UC Berkeley

The ocular lens must maintain transparency and a high refractive index throughout life in order to transmit a clear light image onto the retina. Cataracts, defined as any lens opacity, are one of the major causes for vision impairment in the world. A non-surgical way to either prevent or delay cataract formation will significantly improve vision care and reduce eye Medicare cost. By studying cataractogenesis at the genetic, morphological, histological and biochemical levels, my thesis project has provided some new understanding of the molecular mechanisms that maintain lens homeostasis, and has demonstrated the suppression of a congenital cataract through a genetic method.

My thesis includes three parts: 1) To study the molecular basis for the nuclear cataract formation in gamma;B-S11R mutant mice; 2) To study how does elevation of alpha3 connexin in the lens, expressed from the knock-in (Ki) alpha3 gene, prevent the nuclear cataract caused by the gammaB-S11R mutation; 3) To identify a genetic modifier of the C57BL/6J mouse strain background that dominantly suppresses the nuclear cataract caused by the alpha3 null mutation.

We have determined that a dominant nuclear cataract, linked to the gammaB-crystallin S11R point mutation, was associated with abnormal gamma-crystallin protein aggregation and disrupted membrane-cytoskeleton structures of inner lens fiber cells (Li et al., 2008). Moreover, increases of lens calcium level and calcium-dependent protein degradation were related to the degeneration of inner mature fiber cells and the nuclear cataract formation of gammaB-crystallin S11R mutant lenses, similar to that of alpha3 connexin (Gja3 or Cx46) homozygous knockout lenses. Thus, similar mechanisms probably associated with nuclear cataract formation in both gammaB-S11R crystallin and alpha3 connexin mutant lenses.

Knockin alpha3 (Ki alpha3) connexin under endogenous alpha8 connexin (Gja8 or Cx50) gene promoter is known to improve gap junction formation to suppress cataracts caused by alpha8-G22R connexin mutation (Xia et al., 2006b). In order to evaluate whether improved gap junction communication mediated by Ki alpha3 could suppress the nuclear cataract caused by gammaB-S11R mutation, we have generated Ki alpha3 and gammaB-S11R compound homozygous mutant mice. Unlike the dense nuclear cataracts in gammaB-S11R mutant mice, these compound mutant micedisplayed clear lenses with relatively well-organized fiber cells. Immunohistochemistry data revealed normal distribution of actin filaments and cytosolic gamma-crystallins. Compound mutant lenses had no obvious increase of crystallin protein degradation and total lens calcium level remained similar to that of wild-type lenses. Thus, elevation of gap junction communication via knockin alpha3 connexin prevents lens calcium elevation which probably inhibits calcium-dependent protein degradation and prevents fiber cell degeneration.

It is known that other genetic factor(s) influences the severity of nuclear cataracts in alpha3 connexin knockout mice between 129/SvJ or C57BL/6J strain backgrounds (Gong et al., 1999; Gong et al., 1997). In order to identify the genetic suppressor in C57BL/6J alpha3 knockout mice, we generated alpha3 knockout mice at the C57BL/6J x 129/SvJ hybrid strain backgrounds. Genome-wide genetic linkage testing and fine mapping located a genetic modifier in a region about two centimorgans on chromosome 7. This genetic modifier of C57BL/6J strain background can suppress dense nuclear opacity resulted from a loss of alpha3connexin in the lens.. The severity of nuclear cataracts was correlated to the amount of cleaved forms of crystallin proteins in alpha3 knockout lenses. This genetic suppressor may regulate calcium homeostasis to inhibit the activities of calpain proteases in the lens. by

This thesis demonstrates for the first time that upregulation of gap junction communication can prevent a nuclear cataract caused by a gammaB-crystallin gene mutation. Moreover, a genetic suppressor functions on the upstream of calpain proteases to suppress a nuclear cataract caused by gap junction communication composed by alpha3 connexin in the lens. Understanding the roles of gap junction communication and related genetic modifier(s) are important for developing a potential strategy for nuclear cataract prevention in the future.