Knockin Alpha3 Connexin (50KI46) Alleviates Lens Cataract Caused by Alpha8–G22R Mutation

Abstract

Abstract: : Purpose:To study the molecular basis for how α8–G22R mutation leads to a dominant cataract and how knockin α3 (50KI46) allele prevents the phenotypes caused by alpha8–G22R mutation in the lens. Methods: Two mouse compound mutant lines, α8 (G22R/–) α3 (–/–) and α8 (50KI46/– α3 (–/–), were generated from the breeding of several generations of the original mutation lines, Lop10 (α8–G22R mutation) or knockin α3 (50KI46) with double knockout α8 (–/–) α3 (–/–) mice. The offspring produced from the intercross between these two compound mutant mice were genotyped for three types of mice: α8 (G22R/50KI46) α3 (–/–), α8 (50KI46/–) α3 (–/–), and α8 (G22R/–) α3 (–/–). Morphological changes of these three types of mice were examined by light and electron microscopy, and biochemical changes and properties of gap junctions and connexins were analyzed by Western blot and immunohistochemical staining. Results: We have found thatα8 (G22R/50KI46) α3 (–/–) mice develop lenses with very mild nuclear cataract, while α8 (G22R/G22R) α3 (+/+) or α8 (G22R/–) α3 (–/–) mice develop lenses with severely altered fibers and posterior rupture. Histology shows relative normal morphology in the lenses of α8 (G22R/50KI46) α3 (–/–) mice. Moreover, Western blot and immunohistochemical staining verify an increase of α8–G22R mutant subunits and the presence of gap junction plaques formed by α8–G22R mutant subunits in the lenses of α8 (G22R/50KI46) α3 (–/–) mice. Conclusions: Unlike endogenous wild–type α3 subunits, the knockin wildtype α3 subunits expressed under the promoter of α8 gene somehow stabilize α8–G22R subunits and facilitate the α8–G22R subunits to form gap junctions in the lens fibers. We hypothesize that the knockin α3 subunits restore the functional gap junctions in the fibers and rescue the lens phenotype by interacting with the α8–G22R subunits. We proceed to investigate the molecular basis for this interaction and to explain why endogenous α3 subunits are unable to interact with α8–G22R subunit to form gap junctions in the lens fibers.

Keywords: gap junctions/coupling • cell-cell communication • cataract