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Joshua Hirt, Angela Dixon, Stuart J McKinnon, Paloma Borrajo Liton; Autophagy in the Neuroretina and Angle Region of DBA/2J Mice. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):2449.
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© ARVO (1962-2015); The Authors (2016-present)
To investigate the activation of autophagy in the neuroretina and angle region tissues of DBA/2J mice.
DBA/2J::GFP-LC3 mice were obtained by cross-breeding GFP-LC3 with DBA/2J mice and backcrossed 10 times prior to experimental analysis. DBA/2J, DBA/2J::GFP-LC3, and their controls (C57BL/6 and GFP-LC3, n=10 per group) were aged to 12-months. IOP was measured at monthly basis using a rebound tonometer. Optic nerves were fixed with 4% PFA and resin-embedded for axon counts and electron microscopy. Tissues used for WB and qPCR analysis were immediately processed for protein or mRNA isolation, respectively. Protein expression levels of LC3B, Lamp1, and p62 were evaluated by WB and immunofluoresce. mRNA expression levels of Atg genes were quantified by qPCR. RGC count was performed in whole-flat mounts of neuroretinas using an anti-Brn3a antibody.
DBA/2J demonstrated higher cumulative IOP compared to C57BL/6 (412.6±65.44 vs. 235.7±8.159 mmHg/month, p=0.02), reaching a peak around 9 months of age. DBA/2J::GFP-LC3 showed higher cumulative IOP at younger ages compared to DBA/2J (429.6±69.28 vs. 391.4±54.14 mmHg/month). DBA/2J exhibited a decrease in RGC (390±115 vs 592±98 RGC/mm2) and axon number (45654±11187 vs 55912±6171 axons) compared to C57BL/6. The decrease in RGC and axon count was more pronounced in DBA/2J::GFP-LC3 (197.2±52.1RGC/mm2, 20384±3850 axons). Interestingly, electron micrographs showed the abundance of autophagic bodies in the axons of DBA/2J::GFP-LC3, which were not observed in GFP-LC3 mice. Immunofluorescence analysis demonstrated a qualitatively lower level of p62 expression in the RGC layer and decreased number of GFP-LC3 labeled RGC cells in DBA/2J mice. Decreased LC3-II was also detected by WB in the neuroretina of DBA/2J mice, together with a significant lower amount of LAMP1. In contrast, elevated LC3 and p62 protein levels were observed in the angle region. No significant mRNA changes were detected, with the exception of mTOR, which was upregulated 3.1±0.14 fold in the retina of DBA/2J mice.
Taken together, our results show (1) that autophagy is activated in the angle region and neuroretina tissues of DBA/2J compared to age-matched C57BL/6 controls, indicating that autophagy is a cellular mechanism triggered in response to elevated IOP; and (2) that over-activation of autophagy in vivo may contribute to the RGC loss and neurodegeneration in glaucoma.
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