The principal objective of this research proposal is to test the hypothesis that cerebral hypoxia causes immediate, measurable damage to mitochondrial and nuclear DNA, and activates DNA repair in the rat brain. In humans, cerebral hypoxia is a component of severe brain insults, including trauma, stroke and perinatal asphyxia. These insults may cause long term neuropathological and developmental deficits. We propose to address the premise that DNA damage and efficiency of DNA repair are among the early determinants of cellular survival in the brain, after hypoxic/ischemic injury. To date, DNA damage, as a direct measurable event, has not been addressed in cerebral hypoxia. Likewise, activation of genes involved in DNA repair and correlation of their expression with cell survival or cell death in the brain has not been examined. Our preliminary findings show that hypoxia induces immediate DNA damage, AP-endonuclease (APE), DNA polymerase b and the mammalian homologue of MutY (MYH) glycosylase are activated. Dissimilar repair kinetics for nuclear and mitochondrial DNA are also observed following hypoxia. Furthermore, with hypoxic insult, MYH appears to translocate into the mitochondria. This is the first evidence, to our knowledge, suggestive of activation of DNA repair in the mitochondria after cerebral hypoxia.
Our Specific Aims are: 1) To measure hypoxia-induced damage and repair in nuclear and mitochondrial DNA and to determine the dose response relationship between severity of hypoxia and the extent and distribution of DNA damage. 2) To map in situ, in specific cells populations, distribution of hypoxia-induced DNA damage. 3) To characterize the hypoxia-induced DNA damage response: mechanisms of activation of DNA damage repair enzymes and sensing molecules. Two sensitive Quantitative PCR (QPCR) assays are used to measure DNA damage. One measures the integrity of the mitochondrial genome and the other measures the integrity of nuclear DNA. The later is a novel assay, developed in our laboratory, which utilizes the high copy number of Short Interspersed DNA Elements (SINEs) mammalian genomes, to simultaneously amplify long random segments of nuclear DNA. Due to the high magnitude of attainable signal, we have adapted this assay for in situ detection of DNA damage and repair in specific cell populations in the brain. Our studies are intended to help understand the relevance of DNA damage to neuronal survival and, whether the DNA repair response is consequential to preservation and survival of neuronal cells. This information will augment strategies seeking to reduce neuropathologies of brain hypoxia in humans.

Agency
National Institute of Health (NIH)
Institute
National Institute of Neurological Disorders and Stroke (NINDS)
Type
Research Project (R01)
Project #
5R01NS039449-02
Application #
6394288
Study Section
Special Emphasis Panel (ZRG1-BDCN-3 (01))
Program Officer
Jacobs, Tom P
Project Start
2000-09-01
Project End
2004-05-31
Budget Start
2001-06-01
Budget End
2002-05-31
Support Year
2
Fiscal Year
2001
Total Cost
$298,000
Indirect Cost
Name
University of Texas Medical Br Galveston
Department
Surgery
Type
Schools of Medicine
DUNS #
041367053
City
Galveston
State
TX
Country
United States
Zip Code
77555
Gorgun, Falih Murat; Zhuo, Ming; Singh, Shilpee et al. (2014) Neuroglobin mitigates mitochondrial impairments induced by acute inhalation of combustion smoke in the mouse brain. Inhal Toxicol 26:361-9
Singh, Shilpee; Zhuo, Ming; Gorgun, Falih M et al. (2013) Overexpressed neuroglobin raises threshold for nitric oxide-induced impairment of mitochondrial respiratory activities and stress signaling in primary cortical neurons. Nitric Oxide 32:21-8
Englander, Ella W (2013) DNA damage response in peripheral nervous system: coping with cancer therapy-induced DNA lesions. DNA Repair (Amst) 12:685-90
Singh, Shilpee; Englander, Ella W (2012) Nuclear depletion of apurinic/apyrimidinic endonuclease 1 (Ape1/Ref-1) is an indicator of energy disruption in neurons. Free Radic Biol Med 53:1782-90
Lee, Heung Man; Greeley Jr, George H; Englander, Ella W (2011) Transgenic overexpression of neuroglobin attenuates formation of smoke-inhalation-induced oxidative DNA damage, in vivo, in the mouse brain. Free Radic Biol Med 51:2281-7
Deng, Xiaoling; Vidal, Ruben; Englander, Ella W (2010) Accumulation of oxidative DNA damage in brain mitochondria in mouse model of hereditary ferritinopathy. Neurosci Lett 479:44-8
Lee, Heung M; Hallberg, Lance M; Greeley Jr, George H et al. (2010) Differential inhibition of mitochondrial respiratory complexes by inhalation of combustion smoke and carbon monoxide, in vivo, in the rat brain. Inhal Toxicol 22:770-7
Li, Hui; Swiercz, Rafal; Englander, Ella W (2009) Elevated metals compromise repair of oxidative DNA damage via the base excision repair pathway: implications of pathologic iron overload in the brain on integrity of neuronal DNA. J Neurochem 110:1774-83
Lee, Heung M; Reed, Jason; Greeley Jr, George H et al. (2009) Impaired mitochondrial respiration and protein nitration in the rat hippocampus after acute inhalation of combustion smoke. Toxicol Appl Pharmacol 235:208-15
Englander, Ella W (2008) Brain capacity for repair of oxidatively damaged DNA and preservation of neuronal function. Mech Ageing Dev 129:475-82

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