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Terete Borras, Renekia Elliott, Priyadarsini Asokan, Laura Rodriguez Estevez; IDENTIFYING PSEUDOEXFOLIATION GLAUCOMA MECHANISMS BY OVERPRODUCING WILD-TYPE AND N-TERMINUS-DELETED LOXL1 IN VITRO AND IN VIVO. Invest. Ophthalmol. Vis. Sci. 2018;59(9):3029.
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LOXL1 is present in pseudoexfoliation (PEX) material and its gene has been associated with PEX-Glaucoma (PEX-G). Associated SNPs localize at the protein N-terminus. The N-domain provides the correct alignment of LOXL1 with tropoelastin and it is essential for the formation of elastic fibers. Our goal here was to investigate whether overproducing WT and N-deleted LOXL1 proteins by gene transfer would elucidate and reproduce altered mechanisms associated with the disease.
A secreted, N-terminus-deleted viral vector (scAAV2.ΔN.LOXL1) was designed to contain the secretion peptide, the C-terminus and DDK tag (934 bp). A human WT LOXL1 DDK-tagged vector (Adeno.WT.LOXL1) (Vigene) was purified in our laboratories. Primary human trabecular meshwork (HTM) and iris pigment epithelium (IPE) cells, TaqMan probe Hs00935933_mH (ABI) and monoclonal anti-DDK were used to validate delivery of mRNAs and proteins. Primary rabbit anti-human elastin & anti-human Lamp and secondary donkey anti-mouse Alexa 594 & anti-rabbit Alexa 488 antibodies were used to detect LOXL1, elastin network and lysosomes under permeabilized and non-permeabilized conditions. Wistar rats were injected intracamerally with LOXL1 vectors and harvested at 10 days to 1 month. Morphology was analyzed in paraffin-embedded eyes on 5 μm H&E sections.
Both viruses delivered the correct proteins to HTM and IPE cells. Overexpression of WT.LOXL1, but not that of ΔN.LOXL1, induced a marked cell shape change and a ring structure underneath the cell’s membrane. Intracellularly, the WT localized to the ring structure while the ΔN.LOXL1 extended to the whole cytoplasm. The WT seemed to alter lysosome levels and distribution. Extracellularly, the WT associated with elastin fibers while ΔN.LOXL1 did not. Increased ΔN.LOXL1 also led to elastin fiber reduction and fragmentation. In living animals, delivery of ΔN.LOXL1 induced the formation of an altered structure at the iris/ lens interface and elevated IOP.
The effects of the WT and ΔN.LOXL1 protein levels on cell shape, lysosomes and elastin network in HTM & IPE cells might hold important clues to elucidate mechanisms of PEX-G. The formation of an altered structure at the iris/ lens interface in living animals after scAAV2.ΔN.LOXL1 gene transfer, coupled with an effect on IOP, shows that such strategy could lead to the development of a PEX-G model.
This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.
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