Research ArticleCELL BIOLOGY

Optogenetic stimulation of phosphoinositides reveals a critical role of primary cilia in eye pressure regulation

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Science Advances  29 Apr 2020:
Vol. 6, no. 18, eaay8699
DOI: 10.1126/sciadv.aay8699
  • Fig. 1 Optogenetic modulation of OCRL in subcellular compartments.

    (A) Schematic model of optogenetic recruitment of mCh–CRY2–5-ptaseOCRL to the plasma membrane or primary cilium after blue light activation. Activation of blue light causes recruitment of cytosolic mCh–CRY2–5-ptaseOCRL to its dimerization partner EGFP-CIBN, which can be targeted to subcellular compartments. (B) Confocal images of the peripheral region of hRPE cells. Cells expressing the mCh–CRY2–5-ptaseOCRL and CIBN-EGFP-CAAX were analyzed via Z-stack to monitor mCh–CRY2–5-ptaseOCRL accumulation before and at intervals 10 min after illumination with 20 × 300–ms blue light pulses, and a respective mCh–CRY2–5-ptaseOCRL intensity data graph was plotted (N = 6). A.U., arbitrary units. (C) Representative images of CIBN-EGFP constructs with ciliary targeting domains. RPE cells were transfected with ciliary targeting constructs CIBN-EGFP-(VAPA/SSTR3) and then fixed and stained with a ciliary marker (ARL13b). (D) Representative images of optogenetic mCh–CRY2–5-ptaseOCRL recruitment to ciliary targeting CIBN constructs, VAPA and SSTR3, and nuclear targeting CIBN control (NLS). (E) Confocal images of HTM cells expressing the mCh–CRY2–5-ptaseOCRL and CIBN-EGFP-SSTR3. mCh–CRY2–5-ptaseOCRL accumulation in the ciliary area was measured before and at intervals 10 min after illumination with 20 × 300–ms blue light pulses, and a respective mCh–CRY2–5-ptaseOCRL intensity data graph was plotted (N = 6).

  • Fig. 2 Outflow facility measurement of optogenetically treated mice.

    (A) Schematic design of optogenetic constructs CRY2–5-ptaseOCRL and CIBN-EGFP were packaged into AAV2-s or lentiviral vectors and injected at day 0 into the anterior chamber of C57BL/6J WT mice. Ocular injection was visualized by dilution in Fast Green blue dye. The anterior chamber was punctured with a 32-gauge needle horizontally, injected with air to create a small air bubble, and infused with 2 μl of virus (>1010 plaque-forming units). Days 1 to 28: Viral expression of optogenetic constructs with lentivirus or AAV2-s was allowed for a period of 4 weeks, at which time positive cell transduction in the mouse anterior segment, including the TM, was achieved. The vehicle control expressed no fluorescence. (B) Schematic design of in vivo optogenetic activation. The right eye of each animal was exposed to 10-mW blue light (450-nm laser) for 10 min followed by outflow facility measurement. (C) Perfusion plots of mouse eye injected with AAV2-s–CIBN–EGFP–CAAX and CRY2–5-ptaseOCRL with and without blue light exposure. Eyes with illumination demonstrate higher outflow facility than those without exposure, as represented by a significant difference between slopes (N = 10 eyes). (D) No significant difference was observed in AAV2-s–CIBN–EGFP control (N = 10 eyes) or (E) AAV2-s–CIBN–NLS nuclear targeting constructs (N = 8 eyes). (F) Outflow facility measurement of eyes injected with ciliary targeting CIBN via AAV2-s intraocular delivery shows a significant increase in outflow facility (N = 9 eyes). (G) Membrane targeting: Decrease in IOP compared to nonilluminated control eyes (H). Ciliary targeting: Decrease in IOP compared to nonilluminated control eyes. Statistical analysis: Paired sample t test, where P < 0.05 was considered statistically significant. Error bars represent SEM. (I) Representative eye pressure tracing of light-stimulated WT mice eye treated with AAV2-s–CIBN and CRY2–5-ptaseOCRL.

  • Fig. 3 Phosphoinositide modulation influences calcium regulation and the actin cytoskeleton of TM cells.

    (A) Representative image of LifeAct actin and CRY2–5-ptaseOCRL distribution in hRPE and HTM cells. After expression of the CIBN and CRY2–5-ptaseOCRL constructs, the cells were stimulated with a train of pulses of 488-nm light and the cytoskeletal reorganization was measured. Cell outline was indicated before and 10 to 20 min after CRY2–5-ptaseOCRL recruitment. A significant decrease in cell size parallel to contraction of the internal actin arrangement was detected in plasma membrane–targeted CRY2–5-ptaseOCRL. (B) Cell contraction assay of CRY2–5-ptaseOCRL and CIBN-SSTR3/CAAX–transfected HTM cells after blue light illumination. (C) LifeAct actin CRY2–5-ptaseOCRL and CIBN-CAAX–transfected cells pretreated with BAPTA AM before and after blue light optogenetic activation. Representative image and quantification are shown. (D) Fluorescence intensity graph of BAPTA AM after blue light optogenetic activation. (E) CRY2–5-ptaseOCRL recruitment to plasma membrane, followed by 10-min incubation and phalloidin staining in 4% paraformaldehyde fixation. Recruitment to plasma membrane was associated with increased actin stress fiber fragmentation compared with non–light-activated control. Statistical analysis for cell size change (N = 6), where six treated cell sizes were compared with untreated cell sizes that are normalized to 1, unpaired t test, where P < 0.05 was considered statistically significant. Error bars represent SEM (*P ≤ 0.05 and **P ≤ 0.01).

  • Fig. 4 OCRL modulation of a Lowe syndrome mouse model.

    (A) Left: Retinal imaging and optical coherence tomography of right (OD) and left (OS) eyes of IOB mouse. Right: Vessel imaging of an IOB mouse with severe cataracts. (B) Optic nerve images of human patients with Lowe syndrome and glaucomatous optic nerve cupping. (C) Flat mount and (D) cross sections of normal and IOB mice retinas, which were stained with an RGC marker, RBPMS, and hematoxylin and eosin stain, respectively. A marked decrease in the RGCs layer and number of RGCs is detected in 4-month-old IOB mice compared to the same age WT control. IPL, inner plexiform layer; INL, inner nuclear layer; OPL, outer plexiform layer; ONL, outer nuclear layer. (E) Average outflow facility of perfused eyes in IOB−/− mice (N = 14) or IOB+/Y mice (control, N = 12) and comparison performed with unpaired t test. (F) IOP measurements using TonoLab tonometer. IOP measurements of IOB−/− mice (N = 10) versus IOB+/Y (control) mice (N = 8) are obtained, and comparison was performed with unpaired t test. (G) Perfusion plots of IOB model eyes injected with AAV2-s–CIBN–CAAX and mCh–CRY2–5-ptaseOCRL with and without blue light exposure. Eyes with illumination demonstrate a higher outflow facility and lower IOP than those without exposure, as represented by a significant difference between slopes (N = 4, comparison between left and right eye). Statistical analysis was performed using paired sample t test. (H) Perfusion plots of IOB model eyes injected with AAV2-s–CIBN–SSTR3 and mCh–CRY2–5-ptaseOCRL with and without blue light exposure. Eyes with illumination demonstrate a higher outflow facility and lower IOP than those without exposure, as represented by a significant difference between slopes (N = 4, comparison between left and right eye). Statistical analysis was performed by paired sample t test, where P < 0.05 was considered statistically significant. Error bars represent SEM (*P ≤ 0.05 and **P ≤ 0.01).

  • Fig. 5 Phosphoinositides and primary cilia regulate aqueous outflow facility.

    (A) Anterior chambers of WT mice eyes were injected with 100 μM (YU142670) OCRL inhibitor, and perfusion analysis was performed after 30-min incubation. Perfusion analysis of related, untreated control mice was performed under the same conditions, and outflow comparison is shown as a whisker plot of outflow facility C for YU142670. (B) Schematic representation of drug targets. (C) Perfusion plots of eye anterior chamber injected with 1 μl of 10 μM PI3K inhibitor (GSK1059615) or 50 μM PI3K activator (740-Y-P) per eye. (D) Eye pressure tonometer analysis of GSK1059615- and 740-Y-P–treated mice after 2-hour incubation. (E) Analysis of IFT88fl/fl mice intraocularly injected with CRE lentivirus for primary cilia removal in combination with AAV2-s–CRY2–OCRL + AAV2-s–CIBN–CAAX/SSTR3. (F) Relevant whisker plot of outflow facility C for optogenetically treated IFT88fl/fl/CRE eyes (blue dots, light activated) (N = 4 eyes). Statistical analysis was performed by independent samples t test and paired t test, where P < 0.05 was considered statistically significant. Error bars represent SEM. (G) Schematic representation of 5-ptaseOCRL–dependent optogenetic modulation of IOP in the TM of mouse eye. (*P ≤ 0.05 and **P ≤ 0.01).

Supplementary Materials

  • Supplementary Materials

    Optogenetic stimulation of phosphoinositides reveals a critical role of primary cilia in eye pressure regulation

    Philipp P. Prosseda, Jorge A. Alvarado, Biao Wang, Tia J. Kowal, Ke Ning, W. Daniel Stamer, Yang Hu, Yang Sun

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