A CRYSTALLIN MUTATION WITH ABNORMAL ASTROCYTES AND RETINAL VESSELS

描述(申请人提供)：我们在Spraogue Dawley大鼠身上发现了一种自发突变，具有不寻常的眼睛表型，我们将其命名为Nuc1。在杂合子中，突变表现为单一半显性基因座，具有存活的纯合子和中间表型。导致Nuc1的突变是位于10号染色体上的A3/A1晶状体蛋白基因外显子6上的27bp插入。除Nuc1外，已知人类晶状体蛋白A3/A1基因的几个突变，所有这些突变都会导致显性白内障。在纯合子Nuc1大鼠中，即使在视网膜血管发育之后，胎儿眼内血管仍然存在。在出生后早期发育期间，这些大鼠的视网膜也比正常大鼠厚得多，血管数量过多。随着Nuc1纯合子大鼠的成熟，我们发现了微动脉瘤形成、血管内沉积和一些血管内血流阻塞的证据。我们发现，在视网膜中，A3/A1仅在星形胶质细胞中表达，而在Nuc1纯合子中，星形胶质细胞形态异常。这项研究的目的是解决由于Nuc1突变而导致视网膜星形胶质细胞的正常功能受损的可能性。星形胶质细胞在建立功能性视网膜血管系统中起着重要作用，然而，参与这一过程的细胞和分子机制仍不清楚。将Nuc1建立为遗传工具将提供一个独特的系统来研究星形胶质细胞的生物学，特别是它们与视网膜神经节和内皮细胞的相互作用。我们拟议研究的目标是验证我们的假设，即突变的A3/A1晶体蛋白的表达会影响星形胶质细胞的功能，导致Nuc1大鼠视网膜血管系统的不正确组织和功能。为了验证这一假说，提出了以下具体目标：特定目的1：鉴定和比较Nuc1和野生型大鼠视网膜星形胶质细胞中A3/A1晶体蛋白的结构、亚细胞定位和蛋白质相互作用。特异性目的2：探讨突变型A3/A1晶状体蛋白表达是否干扰星形胶质细胞的增殖或迁移。目的3：探讨突变型A3/A1对视网膜血管形成的影响。尽管对视网膜血管发育的了解取得了很大进展，但关于调控血管发育的机制和信号通路仍有许多问题有待解答。我们相信，对Nuc1大鼠的研究将为调控血管发育的细胞和分子机制提供新的见解，包括神经元、星形胶质细胞和内皮细胞之间的分子相互作用。

DESCRIPTION (provided by applicant): 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.