL2 and BDNF mRNA in response to 6-OHDA treatment (Fig. 4). No changes were observed in the mRNA levels of mitogen-activated protein kinase (MAPK) phosphatase-3 (MKP-3), which lacks CRE sequences in its promoter region, or in the mRNA levels of β-actin. In addition, stimulation of CRE by cAMP prevented the 6-OHDA-induced reduction in mRNA expression of Bcl-2 and BDNF (Fig. 4). Because cAMP also conferred significant protection, these data indicate that 6-OHDA-mediated repression of CRE transactivation results in decreased expression of CRE-controlled survival genes. Electrophoretic Mobility Shift Assay Demonstrated Decreased CRE Binding Activity in 6-OHDA-treated Cells—Nuclear extracts from B65 cells treated with 6-OHDA and/or Bt2cAMP for 3 h were incubated with radiolabeled DNA containing a palindromic CRE promoter sequence. Use of specific (unlabeled probe) and nonspecific poly(dI-dC) competitors revealed a specific protein-CRE doublet. 6-OHDA resulted in decreased CRE binding activity of both bands compared with control and Bt2cAMP-treated cells (Fig. 5A). A supershift assay confirmed the presence of CREB/pCREB in the complex (Fig. 5B). Interestingly, Bt2cAMP co-treatment did n
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