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S Amer Riazuddin, Shahid Yar Khan, Muhammad Ali, Firoz Kabir, Chan Hyun Na, Yinghong Ma, Caihong Qiu, Muhammad Asif Naeem, Sheikh Riazuddin, J Fielding Hejtmancik; The physiological significance of autophagy in lens morphogenesis. Invest. Ophthalmol. Vis. Sci. 2019;60(9):2232.
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© ARVO (1962-2015); The Authors (2016-present)
We previously reported multiple mutations in FYCO1, an autophagy-associated gene responsible for congenital cataracts. The current study was performed to investigate the physiological significance of autophagy in lens morphogenesis.
We developed Fyco1 knockout (Fyco1-/-) mice and examined Fyco1-/- lens at different developmental time points. Next, we developed a CRISPR-mediated knock-in (KI) human lens epithelial (HLE) cells and human embryonic stem cells (hESCs) harboring the FYCO1 mutation (c.2206C>T, p.Q736*). Wild-type (Wt) and KI hESCs were differentiated into lentoid bodies (LB) using the “fried egg” method. Mass spectrometry was performed to identify the differentially expressed proteins in Fyco1-/- lens and KI LB. Western blot was performed to estimate the cellular mass of endoplasmic reticulum (ER) in Fyco1-/- lens and KI LB.
The Fyco1-/- mouse lens recapitulated the cataractous phenotype seen in patients harboring mutations in FYCO1 with persistent nuclei in Fyco1-/- lens fiber cells. Mass spectrometry detected differential expression of autophagy-associated proteins including ATG3, ATG7, ATG12, WIPI1, WIPI2, OPTN, p62, and mTOR1 (p>0.05) in Fyco1-/- lens while examination of the autophagy flux suggested a decreased autophagy in RFP-EGFP-Map1lc3b/Fyco1-/- lens. Next, we investigated endogenous levels of p62, pH-sensitive localization of GFP-LC3, and CYTO-ID-based detection of autophagic vesicles in KI HLE cells, which confirmed decreased autophagy consistent with the data from Fyco1-/- lens. In parallel, differentiation of KI hESCs revealed abridged autophagy with accumulated organelles while proteome analysis identifed accumulation of autophagy-related proteins including p62 (~2-fold: p>0.05) in the KI LB. Importantly, ERGIC1 and ERGIC3, proteins known to be associated with ER-Golgi intermediate compartments were significantly (p>0.01) accumulated in both Fyco1-/- lens and KI LB. Western blot analysis demonstrated a 1.6- and 1.5-fold increased ER mass in Fyco1-/- lens and KI LB, respectively confirming results from the proteome data sets.
Our data strongly suggest that autophagy is important for organelle degradation in the developing lens and while lack thereof may not completely inhibit the formation of the organelle-free zone, belated organelle degradation due to impaired autophagy is physiologically untenable and results in cataractogenesis.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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