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. 2016 Jun 1;57(7):3287-96.
doi: 10.1167/iovs.16-19356.

Harnessing the Endocannabinoid 2-Arachidonoylglycerol to Lower Intraocular Pressure in a Murine Model

Sally Miller  1 , Emma Leishman  1 , Sherry Shujung Hu  2 , Alhasan Elghouche  1 , Laura Daily  1 , Natalia Murataeva  1 , Heather Bradshaw  1 , Alex Straiker  1
Affiliations

Harnessing the Endocannabinoid 2-Arachidonoylglycerol to Lower Intraocular Pressure in a Murine Model

Sally Miller et al. Invest Ophthalmol Vis Sci. .

Abstract

Purpose: Cannabinoids, such as Δ9-THC, act through an endogenous signaling system in the vertebrate eye that reduces IOP via CB1 receptors. Endogenous cannabinoid (eCB) ligand, 2-arachidonoyl glycerol (2-AG), likewise activates CB1 and is metabolized by monoacylglycerol lipase (MAGL). We investigated ocular 2-AG and its regulation by MAGL and the therapeutic potential of harnessing eCBs to lower IOP.

Methods: We tested the effect of topical application of 2-AG and MAGL blockers in normotensive mice and examined changes in eCB-related lipid species in the eyes and spinal cord of MAGL knockout (MAGL-/-) mice using high performance liquid chromatography/tandem mass spectrometry (HPLC/MS/MS). We also examined the protein distribution of MAGL in the mouse anterior chamber.

Results: 2-Arachidonoyl glycerol reliably lowered IOP in a CB1- and concentration-dependent manner. Monoacylglycerol lipase is expressed prominently in nonpigmented ciliary epithelium. The MAGL blocker KML29, but not JZL184, lowered IOP. The ability of CB1 to lower IOP is not desensitized in MAGL-/- mice. Ocular monoacylglycerols, including 2-AG, are elevated in MAGL-/- mice but, in contrast to the spinal cord, arachidonic acid and prostaglandins are not changed.

Conclusions: Our data confirm a central role for MAGL in metabolism of ocular 2-AG and related lipid species, and that endogenous 2-AG can be harnessed to reduce IOP. The MAGL blocker KML29 has promise as a therapeutic agent, while JZL184 may have difficulty crossing the cornea. These data, combined with the relative specificity of MAGL for ocular monoacylglycerols and the lack of desensitization in MAGL-/- mice, suggest that the development of an optimized MAGL blocker offers therapeutic potential for treatment of elevated IOP.

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Figures

Figure 1
Figure 1
2-Arachidonoyl glycerol reduces IOP in a concentration- and CB1-dependent manner. (A) Shows changes in IOP (mm Hg) after 1 hour, with increasing concentrations of 2-AG lowering IOP in a concentration-dependent manner. (B) Responses to 5 mM 2-AG are absent in CB1−/− animals, but present in CB2−/− animals (C).
Figure 2
Figure 2
Monoacylglycerol lipase inhibition can reduce IOP. (A) Topical WIN55212 (1%) reduces IOP in MAGL−/− eyes. (B) 2-Arachidonoyl glycerol (2 mM) treatment in MAGL−/− eyes results in a trend toward IOP reduction (P = 0.0502). (C–E) Topical treatment with KML29 (1 mM) lowers IOP at 1, 4, and 8 hours after treatment. (F) However, KML29 is without effect in CB1−/− mice. *P < 0.05, ϕP = 0.05, paired t-test.
Figure 3
Figure 3
Monoacylglycerol lipase is prominently expressed in ciliary epithelium and iris but not cornea. (A) Overview in a nonpigmented CD1-strain mouse shows MAGL expression in several major anterior eye structures. CE, ciliary epithelium. (A', C'–F') Corresponding differential interference contrast (DIC) images. (B) Monoacylglycerol lipase staining in tissue from MAGL KO anterior eye tissue, taken at same settings. (C) Iris is prominently labelled. (D) Monoacylglycerol lipase is not present in corneal epithelium, endothelium, or stroma. (E) Monoacylglycerol lipase is present in ciliary epithelium (arrow), particularly the inner pigmented layer. (F) Close-up of the angle shows prominent labelling of the ciliary body (lower portion of image) but little labelling of the trabecular meshwork. Scale bars: (A) 150 μm, (B) 50 μm, (C–E) 40 μm, (F) 25 μm.
Figure 4
Figure 4
Monoacylglycerol lipase is expressed in pigmented ciliary epithelium. (A) Top shows phalloidin staining, particularly of ciliary muscle (cm) and trabecular meshwork (tm). Bottom shows MAGL staining absent outside of the ciliary body. (B) Inset from (A) shows MAGL staining in ciliary epithelium. (B1) Inset from (B) shows staining is limited to the inner pigmented epithelium (pe) and absent from nonpigmented epithelium (npe). (B2) Second inset shows that some staining appears to be membrane associated (sideways arrows) but other MAGL staining appears to be intracellular, in some cases with a striated pattern (angled arrows). Scale bars: (A) 50 μm, (B) 20 μm, (C, D) 5 μm.
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