We have discovered a spontaneous mutation in the Sprague Dawley rat with an unusual eye phenotype that we have named Nuc1. The mutation behaves as a single semi-dominant locus with a viable homozygote and an intermediate phenotype in the heterozygotes. The mutation causing Nuc1 is a 27 base pair insertion in exon 6 of the ?A3/A1 crystallin gene on chromosome 10. In addition to Nuc1 several human mutations in ?A3/A1 crystallin are known, all of which cause dominant cataract. In homozygous Nuc1 rats the fetal intraocular vessels persist even after development of the retinal vessels. During early post-natal development these rats also have a much thicker retina than normal with an excess number of vessels. As the Nuc1 homozygote rat matures, we find evidence of microaneurysm formation, intravascular deposits and blockage of blood flow inside some vessels. We have found that in the retina, ?A3/A1 is expressed only in astrocytes and that in the Nuc1 homozygotes the astrocytes are morphologically abnormal. The purpose of this study is to address the possibility that the normal functioning of the retinal astrocytes is compromised as a consequence of the Nuc1 mutation. It is now accepted that astrocytes play a major role in the establishment of a functional retinal vasculature, however, the cellular and molecular mechanisms involved in this process remain elusive. Establishing Nuc1 as a genetic tool will provide a unique system in which to study the biology of astrocytes, in particular their interactions with retinal ganglion and endothelial cells. Our goal for the proposed studies is to test our hypothesis that expression of mutant ?A3/A1 crystallin affects astrocyte function, leading to improper organization and function of the retinal vasculature in the Nuc1 rat. To test this hypothesis, the following specific aims are proposed:
SPECIFIC AIM 1 : To characterize and compare the structure, sub-cellular localization and protein interactions of ?A3/A1 crystallin protein in retinal astrocytes from Nuc1 and wild type rats.
SPECIFIC AIM 2 : To investigate if the proliferation or migration of astrocytes is disrupted by expression of mutant ?A3/A1 crystallin SPECIFIC AIM 3: To demonstrate the effect of astrocytes expressing mutant ?A3/A1 on retinal vasculature. Despite rapid progress made in understanding the development of the retinal vasculature, many questions remain to be answered about the mechanisms and signaling pathways regulating vascular development. We believe that studies on the Nuc1 rat will provide new insights into the cellular and molecular mechanisms that regulate vascular development including the molecular interactions among neurons, astrocytes and endothelial cells.
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