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J Mario Wolosin, Zheng Wang, Stephanie Gillespie, Shozer Dawn, Robert Ritch, Audrey M Bernstein; LOXL1 And Misfolded Protein Processing Pathways In XFS Glaucoma Cells.. Invest. Ophthalmol. Vis. Sci. 2017;58(8):4601.
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© ARVO (1962-2015); The Authors (2016-present)
Tenon fibroblasts (TFs) from eyes with exfoliation syndrome (XFS) and glaucoma display autophagy impairment features similar to age-related neurodegenerative diseases ( PLoS ONE 2016, 11: e0157404 ). The latter are frequently linked to high misfolding frequency in protein variants. Since XFS pathology is associated with two non-synonymous variants of LOXL1, we examined whether in XFS cells LOXL1 polypeptides are processed by misfolding pathways.
TF cultures were derived from XFS and primary open angle glaucoma (POAG, control) patient tissue. Intracellular protein aggregates were visualized with ProteostatTM. Autophagy was accelerated by FBS removal (starvation). Co-IP was used to identify interaction between USP19 and LOXL1. Analytical methods included Western blot, immunostaining and electroporation.
Proteostat staining demonstrated the presence of aggregated protein only in XFS cells. Aggregate presence in XFS TFs was also inferable from the much higher expression of clusterin, a protein expressed in response to aggregations (XFS/POAG = 1.9 fold, p<0.005). Total inhibition of autophagy by Spautin or Bafilomycin-A increased the LOXL1 concentration in XFS TF by 1.63±0.18, (p<0.01) and 1.41±0.01, (p<0.005), respectively. There was no comparable effect in POAG TFs, indicating that processing of LOXL1-containing aggregates by autophagy takes place only in XFS cells. Autophagy requires the close approximation of autophagosomes and lysosomes at the microtubule organizing center. The latter is held together by a Y shaped γ-tubulin complex (γ-TuSK) and anchored to the nuclear centrosomes via Ninein. Immunostaining for these proteins revealed that in XFS cells the complex is small and mislocalized compared to the well-developed, mature γ-TuSK seen in POAG cells. A USP19-dependent export mechanism for misfolded proteins has been recently described ( Nat Cell Biol. 2016;18:765). Co-IP studies demonstrated association of USP19 with LOXL1 in the XFS conditioned medium but not in the POAG medium. Additionally, knockdown of USP19 (>80%) by siRNA significantly reduced the amount of LOXL1 exported, indicating that USP19 acts as a chaperone for LOXL1 in XFS cells.
Cellular pathways that handle misfolded proteins are actively clearing LOXL1 polypeptides in XFS cells.
This is an abstract that was submitted for the 2017 ARVO Annual Meeting, held in Baltimore, MD, May 7-11, 2017.
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