Phosphodiesterase inhibition reduces outer segment shedding of zebrafish rod photoreceptors

Abstract

Purpose: A common characteristic of inherited retinal dystrophies is the progressive degeneration of photoreceptor outer segments, and yet the molecular mechanisms that regulate outer segment length are largely unknown. We previously described a genetically encoded marker of zebrafish rod photoreceptor outer segment (ROS) renewal, which will aid in identifying the molecular mechanisms that regulate ROS length. Given that ROS shedding follows daily illumination, we hypothesized that pharmacological inhibition of phosphodiesterase activity, which should mimic the dark state by maintaining high cGMP concentration, would inhibit ROS shedding.

Methods: Transgenic zebrafish that express GFP in rods and a transmembrane-bound mCherry protein under the control of a heat shock promoter were heat shocked at 5 days post fertilization (dpf) and treated for 3 days post heat shock (dpHS) with a range of concentrations of phosphodiesterase inhibitor (sildenafil, tadalafil, or vardenafil). Following drug treatment, retinal sections were immunostained and confocal z-stacks were collected of the photoreceptor layer. Individual rod photoreceptors (n=50-120 per treatment) were analyzed by measuring the distance from the mCherry stripe to the ROS tip (D^S). D^S from drug-treated fish were compared to DMSO-treated control fish. Statistical analyses were performed using Student’s t-test.

Results: As compared to DMSO-treated control (5.3 µm), D^S was significantly increased (p<0.001) in 50 µM sildenafil (7.6 µm) and 50 µM vardenafil-treated (6.8 µm) larvae but was decreased in 50 µM tadalafil-treated larvae (4.4 µm). Rod morphology was noticeably affected by vardenafil as seen by a compaction of the myoid region of the inner segment. D^S was also significantly increased in adult fish treated with 50 µM sildenafil for 6 dpHS as compared to control (22.5 µm vs. 16.5 µm; p<0.001).

Conclusions: The increases in D^S with sildenafil and vardenafil treatments are consistent with our hypothesis that phosphodiesterase inhibition would inhibit ROS shedding. Further investigation of cGMP concentration as a mechanism for the regulation of ROS length is needed. In addition, investigation of sildenafil treatment on models of human retinal degeneration disease may provide a basis for potential treatment of blinding disorders that are characterized by photoreceptor degeneration.