Cataracts, Connexin Mutants and Genetic Modifiers

 描述(申请人提供)：这项研究计划旨在阐明缝隙连接和细胞骨架组件如何协调调节纤维细胞组装和细胞表面互锁结构以实现晶状体的基本光学和生物力学性能的机制。由膜/细胞骨架结构提供的晶状体纤维细胞互锁结构对于晶状体止血、弹性和透明性是必不可少的。在人和小鼠晶状体中，晶状体纤维细胞的缝隙连接主要由Cx46(Gja3)和CX50(Gja8)连接蛋白组成。Gja3和Gaj8突变会导致人类和小鼠各种类型的白内障。这些连接蛋白突变导致的白内障发生与缝隙连接通道功能障碍、晶状体内环境平衡受损和晶体蛋白降解有关。然而，这些病理事件的连续步骤背后的分子和细胞机制还不是很清楚。目前尚不清楚连接蛋白基因突变是如何导致各种类型的白内障的，特别是当一个相同的基因突变导致不同类型的白内障在人类个体和不同品系的小鼠背景中时。我们最近的研究结果表明，在Gja3(-/-)晶状体的成熟纤维中，表面的舌槽结构被消除，对应于核性白内障。细胞骨架蛋白包括CP49和周轴素作为遗传修饰物调节不同品系背景下Gja3(-/-)小鼠的白内障严重程度。相反，CX50(Gja8)的缺失损害了外周分化纤维细胞的球窝结构，从而延迟了纤维细胞的伸长，并破坏了晶状体的动态平衡，导致晶状体变小。这个项目的具体目的是测试我们的假设，即缝隙连接和细胞骨架组件之间的功能协调控制着晶状体纤维到纤维互锁结构，包括短端上的突起 晶状体皮质中六角形纤维细胞的长边和球窝，晶状体核心中成熟纤维细胞中的舌状/沟槽结构。具体目标1将研究Cx46(Gja3)缝隙连接、CP49和外轴蛋白如何协调控制纤维细胞表面联锁结构，以维持晶状体的完整性、动态平衡和透明度。我们将进一步确定与Cx46(Gja3)间隙连接/细胞骨架相关的蛋白质组分，这些蛋白质组分对维持突起等互锁结构非常重要。具体目标2旨在阐明CX50(Gja8)缝隙连接如何在晶状体生长所需的六角形纤维细胞的长边建立球窝结构的分子基础。我们将确定特定的含有PDZ结构域的蛋白质，这些蛋白质与CX50的C末端结合，并与细胞骨架相连，以建立球窝结构。特殊目的3将鉴定和表征第二染色体上的第三个遗传修饰物，该修饰物可以抑制129-周轴素在白内障发生过程中的作用。我们将进一步确定与核性白内障相关的特定生化、生理和生物力学变化。

 DESCRIPTION (provided by applicant): This research proposal aims to elucidate the mechanisms for how gap junctions and cytoskeletal components coordinately regulate fiber cell assembly and cell surface interlocking structures to achieve fundamental optical and biomechanical properties of the lens. Lens fiber cell interlocking structures provided by membrane/cytoskeletal structures are essential for lens hemostasis, elasticity and transparency. Gap junctions in lens fiber cells predominantly consist of Cx46 (Gja3) and Cx50 (Gja8) connexins in human and mouse lenses. Gja3 and Gaj8 mutations cause various types of cataracts in humans and mice. Cataractogenesis caused by these connexin mutations is associated with dysfunctional gap junction channels, impaired lens homeostasis and degradation of crystallin proteins. However, the molecular and cellular mechanisms underlying the sequential steps of these pathological events are not well understood. It is unknown how connexin gene mutations lead to various types of cataracts, especially when an identical gene mutation leads to different types of cataracts in human individuals and in different mouse strain backgrounds. Our recent results show that surface tongue-and-groove structures are eliminated in lens mature fibers of Gja3(-/-) lenses, corresponding to nuclear cataracts. Cytoskeletal proteins including CP49 and periaxin act as genetic modifiers to modulate cataract severity of Gja3(-/-) mice in different strain backgrounds. In contrast, a loss of Cx50(Gja8) impairs the ball-and-socket structures of peripheral differentiating fiber cells to delay the fiber cell elongation nd disrupt lens homeostasis to lead to smaller lenses. Specific aims of this project are designed to test our hypothesis that functional coordination between gap junctions and cytoskeletal components controls lens fiber-to-fiber interlocking structures, including protrusions on the short sides and ball-and-sockets on the long sides of hexagonal shaped fiber cells in lens cortex and the tongue/groove structures in mature fiber cells in the lens core. Specific aim 1 will study how Cx46 (Gja3) gap junctions, CP49 and periaxin coordinately control fiber cell surface interlock structures to maintain lens integrity, homeostasis and transparency. We will further identify protein components associated with Cx46 (Gja3) gap junctions/cytoskeleton that are important for maintaining interlocking structures such as protrusions. Specific aim 2 aims to elucidate the molecular basis for how Cx50 (Gja8) gap junctions establish ball-and-socket structures on the long sides of hexagonal shaped fiber cells needed for lens growth. We will identify specific PDZ-domain containing proteins that bind to the C-terminal ends of Cx50 and link to cytoskeleton for establishing ball-and-socket structures. Specific aim 3 will identify and characterize the third genetic modifier on Chromosome 2, which can suppress the effect of 129-periaxin during cataractogenesis. We will further determine specific biochemical, physiological and biomechanical changes associated with nuclear cataracts.