Cataracts, Connexin Mutants and Genetic Modifiers

 描述（由申请人提供）：本研究计划旨在阐明间隙连接和细胞骨架组分如何协调调节纤维细胞组装和细胞表面互锁结构以实现透镜的基本光学和生物力学特性的机制。由膜/细胞骨架结构提供的透镜纤维细胞互锁结构对于透镜止血、弹性和透明度是必不可少的。在人和小鼠晶状体中，透镜纤维细胞中的间隙连接主要由Cx46（Gja 3）和Cx 50（Gja 8）连接蛋白组成。Gja 3和Gaj 8突变导致人类和小鼠的各种类型的白内障。由这些连接蛋白突变引起的白内障与间隙连接通道功能障碍、透镜稳态受损和晶状体蛋白降解有关。然而，这些病理事件的连续步骤的分子和细胞机制还没有很好地理解。目前尚不清楚连接蛋白基因突变如何导致各种类型的白内障，特别是当相同的基因突变导致人类个体和不同小鼠品系背景中不同类型的白内障时。我们最近的研究结果表明，Gja 3（-/-）晶状体的透镜成熟纤维中的表面舌槽结构被消除，对应于核性白内障。细胞骨架蛋白CP 49和periaxin作为遗传修饰剂调节不同品系背景的Gja 3（-/-）小鼠白内障的严重程度。相反，Cx 50（Gja 8）的缺失损害外周分化纤维细胞的球窝结构以延迟纤维细胞伸长并破坏透镜体内平衡以导致更小的晶状体。本项目的具体目的是为了验证我们的假设，即间隙连接和细胞骨架成分之间的功能协调控制透镜纤维与纤维的互锁结构，包括短纤维上的突起。 透镜皮质的六角形纤维细胞长边有球窝状结构，透镜核的成熟纤维细胞有舌/沟结构。具体目标1将研究Cx46（Gja 3）间隙连接、CP 49和轴周蛋白如何协调控制纤维细胞表面互锁结构以维持透镜完整性、稳态和透明度。我们将进一步鉴定与Cx46（Gja 3）间隙连接/细胞骨架相关的蛋白质组分，这些蛋白质组分对于维持诸如突起的联锁结构是重要的。具体目标2旨在阐明Cx 50（Gja 8）间隙连接如何在透镜生长所需的六边形纤维细胞的长边上建立球窝结构的分子基础。我们将确定特定的PDZ结构域含有蛋白质，结合到Cx 50的C-末端，并连接到细胞骨架建立球窝结构。具体目标3将鉴定和表征2号染色体上的第三种遗传修饰剂，其可以抑制白内障发生过程中129-periaxin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.