Optogenetic Regulation of Aqueous Outflow in Mouse Trabecular Meshwork

Abstract

Purpose : Trabecular meshwork cells are critical to maintaining aqueous outflow and intraocular pressure. In tissues such as the kidney, bones, and cartilage, a small cellular appendage known as the primary cilium plays a mechanosensory role in fluid flow. Our previous findings demonstrate that phosphoinositide lipids within primary cilium may be an important regulator of aqueous humor dynamics. In particular, a mouse model deficient in OCRL, an inositol polyphosphate 5-phosphatase which dephosphorylates PI(4,5)P₂ and localizes to the primary cilium of trabecular meshwork, demonstrated elevated IOP levels and lower outflow rate. The purpose of this study is to explore the potential for cilia of trabecular meshwork cells to serve a mechanosensory role in the eye.

Methods : A novel optogenetic system was used to selectively direct protein translocation and activation of ciliary inositol phosphatases. Ciliary targeting GFP-CIBN-VAPA and light-sensitive mCherry-CRY2-OCRL constructs were transduced into HTM cells. C57bl/6J wildtype mice were injected with the same constructs via lentiviral intraocular delivery and allowed to express the constructs. Blue light emitting LED was used to stimulate the localization of the optogenetic module of CIBN/CRY2-OCRL to the cilia. An anterior chamber perfusion system was built to measure conventional outflow facility in mouse eyes via a computer-controlled flow pump.

Results : HTM cells transduced with GFP-CIBN-VAPA demonstrated proper ciliary targeting, where GFP expression was detected along cilia. Upon blue light stimulation of HTM cells transduced with both CIBN and CRY2 modules, mCherry-OCRL expression was strongly detected along GFP-targeted cilia. Furthermore, TM cells of mice injected with GFP lentivirus strongly expressed GFP. When injected with both CIBN and CRY2 constructs, increased outflow facility (p < 0.001, student t-test) was detected in mice after exposure to blue light, as compared to mice expressing only the CIBN construct (N=18; control N=14). There was no statistical significance (p = 0.0029, student t-test) between mice expressing only CIBN or CRY2 when exposed to blue light (N=14; N=7).

Conclusions : Alterations in PIP₂ dephosphorylation via OCRL recruitment increase outflow facility in mice by optogenetic stimulation of inositol phosphatase OCRL. Our results support an important role for ciliary phosphoinositides in the regulation of aqueous outflow.

This is an abstract that was submitted for the 2018 ARVO Annual Meeting, held in Honolulu, Hawaii, April 29 - May 3, 2018.