Molecular Mechanisms of Exfoliation Glaucoma

摘要-豪瑟/斯塔默R 01。 假性剥脱性青光眼是一种以不溶性蛋白质为特征的致盲性青光眼 沉积在眼前房中，是OAG最常见的可识别的继发性形式， 全球约有700万例。全基因组关联研究已经确定了遗传变异， 赖氨酰氧化酶样-1（LOXL 1）基因座与PEX风险密切相关。不幸的是， 该位点导致PEX青光眼的机制尚不清楚。我们最近的报告描述了一个 LOXL 1基因座内的长非编码RNA（在本文中表示为PEXpress）。我们发现基因变异 改变PEXpress的启动子强度，并与PEX青光眼密切相关。我们延长 初步实验中的这些观察结果表明，PEXpress的敲低改变了表达， 数百个下游靶基因，分析揭示了导致眼部疾病的途径的改变。 高血压包括细胞外基质重塑和机械传导。使用无偏质量 光谱和直接结合试验，我们发现PEXpress特异性结合mRNA加工 蛋白质，hnRNPL通过PEXpress中的14个碱基对结合区，并且内源性hnRNPL与 内源性PEXpress。基于这些观察，我们假设， PEXpress/hnRNPL复合物导致下游靶基因的失调，导致小梁改变， 网（TM）和施莱姆管（SC）细胞生物学，传统的外流功能，并最终，增加 眼压为了验证这一假设，我们构建了三个具体目标。在第一个目标中，我们将 确定PEXpress/hnRNPL复合物在人类常规免疫中对基因和蛋白质调控的作用。 流出细胞目的2旨在确定PEXpress/hnRNPL复合物对人体的功能影响 传统的流出细胞。目的3将使用灌注的人来确定PEXpress对流出设施的影响 器官培养中的前节。作为这项研究的成果，我们希望（i）确定基因和蛋白质 靶点加上由PEXpress /hnRNPL复合物调节的信号通路， 流出阻力的调节，（ii）确定PEXpress/hnRNPL在TM和SC细胞信号传导、收缩性 和屏障功能，加上（iii）确定PEXpress在完整模型中对常规流出功能的作用 系统

.ABSTRACT – Hauser/Stamer R01 . Pseudoexfoliation (PEX) glaucoma is a blinding form of glaucoma clinically characterized by insoluble protein deposits in the anterior chamber of the eye, and it is the most common identifiable secondary form of OAG, accounting for ~7 million cases worldwide. Genome wide association studies have identified genetic variants in the lysyl oxidase-like-1 (LOXL1) locus that are strongly associated with risk of PEX. Unfortunately, the functional mechanisms by which this locus contribute to PEX glaucoma are unknown. Our recent report characterized a long non-coding RNA (denoted herein as PEXpress) within the LOXL1 locus. We found that genetic variants alter the promoter strength of PEXpress, and are strongly associated with PEX glaucoma. We have extended these observations in preliminary experiments, revealing that knock down of PEXpress changes the expression of hundreds of downstream target genes, and analysis reveals modifications in pathways that lead to ocular hypertension including extracellular matrix remodeling and mechanotransduction. Using unbiased mass spectrometry and direct binding assays, we discovered that PEXpress specifically binds to the mRNA processing protein, hnRNPL via a 14 base pair binding region in PEXpress, and that endogenous hnRNPL complexes with endogenous PEXpress in cell culture. Based upon these observations, we hypothesize that alterations in the PEXpress/hnRNPL complex result in the dysregulation of downstream target genes, leading to altered trabecular meshwork (TM) and Schlemm’s canal (SC) cell biology, conventional outflow function and ultimately, increased intraocular pressure. To test this hypothesis, we have constructed three specific aims. In the first aim, we will determine the role of the PEXpress/hnRNPL complex on gene and protein regulation in human conventional outflow cells. Aim 2 is designed to determine the functional effects of the PEXpress/hnRNPL complex on human conventional outflow cells. Aim 3 will determine the effects of PEXpress on outflow facility using perfused human anterior segments in organ culture. As outcomes of this research we expect to (i) identify gene and protein targets plus signaling pathways regulated by PEXpress /hnRNPL complex in cell types responsible for the regulation of outflow resistance, (ii) identify role of PEXpress/hnRNPL in TM and SC cell signaling, contractility and barrier function, plus (iii) determine the role of PEXpress on conventional outflow function in an intact model system.