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.[unreadable] [unreadable] 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).[unreadable] [unreadable] We have previously shown that the lenses of DKO mice exhibit disintegration of fiber cells surrounding the lens nucleus, and that these morphological abnormalities are consistent with, and likely result from, elevated DEVDase and VEIDase activities in lenses lacking alpha-crystallin, suggesting involvement of the apoptosis pathway in this patholology. [unreadable] [unreadable] The process of secondary lens fiber cells maturation parallels many classical apoptosis events: chromatin condensation, DNA fragmentation, nucleopore clustering with subsequent nuclear breakdown, and disappearance of mitochondria and other organelles. However, lens fiber cell differentiation shows signs of attenuated or arrested apoptosis: breakdown of nuclei occurs in days, not hours; the cytoskeleton remains intact throughout the remaining life of the organism; and the lens fiber cells show increased resistance to apoptotic agents such as staurosporine and etoposide.[unreadable] [unreadable] Changing of cell morphology, elevated caspase activity, and fragmentation of nuclear DNA are hallmark characteristics of apoptotic cells. We show that the absence of alpha-crystallin in the lens of DKO causes elevated caspase activity in lens secondary fiber cells and almost every nucleated secondary fiber cell in the lens of DKO mice, regardless of the location, was TUNEL-positive. As a result, lens secondary fiber cells fail to halt their apoptosis-like maturation program after degradation of the cell nuclei, mitochondria and all other organelles, and progress to a stage of cell disintegration as would most apoptotic cells. [unreadable] [unreadable] Our data suggest that alpha-crystallin plays a role in suppressing caspase activity, resulting in retention of lens fiber cell integrity. The mechanism by which alphaA- and alphaB-crystallin inhibit caspase activity is unknown. We are trying to understand possible links between alpha-crystallin and caspase proteins. Earlier, using a pull-down assay, we were able to demonstrate interaction between caspase 6 and alphaA-, but not alphaB-crystallin. Recently using same assay, we did not find any interaction between caspase 8 or 12/17 kDa form of active caspase 3 and alphaA- or alphaB-crystallin. [unreadable] [unreadable] Binding of partially processed or intact procaspase 3 to alphaB-crystallin was reported by other laboratories. Interestingly, the pull-down assay demonstrates interaction between caspase 9 and alphaA-crystallin in lens extract and direct interaction between purified caspase 9 and purified alphaA-crystallin. Covalently bound caspase 9 inhibitor shows no significant effect on the binding of caspase 9 to alphaA-crystallin, suggesting that alphaA-crystallin does not block the active center of caspase 9. These data are consistent with our observation that alpha-crystallin and HSP-27 have little or no effect on the LEHDase activity of caspase 9.[unreadable] [unreadable] However, alpha-crystallin complex and individual alphaA-crystallin and alphaB-crystallins inhibit cleavage of procaspase 3 to the active form of caspase 3 in a cell-free system. Activation of procaspase 3 by caspase 9 in the presence of small heat shock proteins resulted in 10 fold or greater inhibition of caspase-3 activity. Cleavage of procaspase-3 by caspase-9 in the presence of alphaA-crystallin resulted in formation of 26 kDa subunit. Similar accumulation of 24/26 kDa caspase 3 peptide was reported by other laboratories in ther presence of alpha-crystallin. Our data, along with data of others, suggest that alpha-crystallin interferes with the caspase cascade in the apoptotic pathway triggered by cytochrome c/ATP. Further elucidation of the mechanisms regulating caspase activities is essential.[unreadable] [unreadable] A project initiated two years ago to do a large (50 kb) targeted deletion in the mouse genome, spanning 5 closely related genes expressed in the visual system, failed to produce a correctly targeted ES cell clone in the 300 clones tested. The Board of Scientific Counselors suggested the project be dropped, and it has been.[unreadable] [unreadable] In collaboration with Allen Taylor, we are examining the role of the ubiquitin pathway in lens development.[unreadable] [unreadable] In collaboration with David Hinton, Ram Kannan and colleagues, we are examining the role of alpha-crystallin in the retina and RPE.[unreadable] [unreadable] In collaborations with Arthur Vandenbark and Hans VanNoort, the role of alphaB-crystallin in induction and pathogenesis of multiple sclerosis is being investigated.