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...

The intracellularly acting protein toxin of Pasteurella multocida (PMT) causes numerous effects in cells, including activation of inositol 1,4,5-trisphosphate (IP 3 ) signaling, Ca 2؉ mobilization, protein phosphorylation, morphological changes, and DNA synthesis. The direct intracellular target of PMT responsible for activation of the IP 3 pathway is the G q/11 ␣-protein, which stimulates phospholipase C (PLC) ␤1. The relationship between PMT-mediated activation of the G q/11 -PLC-IP 3 pathway and its ability to promote mitogenesis and cellular proliferation is not clear. PMT stimulation of p42/p44 mitogen-activated protein kinase occurs upstream via G q/11 -dependent transactivation of the epidermal growth factor receptor. We have further characterized the effects of PMT on the downstream mitogenic response and cell cycle progression in Swiss 3T3 and Vero cells. PMT treatment caused dramatic morphological changes in both cell lines. In Vero cells, limited multinucleation, nuclear fragmentation, and disruption of cytokinesis were also observed; however, a strong mitogenic response occurred only with Swiss 3T3 cells. Significantly, this mitogenic response was not sustained. Cell cycle analysis revealed that after the initial mitogenic response to PMT, both cell types subsequently arrested primarily in G 1 and became unresponsive to further PMT treatment. In Swiss 3T3 cells, PMT induced up-regulation of c-Myc; cyclins D1, D2, D3, and E; p21; PCNA; and the Rb proteins, p107 and p130. In Vero cells, PMT failed to up-regulate PCNA and cyclins D3 and E. We also found that the initial PMT-mediated up-regulation of several of these signaling proteins was not sustained, supporting the subsequent cell cycle arrest. The consequences of PMT entry thus depend on the differential regulation of signaling pathways within different cell types.The protein toxin from Pasteurella multocida (PMT) is the primary etiological agent of progressive atrophic rhinitis (11), and purified PMT experimentally induces all of the major symptoms of atrophic rhinitis in animals (2, 23). PMT appears to bind to and enter mammalian cells via receptor-mediated endocytosis (30, 33), although the details of this process are still unclear. In fibroblasts and osteoblasts, PMT acts intracellularly to enhance inositolphospholipid hydrolysis (25, 26), mobilize intracellular Ca 2ϩ pools (40), increase protein phosphorylation (41), stimulate cytoskeletal changes such as stress fiber formation and focal adhesion assembly (5, 21), and initiate DNA synthesis (33). In contrast to the case for fibroblast cells, PMT exerts cytotoxic or cytopathic effects on Vero cells (2, 28), embryonic bovine lung cells (34), and canine osteosarcoma cells (30).PMT exerts its effects on these cellular processes by acting on the free, monomeric ␣ subunit of the G q/11 family of heterotrimeric G-proteins (46), which stimulates phospholipase C (PLC) ␤1 to hydrolyze phosphatidylinositol 4,5-bisphosphate to inositol 1,4,5-trisphosphate (IP 3 ) and diacylglycerol. Accordingly, the releas...

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