ATM localization and gene expression in the adult mouse eye
| Autor: | Leemput, J., Masson, C., Bigot, K., Errachid, A., Dansault, A., Provost, A., Gadin, S., Said AOUFOUCHI, Menasche, M., Abitbol, M. |
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| Jazyk: | angličtina |
| Rok vydání: | 2009 |
| Předmět: |
Cytoplasm
Gene Expression Cell Cycle Proteins Ataxia Telangiectasia Mutated Proteins Protein Serine-Threonine Kinases Eye Retina Cornea Histones Mice Cerebellum Lens Crystalline Animals RNA Messenger In Situ Hybridization Cell Nucleus Microscopy Confocal Reverse Transcriptase Polymerase Chain Reaction Tumor Suppressor Proteins Ciliary Body Deoxyguanosine Immunohistochemistry DNA-Binding Proteins Mice Inbred C57BL Oxidative Stress 8-Hydroxy-2'-Deoxyguanosine Research Article DNA Damage |
| Zdroj: | Molecular Vision Scopus-Elsevier |
| ISSN: | 1090-0535 |
| Popis: | Purpose High levels of metabolism and oxygen consumption in most adult murine ocular compartments, combined with exposure to light and ultraviolet (UV) radiation, are major sources of oxidative stress, causing DNA damage in ocular cells. Of all mammalian body cells, photoreceptor cells consume the largest amount of oxygen and generate the highest levels of oxidative damage. The accumulation of such damage throughout life is a major factor of aging tissues. Several multiprotein complexes have recently been identified as the major sensors and mediators involved in the maintenance of DNA integrity. The activity of these complexes initially seemed to be restricted to dividing cells, given their ultimate role in major cell cycle checkpoints. However, it was later established that they are also active in post-mitotic cells. Recent findings demonstrate that the DNA damage response (DDR) is essential for the development, maintenance, and normal functioning of the adult central nervous system. One major molecular factor in the DDR is the protein, ataxia telangiectasia mutated (ATM). It is required for the rapid induction of cellular responses to DNA double-strand breaks. These cytotoxic DNA lesions may be caused by oxidative damage. To understand how ATM prevents oxidative stress and participates in the maintenance of genomic integrity and cell viability of the adult retina, we determined the ATM expression patterns and studied its localization in the adult mouse eye. Methods Atm gene expression was analyzed by RT–PCR experiments and its localization by in situ hybridization on adult mouse ocular and cerebellar tissue sections. ATM protein expression was determined by western blot analysis of proteins homogenates extracted from several mouse tissues and its localization by immunohistochemistry experiments performed on adult mouse ocular and cerebellar tissue sections. In addition, subcellular localization was realized by confocal microscopy imaging of ocular tissue sections, with a special focus on retinal cells. Results Using RT–PCR, we detected a band of the expected size, with its sequence matching the amplified Atm cDNA sequence. Atm mRNA was detected in most cell bodies of the adult mouse eye by in situ hybridization of ocular tissue sections with specific digoxigenin-labeled PCR-amplified cDNA probes. Western blotting with different specific antibodies revealed bands corresponding to the expected sizes of ATM and its active forms (ATMp). These bands were not observed in the analysis of protein homogenates from Atm-deficient mouse tissues. ATM immunoreactivity was detected in the nucleus of all adult mice retinal cells and in most non-neuronal ocular cell types. The active phosphorylated form of ATM was also present in the retina as well as in non-neuronal cells of the adult mouse eye. However, its subcellular localization differed as a function of the cell type examined. A major finding of this study was that ATMp immunostaining in photoreceptor cells was exclusively in the cytoplasm, whereas ATM immunostaining was only in the nucleus of these cells. Furthermore, the specific and distinct ATM and ATMp immunolabeling patterns in photoreceptor cells were identical to those observed in the adult mouse cerebellar granule cells. Conclusions We report the expression profile of Atm gene and protein in the adult mouse eye. In particular, we observed a difference between the localization patterns of the active and inactive forms of ATM in photoreceptor cells. These localization patterns suggest that ATM and its phosphorylated activated form may be involved in both the protection of cells from oxidative damage and the maintenance of ocular cell structure and function. The protection mechanisms mediated by the two forms of ATM appear to be particularly important in maintaining photoreceptor integrity. |
| Databáze: | OpenAIRE |
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