Molecular Mechanisms of Exfoliation Glaucoma

.ABSTRACT-HAUSER/STAMER R01 摘要假性剥脱性青光眼是一种致盲的青光眼，临床上以不溶性蛋白为特征。 位于眼前房，是OAG最常见的可识别的继发性形式， 占全球约700万例。全基因组关联研究已经确定了 赖氨酰氧化酶样-1(LOXL1)基因座与PEX的风险密切相关。不幸的是，功能性的 该基因座导致PEX青光眼的机制尚不清楚。我们最近的报告描述了 LOXL1基因座内的长非编码RNA(这里表示为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。我们已经延长了 这些观察在初步实验中揭示了PEXPRESS的击倒改变了表达 数以百计的下游靶基因，分析揭示了导致眼睛 高血压包括细胞外基质重塑和机械转导。使用无偏质量 光谱分析和直接结合分析发现，PEXPRESS与mRNA的加工有特异性结合 通过PEXpress中14个碱基对结合区的蛋白质、hnRNPL以及内源性hnRNPL与 细胞培养中的内源性PEXpress。基于这些观察结果，我们假设 PEXPRESS/hnRNPL复合体导致下游靶基因调控失调，导致小梁改变 Nethwork(TM)和Schlemm氏管(SC)细胞生物学，常规流出功能并最终增加 眼压。为了检验这一假设，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复合体在人类常规基因和蛋白质调控中的作用 流出单元格。目的2旨在确定PEXpress/hnRNPL复合体对人类的功能效应 传统的流出细胞。目标3将确定PEXPRESS对使用灌流人的流出设施的影响 器官培养中的前段。作为这项研究的结果，我们希望(I)鉴定基因和蛋白质 靶点加上由PEXpress/hnRNPL复合体调控的信号通路在细胞类型中负责 外流阻力的调节，(Ii)确定PEXpress/hnRNPL在TM和SC细胞信号转导和收缩中的作用 和屏障函数，加上(Iii)确定PEXpress在完整模型中对常规流出函数的作用 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.