A CRYSTALLIN MUTATION WITH ABNORMAL ASTROCYTES AND RETINAL VESSELS

晶状体蛋白突变导致星形胶质细胞和视网膜血管异常

• 批准号: 7350844
• 负责人: DEBASISH SINHA
• 金额: $ 41万
• 依托单位: JOHNS HOPKINS UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-07-01 至 2011-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7350844
• 关键词: Address; Adult; Affect; Antibodies; Apoptosis; Astrocytes; Axon; Axonal Transport; Base Pairing; Binding; Biochemistry; Biological Assay; Biology; Blood Vessels; Blood flow; Bromodeoxyuridine; Cataract; Cell physiology; Cells; Chromosomes, Human, Pair 10; Coculture Techniques; Colchicine; Communication; Complement; Crystallins; Deposition; Development; Dyes; Electrical Resistance; Endothelial Cells; Endothelium; Evans blue stain; Exons; Eye; Genes; Genetic; Glial Cell Proliferation; Glial Fibrillary Acidic Protein; Goals; Growth; Heterozygote; Homozygote; Hour; Human; In Situ Nick-End Labeling; Injection of therapeutic agent; Intermediate Filaments; Label; Measures; Microaneurysm; Molecular; Mutation; Names; Neurons; Optic Nerve; Phenotype; Photography; Play; Process; Proteins; Rattus; Retina; Retinal; Retinal Ganglion Cells; Role; Signal Pathway; Sprague-Dawley Rats; Staining method; Stains; Structure; Surface; System; Techniques; Testing; Thick; Tight Junctions; Vascular Permeabilities; Vimentin; Yeasts; anterior chamber; base; cell motility; fetal; insight; migration; mutant; nestin protein; novel; research study; retina blood vessel structure; time use; tool; yeast two hybrid system

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.

描述（由申请人提供）：我们在Sprague道利大鼠中发现了一种自发突变，具有异常的眼睛表型，我们将其命名为Nuc 1。该突变表现为单个半显性基因座，具有可存活的纯合子和杂合子中的中间表型。导致Nuc 1的突变是在10号染色体上的<$A3/A1晶体蛋白基因的外显子6中插入27个碱基对。除了Nuc 1之外，还已知<$A3/A1晶体蛋白中的几种人类突变，所有这些突变都会导致显性白内障。在纯合子Nuc 1大鼠中，胎儿眼内血管即使在视网膜血管发育后仍持续存在。在出生后的早期发育过程中，这些大鼠的视网膜也比正常人厚得多，血管数量过多。随着Nuc 1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。我们已经发现，在视网膜中，<$A3/A1仅在星形胶质细胞中表达，并且在Nuc 1纯合子中，星形胶质细胞形态异常。本研究的目的是解决视网膜星形胶质细胞的正常功能受到损害的可能性，作为Nuc 1突变的结果。目前认为星形胶质细胞在视网膜血管系统的形成中起着重要作用，然而，参与这一过程的细胞和分子机制仍不清楚。将Nuc 1作为一种遗传工具将提供一个独特的系统来研究星形胶质细胞的生物学，特别是它们与视网膜神经节和内皮细胞的相互作用。我们提出的研究目标是检验我们的假设，即突变型<$A3/A1晶体蛋白的表达影响星形胶质细胞功能，导致Nuc 1大鼠视网膜血管的组织和功能不正常。为了验证这一假设，提出了以下具体目标：具体目标1：表征和比较Nuc 1和野生型大鼠视网膜星形胶质细胞中<$A3/A1晶体蛋白的结构、亚细胞定位和蛋白质相互作用。具体目标2：研究突变型<$A3/A1晶体蛋白的表达是否会破坏星形胶质细胞的增殖或迁移特异性目的3：证明表达突变型<$A3/A1的星形胶质细胞对视网膜血管系统的影响。尽管在了解视网膜血管系统的发育方面取得了快速进展，但关于调节血管发育的机制和信号通路仍有许多问题有待回答。我们相信，对Nuc 1大鼠的研究将为调节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。