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Jorge Antonio Alvarado, Philipp Prosseda, ke ning, Tia Kowal, BIAO WANG, Yang Hu, Yang Sun; The Role of Phosphoinositides in Aqueous Humor Dynamics via Optogenetic Stimulation in the Trabecular Meshwork. Invest. Ophthalmol. Vis. Sci. 2019;60(9):5131.
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Glaucoma is the leading cause of blindness worldwide, often associated with elevated intraocular pressure (IOP) and reduced aqueous outflow facility. Defects in the trabecular meshwork (TM) result in aqueous humor build-up and subsequent rise in IOP. Previously we found that changes in OCRL, an inositol polyphosphate 5-phosphatase that localizes to primary cilia of the TM, affects aqueous humor dynamics; in particular, Ocrl-deficient mice had elevated IOP and lower outflow facility. The purpose of this study is to explore the potential for cilia in TM cells to maintain aqueous humor homeostasis.
In vivo, a CIBN/CRY2 optogenetic system was used to recruit light-sensitive CRY2-OCRL to the cilia via CIBN-VAPA or -SSTR3; to the cell membrane via CIBN-CAAX; or to the nucleus via CIBN-NLS. AAV viral particles of this CIBN/CRY2 module were injected into the anterior chamber of Ocrl-deficient or dexamethasone-treated mice, and after blue light stimulation, an anterior chamber perfusion system was used to record conventional outflow facility. In vitro, LifeAct Actin and CIBN/CRY2 constructs were transfected to HTM cells and stimulated with blue light. Additionally, cells were treated with OCRL inhibitor or transfected with CRY2/CIBN and stained for phalloidin after light stimulation.
After CIBN/CRY2 expression, increased outflow facility and lower IOP (p < 0.05, t-test) were recorded in both Ocrl-deficient and steroid-induced glaucoma eyes (N=3; N=4) upon blue light stimulation compared to non-stimulated controls. HTM cells transfected with CAAX had fewer actin stress fibers and a significant decrease in cell size (p < 0.05) after light exposure, while VAPA, NLS, and phosphatase-dead mutant OCRL did not produce cytoskeletal defects (N=6). Cells treated with OCRL inhibitor had a disorganized actin structure in the phalloidin staining when compared to untreated.
Defects in outflow facility were rescued in Ocrl-deficient and steroid-induced glaucoma mouse models via optogenetic OCRL recruitment to primary cilia and cellular membrane. Cell contraction and actin stress fiber reduction were observed when OCRL with active phosphatase domain was targeted to the cell membrane, in addition to actin disarray when OCRL was inhibited. Our results support an important role for OCRL in cytoskeletal organization, which in turn modulates aqueous humor dynamics.
This abstract was presented at the 2019 ARVO Annual Meeting, held in Vancouver, Canada, April 28 - May 2, 2019.
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