Genetically altered mice to study ocular function and pa

National Eye Institute

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Abstract

Proper development and function of the eye, and indeed of an entire living organism, involves the correct and precise expression of a very large number of genes located on the organism's chromosomes. A great amount can be learned about these genes and their effects through in vitro laboratory experimentation and tissue culture techniques, however their role and importance in the development and health of an intact organism can be assessed only when studied in an intact organism. A myriad of knowledge has been obtained from transgenic mice, gene knockout and knockin mice. We have previously generated alpha-crystallin gene knockout mice to study the in vivo function of these remarkable proteins. The alpha-crystallins comprise a large fraction of the soluble protein in the vertebrate lens where they were, for many years, believed to function solely as structural proteins. Lenticular alpha-crystallin is comprised of two similar subunits alphaA and alphaB, each encoded by a single gene. They are related to the small heat shock proteins, and in vitro they exhibit molecular chaperone activity, autokinase activity, and interact with, and affect the state of, several cytoskeletal components. alpha-Crystallin, especially alphaB-crystallin, has been shown to be a normal constituent of many non-lenticular tissues, and has been detected in cytoplasmic inclusion bodies found in several human pathological conditions. Toward understanding the major roles of alpha-crystallin in vivo, we previously generated alphaA- and alphaB-crystallin gene knockout mice and alphaA-/alphaB-crystallin gene double knockout mice (DKO). The lenses of DKO mice exhibit disintegration of fiber cells surrounding the lens nucleus. We showed that morphological abnormalities in the lens secondary fiber cells of DKO mice are consistent with, and likely result from, elevated DEVDase and VEIDase activities, corresponding to caspase 3 and caspase 6 respectively. Caspase 3 and caspase 6 activities are higher in the lenses of DKO mice, than of wild type mice, under all conditions tested. Caspase 3 and 6 activity levels in wild type mouse lenses remained relatively constant at all ages tested. However, activity levels of caspase 3 and caspase 6 in DKO mouse lenses fluctuated with the animal?s age, and changes in caspase activities were consistent with changes in lens morphology. Immunofluorescent microscopy revealed an increase in caspase 6 and the active form of caspase 3 in lens secondary fiber regions in DKO mice. Elevated caspase activities in this area are consistent with the site of the cell disintegration. Based on our novel biotin-inhibitor tagging isolation technique, the DKO mouse lens has 50% more caspase 6 than wild type, however, we were unable to detect caspase 3 at the protein level. Caspase 6 had not previously been implicated in the lens secondary fiber cell maturation, nor had its possible inhibition by alpha-crystallin been described previously. However, according to our screening experiments with a wide spectrum of caspase inhibitors, caspase 6 plays a similar, or even more important, role than caspase 3 in secondary lens fiber cells maturation. Our data suggest that alpha-crystallin plays a role in suppressing caspase activity, resulting in retention of lens fiber cell integrity following degradation of mitochondria and other organelles, which occurs during the apoptosis-like pathway of lens cell terminal differentiation. A collaboration with the laboratory of Chris Glembotski confirmed our initial observations that the hearts of alphaB-crystallin/HSPB2 knockout mice are morphologically normal. It further demonstrated that cardiac function in these mice was normal under non-stressful conditions, demonstrating that these two small heat shock proteins are not essential for proper development and basal function of the heart. However, ischemia/reperfusion-induced stress, which mimics a heart attack, caused a marked decrease in recovery of cardiac function, compared to wild type mouse heart, and a marked increase in the amount of heart cell death. This demonstrates that one or both of these two small heat shock proteins, alphaB-crystallin and HSPB2, are essential for protecting the heart under stressful conditions. We have initiated a project to do a large (50 kb) targeted deletion in the mouse genome, spanning 5 closely related genes expressed in the visual system. Using state of the art recombineering techniques, we quickly generated the 50 kb deletion in a 190 kb BAC clone. Transfection and antibiotic selection yielded 300 ES cell clones, which are currently being analyzed for presence of the correctly targeted mutation. In collaboration with the laboratories of Xuejun Wang and Ivor Benjamin, we are further investigating the role of alphaB-crystallin and HSPB2 in cardiac function. In collaboration with Usha Andley, we are investigating the role of alphaB-crystallin in maintaining genomic stability and control of cell proliferation. In collaboration with Allen Taylor, we are examining the role of the ubiquitin pathway in lens development. In collaboration with Joseph Horwitz (UCLA) we are reexamining the constituents of inclusion bodies in lenses of alphaA knockout mice. There appears to be a significant amount of gamma-crystallin in the inclusion body fraction, which increases with age, and a gradual, age-dependent loss of many crystallins in the alphaA knockout lenses, some of the changes observed in age-related cataracts.