LOXL1 protein aggregation in Exfoliation Glaucoma

Abstract

Purpose : Tenon fibroblasts (TF) from exfoliation glaucoma (XFG) patients display cellular impairments resembling those observed in age-related neurodegenerative diseases (PLoS ONE 2016, 11: e0157404). The latter are related to intrinsically disorganized polypeptide (IDP) domains which frequently misfold and aggregate. We examined whether LOXL1 protein contains IDP domains and demonstrates increased processing through autophagy pathways in XFG TFs.

Methods : Autophagy was accelerated by FBS removal. Intracellular protein aggregates were visualized with Proteostat^TM. Skin fibroblasts were transduced with a lentivector for overexpression of FLAG-tagged LOXL1. Microtubule dynamics were measured with live cell scanning confocal recordings of cells expressing EB1-mCherry, which tracks the plus ends of growing microtubules. Additional analytical methods included Western blot, immunostaining and flow cytometry for cell size and relative MMPT using JC1.

Results : Multiple IDP domains are present in the LOXL1 N-terminus (DisEMBL [EMBL]), while no IDP was present in LOX or LOXL2-4. Proteostat staining demonstrated aggregated protein only in XFG TF. Aggregate presence in XFS TF was also inferable from the much higher expression of clusterin (XFG/POAG = 1.9 fold, p<0.005). Inhibition of autophagy by Spautin or Bafilomycin-A increased LOXL1 protein in XFG 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 TF, indicating that processing of LOXL1-containing aggregates by autophagy takes place only in XFG cells. Autophagy is coordinated with microtubule dynamics. In XFG TF “dynamicity” (total plus end movement) and speed (um/min) of microtubule growth were both 2-fold higher compared to controls (p < 0.05). Additionally, cells overexpressing LOXL1-FLAG accumulated as amorphous aggregations in the perinuclear zone and caused a) cell shape change; b) increase in cell diameter (29 ± 3 %; p < 0.01); c) increase in MMPT signal (12 % ± 5 (p < 0.05) indicative of cell stress; and d) a higher proportion of cells with faster growing microtubules.

Conclusions : Cellular pathways that handle misfolded proteins are actively attempting to clear LOXL1 proteins in XFG cells. LOXL1 may be an aggregation-prone protein.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.

 

Figure 1. LOXL1 overexpressing cells have aggregated LOXL1. Western blot and immunostaining for LOXL1 in immortalized skin fibroblasts transduced with a CMV-LOXL1-FLAG lentivector or empty vector control. Bar=50um.