Dissecting the mechanisms for intercellular protein transport and transparency in the lens: Functional studies of gap junction communication and alpha-crystallins

Cataracts are the leading cause of blindness in the world. This thesis focuses on studies of the roles of gap junction communication and of alpha-crystallins in establishing and maintaining lens transparency by using unique mouse genetic models for cataractogenesis. Intercellular gap junction communication is essential for maintaining the stability and solubility of lens crystallins. This work provides the first experimental evidence that gap junction communication plays a key role in the regulation of intercellular protein distribution in the lens. The mechanisms that control orderly arrangement and distribution of crystallin proteins between lens fiber cells, which is required for lens transparency and high refractive index, remain unclear. Intercellular gap junction channels formed by alpha3 and alpha8 connexins are known to transport only small molecules, ions and water, but not macromolecules and proteins. Using green fluorescent protein as a protein transport sensor in vivo, we have demonstrated that gap junction communication, formed by either alpha3 or alpha8 connexin, is needed for the protein exchange pathway between lens fibers. Presumably, uniform distribution of crystallin proteins requires proper gap junction communication.;Crystallins, alpha, beta and gamma classes, make up over 90% of total lens proteins. It has been hypothesized that lens transparency requires alpha-crystallins, consisting of alphaA and alphaB subunits, to function as molecular chaperones to prevent aggregation of denatured lens proteins and cataract formation. Using alpha-crystallin mutant mice, we have found that mutant alpha-crystallins with either increased or decreased chaperone-like activity can lead to a variety of cataracts. For comparing the degree of cataract severity between different mouse lenses, we have developed the first quantitative method to measure light scattering in cataractous lenses using a fiber optic cable and spectrometer. Increased chaperone-like activity of alphaA-Y118D mutant proteins causes abnormal protein aggregation and a delay in the formation of the macromolecular exchange pathway. Mutant alphaA-Y118D proteins also inhibit lens fiber cell elongation to reduce lens size in mutant mice.;This work suggests that appropriate chaperone-like activity of alpha-crystallins and gap junction communication are essential for lens growth, transparency and high refractive index as well as cataract prevention.