Induction of autophagy in rats upon overexpression of wild-type and mutant optineurin gene

Optineurin is a gene associated with normal tension glaucoma and amyotrophic lateral sclerosis. It has been reported previously that in cultured RGC5 cells, the turnover of endogenous optineurin involves mainly the ubiquitin-proteasome pathway (UPP). When optineurin is upregulated or mutated, the UPP function is compromised as evidenced by a decreased proteasome β5 subunit (PSMB5) level and autophagy is induced for clearance of the optineurin protein.

The UPP function was reduced and autophagy was induced when wild-type and E50K optineurin was overexpressed in rat eyes. This study validates the in vitro findings, confirming that UPP impairment and autophagy induction also occur in vivo. In addition, rapamycin is demonstrated to clear the accumulated mutant optineurin. This agent may potentially be useful for rescuing of the adverse optineurin phenotypes in vivo.

Adeno-associated type 2 viral (AAV2) vectors for green fluorescence protein (GFP) only, GFP-tagged wild-type and Glu50Lys (E50K) mutated optineurin were intravitreally injected into rats for expression in retinal ganglion cells (RGCs). Following intravitreal injections, eyes that received optineurin vectors exhibited retinal thinning, as well as RGC and axonal loss compared to GFP controls. By immunostaining and Western blotting, the level of PSMB5 and autophagic substrate degradation marker p62 was reduced, and the level of autophagic marker microtubule associated protein 1 light chain 3 (LC3) was enhanced. The UPP impairment and autophagy induction evidently occurred in vivo as in vitro. The optineurin level, RGC and axonal counts, and apoptosis in AAV2-E50K-GFP-injected rat eyes were averted to closer to normal limits after treatment with rapamycin, an autophagic enhancer. Conclusions: The UPP function was reduced and autophagy was induced when wild-type and E50K optineurin was overexpressed in rat eyes. This study validates the in vitro findings, confirming that UPP impairment and autophagy induction also occur in vivo. In addition, rapamycin is demonstrated to clear the accumulated mutant optineurin. This agent may potentially be useful for rescuing of the adverse optineurin phenotypes in vivo.