March 2012
Volume 53, Issue 14
ARVO Annual Meeting Abstract  |   March 2012
ASB10 Expression in Trabecular Meshwork Cells and the Effects of Gene Silencing on Outflow Facility
Author Affiliations & Notes
  • Yong-Feng Yang
    Ophthalmology, Casey Eye Institute-OHSU, Portland, Oregon
  • Mary K. Wirtz
    Ophthalmology, Casey Eye Institute-OHSU, Portland, Oregon
  • Ted S. Acott
    Ophthalmology, Casey Eye Institute-OHSU, Portland, Oregon
  • Kate E. Keller
    Ophthalmology, Casey Eye Institute-OHSU, Portland, Oregon
  • Footnotes
    Commercial Relationships  Yong-Feng Yang, None; Mary K. Wirtz, None; Ted S. Acott, None; Kate E. Keller, None
  • Footnotes
    Support  EY003279, EY008247, EY010572, EY019643, EY011650, Research to Prevent Blindness
Investigative Ophthalmology & Visual Science March 2012, Vol.53, 3237. doi:
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      Yong-Feng Yang, Mary K. Wirtz, Ted S. Acott, Kate E. Keller; ASB10 Expression in Trabecular Meshwork Cells and the Effects of Gene Silencing on Outflow Facility. Invest. Ophthalmol. Vis. Sci. 2012;53(14):3237.

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

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Purpose: : ASB10 (ankyrin repeat and SOCS box containing protein-10) was recently identified as the primary open-angle glaucoma (POAG) gene at the GLC1F locus. A new ASB10 splice variant was identified in POAG patients, which resulted in production of a truncated ASB10 protein. We hypothesize that loss of full-length ASB10 contributes to elevated intraocular pressure (IOP). Here, we investigate ASB10 protein expression in cultured trabecular meshwork (TM) cells and evaluate the effects of knocking down full-length ASB10 transcripts and protein on outflow facility using silencing lentivirus.

Methods: : Human TM cells were derived from donor eyes. Immunofluorescence and confocal microscopy was performed using antibodies to ASB10, fibronectin, HDAC6, caveolin-1 (CAV1), and ubiquitin (Ub). Two antibodies to ASB10 are available: one that recognizes the V1 alternatively spliced N-terminal domain and one that recognizes all full-length ASB10 isoforms. shRNA lentivirus was generated to target full-length ASB10 and the effects on outflow facility were determined in an ocular perfusion culture model.

Results: : The two ASB10 antibodies showed different localizations by immunofluorescence. The V1 antibody stained in a predominantly fibrillar pattern, which partially colocalized with fibronectin. Conversely, the antibody to all isoforms stained intracellular vesicles and little fibrillar staining was observed. The vesicular staining colocalized with HDAC6, a specific marker of aggresomes, and partially colocalized with CAV1 and Ub. Western blotting showed that both antibodies recognized a band at the predicted size (49 kDa), but other bands were also observed. Application of shASB10 lentivirus targeting full-length transcripts to human perfusion culture caused an approximately 50% reduction in outflow facility compared to control lentivirus.

Conclusions: : Colocalization with fibronectin suggests that a portion of ASB10 variant 1 is extracellular, while colocalization with HDAC6, CAV1 and Ub suggests that ASB10 could play a role in the degradation pathway, a function similar to other ASB proteins. ASB10 silencing studies indicate that reduction of full-length ASB10 may contribute to elevated IOP in POAG patients.

Keywords: trabecular meshwork • outflow: trabecular meshwork • immunohistochemistry 

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