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Anna Kyunglim Park, D J Sidjanin; Functional loss of TBC1D20 disrupts autophagy resulting in cataracts. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):4839.
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© ARVO (1962-2015); The Authors (2016-present)
Mutations in TBC1D20 cause congenital cataracts in Warburg Micro syndrome patients and blind-sterile (bs) mice. TBC1D20 is an ER associated protein that functions as a GTPase activating protein (GAP) enhancing the rate of GTP hydrolysis when bound to RAB1. How functional loss of TBC1D20 results in congenital cataracts in humans and mice has not yet been established. RAB1, the TBC1D20 substrate, is an established regulator of autophagy, a cellular process required for lens development and transparency. As a part of this study, we set out to evaluate if the functional loss of TBC1D20 disrupts lens autophagy.
Lenses from WT and bs mice were evaluated using slit lamp biomicroscopy, H&E, immunohistochemistry and western blotting. For evaluation of autophagy, primary mouse embryonic fibroblasts (MEFs) were generated from WT and bs embryos on the same genetic background and subsequently analyzed with transmission electron microscopy (TEM), western blotting, as well as molecular and immunocytochemical approaches.
bs lenses exhibited an accumulation of p62 suggesting disrupted autophagy. bs MEFs also exhibited an accumulation of p62, but also exhibited an accumulation of organelles constitutively turned over by autophagy. Additionally, an accumulation of LC3 positive structures in bs MEFs suggested a defect in maturation of autophagic membranes. TEM evaluation of bs MEFs revealed an accumulation of multi-lamellar, enclosed structures with electron-light contents indicating a failure in the formation of autolysosomes. bs lenses did not alter the organelle degradation process of maturing lens fiber cells, but resulted in an accumulation of TUNEL-positive and ubiquitin-positive debris in bs lens fiber cells.
Our results show that TBC1D20 mediates the formation of amphisomes. TBC1D20 functional loss results in an accumulation of autophagosomes that fail to fuse with lysosomes resulting in disruption of the autophagic flux. In the lens, TBC1D20-mediated autophagy is indispensable for lens development and transparency by mediating the removal of the cellular debris most likely generated by the organelle degradation process during the terminal lens fiber cell differentiation process. Current studies focus on establishing if treatments of bs mice with autophagic stimulators can rescue disrupted autophagy and consequently rescue or delay the onset of cataracts in bs mice.
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