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）调节液体流动的机制，并最终利用这些知识开发预防原发性开角型青光眼（POAG）进展的有效疗法。 这个竞争性更新项目的直接目标是测试关于TM细胞感知ECM中的流体流动波动的分子机制的假设，并且通过该分子机制将这种感觉转换为改变细胞形状和运动性以增加或减少TM孔径，从而调节流体通过TM。特别是，我们专注于cochlin，分泌的ECM蛋白，因为质谱分析已确定cochlin专门在人类肿瘤TM，但不是在正常TM。我们已经表明，在体外cochlin经历聚集体的形成和多聚化时，受到流体剪切波动表明cochlin是能够mechanosensing。我们的组织假设是，cochlin mechanosensing，在脑胶质瘤TM，与跨膜蛋白，以调节TM细胞的形状和运动，导致ECM中的流体流动失调。 因此，cochlin在眼内压（IOP）升高中起关键作用。 目的1是检验在IOP失调开始时发生异常cochlin过表达的假设。 我们将使用新开发的基于试剂的光谱（SD）和磁动（MM）光学相干断层扫描（OCT），在不同年龄的活的青光眼DBA/2 J小鼠中确定真实的时间cochlin和TREK-1水平和IOP失调（IOP的早期和持续异常升高），并与对照DBA/2 J-Gpnmb +/SjJ小鼠进行比较。目的2是检验以下假设：cochlin机械感测信号通过与跨膜蛋白（如TREK-1）相互作用进行转导，导致细胞骨架变化，从而调节穿过TM过滤器的流体流动。 目的3是检验cochlin的慢性异常表达受一组转录因子（Barx 2、Nrf 2和Brn 3a）调节的假设。我们将使用原代TM细胞，尸体TM组织和DBA/2 J小鼠，以确定转录因子的相对水平（这是响应于压力/拉伸周期），其水平调节伴随着cochlin过表达。Cochlin是第一个与TM ECM中流体剪切变化的机械感测机械相关的分子。明确该蛋白在异常房水流出调节中的作用对于理解眼压调节、POAG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.