Molecular Mechanisms of Exfoliation Glaucoma

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.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.