This lab has 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. alphaA-Crystallin, and indeed many of the formerly """"""""lens-specific"""""""" crystallins, has also recently been shown to be expressed on non lenticular tissues. 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).? ? We have previously shown that the lenses of DKO mice exhibit disintegration of fiber cells surrounding the lens nucleus, and have shown that these morphological abnormalities result from elevated DEVDase and VEIDase activities in lenses lacking alpha-crystallin, suggesting involvement of the apoptosis pathway in this pathology. ? ? To analyze alpha-crystallin's possible regulation of genes in the apoptotic pathway, we employed a PCR array strategy, using lenses from alpha-crystallin-null mice, A/B-crystallin/caspase-3 triple KO mice, A/B-crystallin/caspase-6 triple KO mice, and wild type mice. These same two lines of triple KO mice were used to assess the roles of caspase-3 and caspase-6 in the lens disintegration process observed in alpha-crystallin-null mice, and attempt the rescue of this cell disintegration phenomenon by elimination of these individual caspases. PCR array analysis of apoptotic gene expression from lenses of alpha-crystallin-null mice revealed a predominant down-regulation in most functional gene groups including Anti-apoptosis, CIDE Domain, IAP, Caspase, Bcl-2, and the TNF Ligand Family. Interestingly, CARD gene family anti-apoptotic genes were down-regulated and pro-apoptotic genes were up-regulated. PCR array studies of lenses from triple knockout mice showed only minor, if any, difference compared to alpha-crystallin-null mice. This observation is consistent with a histological study demonstrating that elimination of either caspase-3 alone or caspase-6 alone failed to rescue the lens secondary fiber cell disintegration phenotype in alpha-crystallin-null mice. We have hypothesized that, in secondary lens fiber cells, alpha-crystallin interferes with the apoptosis-like maturation program, halting it after organelle removal, but before cellular disintegration. Our new data, along with data from others, suggest that alpha-crystallin interferes with the caspase cascade in the apoptotic pathway. Caspase activity regulation could be at the protein-protein level by direct interaction as has recently been demonstrated by several labs, and/or at the level of gene expression regulation. Changes in apoptotic pathway gene expression patterns in lenses of alpha-crystallin-null mice, reported here, suggest a novel role for alpha-crystallin as a regulator of gene expression in the apoptotic pathway.? ? In a collaborative effort, Lena Claesson-Welch and Anna Dimberg utilized our alphaB-crystallin-null mice to study the effects of alphaB-crystallin on tumor angiogenesis. In the alphaB-crystallin-null mice, endothelial cells infiltrating a transplanted tumor (containing normal alphaB) displayed a higher level of apoptosis, leading to leaky, defective vessels in the tumor, and death of portions of the tumor. As well as being a striking finding in itself, this adds to the mounting evidence that alphaB is an antiapoptotic agent in many cell types. ? ? In a continuing collaboration, David Hinton and Ram Kannan used our alphaA-crystallin-null mice and alphaB-crystallin-null mice to study the roles of these small heat shock proteins in the retina, when exposed to oxidative stress in the form of an intravitreal injection of cobalt chloride. Retinal degeneration in response to cobalt chloride injection was much more severe, and began earlier, in both alphaA-crystallin-null mice and alphaB-crystallin-null mice, compared to wild type controls, and resembled several retinal degeneration diseases. This further illustrates the importance of alpha-crystallin in the retina, especially in protection against oxidative stress.? ? Similarly, in a collaborative effort, Narsing Rao and Suraj Bhat used our alphaA-crystallin-null mice to study alphaA-crystallin function in experimental autoimmune uveitis, a rodent model of uveitis, and found that alphaA is critical in protecting photoreceptor cells from the oxidizing environment produced early during uveitis. In the absence of alphaA, symptoms of uveitis began earlier, and extent of retinal damage was much greater than in wild type mice. AlphaA was shown to bind to procaspase 3 and to nitrated cytochrome c, both of which could inhibit initiation of apoptosis in photoreceptor cells.? ? A new mouse modeling project in the lab is a collaboration with Dr. Debasish Sinha of Johns Hopkins University, who has been on sabbatical (IPA) in our laboratory for all of this year, and parts of the previous and next fiscal years. This modeling project investigates the non-lens functions of betaA3/A1-crystallin, originally isolated as a lens protein. Dr. Sinha and associates have shown that a naturally occurring mutation of this gene causes abnormalities in astrocytes, which leads to altered development of the neural retina and retinal vasculature, with similar defects in the brain. Using an astrocyte-specific GFAP promoter, we have made transgenic mouse models in which expression of the mutant rat betaA3/A1 is restricted to astrocytes. Initial characterization of these mice reveals that astrocyte arrangement surrounding retinal blood vessels is abnormal, compared to wild type. The lenses however remain transparent and intact, achieving one of the primary goals of this modeling project. The disintegrating lens in the original mutant rat had raised concerns that the retinal phenotype may not be caused by expression of the mutant crystallin in astrocytes, but a secondary effect of lens debris and associated processes. These initial studies suggest that, like a similar phenotype observed in brain, the retinal phenotype does indeed result from mutant betaA3/A1 expression in astrocytes, and not from lens degradation. Further analysis of these mice is ongoing.? ? We also began a collabration with Dr. Lijin Dong of the NEI Genetic Engineering Facility, to generate conditional knockout mice in which the betaA3/A1 gene is specifically disrupted in astrocytes. Again, this approach avoids interference or complicating secondary effects from a disintegrating lens. The approach has been to insert loxP recombinase sites flanking exons 4 and 5 of the mouse betaA3/A1-crystallin gene, and excising this portion of the gene by astrocyte-specific expression of cre recombinase, again using the GFAP promoter. The targeting construct has been completed. Many putative targeted ES cell clones have been identified by long range PCR. Several of these clones were confirmed by Southern blot to have the correct chromosomal replacement, and a few clones are awaiting karyotype analysis prior to being used for microinjection to generate chimeric mice.
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