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Alexandra Fuller, Shu-Yu Wu, Sanjay Mishra, Ping Zou, Hassane S Mchaourab; Destabilized human γD-crystallins form aggregation in the zebrafish lens. Invest. Ophthalmol. Vis. Sci. 201657(12):.
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© ARVO (1962-2015); The Authors (2016-present)
The precise refractive properties of the lens are achieved, primarily, through the proper packing of crystallins. Cataract formation is thought to arise when resident lens chaperones are unable to completely suppress the aggregation of damaged or modified proteins. γD-crystallin is one of the most abundant crystallins in the human lens, and mutations in γD-crystallin have been associated with congenital cataracts. Based on in vitro studies, cataract-linked γD-crystallin mutants were found destabilized and may be capable of escaping quality control by α-crystallin chaperones. To better understand the cellular responses induced by these disease-associated γD-crystallins in vivo, we analyze the protein dynamics of human γD-crystallin (Hsa.Crygd) mutants in zebrafish lens fiber cells by genetic approaches.
The Gal4/UAS targeted gene expression system was utilized to create lens fiber mosaics in zebrafish. UAS responder constructs expressing fluorescently tagged human γD-crystallin (Hsa.Crygd-mCherry), including wild-type and three variants, I4F, V76D and I4F/V76D double mutant, were generated by Gateway Cloning. Embryos derived from a lens-specific transgenic driver line, Tg[cryaa:Gal4], were injected with the constructs to achieve lens-specific overexpression of the mCherry tagged γD-crystallins. Aggregate formation and lens defects were examined in zebrafish larvae (4 dpf) using fluorescence and differential interference contrast (DIC) microscopy, respectively.
We observed a strong relationship between in vitro γD-crystallin stability and lens defects, such that decreased γD-crystallin stability corresponds to an increase in lens defect severity. Likewise, the occurrence of fluorescent punctates in the lens also exhibited the same relationship: embryos expressing the most destabilized γD-crystallin (I4F/V76D) exhibit the highest frequency of punctate formation. Intriguingly, most punctates co-localized with lens abnormalities.
Although preliminary, our results suggest that a relationship exists between the stability of γD-crystallin protein and the observation of aggregation and lens defect phenotypes. We argue that our study rationalizes the use of transgenic zebrafish expressing γD-crystallin mutants to be a disease-relevant model that can offer valuable insights into the molecular mechanisms of cataract formation.
This is an abstract that was submitted for the 2016 ARVO Annual Meeting, held in Seattle, Wash., May 1-5, 2016.
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