June 2015
Volume 56, Issue 7
Free
ARVO Annual Meeting Abstract  |   June 2015
Impaired Lysosomal and Mitochondrial Function in Exfoliation Glaucoma
Author Affiliations & Notes
  • Andrew Want
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY
    Einhorn Clinical Research Center, New York Eye and Ear Infirmary of Mount Sinai, New York, NY
  • Stephanie Gillespie
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY
  • J Mario Wolosin
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY
  • Robert Ritch
    Einhorn Clinical Research Center, New York Eye and Ear Infirmary of Mount Sinai, New York, NY
  • Audrey Bernstein
    Ophthalmology, Icahn School of Medicine at Mount Sinai, New York, NY
  • Footnotes
    Commercial Relationships Andrew Want, None; Stephanie Gillespie, None; J Mario Wolosin, None; Robert Ritch, None; Audrey Bernstein, None
  • Footnotes
    Support None
Investigative Ophthalmology & Visual Science June 2015, Vol.56, 1695. doi:
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    • Get Citation

      Andrew Want, Stephanie Gillespie, J Mario Wolosin, Robert Ritch, Audrey Bernstein; Impaired Lysosomal and Mitochondrial Function in Exfoliation Glaucoma. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):1695.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract
 
Purpose
 

In the eye, exfoliation syndrome (XFS) is characterized by the aggregation of disorganized microfibrils (exfoliation material, XFM). Deposition of XFM and pigment in the aqueous outflow pathway leads to chronic intraocular pressure elevation leading in turn to glaucoma. Similar to other age-related diseases in which protein aggregates cause disease, we hypothesize that lysosomal and mitochondrial dysfunction contributes to the formation of XFM aggregates.

 
Methods
 

Tenon fibroblasts (TFs) were explanted from tissue discards obtained from older, age-matched XFS and primary open-angle glaucoma (POAG) patients who underwent trabeculectomy surgery and from young healthy donors who underwent strabismus surgery. Experiments were performed in supplemented serum-free media on collagen or in 1% FBS-containing media. Cell size and mitochondrial membrane potential (MMPT, JC1 dye) were quantified by flow cytometry. Lysosomes and microtubules were immunodetected with Lamp-1 and β-tubulin antibody, respectively. Culturing TFs in media with stabilized vitamin C for 1 month generated self-synthesizing 3D gels.

 
Results
 

Normally, under conditions of nutrient deprivation, lysosomes become peri-nuclear, where they fuse with autophagosomes, clearing the cells of waste. In XFS TFs compared to POAG TFs and healthy TFs, lysosomes did not relocalize in response to changes in nutrient conditions, suggesting that lysosomal degradation is impaired in these cells (Figure 1). In 3D culture, XFS TFs demonstrated a disorganized morphology with elevated expression of XFM-containing proteins LOXL1 and Fibulin-5. Consistent with impaired lysosomal degradation a) the percent of cells displaying depolarized mitochondria was 10x higher in XFS than in POAG TFs (26 % vs. 2%, p < 0.01) and b) the build up of intracellular organelles led to a 1.7-fold increase in XFS cell size.

 
Conclusions
 

Our findings suggest that lysosomes and mitochondria are compromised in XFS TFs, leading to a toxic environment. This may lead to reduced degradation and increased secretion of XFM aggregates.  

 
Figure 1. XFS TFs display dysfunctional lysosomes. XFS (A), POAG (B), and young healthy (C) TFs were seeded on collagen-coated coverslips in supplemented serum-free media. After 24h cells were fixed and immunostained for lysosomes, LAMP1 (pink), microtubules, β-tubulin (green), nucleus, DAPI (blue). Arrow indicates lysosome distribution. N=3 for each patient type. Bar =50um.
 
Figure 1. XFS TFs display dysfunctional lysosomes. XFS (A), POAG (B), and young healthy (C) TFs were seeded on collagen-coated coverslips in supplemented serum-free media. After 24h cells were fixed and immunostained for lysosomes, LAMP1 (pink), microtubules, β-tubulin (green), nucleus, DAPI (blue). Arrow indicates lysosome distribution. N=3 for each patient type. Bar =50um.

 
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