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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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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.
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.
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.
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.
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