Mitochondrial Cyclic AMP Response Element-binding Protein (CREB) Mediates Mitochondrial Gene Expression and Neuronal Survival

Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders. Cyclic AMP response element-binding protein (CREB) is a widely expressed transcription factor whose role in neuronal protection is now well established. Here we report that CREB is present in the mitochondrial matrix of neurons and that it binds directly to cyclic AMP response elements (CREs) found within the mitochondrial genome. Disruption of CREB activity in the mitochondria decreases the expression of a subset of mitochondrial genes, including the ND5 subunit of complex I, down-regulates complex I-dependent mitochondrial respiration, and increases susceptibility to 3-nitropropionic acid, a mitochondrial toxin that induces a clinical and pathological phenotype similar to Huntington disease. These results demonstrate that regulation of mitochondrial gene expression by mitochondrial CREB, in part, underlies the protective effects of CREB and raise the possibility that decreased mitochondrial CREB activity contributes to the mitochondrial dysfunction and neuronal loss associated with neurodegenerative disorders. The cAMP response element-binding protein (CREB) 3The abbreviations used are: CREBcAMP response element-binding proteinCRECREB response elementATFactivating transcription factorNDNADH dehydrogenaseEMSAelectric mobility shift assaymtHttmutant huntingtinPBSphosphate-buffered salineECFPenhanced cyan fluorescent proteinRTreverse transcriptionHDHuntington disease3-NP3-nitropropionic acid. is a transcription factor known to mediate stimulus-dependent expression of genes critical for the plasticity, growth, and survival of neurons (1Lonze B.E. Ginty D.D. Neuron. 2002; 35: 605-623Abstract Full Text Full Text PDF PubMed Scopus (1748) Google Scholar). A variety of stimuli alter levels of intracellular second messengers in neurons, such as cAMP and calcium, and activate CREB by leading to phosphorylation at its critical regulatory site, serine 133 (2Mayr B. Montminy M. Nat. Rev. Mol. Cell Biol. 2001; 2: 599-609Crossref PubMed Scopus (2072) Google Scholar, 3Impey S. Goodman R.H. Sci. STKE. 2001; 82: PE1Google Scholar). Overexpression of constitutively active CREB prevents cell death induced by growth factor deprivation, while expression of a dominant negative form of CREB leads to apoptosis in both sympathetic neurons and cerebellar granule cells (4Riccio A. Ahn S. Davenport C.M. Blendy J.A. Ginty D.D. Science. 1999; 286: 2358-2361Crossref PubMed Scopus (695) Google Scholar, 5Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1680) Google Scholar). A recent report that CREB is present in the mitochondria raises the possibility that CREB could mediate mitochondrial gene expression (6Cammarota M. Paratcha G. Bevilaqua L.R. Levi de Stein M. Lopez M. Pellegrino de Iraldi A. Izquierdo I. Medina J.H. J. Neurochem. 1999; 72: 2272-2277Crossref PubMed Scopus (81) Google Scholar). Nonetheless, the function of mitochondrial CREB is not known. The present study confirms the presence of CREB in the mitochondria and addresses the role of CREB in mitochondrial gene expression and neuronal survival. The results raise the possibility of a novel mechanism for CREB dysfunction in the pathogenesis of neurodegenerative disorders. cAMP response element-binding protein CREB response element activating transcription factor NADH dehydrogenase electric mobility shift assay mutant huntingtin phosphate-buffered saline enhanced cyan fluorescent protein reverse transcription Huntington disease 3-nitropropionic acid. Isolation of Mitochondria—Mitochondria were isolated from primary cultured cortical neurons and adult rat brains by sucrose density gradient centrifugation (6Cammarota M. Paratcha G. Bevilaqua L.R. Levi de Stein M. Lopez M. Pellegrino de Iraldi A. Izquierdo I. Medina J.H. J. Neurochem. 1999; 72: 2272-2277Crossref PubMed Scopus (81) Google Scholar). Confocal Microscopy—Indirect labeling methods were used to determine the levels of CREB, phosphorylated CREB (pCREB), and neurofilament (200 kDa) in cortical neuronal cultures and human and rat brain tissues as described previously (7Ryu H. Lee J. Olofsson B.A. Mwidau A. Dedeoglu A. Escudero M. Flemington E. Azizkhan-Clifford J. Ferrante R.J. Ratan R.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4281-4286Crossref PubMed Scopus (221) Google Scholar). Immunogold Labeling and Electron Microscopy— Frozen samples were sectioned at -120 °C, and the sections were transferred to Formvar/carboncoated copper grids. Samples were incubated with antibody in 1% bovine serum albumin for 30 min. After rinsing the samples four times with PBS, protein A-gold (10 nm) in 1% bovine serum albumin was added for 20 min. Contrasting stain procedures were carried out using 2% methyl cellulose: 3% uranyl acetate (9:1) for 10 min on ice. To dry the samples, grids were picked up with a loop and excess liquid was removed using filter paper. DNase I Footprinting Analysis—The mitochondrial DNA fragment encompassing 15858/16063 bp (GenBank™ accession number J01420) was prepared by PCR and used as a probe in the DNase I footprinting experiment (8Kim C.H. Hwang D.Y. Park J.J. Kim K.S. J. Neurosci. 2002; 22: 2579-2589Crossref PubMed Google Scholar). Electrophoretic Mobility Shift Assay (EMSA)—We performed EMSAs on mitochondrial extracts from rat brain tissues and cortical neurons using a 32P-labeled oligonucleotide containing a wild-type CREB-binding site as described previously (7Ryu H. Lee J. Olofsson B.A. Mwidau A. Dedeoglu A. Escudero M. Flemington E. Azizkhan-Clifford J. Ferrante R.J. Ratan R.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4281-4286Crossref PubMed Scopus (221) Google Scholar). Mitochondrial D-loop CRE oligonucleotides were designed from the CRE I-III sequences shown in Fig. 2B. Supershifts were performed with pCREB-specific antibody for the Ser-133 residue (Upstate Biotechnology Inc., Lake Placid, NY), ATF-1/CREB (25C10G; Santa Cruz Biotechnology), or CREB-1 (24H4B and 240; Santa Cruz Biotechnology). Mitochondrial DNA and Protein Cross-linking and Immunoprecipitation—Mitochondrial DNA and protein cross-linking and immunoprecipitation analysis for CREB binding to mitochondrial DNA was performed using a chromatin immunoprecipitation assay kit (Upstate Biotechnology Inc.). Mitochondrial fraction pellets or HT-22 cells transfected with pDs-Red2-Mito empty vector, pDs-Red2-Mito-wt-CREB, and pDsRed2-A-CREB for 24 h were cross-linked with 1% formaldehyde for 20 min at room temperature. PCR amplification was carried out for 35 cycles, and PCR products were separated on 2% agarose gels. Three primers were used to amplify the segment flanking the three or two CRE-like sites in the D-loop of mitochondria. The forward primers were 5′-GTGGTGTCATGCATTTGGTATCT-3′ and 5′-ATCAACATAGCCGTCAAGGCATG-3′, and the reverse primer was 5′-TCACCGTAGGTGCGTCTAGACTGT-3′. Normal rabbit IgG served as a negative control. Construction of Plasmids—To generate mitochondrially targeted fusion proteins, wt-CREB and A-CREB (9Ahn S. Olive M. Aggarwal S. Krylov D. Ginty D.D. Vinson C. Mol. Cell. Biol. 1998; 18: 967-977Crossref PubMed Scopus (448) Google Scholar) were subcloned into pECFP-Mito and pDs-Red2 Mito vector (CLONTECH Laboratories, Inc., Palo Alto, CA). Real-time PCR and Conventional RT-PCR—To quantify the copy number of mRNA of the ND5 and ND6 genes, real-time PCR was performed using a DNA Engine Opticon System (MJ Research Inc., Las Vegas, NV). For the detection of mitochondria-encoded gene expression, total cellular RNA digested with RNase-free DNase was reverse-transcribed with SuperScript RT-PCR kit (Invitrogen). The probe and primers designed to amplify mitochondrial transcripts were as follows: human ND2, 4704-5103; human ND4, 11479-11929; human ND5, 13569-13917, human cytochrome b, 15494-15748; human ATPase 6, 8854-9087; human complex IV, 6188-6377, human mitochondrial 12 S rRNA, 576-422; human 18 S RNA, 5′-CCGAGATTGAGCAATAACAGG-3′ (forward) and 5′-AGTTCGACCGTCTTCTCAGG-3′ (reverse). Measurement of Mitochondrial Enzyme Activities—Respiratory activities were measured polargraphically as described previously (10Jazayeri M. Andreyev A. Will Y. Ward M. Anderson C.M. Clevenger W. J. Biol. Chem. 2003; 278: 9823-9830Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar). Western Blot Analysis—Western blot was performed using subcellular fractions and cell or tissue lysates as described previously (7Ryu H. Lee J. Olofsson B.A. Mwidau A. Dedeoglu A. Escudero M. Flemington E. Azizkhan-Clifford J. Ferrante R.J. Ratan R.R. Proc. Natl. Acad. Sci. U. S. A. 2003; 100: 4281-4286Crossref PubMed Scopus (221) Google Scholar). In the neurons of the adult rat cortex, we found that CREB and pCREB (Ser-133) were partially extranuclear. Furthermore, the extranuclear CREB colocalized with the mitochondrial marker cytochrome c (Fig. 1A, panels a-f). In cultured immature embryonic cortical neurons, CREB and pCREB partially colocalized with the mitochondrial marker MitoTracker and were found within the proximal segments of dendrites (Fig. 1A, panels g-i). CREB staining was most prominent in neurofilament-200 (NF-200)-positive cells (data not shown). Moreover, both nuclear and extranuclear CREB and pCREB immunoreactivity was absent in neurons derived from CREB null mice (Fig. 1B). In addition, we found that CREB-binding protein is not localized in the mitochondria (supplemental Fig. 1). To pinpoint the subcellular localization of extranuclear CREB, we performed immuno-electron microscopy using cultured cortical neurons. This analysis demonstrated that pCREB is present within both the nucleus and the mitochondrial matrix of these neurons (Fig. 1C). We further verified the localization of CREB to the mitochondrial fraction using subcellular fractionation of adult rat brain tissue by sucrose density centrifugation (Fig. 1D). The presence of complex I (NADH oxidoreductase) immunoreactivity, together with the absence of immunoreactivity of the endoplasmic reticulum marker Bip (GRP78) or the nuclear marker Sp1, confirmed that the fractions contained only mitochondria. Mitochondrial pCREB was resistant to proteinase K treatment in the absence of detergent, suggesting that pCREB is located within the mitochondrial matrix (Fig. 1, C and E). We employed EMSA to determine whether mitochondrial CREB is capable of binding to a canonical CRE DNA sequence (5′-TGACGTCA-3′). Consistent with the immunoblot and immunolocalization experiments, mitochondrial extracts from rat embryonic cortical neurons and adult rat brain tissue revealed the presence of CRE-binding activities (Fig. 1F). Although CREB lacks a classical mitochondrial targeting sequence, alternative pathways exist for targeting proteins to the mitochondria (11Marchenko N.D. Zaika A. Moll U.M. J. Biol. Chem. 2000; 275: 16202-16212Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 12Merrick B.A. He C. Witcher L.L. Patterson R.M. Reid J.J. Pence-Pawlowski P.M. Selkirk J.K. Biochim. Biophys. Acta. 1996; 1297: 57-68Crossref PubMed Scopus (58) Google Scholar). To determine whether chaperone molecules may play a role in the mitochondrial targeting of CREB, we performed a coimmunoprecipitation assay using lysates from the mitochondrial fraction to assess the degree of association of CREB and mitochondria (mt) HSP70 (GRP 75) (11Marchenko N.D. Zaika A. Moll U.M. J. Biol. Chem. 2000; 275: 16202-16212Abstract Full Text Full Text PDF PubMed Scopus (786) Google Scholar, 12Merrick B.A. He C. Witcher L.L. Patterson R.M. Reid J.J. Pence-Pawlowski P.M. Selkirk J.K. Biochim. Biophys. Acta. 1996; 1297: 57-68Crossref PubMed Scopus (58) Google Scholar). We found that CREB coprecipitated with mtHSP70 in human and rat brain tissues and in embryonic neurons (supplemental Fig. 2). The data support the possibility that chaperone molecules are involved in delivering CREB to the mitochondria. The D-loop is the control site for both transcription and DNA replication in the mitochondrial genome. Within this region, we identified variant CRE-like sequences by in vitro footprinting analysis (Fig. 2, A and B) and observed that these sites formed specific DNA-protein complexes with CREB (Fig. 2C) and that complex formation by mitochondrial CRE elements did not compete with NFκB, Sp1, or USF-1 cis-elements (Fig. 2D). Furthermore, we performed chromatin immunoprecipitation with a CREB antibody to demonstrate that CREB is bound to these mitochondrial DNA sites in the intact cell (Fig. 2E) (supplemental Fig. 3). To evaluate the role of CREB in mitochondrial gene expression and survival, we expressed a mitochondrially targeted form of either CREB (mito-wt-CREB-ECFP) or its dominant negative (mito-A-CREB). Transiently expressed mito-wt-CREB-ECFP protein colocalized with mitotracker staining, confirming that the heterologous protein is localized in the mitochondria (Fig. 3A). Immunoblotting verified that mito-wt-CREB (∼80 kDa), mito-A-CREB (∼30 kDa), and mito-ECFP (∼28 kDa) fusion proteins were expressed at similar levels (Fig. 3B). EMSA analysis demonstrated that the dominant negative mito-A-CREB disrupts the mitochondrial DNA-binding activity of CREB (Fig. 3, C and D). To more definitively address the function of CREB targeted to mitochondria, we prepared cell lines that stably express either mito-wt-CREB or mito-A-CREB. We used real-time PCR and RT-PCR to determine to what extent mitochondrial gene expression was altered in the stable cell lines (Fig. 3E and supplemental Table 1). We found that mito-wt-CREB and mito-A-CREB inversely regulate the expression of some mitochondrial genes. Mito-wt-CREB increased levels of transcripts of the ND2, ND4, and ND5 mitochondrial genes, while mito-A-CREB decreased them. Interestingly, ND5 expression was significantly reduced in mito-A-CREB cells. Consistent with reduced expression of ND5 (a complex I subunit), we also observed a relative reduction of complex I activity in mito-A-CREB cells (Fig. 3F). We monitored levels of the c-fos gene, a transcript regulated by nuclear CREB levels, to verify that mito-A-CREB does not affect nuclear CREB activity. As expected, we found that neither mito-wt-CREB or A-CREB influences c-fos expression as compared with control (supplemental Fig. 4). Our results that mito-A-CREB down-regulates several of the mitochondrial genes, in part, likely reflect diminished mito-CREB transcriptional activity. However, the failure to detect a decrease in levels of some mitochondrial genes, such as the cytochrome b or ATPase 6 genes that are also encoded on the H strand, could be due to other factors, such as differences in mRNA stability. Indeed, mutations in the mitochondrial RNA binding protein, LRPPRC (leucine-rich pentatricopeptide repeat cassette) are responsible for a French Canadian form of Leigh's syndrome. In this syndrome, cytochrome c oxidase mRNAs are selectively decreased as compared with other mRNAs encoded in the mitochondrial H-strand (13Xu F. Morin C. Mitchell G. Ackerley C. Robinson B.H. Biochem. J. 2004; 382: 331-336Crossref PubMed Scopus (139) Google Scholar). Previous studies have established that decreasing ND5 expression corresponds with decreasing complex I-dependent respiration, suggesting that ND5 transcript levels may tightly control mitochondrial respiration rate (14Bai Y. Shakwley R.M. Attardi G. Mol. Cell Biol. 2000; 20: 805-815Crossref PubMed Scopus (103) Google Scholar). Indeed, mitochondrial DNA mutations in the genes encoding the ND5 subunit of complex I are associated with mitochondrial myopathy and Lebers hereditary optic neuropathy, which show defects in complex I activity (14Bai Y. Shakwley R.M. Attardi G. Mol. Cell Biol. 2000; 20: 805-815Crossref PubMed Scopus (103) Google Scholar, 15Bentlage H.A. Janssen A.J. Chomyn A. Attardi G. Walker J.E. Schagger H. Sengers R.C. Trijbels F.J. Biochim. Biophys. Acta. 1995; 1234: 63-73Crossref PubMed Scopus (20) Google Scholar, 16Wissinger B. Besch D. Baumann B. Fauser S. Christ-Adler M. Jurklies B. Zrenner E. Leo-Kottler B. Biochem. Biophys. Res. Commun. 1997; 234: 511-515Crossref PubMed Scopus (56) Google Scholar). Thus, CREB regulation of expression of the ND5 and/or ND6 subunit may influence mitochondrial respiration (17Enriquez J.A. Fernandez-Silva P. Garrido-Perez N. Lopez-Perez M.J. Perez-Martos A. Montoya J. Mol. Cell Biol. 1999; 19: 657-670Crossref PubMed Scopus (143) Google Scholar). Interestingly, however, intracellular ATP levels were not decreased by mito-A-CREB expression (supplemental Table 2). There are a number of mitochondrial inhibitors that affect complexes of the electron transport chain by reducing cellular levels of ATP, resulting in energy deficiency and pathogenesis M. N. Y. Acad. Sci. PubMed Scopus Google Scholar, E. Ferrante R.J. E. J. Neurosci. PubMed Google Scholar, Ferrante R.J. S. P. J. Neurochem. 1998; PubMed Scopus Google Scholar). such 3-nitropropionic is of dehydrogenase and both the and complex activity of the electron transport chain E. Ferrante R.J. E. J. Neurosci. PubMed Google Scholar). is associated with in both and as used as for Ferrante R.J. S. P. J. Neurochem. 1998; PubMed Scopus Google Scholar). We further that mitochondria may be more to mitochondrial in the presence of a dominant negative or mutant mitochondrial To to address this we the of on cell lines stably either mito-wt-CREB or mito-A-CREB. Mito-wt-CREB cells were resistant to mito-A-CREB cells were more (Fig. A and in mito-A-CREB cells was associated with of cytochrome c compared with mito-wt-CREB and mito-ECFP cells (Fig. These results support the that defects in mitochondrial transcription are associated with increased to mitochondrial We further found of neurons in the mice measured by staining (1Lonze B.E. Ginty D.D. Neuron. 2002; 35: 605-623Abstract Full Text Full Text PDF PubMed Scopus (1748) Google Scholar). The data directly support a survival and/or role for CREB in neurons (supplemental Fig. H. A. F. C. D. J. C. W. G. Nat. 2002; PubMed Scopus Google Scholar). Interestingly, a decreased of mitochondrial CREB proteins was found to with the of the in mice (supplemental Fig. the phenotype of mice is of H. A. F. C. D. J. C. W. G. Nat. 2002; PubMed Scopus Google data are with a loss of function of mitochondrial CREB as a of and/or loss in To determine whether is of mitochondrial transcription in a we mitochondrial transcript levels in Mitochondrial ND5 and ND6 mRNA were significantly decreased in similar to the induced by a dominant negative CREB targeted to the mitochondria (supplemental Fig. 6, A and and Fig. 3). Furthermore, cell and neuronal loss in mice were in control mice in response to (supplemental Fig. These results support the that defects in mitochondrial transcription are associated with increased to mitochondrial in as well as in vitro (Fig. 4). In with studies mitochondrial function in and mice E. Ferrante R.J. E. J. Neurosci. PubMed Google Scholar, E. P. Ferrante R.J. A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, M.A. 1999; PubMed Scopus Google that loss of mitochondrial CREB may play a role in the of that mitochondrial dysfunction a role in and in neurodegenerative such as and disease M. N. Y. Acad. Sci. PubMed Scopus Google Scholar, E. Ferrante R.J. E. J. Neurosci. PubMed Google Scholar, Ferrante R.J. S. P. J. Neurochem. 1998; PubMed Scopus Google Scholar, E. P. Ferrante R.J. A. Proc. Natl. Acad. Sci. U. S. A. 1995; PubMed Scopus Google Scholar, 1995; PubMed Scopus Google Scholar, M.A. 1999; PubMed Scopus Google Scholar, J. Res. Mol. Res. PubMed Scopus Google Scholar, Science. 1998; PubMed Google Scholar). Our data demonstrate that CREB is present within the mitochondria and to regulate mitochondrial gene expression in neurons. transcription factors, such as and the in part, to mitochondria and effects with or the expression of the mitochondrial genes M. S. Zaika A. P. Moll U.M. Mol. Cell. 2003; Full Text Full Text PDF PubMed Scopus Google Scholar, H. J. B. G. J. F. J.A. Science. 2000; PubMed Scopus Google Scholar). In the present mitochondrial CREB to regulate at in part, by mitochondrial genes whose expression may be by CRE-like elements within the D-loop of the mitochondrial F. P. A. I. S. R.J. G. C. Mol. Cell. Biol. 1999; 19: PubMed Google Scholar, H. Lee J. S. Ratan R.R. Ferrante R.J. Proc. Natl. Acad. Sci. U. S. A. PubMed Scopus Google Scholar). Our data also that of mitochondrial gene expression role in to cell death a CREB and a mitochondrial toxin known to a phenotype similar to This is in with studies that of a dominant negative CREB induces apoptosis (4Riccio A. Ahn S. Davenport C.M. Blendy J.A. Ginty D.D. Science. 1999; 286: 2358-2361Crossref PubMed Scopus (695) Google Scholar, 5Bonni A. Brunet A. West A.E. Datta S.R. Takasu M.A. Greenberg M.E. Science. 1999; 286: 1358-1362Crossref PubMed Scopus (1680) Google Scholar, J. B. A. I. A. A. M. J. J. Neurosci. 2003; PubMed Google Scholar) and that of both CREB and leads to similar to that observed in H. A. F. C. D. J. C. W. G. Nat. 2002; PubMed Scopus Google Scholar). However, these studies have that the is by a loss of nuclear CREB activity. We show that mutant huntingtin CREB (supplemental Fig. Thus, mutant huntingtin the to CREB and decrease mitochondrial transcriptional activity M. C. S. Y. H. A. M. Y. I. Y. M. J. I. I. N. H. S. Nat. 2000; PubMed Scopus Google Scholar, A. J. H. J. J. M. A. Mol. 2001; PubMed Google Scholar, M. H. J.K. M. H. S. J. Science. 2001; PubMed Scopus Google Scholar, S. E. E. F. F. M.E. Mol. 2003; PubMed Scopus Google Scholar, Nat. Neurosci. 2002; PubMed Scopus Google Scholar). In to the effects that the loss of CREB may in nuclear huntingtin proteins may affect the mitochondrial CREB Our the function may be in Huntington disease. We for support and F. for with

Fetched live from OpenAlex and de-inverted. Abstracts are not stored in this database: the inverted indexes are 8.6 GB of the frame’s 9.3 GB of text, and the host has 13 GB free.