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Edith Arnold, Patrice Fort, Steven Abcouwer, Thomas Gardner; Role of Autophagy in Diabetic Retinopathy and Metabolic Stress. Invest. Ophthalmol. Vis. Sci. 2013;54(15):3688.
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© ARVO (1962-2015); The Authors (2016-present)
Metabolic stress such as growth factor and nutrient deprivation participate in neuronal cell death in the retina in diabetes. Autophagy contributes to cell energy homeostasis and repair during basal and nutrient stress conditions and its malfunction contributes to the pathogenesis of human neuronal disorders. In this work we investigated if autophagy is altered in diabetic retinopathy in response to the metabolic stress.
Retinas from 4, 6 and 8 months old diabetic Ins2Akita and control mice were harvested. R28 retinal neuronal cells were subjected to 2h serum and/or amino acids (AA) deprivation with and without chloroquine (CQ, 10μM), an autophagy inhibitor, to evaluate the role of autophagy in the response of retinal neuronal cells to metabolic stress. Retinal homogenates and cell lysates were analyzed by Western blot and the content of autophagy proteins LC3 I and II was determined. In addition Atg5, caspase-3 and PARP cleavage products were immunodetected in the R28 cell lysates. R28 cell death was measured using a DNA fragmentation ELISA.
The ratio of LC3-II/LC3-I increased in the retinas of 6 months old diabetic Ins2Akita mice compared to control retinas, while no change was observed in the retinas of 4 and 8 months old diabetic Ins2Akita mice. Total LC3 (I+II) content was significantly decreased in the retinas of 8 months old diabetic Ins2Akita mice relative to the control retinas. Serum deprivation of R28 cells significantly reduced the content of LC3 and Atg5 and increased apoptotic cell death compared to control conditions, while AA deprivation alone did not produce these effects. However, when serum deprivation of R28 cells was accompanied by AA deprivation, apoptotic cell death and the amount of cleaved caspase-3 and cleaved PARP were significantly enhanced compared to serum deprivation alone. Moreover, inhibition of autophagy with CQ significantly exacerbated apoptotic cell death and the content of apoptotic proteins in serum-starved and AA-deprived cells.
These results show that autophagy is altered in the diabetic retina during the progression of diabetes. This study also demonstrates that the combination of growth factor and AA deprivation leads to neuronal cell death and reveal the protective role of autophagy in R28 retinal neuronal cells during metabolic stress. Autophagy modulation may represent a novel therapeutic strategy to prevent neurodegeneration in diabetic retinopathy.
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