Antioxidants modulate mitochondrial PKA and increase CREB binding to D-loop DNA of the mitochondrial genome in neurons

Abstract

The protein kinase A (PKA) and the cAMP response element (CRE) binding protein (CREB) signaling pathways mediate plasticity and prosurvival responses in neurons through their ability to regulate gene expression. The PKA-CREB signaling mechanism has been well characterized in terms of nuclear gene expression. We show that the PKA catalytic and regulatory subunits and CREB are localized to the mitochondrial matrix of neurons. Mitochondrial CRE sites were identified by using both serial analyses of chromatin occupancy and chromatin immunoprecipitation. Deferoxamine (DFO), an antioxidant and iron chelator known to inhibit oxidative stress-induced death, activated mitochondrial PKA and increased mitochondrial CREB phosphorylation (Ser-133). DFO increased CREB binding to CRE in the mitochondrial D-loop DNA and D-loop CRE-driven luciferase activity. In contrast, KT5720, a specific inhibitor of PKA, reduced DFO-mediated neuronal survival against oxidative stress induced by glutathione depletion. Neuronal survival by DFO may be, in part, mediated by the mitochondrial PKA-dependent pathway. These results suggest that the regulation of mitochondrial function via the mitochondrial PKA and CREB pathways may underlie some of the salutary effects of DFO in neurons. P rotein kinase A (PKA) and cAMP response element (CRE) binding protein (CREB) play important roles in neuronal plasticity by altering expression of target genes in response to environmental stimuli (1-3). The PKA and CREB pathway is involved in neuronal survival of the central and peripheral nervous systems (4, 5). PKA is a tetramer consisting of two regulatory (Reg) and two catalytic (Cat) subunits. The binding of cAMP to a Reg subunit dissociates the holoenzyme into two free Cat subunits, which induce the phosphorylation of cellular substrates and a Reg subunit dimer. PKA phosphorylates CREB at Ser-133. Phosphorylated CREB (pCREB) subsequently binds to CRE and recruits the CREB binding protein, a transcriptional coactivator, to activate gene expression (6-8). It has been shown that PKA and CREB molecules localize into subcellular organelles (8-13). Interestingly, PKA-anchoring proteins in dendrites have been shown to tether PKAII-␣ to the outer membrane of mitochondria (8). Furthermore, the localization of the Cat subunit of PKA, and Reg subunit I (RI) or II (RII), has been found in the mitochondrial matrix (6,(9)(10)(11)(12). CREB is also present in the mitochondria of the adult rat brain (7). Mitochondrial CREB has been implicated in plasticitydependent changes associated with behavioral training (8). Thus, the localization of PKA and CREB in the mitochondria raises the possibility that they may play a role in neuronal plasticity and cell survival by regulating mitochondrial function via the mitochondrial PKA and CREB-dependent pathway (13).Deferoxamine (DFO), an iron chelator and antioxidant, is neuroprotective, reducing the level of toxic radical species such as redox-reactive irons (14, 15). We have previously shown that neuronal protection from oxidative st...

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“…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).…”

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ATRX induction by mutant huntingtin via Cdx2 modulates heterochromatin condensation and pathology in Huntington's disease

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