Mitochondrial cyclic AMP response element-binding protein (CREB) mediates mitochondrial gene expression and neuronal survival

Abstract

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.

FIGURE 1. Identification of mitochondria as a site of CREB localization in neurons

A, both CREB (panels a– c) and pCREB (Ser-133) (panels d–i) colocalize with mitochondria in the cerebral cortex of rat adult brain in situ (panels a–f) and primary cultured cortical neurons (panels g–i). White arrows indicate colocalization with punctate staining of pCREB and mitochondria in a single cortical neuron. B, pCREB immunoreactivity (panels a and d) with cytochrome c (Cyto. c) (panels b and e) is impaired in dorsal root ganglia in CREB null (^−/−) mice (panels a– c) but not in CREB^+/− (panels d–f). C, immunogold electron microscopy shows pCREB in the mitochondrial matrix of cortical neurons. Mito, mitochondria; Nu, nucleus. Scale bar: 200 nm. D, CREB is found in the mitochondrial subcellular fraction. pCREB was detected as a 43-kDa band. The same blot was then stripped and reblotted with anti-complex I (mitochondrial NADH oxidoreductase (OR), 37-kDa subuint) (mitochondrial marker), Bip (GRP78) (endoplasmic reticulum marker), and Sp1 (nucleus marker), each of which were also detected. N, nuclear fraction; C1, cytoplasmic fraction 1; P1, pellet 1 mitochondria-rich fraction; P2, pellet 2 mitochondria fraction. E, proteinase K assay using the mitochondrial fraction shows that pCREB is localized to the mitochondrial matrix. F, mitochondrial fraction (MF) of cortical neurons (CN) and adult rat brain show specific CREB DNA binding activity by EMSA. EMSA was performed with mitochondrial and nuclear extracts using ³²P-labeled CRE oligonucleotides. Supershift analysis for identification of CREB complex used antibodies (Ab) for pCREB (Ser-133), ATF-1, and ATF-2. NF, nuclear fraction.

FIGURE 2. CREB binds to the non-coding region of mitochondrial DNA

A, in vitro footprinting analysis reveals three putative CRE-like sites in the D-loop of mouse mitochondrial DNA. B, sequences of CRE-like sites in the D-loop are presented. Three CRE-like sequences (I–III) as underlined are found in the D-loop. Arrows indicate the direction of CREB binding to the putative CRE. C, EMSA shows that CREB binds to mitochondrial CRE-like sites (I–III). A canonical CRE site in a nuclear gene, tyrosine hydroxylase (TH), promoter was used as a standard of CREB binding activity to mitochondrial DNA. D, nonspecific competitor analysis confirmed that CREB is specifically associated with mitochondrial CRE-like sequences. SS, supershift analysis. E, CREB association with mitochondrial DNA was further examined using DNA-protein cross-linking, immunoprecipitation of pCREB, and PCR methods (see supplemental methods). The mitochondria pellet was cross-linked by 1% formaldehyde, sonicated, and immunoprecipitated. Immuoprecipitated and eluted mitochondrial DNA with CREB antibody and IgG were amplified with primers designed for CRE sites in D-loop sequences and non-CRE sites in ND6 sequences. Lanes 1 and 3, IgG immunoprecipitation; lanes 2 and 4, CREB antibody immunoprecipitation; lane 5, 50-bp molecular marker. The arrow indicates an amplified signal with CRE site primers (15896 –15996).

FIGURE 3. Mitochondrially targeted CREB directly regulates the expression of mitochondrial genes

A, confocal microscopy showed the expression of mito-wt-CREB-ECFP vector (panel a; green) and mitotracker (panel b; red) staining and overlaid images (panel c) in SH-SY5Y cells. ECFP fusion proteins were visualized by indirect labeling using mouse anti-ECFP antibody and goat anti-mouse IgG antibody conjugated with fluorescein isothiocyanate. B, Western blot analysis of mito-ECFP (lane 1), mito-wt-CREB-ECFP (lane 2), and mito-A-CREB-ECFP fusion protein (lane 3). C, A-CREB inhibits CREB DNA binding to the mitochondrial EMSA of mitochondrial extracts from mito-ECFP (lane 1), mito-wt-CREB-ECFP (lane 2), and mito-A-CREB-ECFP (lane 3) or mito-wt-CREB-ECFP and mito-A-CREB-ECFP co-transfected (lane 4) cells. Anti-CREB antibody (Ab) was used for supershift analysis (lane 5). D, mitochondrially targeted A-CREB inhibits mito-wt-CREB association with mitochondrial D-loop CRE sequences. HT-22 cells were transfected with mito-Red2 (lane 1), mito-wt-CREB-Red2 (lane 2), and mito-A-CREB-Red2 and mito-wt-CREB-Red2 (lane 3) for 24 h. E, RT-PCR analysis of the expression of mitochondrial DNA-encoded genes in mito-ECFP (lane 1), mito-wt-CREB-ECFP (lane 2), and mito-A-CREB-ECFP (lane 3) in SH-SY5Y cell lines. Complex I (ND2, ND4, and ND5), complex III (cytochrome b (Cyto B)), complex IV (cytochrome c oxidase subunit III (COXIII)), complex V (ATPase 6), and 12 S RNA gene fragments were examined for mitochondrial gene expression. 18 S RNA was used as a nuclear-encoded gene control. The representative data are shown from three separate experiments (see numerical and real-time PCR data in the supplemental Table 1 and Fig. 4). F, the ratio of complex I-dependent respiration on glutamate/malate compared with complex II-dependent respiration on succinate (plus rotenone) was significantly decreased in permeabilized mito-A-CREB-ECFP cells compared with mito-ECFP and mito-wt-CREB-ECFP cells. Data are expressed as the mean ± S.E. of three to five separate experiments.

FIGURE 4. Mitochondrial CREB is involved in the neuronal survival

A, mito-A-CREB cells (panels e and f) were highly susceptible to 3-NP-induced cytotoxicity compared with mito-ECFP (panels a and b) and mito-wt-CREB cells (panels c and d). Cells were treated with vehicle (control) and 3-NP (0.5–5 mM) for 72 h. B, cell viability was measured by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Data are the average ± S.E. of three separate experiments: significant at p < 0.05 (*) and p < 0.01 (**). C, cytochrome c release is higher in mito-A-CREB cells (lane 3) compared with mito-ECFP (lane 1) and mito-wt-CREB cells (lane 2) in response to 3-NP. Cytosol fractions were prepared from cells treated with vehicle (control) or 3-NP (0.5 mM) for 72 h.