Trabecular Meshwork Proteins in Glaucoma

描述(由申请人提供)：长期目标是了解小梁网络(TM)细胞外基质(ECM)中液体流动的调节机制，并最终利用这一知识开发预防原发性开角型青光眼(POAG)进展的有效疗法。这个相互竞争的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说，以及通过这种感觉被传导到改变细胞形状和运动性以增加或减小TM孔大小，从而调节液体通过TM的分子机制。特别是，我们关注的是cochlin，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cochlin，而在正常TM中没有。我们在体外已经表明，当受到流体剪切波动时，Cochlin经历了聚集体形成和多聚化，这表明Cochlin具有机械传感的能力。我们的组织假说是，在青光眼TM中，Cochlin机械传感与跨膜蛋白沟通，调节TM细胞的形状和运动，导致ECM中液体流动的失调。因此，Cochlin在眼压升高中起着关键作用。目的1是验证眼压失调初期异常纤毛虫蛋白过度表达的假说。我们将使用新开发的试剂光谱(SD)和磁动力(MM)光学相干断层扫描(OCT)技术，在不同年龄的活体青光眼DBA/2J小鼠中实时检测Cochlin和Trek-1的水平以及眼压的失调(早期和持续的眼压异常上升)，并与对照组DBA/2J-GPNMB+/SjJ小鼠进行比较。目的2是为了验证这样的假设，即Cochlin机械传感信号是通过与跨膜蛋白(如Trek-1)的相互作用而传递的，从而导致细胞骨架的变化，从而调节流经TM过滤器的液体。目的3验证Cochlin慢性异常表达受一组转录因子(Barx2、Nrf2和Brn3a)调控的假说。我们将使用原代TM细胞、身体TM组织和DBA/2J小鼠来确定转录因子(对压力/拉伸周期做出反应)的相对水平，其水平调节伴随着Cochlin的过度表达。Cochlin是第一个与TM ECM中流体剪切变化的机械传感有关的分子。确定该蛋白在异常房水流出调节中的功能对于理解眼压调节、POAG的发病机制和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.