Molecular mechanism underlying autosomal-dominant cataract in the Philly mouse: a βB2-crystallin mutant

Abstract

Cataracts, the clouding of the transparent lens, are the leading cause of blindness worldwide. While many cataracts are age-related, genetic mutations, especially in lens proteins such as crystallins, are a major cause of congenital cataracts. Crystallins are highly expressed proteins in the lens that help maintain transparency and high refractive index. βB2-crystallin, encoded by Crybb2, is the most abundantly expressed β-crystallin in the postnatal mouse lens and has traditionally been viewed as a structural protein. However, its expression in non-lens tissues such as the brain, retina, testis, and ovary, which are the organs that do not require refractive properties, suggests its potential non-refractive roles. ☐ This thesis investigates the non-refractive functions of βB2-crystallin using the Crybb2Phil/Phil mouse (Philly mouse), which carries a 12-bp in-frame deletion in exon 6 of Crybb2. To understand how this mutation affects the lens at the molecular level, RNA sequencing and enrichment analyses were conducted using whole lenses from 7-week-old Crybb2Phil/Phil mutant and wild-type mice, and immunofluorescence staining was performed across different ages from postnatal day 0 to 20 weeks. ☐ RNA-seq data revealed upregulation of cell proliferation-related genes like Mki67 and Pcna, supported by Ki-67 staining showing increased cell proliferation in both the germinative zone and anterior epithelial regions near the fibrotic lesion sites. Apoptosis-related genes were also altered, with elevated cleaved caspase-3 staining in lesion-adjacent epithelial cells. EMT-associated changes in both RNA and protein levels were examined. Although Acta2 (α-SMA) was unexpectedly downregulated at the transcript level, immunostaining revealed α-SMA upregulation in subcapsular fibrotic regions at 9 and 20 weeks. Epithelial marker E-cadherin was progressively reduced, and TGF-β1 was increased, colocalizing with α-SMA. These findings indicate EMT and fibrotic changes in the Crybb2Phil/Phil mutant lens. In addition, dysregulation of autophagy-related genes, including Sqstm1, Map1lc3b, and Lamp2 was observed, and accumulation of P62 protein in mutant fiber cells suggests impaired autophagic flux. This result is similar to previous findings in βA3/A1-crystallin, which regulates autophagy in the retinal pigment epithelium (RPE), raising the possibility that βB2-crystallin plays a similar role in the lens. ☐ Altogether, these results suggest that βB2-crystallin has broader functions beyond its refractive role. The Crybb2Phil/Phil mutation disrupts multiple pathways such as cell proliferation, apoptosis, EMT, and autophagy, that are essential for lens development and maintenance. This study reveals non-refractive functions of βB2-crystallin and shows how their disruption contributes to cataract formation.