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. 2010 Dec 3;285(49):38534-42.
doi: 10.1074/jbc.M110.145896. Epub 2010 Oct 2.

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain

Nnennaya Kanu  1 , Kay Penicud, Mariya Hristova, Barnaby Wong, Elaine Irvine, Florian Plattner, Gennadij Raivich, Axel Behrens
Affiliations

The ATM cofactor ATMIN protects against oxidative stress and accumulation of DNA damage in the aging brain

Nnennaya Kanu et al. J Biol Chem. .

Abstract

Progressive accumulation of DNA damage is causally involved in cellular senescence and organismal aging. The DNA damage kinase ATM plays a central role in maintaining genomic stability. ATM mutations cause the genetic disorder ataxia telangiectasia, which is primarily characterized by progressive neurodegeneration and cancer susceptibility. Although the importance of ATM function to protect against oxidative DNA damage and during aging is well described, the mechanism of ATM activation by these stimuli is not known. Here we identify ATM interactor (ATMIN) as an essential component of the ATM signaling pathway in response to oxidative stress and aging. Embryos lacking ATMIN (atmin(Δ/Δ)) died in utero and showed increased numbers of cells positive for phosphorylated histone H2aX, indicative of increased DNA damage. atmin(Δ/Δ) mouse embryonic fibroblasts accumulated DNA damage and prematurely entered senescence when cultured at atmospheric oxygen levels (20%), but this defect was rescued by addition of an antioxidant and also by culturing cells at physiological oxygen levels (3%). In response to acute oxidative stress, atmin(Δ/Δ) mouse embryonic fibroblasts showed slightly lower levels of ATM phosphorylation and reduced ATM substrate phosphorylation. Conditional deletion of ATMIN in the murine nervous system (atmin(ΔN)) resulted in reduced numbers of dopaminergic neurons, as does ATM deficiency. ATM activity was observed in old, but not in young, control mice, but aging-induced ATM signaling was impaired by ATMIN deficiency. Consequently, old atmin(ΔN) mice showed accumulation of DNA damage in the cortex accompanied by gliosis, resulting in increased mortality of aging mutant mice. These results suggest that ATMIN mediates ATM activation by oxidative stress, and thereby ATMIN protects the aging brain by preventing accumulation of DNA damage.

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Figures

FIGURE 1.
FIGURE 1.
Accumulation of DNA damage in ATMIN-deficient embryos and cells. A, H&E staining of sagittal sections through E12.5 ATMIN+/+ and ATMINΔ/Δ embryonic brains and high magnification of the indicated region. Lower panel shows pS139-γH2aX staining of a representative region and the histogram shows quantification of cells with pS139-γH2aX foci staining, n = 3. B, ATMIN+/+ and ATMINΔ/Δ fibroblasts were cultured at 3 or 20% oxygen for either 2 (P2) or 4 (P4) passages and a growth curve was performed to monitor cell proliferation. C, cells were cultured as above for 4 passages followed by fixation with 4% PFA and staining for pS139-γH2aX. The percentage of positive cells was quantitated.
FIGURE 2.
FIGURE 2.
ATMINΔ/Δ fibroblasts are highly susceptible to oxidative damage and exhibit aberrant DNA damage signaling. A, ATMIN+/+ and ATMINΔ/Δ fibroblasts were cultured at 3 or 20% oxygen for 1 passage (P1) or up to 4 passages (P4). Cells were fixed and stained for senescence-associated β-galactosidase. The histogram shows quantification of staining. B, ATMIN+/+ and ATMINΔ/Δ fibroblasts were treated as in A for 48 h following which cells were harvested and proteins were blotted for p21. C, ATMIN+/+ and ATMINΔΔ fibroblasts were treated as in A for 4 passages either in the absence or presence of 100 μm NAC. Cells were fixed and stained with senescence-associated β-galactosidase and the percentage of positive cells was counted. The histogram shows quantification from two experiments. D, ATMIN+/+ and ATMINΔΔ fibroblasts were cultured at 20% oxygen for 4 passages followed by fixation with 4% PFA and staining with pS139-γH2aX and pS1987-ATM. Colocalizing and noncolocalizing foci are indicated by yellow and white arrowheads, respectively. The histogram shows quantification of the percentage of pS139-γH2aX-positive cells that do not have colocalizing pS1987-ATM foci. E, ATMIN-deficient fibroblasts display reduced DNA damage signaling in response to treatment with paraquat. Cells were treated with 10 μm paraquat for 10 h and cell extracts were blotted for pS1987-ATM, ATM, pS824-Kap1, Kap1, pS15-P53, P53, pT68-Chk2, Chk2, and β-actin.
FIGURE 3.
FIGURE 3.
Characterization of ATMINΔN mice. A, in situ hybridization for ATMIN demonstrates its normal localization throughout the cortex, hippocampus, and dentate gyrus. The signal is significantly diminished in the brains of ATMINΔN mice. B, representative pictures of ATMINF/F and ATMINΔN mice at 3 months of age demonstrating severe growth retardation. C, ATMINΔN mice exhibit reduced body weight from birth. A cohort of ATMINF/F and ATMINΔN mice were weighed weekly over a period of 7 weeks. n = at least 10 mice per genotype. D, Kaplan-Meier curve for survival of ATMINΔN mice over a period of 18 months. E, sections through the substantia nigra pars compacta of ATMINF/F and ATMINΔN mice were stained with anti-tyrosine hydroxylase antibody to measure the number of dopaminergic neurons in this region. The density of tyrosine hydroxylase-positive cells was counted and corrected using Abercrombie correction.
FIGURE 4.
FIGURE 4.
Increased neurodegeneration in ATMINΔN brains. A, H&E stain of coronal sections through the midbrain of 18-month-old ATMINF/F and ATMINΔN mice showing similar morphology. B, sections from 2- and 18-month-old ATMINF/F and ATMINΔN mice were stained with NeuN to quantify neuronal density. The percentage loss in neuronal density through the cortex of ATMINF/F and ATMINΔN mice was calculated. C, sections from 18-month-old mice were stained with anti-glial fibrillary acidic protein antibody to measure astrocytic gliosis and with anti-Iba1 antibody for activated microglia (D). All numbers were corrected using the Abercrombie method. The histograms represent the quantification of staining from three mice.
FIGURE 5.
FIGURE 5.
Aging related DNA damage signaling in ATMIN-deficient mice. Mice were sacrificed at 2 (A) or 18 months (B) of age and coronal sections through the cortex were cut. Representative sections were stained with pS1987-ATM, pS957-SMC1, pS139-γH2aX, and p53 and the percentage of positive cells in an equivalent area was counted. Cells positive for pS1987-ATM are highlighted with red arrowheads. The histograms represents the mean percentage of positive cells from three to five mice. C, the brains of the aged mice in B were lysed and proteins were blotted for pS1987-ATM, ATM, pS957-SMC1, SMC1, pS15-p53, p53 and β-actin.
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