Trabecular Meshwork Proteins in Glaucoma

描述（由申请人提供）：长期目标是了解通过小梁网（TM）细胞外基质（ECM）的流体流动的调节机制，并最终利用这些知识开发有效的治疗方法来预防原发性开角型青光眼（POAG）的进展。这个竞争性更新项目的直接目标是测试关于TM细胞感知ECM中流体流动波动的分子机制的假设，以及这种感觉被转导到改变细胞形状和运动以增加或减少TM孔径，从而调节流体通过TM的假设。我们特别关注耳蜗蛋白，这是一种分泌的ECM蛋白，因为质谱分析已经在人类青光眼TM中发现了耳蜗蛋白，而在正常TM中没有发现。我们已经在体外表明，当受到流体剪切波动时，耳蜗会经历聚集体的形成和多聚化，这表明耳蜗具有机械感应能力。我们的组织假设是，青光眼TM中的耳蜗机械感应与跨膜蛋白沟通，调节TM细胞的形状和运动，导致ECM中流体流动失调。因此，耳蜗在眼压升高中起关键作用。目的1是验证异常耳蜗过表达发生在IOP失调发病的假设。我们将使用新开发的基于光谱（SD）和磁动力（MM）光学相干断层扫描（OCT）技术，实时测定不同年龄的活青光眼DBA/2J小鼠的耳蜗素和TREK-1水平以及IOP失调（早期和持续的IOP异常升高），并与对照DBA/2J- gpnmb +/SjJ小鼠进行比较。目的2是验证耳蜗机械感应信号是通过与跨膜蛋白（如TREK-1）的相互作用而转导的假设，从而导致细胞骨架的变化，从而调节流经TM过滤器的流体流动。目的3是验证耳蜗蛋白慢性异常表达受一组转录因子（Barx2、Nrf2和Brn3a）调控的假设。我们将使用原代TM细胞、尸体TM组织和DBA/2J小鼠来确定转录因子（对压力/拉伸周期有反应）的相对水平，其水平调节伴随着科克林的过表达。Cochlin是第一个与TM的ECM中流体剪切变化的机械传感机制相关的分子。确定该蛋白在异常水流出调节中的功能，对了解IOP调节、POAG发病机制以及潜在的干预策略具有重要意义。

DESCRIPTION (provided by applicant): The long-term goals are to understand the mechanisms of regulation of fluid flow through the extracellular matrix (ECM) of the trabecular meshwork (TM) and to eventually use this knowledge to develop effective therapies for preventing primary open angle glaucoma (POAG) progression. The immediate goal of this competing renewal project is to test hypotheses about the molecular mechanisms by which TM cells sense the fluid flow fluctuations in the ECM and by which the sensation is transduced to change cell shape and motility to increase or decrease TM pore size, thus regulating the passing of fluid through TM. In particular, we focus on cochlin, a secreted ECM protein, because mass spectrometric analyses have identified cochlin exclusively in human glaucomatous TM but not in normal TM. We have shown in vitro that cochlin undergoes aggregate formation and multimerization when subjected to fluid shear fluctuations indicating that cochlin is capable of mechanosensing. Our organizing hypothesis is that cochlin mechanosensing, in glaucomatous TM, communicates with transmembrane proteins to modulate TM cell shape and motility leading to dysregulation of fluid flow in ECM. Thus, cochlin plays a key role in intraocular pressure (IOP) elevation. Aim 1 is to test the hypothesis that aberrant cochlin over-expression occurs at the onset of IOP dysregulation. We will determine real time cochlin and TREK-1 levels and dysregulation of IOP (early and continuous abnormal rise in IOP) across different ages in live glaucomatous DBA/2J mice and compare with control DBA/2J-Gpnmb+/SjJ mice using newly developed reagent-based spectral (SD) and magnetomotive (MM) optical coherence tomography (OCT). Aim 2 is to test the hypothesis that the cochlin mechanosensing signal is transduced via interaction with transmembrane proteins (such as TREK-1), leading to the cytoskeleton changes that modulates fluid flow across the TM filter. Aim 3 is to test the hypothesis that chronic aberrant expression of cochlin is regulated by a set of transcription factors (Barx2, Nrf2 and Brn3a). We will use primary TM cells, cadaver TM tissues and DBA/2J mice to determine the relative levels of transcription factors (that are responsive to pressure/stretch cycles) whose level modulation is accompanied with cochlin overexpression. Cochlin is the first molecule mechanistically linked to mechanosensing of fluid shear change in the ECM of TM. Establishing this protein's function in aberrant aqueous outflow regulation has great significance for understanding IOP regulation, POAG pathogenesis, and potential intervention strategies.