Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts
Hao Yang, … , Wei Li, Xingchao Shentu
Hao Yang, … , Wei Li, Xingchao Shentu
Published September 17, 2024
Citation Information: J Clin Invest. 2024;134(18):e169666. https://doi.org/10.1172/JCI169666.
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Research Article Ophthalmology

Reversible cold-induced lens opacity in a hibernator reveals a molecular target for treating cataracts

Abstract

Maintaining protein homeostasis (proteostasis) requires precise control of protein folding and degradation. Failure to properly respond to stresses disrupts proteostasis, which is a hallmark of many diseases, including cataracts. Hibernators are natural cold-stress adaptors; however, little is known about how they keep a balanced proteome under conditions of drastic temperature shift. Intriguingly, we identified a reversible lens opacity phenotype in ground squirrels (GSs) associated with their hibernation-rewarming process. To understand this “cataract-reversing” phenomenon, we first established induced lens epithelial cells differentiated from GS-derived induced pluripotent stem cells, which helped us explore the molecular mechanism preventing the accumulation of protein aggregates in GS lenses. We discovered that the ubiquitin-proteasome system (UPS) played a vital role in minimizing the aggregation of the lens protein αA-crystallin (CRYAA) during rewarming. Such function was, for the first time to our knowledge, associated with an E3 ubiquitin ligase, RNF114, which appears to be one of the key mechanisms mediating the turnover and homeostasis of lens proteins. Leveraging this knowledge gained from hibernators, we engineered a deliverable RNF114 complex and successfully reduced lens opacity in rats with cold-induced cataracts and zebrafish with oxidative stress–related cataracts. These data provide new insights into the critical role of the UPS in maintaining proteostasis in cold and possibly other forms of stresses. The newly identified E3 ubiquitin ligase RNF114, related to CRYAA, offers a promising avenue for treating cataracts with protein aggregates.

Authors

Hao Yang, Xiyuan Ping, Jiayue Zhou, Hailaiti Ailifeire, Jing Wu, Francisco M. Nadal-Nicolás, Kiyoharu J. Miyagishima, Jing Bao, Yuxin Huang, Yilei Cui, Xin Xing, Shiqiang Wang, Ke Yao, Wei Li, Xingchao Shentu

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Figure 6

RNF114 reverses cataracts in non-hibernators.

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RNF114 reverses cataracts in non-hibernators. (A and B) CCK8 assay was p...
(A and B) CCK8 assay was performed to evaluate cell viability of RNF114-knockin or -knockdown HLECs and GS iLECs under low-temperature treatment, followed by statistical analysis (n = 3 independent experiments). (C) Immunofluorescence images depict localization of TAT-RNF114 in HLECs and rat lenses after 30 minutes of TAT-RNF114 pretreatment. Scale bars: 20 μm. Rat lens slices: original magnification, ×4. (D) Live-cell imaging showing intracellular localization of GFP-CRYAA(Y118D) in HLECs after 30 minutes of pretreatment. GFP–WT CRYAA: treated with TAT-RNF114 (100 μM); GFP-CRYAA(Y118D): treated with TAT-RNF114 (100 μM) or TAT (100 μM). Red arrows indicate typical protein aggregate degradation. Intensity of intracellular fluorescence spots was also quantified (2-tailed Student’s t tests followed by Holm-Šidák correction, n = 3, 5 fields of view per experiment). (E) Photographs of dissected rat lenses pretreated with TAT (100 μM) or TAT-RNF114 (100 μM) (awake group, 4°C for 24 hours, and rewarmed at 37°C for 30 minutes). For TAT and TAT-RNF114 treatments: TAT or TAT-RNF114 was added to the culture medium containing dissected rat lenses in the last 30 minutes of low-temperature treatment. Control and TAT groups: bottom light source imaging; TAT-RNF114: both bottom and side light source imaging. Red arrows indicate noticeable lens opacities. Original magnification, ×4. Quantitative analysis of relative light transmittance in dissected lenses (2-tailed Student’s t tests followed by Holm-Šidák correction, 4 lenses per group). (F) TAT (100 μM), TAT-RNF114ΔC (100 μM), or TAT-RNF114 (100 μM) was added to the live zebrafish culture dish induced with H2O2 to observe lenses after 0 and 12 hours of drug treatment under a Leica DM8000 microscope. Scale bar: 1.0 mm. Original magnification, ×2. Qualitative scoring of zebrafish lens transparency improvement based on LOCS III grading system through visual analysis, and statistical representation showing changes in lens transparency for each zebrafish (2-tailed Student’s t test, 1 eye from 5 zebrafish per group). (All values are presented as mean ± SD, *P < 0.05, **P < 0.01, ***P < 0.001.)