Abstract
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