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.The cAMP response element-binding protein (CREB) 3 is a transcription factor known to mediate stimulus-dependent expression of genes critical for the plasticity, growth, and survival of neurons (1). 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 (2, 3). 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 (4,5). A recent report that CREB is present in the mitochondria raises the possibility that CREB could mediate mitochondrial gene expression (6). 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. MATERIALS AND METHODSIsolation of Mitochondria-Mitochondria were isolated from primary cultured cortical neurons and adult rat brains by sucrose density gradient centrifugation (6).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 (7).Immunogold Labeling and Electron Microscopy-Frozen samples were sectioned at Ϫ120°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, grid...

SIRT3 deregulation is linked to mitochondrial dysfunction in Alzheimer's disease

“…In this context, SIRT3 may modulate p53 binding to mtDNA by deacetylating p53 at K320 and thereby coordinate mitochondrial gene expression. We previously reported that cAMP response element binding protein (CREB) is localized to neuronal mitochondria and specifically binds to CREB response elements in the mitochondrial genome that regulate mitochondrial‐encoded Complex I gene expression (Lee et al ., 2005; Ryu et al ., 2005). In the current study, we provide another layer of mechanism that increased mito‐p53 level leads to ROS accumulation and reduces the rate of mitochondrial oxygen consumption.…”

“…Immunogold labeling and TEM were performed with some modification as previously described (Lee et al ., 2005; Ryu et al ., 2005). The tissues were fixed with 2.5% glutaraldehyde dissolved in 0.1M cacodylate buffer overnight at 4°C and with 2% osmium tetroxide for 1 h. The cells were dehydrated with ethanol series, infiltrated with Spurr's resin series, and polymerized at 60°C for 8 h. The embedded cell was cut with a diamond knife on ultramicrotome (Ultracut S, Leica).…”

Pinacidil protects osteoblastic cells against antimycin A-induced oxidative damage

“…Mitochondrial CREB was hypothesized to mediate pro-survival effects by regulating various mitochondrial genes, the expression of which may be directed by CRE-like elements located in the D-loop of the mitochondrial genome (22). Furthermore, Lee et al (23) demonstrated that the binding of CREB to CRE sequences in the D-loop of mitochondrial DNA enhanced the transcript levels of mitochondrial genes. Therefore, the results of the present study suggested that the protective effects of pinacidil in osteoblasts may be associated with PI3K, Akt and CREB signaling.…”

Consequences of Mitochondrial Dysfunction in Huntington's Disease and Protection via Phosphorylation Pathways

“…CREB is widely expressed and has a well-established role in neuronal protection (Lee et al 2005). mHtt was shown to interfere with CREB transcriptional processes, through direct interaction with CREB-binding protein (CBP) (Steffan et al 2000) and with TATA boxbinding protein (TBP)-associated factor TAF4/TAFII130 (Dunah et al 2002;Shimohata et al 2000), leading to an increase in mHtt-induced cytotoxicity (Steffan et al 2001).…”