Genetically altered mice--Ocular function and pathology

National Eye Institute

Investigators

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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. The lenses of alphaA-/alphaB-crystallin double knockout mice exhibit a strange degeneration of fiber cells surrounding the lens nucleus. We postulated that since the maturation of lens fiber cells involves a process paralleling the events of apoptosis, the cellular disintegration may be due to a failure of the cells to abort the apoptotic pathway after removal of the cell nuclei and other subcellular organelles. Additionally, we believe that the absence of alphaA- and alphaB-crystallin may lead directly to this failure of the lens cells to arrest the denerative process in the knockout mice. Our suspicions were strengthened by a report that alphaB-crystallin can apparently inhibit the autocatalytic activation of caspase 3, a critical protease in apoptosis. AlphaA-crystallin and the alphaA-/alphaB-crystallin complex had not been examined for such inhibitory activity. We have therefore begun investigating whether alphaA- and/or alphaB-crystallin act during lens cell maturation to inhibit cellular disintegration in this apoptosis-like pathway, while permitting organelle removal. Our investigation has thus far revealed that caspase 3 activity is two to four fold (depending on age) higher in the alpha double knockout lenses than wild type lenses, supporting our hypothesis. Moreover, the activity of caspase 6, another protease involved in apoptosis, is two fold higher in the lenses of alpha double knockout mice. Immunoflourescence analysis of lens sections also reveals an increased amount of active caspase 3 in the knockout lenses. The preliminary data support a role for alpha-crystallin in modulating the apoptosis-like process of lens cell fifferentiation and maturation. In collaboration with Deborah Carper's lab in the NEI, and the Children's National Medical Center Microarray Center, we performed gene array analysis on wild type, alphaA knockout and alphaB knockout mouse lenses to define differences in gene expression patterns responsible for the cataractous phenotype in alphaA knockout mice versus the non-cataractous phenotype in alphaB knockout mice. Many of the genes consistently showing different levels of expression in the Affymetrix gene chip analysis were determined to be false positives by quantitative PCR. However, several of the identified genes have indeed been proven to be up or down regulated in the precataractous alphaA knockout lens. Further studies on the expression of these genes as a function of age and spatial distribution within the lens, are ongoing. Also, confirmation of additional differentially regulated genes, identified by Affymetrix screening, is ongoing. In collaboration with the laboratory of Chris Glembotski, and the laboratory of Ivor Benjamin, we are investigating the role of alphaB-crystallin and HSPB2 in cardiac function. 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. We are currently involved in collaborations with laboratories around the world studying the functions of alphaB in the heart, muscle, nervous system, immune system, and eye.