A Novel Hypomorphic Apex1 Mouse Model Implicates Apurinic/Apyrimidinic Endonuclease 1 in Oxidative DNA Damage Repair in Gastric Epithelial Cells.

Aims: Though best known for its role in oxidative DNA damage repair, apurinic/apyrimidinic endonuclease 1 (APE1) is a multifunctional protein that regulates multiple host responses during oxidative stress, including the reductive activation of transcription factors. As knockout of the APE1-encoding gene, Apex1, is embryonically lethal, we sought to create a viable model with generalized inhibition of APE1 expression. Results: A hypomorphic (HM) mouse with decreased APE1 expression throughout the body was generated using a construct containing a neomycin resistance (NeoR) cassette knocked into the Apex1 site. Offspring were assessed for APE1 expression, breeding efficiency, and morphology with a focused examination of DNA damage in the stomach. Heterozygotic breeding pairs yielded 50% fewer HM mice than predicted by Mendelian genetics. APE1 expression was reduced up to 90% in the lungs, heart, stomach, and spleen. The HM offspring were typically smaller, and most had a malformed tail. Oxidative DNA damage was increased spontaneously in the stomachs of HM mice. Further, all changes were reversed when the NeoR cassette was removed. Primary gastric epithelial cells from HM mice differentiated more quickly and had more evidence of oxidative DNA damage after stimulation with Helicobacter pylori or a chemical carcinogen than control lines from wildtype mice. Innovation: A HM mouse with decreased APE1 expression throughout the body was generated and extensively characterized. Conclusion: The results suggest that HM mice enable studies of APE1's multiple functions throughout the body. The detailed characterization of the stomach showed that gastric epithelial cells from HM were more susceptible to DNA damage. Antioxid. Redox Signal. 38, 183-197.

PXF-1 promotes synapse development at the neuromuscular junction in Caenorhabditis elegans.

Guanine nucleotide exchange factors (GEFs) are a family of proteins that modulate small G protein signaling. Mutations in a subfamily of GEFs that act on Rap, known as RapGEFs, have been associated with neurological disorders, and knockout mice display impairments in neuronal activity. However, the precise functions of RapGEFs in the nervous system remain unclear. Here, we have used the Caenorhabditis elegans neuromuscular junction, to investigate how the RapGEF homolog, PXF-1, regulates synaptic function. We found that loss of function mutations in pxf-1 reduced cholinergic activity at the neuromuscular junction. We observed that PXF-1 is expressed in the nervous system, and its expression in neurons is sufficient to promote synaptic activity. In pxf-1 mutant animals, there is a reduction in the levels of synaptic vesicles in cholinergic motor neurons but no change in the overall synapse numbers. In addition to synaptic vesicles proteins, we also found that filamentous actin, a scaffold for nascent synapses, was reduced at developing cholinergic synapses in pxf-1 mutant animals. Our studies indicate that PXF-1 regulates neuromuscular function by promoting the formation of actin filaments to support the development of motor neuron synapses.

DNA polymerase gamma (Polγ) deficiency triggers a selective mTORC2 prosurvival autophagy response via mitochondria-mediated ROS signaling.

Autophagy is a highly regulated evolutionarily conserved metabolic process induced by stress and energy deprivation. Here, we show that DNA polymerase gamma (Polγ) deficiency activates a selective prosurvival autophagic response via mitochondria-mediated reactive oxygen species (ROS) signaling and the mammalian target of rapamycin complex 2 (mTORC2) activities. In keratinocytes, Polγ deficiency causes metabolic adaptation that triggers cytosolic sensing of energy demand for survival. Knockdown of Polγ causes mitochondrial stress, decreases mitochondrial energy production, increases glycolysis, increases the expression of autophagy-associated genes, and enhances AKT phosphorylation and cell proliferation. Deficiency of Polγ preferentially activates mTORC2 formation to increase autophagy and cell proliferation, and knocking down Rictor abrogates these responses. Overexpression of Rictor, but not Raptor, reactivates autophagy in Polγ-deficient cells. Importantly, inhibition of ROS by a mitochondria-selective ROS scavenger abolishes autophagy and cell proliferation. These results identify Rictor as a critical link between mitochondrial stress, ROS, and autophagy. They represent a major shift in our understanding of the prosurvival role of the mTOR complexes and highlight mitochondria-mediated ROS as a prosurvival autophagy regulator during cancer development.

Polyubiquitination of apurinic/apyrimidinic endonuclease 1 by Parkin.

Apurinic/apyrimidinic endonuclease 1 (APE1) is an essential protein crucial for repair of oxidized DNA damage not only in genomic DNA but also in mitochondrial DNA. Parkin, a tumor suppressor and Parkinson's disease (PD) associated gene, is an E3 ubiquitin ligase crucial for mitophagy. Although DNA damage is known to induce mitochondrial stress, Parkin's role in regulating DNA repair proteins has not been elucidated. In this study, we examined the possibility of Parkin-dependent ubiquitination of APE1. Ectopically expressed APE1 was degraded by Parkin and PINK1 via polyubiquitination in mouse embryonic fibroblast cells. PD-causing mutations in Parkin and PINK1 abrogated APE1 ubiquitination. Interaction of APE1 with Parkin was observed by co-immunoprecipitation, proximity ligation assay, and co-localization in the cytoplasm. N-terminal deletion of 41 amino acid residues in APE1 significantly reduced the Parkin-dependent APE1 degradation. These results suggested that Parkin directly ubiquitinated N-terminal Lys residues in APE1 in the cytoplasm. Modulation of Parkin and PINK1 activities under mitochondrial or oxidative stress caused moderate but statistically significant decrease of endogenous APE1 in human cell lines including SH-SY5Y, HEK293, and A549 cells. Analyses of glioblastoma tissues showed an inverse relation between the expression levels of APE1 and Parkin. These results suggest that degradation of endogenous APE1 by Parkin occur when cells are stressed to activate Parkin, and imply a role of Parkin in maintaining the quality of APE1, and loss of Parkin may contribute to elevated APE1 levels in glioblastoma. © 2016 Wiley Periodicals, Inc.