A small molecule screen to identify molecular mechanisms of rod outer segment renewal

Abstract

 

Purpose

 

In many retinal degeneration diseases photoreceptor outer segments progressively shorten before cell death. The question remains whether outer segment loss causes photoreceptor cell death. A continuous renewal process of growth at the outer segment base and shedding from the tip is required for function of photoreceptors, yet we know very little about how these cells regulate renewal. The long-term goal of our research is to identify and define the molecular pathways and cellular mechanisms that regulate photoreceptor maintenance. To this end we are screening a library of small molecules of known bioactivity to identify these molecular pathways and mechanisms of outer segment renewal in zebrafish.

 

Methods

 

The screening platform uses our recently developed Tg(hsp70:mCherry^TM) zebrafish line, which, following heat-shock, expresses transmembrane (TM) mCherry. In rod photoreceptors, the mCherry^TM localizes to a small number of discs in the outer segment and is displaced apically until it is shed from the distal tip. Using bath application, we treat heat-shocked 6-day post fertilization (dpf) zebrafish with drugs for 4 days. At 10 dpf, retinal sections from treated fish are immunostained with anti-Rhodopsin. Confocal z-projections are acquired and measured for growth and shedding rates using mCherry^TM. We expect to screen about 1800 small molecules in two years.

 

Results

 

We will present data showing that this platform allows us to quantitatively measure rates of rod outer segment growth and shedding and also offers the added opportunity to concurrently screen for small molecules that affect phagocytosis and digestion of shed rod outer segment material by the neighboring retinal pigmented epithelium, rod outer segment disc integrity, Rhodopsin trafficking and localization, and rod differentiation and morphogenesis.

 

Conclusions

 

If, as expected, chemical compounds are discovered that stimulate outer segment growth, these can then be tested in zebrafish and mouse models of human retinal degeneration disease for their ability to stimulate outer segment growth, promote photoreceptor survival and prolong vision.  

 

Rod outer segment (ROS) growth assay. (A) Diagram of heat-shock inducted mCherry^TM localized to discs that are displaced distally as new discs are added. (B) Z-projection of an 8dpf retina heat-shocked at 5dpf showing GFP⁺ rods with mCherry^TM stripes in anti-Rho labeled ROSs.