Impairment of Autophagy Causes Superoxide Formation and Caspase Activation in 661 W Cells, a Cell Line for Cone Photoreceptors, under Hyperglycemic Conditions

Abstract

Microvascular changes are the earliest adverse events in diabetic retinopathy, but recent studies have shown that oxidative stress induced by photoreceptors is associated with the development of the retinopathy. The purpose of this study was to determine the roles played by superoxides formed by photoreceptors under hyperglycemic conditions on autophagy. To accomplish this, we cultured 661 W cells, a transformed murine cone cell line, with 5.5 or 25 mM glucose in the presence or absence of 3 methyl adenine (3MA) or rapamycin. The superoxides were determined by flow cytometry using hydroethidine as a fluorescence probe. The autophagy activity was determined by changes in the expression of LC3B2 and P62 by immunoblotting. The degree of mitophagy was determined by the accumulation of mitochondria and lysosomes. Apoptotic changes of 661 W cells were determined by the caspase 3/7 activities. Our results showed higher levels of P62 and superoxides in cells cultured in 25 mM glucose than in 5.5 mM glucose. Addition of 3MA caused a significant increase of P62, superoxides, and caspase 3/7 activities in the 661 W cells cultured in high glucose but not in low glucose. These findings suggest that autophagy is important for the functioning and survival of 661 W cells under hyperglycemic conditions.

Autophagy: A Novel Pharmacological Target in Diabetic Retinopathy

“…In a recent study, Taki and co-workers (2020), using 661W cells, a transformed murine cone cell line, observed that HG treatment (25 mM, 48 h) induced changes in mitophagy and autophagy with mitochondria accumulation and upregulation of p62. Treatment with 3-MA caused a greater increase of p62, superoxides and caspase 3/7 activation suggesting that impairment of the autophagy pathway correlates with superoxide formation and induction of apoptosis (Taki et al, 2020).…”

Diabetic retinopathy: Involved cells, biomarkers, and treatments

“…Although the mechanism by which autophagy protects photoreceptors and the relationship between DR and autophagy are ambiguous, more recent studies have corroborated the importance of autophagic flux. For example, impairment of autophagy increases superoxide formation and apoptosis in 661 W cells (a cone cell line) under hyperglycemic conditions ( Taki et al, 2020 ).…”

mTOR Signalling Pathway: A Potential Therapeutic Target for Ocular Neurodegenerative Diseases

“…Abbreviations: AMD, age-related macular degeneration; CNV, choroidal neovascularization; EGCG, epigallocatechin gallate; HG, high glucose; GA, geographic atrophy; MMXM, Mingmu Xiaomeng; OCT, octreotide; OPTN, optineurin; RGC, retinal ganglion cell; rMCs, retinal Müller cells. References in the figure: 1992 [ 55 ], 1990 [ 60 ], 1991 [ 1 ], 1997 [ 63 ], 2002 [ 61 ], 2009 [ 159 ], 2010 * [ 162 ], 2010 ** [ 161 ], 2013 * [ 160 ], 2013 ** [ 123 ], 2014 * [ 146 ], 2014 ** [ 16 ], 2015 * [ 44 ], 2015 ** [ 94 ], 2016 * [ 120 ], 2016 ** [ 83 ], 2018 [ 89 ], 2019 * [ 19 ], 2019 ** [ 136 ], 2021 * [ 153 ], 2021 ** [ 28 ], 2021 *** [ 91 ].…”