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Brian Link, Amira L Pavlovich, Jason Bader, Joseph C Besharse, Ross F Collery; lrp2-/- zebrafish eyes show modulation of multiple genetic pathways, including Bmp signaling, that cause myopia. Invest. Ophthalmol. Vis. Sci. 2016;57(12):3610.
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© ARVO (1962-2015); The Authors (2016-present)
LRP2 mutations are associated with myopia in human and animal models, causing Donnai-Barrow syndrome in affected patients and the buphthalmic bugeye phenotype in zebrafish. Bmp4 binds to Lrp2 in vitro, and is associated with refractive error and other eye defects. We show that Bmp4 binds to the first LDLA subdomain of Lrp2 in vivo, that overexpression of Bmp antagonists from the RPE leads to increased myopia, and that overexpression of a soluble BMP4-binding domain of Lrp2 leads to increased myopia. Consistent with the idea that a sheddase might regulate Lrp2 activity by promoting release of the BMP4-binding domain, we confirmed extracellular cleavage of Lrp2 which was reduced by inhibition of BACE1 metalloprotease. Finally, we used RNAseq from dissected eye tissues to study the tissue-specific repertoire of transcripts altered by absence of Lrp2.
Transgenic zebrafish used the rpe65a promoter to express genes of interest from the RPE. Eye size and refractive state were measured using SD-OCT. Lrp2 subdomains with fluorescent tags were expressed in HEK293T cells for cleavage analysis and pharmacological inhibition of candidate sheddases. RNA from zebrafish eye tissues were extracted for RNAseq.
RPE-driven expression of Bmp antagonists, as well as a soluble domain of Lrp2, cause myopia. Lrp2 extracellular cleavage was demonstrated and found to be reduced by inhibition of BACE1 metalloprotease. Bmp signaling as well as several other signaling pathways were modulated in RPE, sclera/choroid and retinas of lrp2-/- zebrafish eyes.
Though loss of Lrp2 has been associated with myopia, the mechanisms of Lrp2 regulation, its precise role in signaling, and effects on eye tissue transcriptomes has not been studied. We show show that the BMP-binding domain of Lrp2 is cleaved extracellularly, suggesting a mechanism for how Lrp2 modulates BMP signaling, and perhaps other pathways in the eye. Phenotype analyses of Lrp2 transgenic and mutant zebrafish suggests Lrp2 differentially regulates BMP signaling depending on whether it is membrane-bound or soluble. Proteolysis may fine-tune levels of Bmp signaling and regulate emmetropization. RNAseq analyses of Lrp2 mutants confirmed alterations to Bmp signaling, but also showed changes to Shh and FGF signaling, suggesting Lrp2 coordinates multiple pathways to maintain proper eye size.
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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