A role for vascular deficiency in retinal pathology in a mouse model of ataxia-telangiectasia

Abstract

Ataxia-telangiectasia is a multifaceted syndrome caused by null mutations in the ATM gene, which encodes the protein kinase ATM, a key participant in the DNA damage response. Retinal neurons are highly susceptible to DNA damage because they are terminally differentiated and have the highest metabolic activity in the central nervous system. In this study, we characterized the retina in young and aged Atm-deficient mice (Atm(-/-)). At 2 months of age, angiography revealed faint retinal vasculature in Atm(-/-) animals relative to wild-type controls. This finding was accompanied by increased expression of vascular endothelial growth factor protein and mRNA. Fibrinogen, generally absent from wild-type retinal tissue, was evident in Atm(-/-) retinas, whereas mRNA of the tight junction protein occludin was significantly decreased. Immunohistochemistry labeling for occludin in 6-month-old mice showed that this decrease persists in advanced stages of the disease. Concurrently, we noticed vascular leakage in Atm(-/-) retinas. Labeling for glial fibrillary acidic protein demonstrated morphological alterations in glial cells in Atm(-/-) retinas. Electroretinographic examination revealed amplitude aberrations in 2-month-old Atm(-/-) mice, which progressed to significant functional deficits in the older mice. These results suggest that impaired vascularization and astrocyte-endothelial cell interactions in the central nervous system play an important role in the etiology of ataxia-telangiectasia and that vascular abnormalities may underlie or aggravate neurodegeneration.

Figure 1

Attenuated blood vessels in retinas of Atm^−/− mice. Flat-mount retinas of 2-month-old WT (A, n = 5) and Atm^−/− (B, n = 5) mice imaged with a fluorescent microscope following intracardial perfusion with dextran-fluorescein. Insets show larger magnification of optic nerve region (white frame) and typical peripheral region (yellow frame).

Figure 2

Increased VEGF levels in Atm^−/− mice. Real-time RT-PCR (A) and Western blot (B) analyses reveal significantly higher levels of VEGF in eyecups of Atm^−/− mice relative to VEGF levels in WT control mice. Statistical values were calculated by 2-tailed Student's t-test. *P < 0.05.

Figure 3

Reduced levels of occludin in Atm^−/− mice. A: Real time RT-PCR analysis shows significantly lower levels of occludin in eyecups of Atm^−/− mice relative to WT control mice at 2 months of age. Statistical values were calculated by 2-tailed Student's t-test. *P < 0.0025. B: Confocal images of flat-mount retinas of 6-month-old WT and Atm^−/− mice immunolabeled for CD31 (red) and occludin (green). Cell nuclei are labeled with Sytox Blue (blue). Upper scale bar: 50 µm, lower scale bar: 10 µm.

Figure 4

Increased fibrinogen expression in retinas of Atm^−/− mice. A: Western blot analysis depicts a statistically significant increase of 60% in fibrinogen level in the Atm^−/− retinas in comparison to age-matched controls. Statistical values were calculated by 2-tailed Student's t-test. *P < 0.05. B: Confocal images of flat-mount retinas of mice at 2 months of age show fibrinogen (green) in blood vessels (stained with the pan-endothelial marker CD31, red) of Atm^−/− mice, but no labeling in WT controls. Cell nuclei are stained with Sytox Blue (blue). C: Similar imaging at 6 months of age reveals fibrinogen labeling external to blood vessels in a representative retina from an Atm^−/− mouse. In the WT, fibrinogen labeling is absent or confined to the vessel. Upper scale bar: 50 µm, middle scale bar: 10 µm, lower scale bar: 20 µm.

Figure 5

Vascular leakage in retinas of Atm^−/− mice. Whole-mount retinas of WT and Atm^−/− mice were stained for hemosiderin to indicate deposits resulting from microhemorrhages. The lower panel shows a larger magnification of the area marked with a rectangle in the image of Atm^−/− retina. Blue deposits of hemosiderin were evident in retinas of Atm^−/− mice (several indicated with arrows), but scarcely in retinas of WT mice.

Figure 6

Glial cell alterations in retinas of Atm^−/− mice. Confocal images of flat-mount retinas from WT and Atm^−/− mice, labeled for CD31 (red) and GFAP (green). Insets depict characteristic regions marked with a yellow frame at a larger magnification.

Figure 7

Retinal functional deficits in Atm^−/− mice. A: Representative dark- and light-adapted responses of 6-month-old WT and Atm^−/− mice. B: Average responses of WT (full gray line, circles, n = 5) and Atm^−/− (broken black line, squares, n = 8) mice at 2 months and 6 months (WT n = 5, Atm^−/−n = 5) of age. Average and SE bars are shown for a-wave (open symbols) and b-wave (filled symbols). The a- and b-wave amplitudes were significantly higher in dark-adapted responses of WT mice compared to Atm^−/− mice at all stimulus intensities at 6 months. An asterisk denotes statistically significant amplitude difference at individual stimulus intensities in the graph of light-adapted responses. Statistical values were calculated by 2-tailed Student's t-test. *P < 0.05.