Purpose: Retinal degenerations are a heterogeneous group of diseases in which there is slow but progressive loss of photoreceptors (PR). There are currently no approved therapies for treating retinal degenerations. In an effort to identify novel small molecules that are (1) neuroprotective and (2) promote PR differentiation, we have developed microscale (1,536 well) cell culture assays using primary retinal neurons.
Methods: Primary murine retinal cells are isolated, seeded, treated with a 1,280 compound chemical library in a 7 point titration and then cultured under conditions developed to assay protection against an introduced stress or enhance PR differentiation. In the protection assays a chemical insult is introduced and viability assessed after 72 h using CellTiterGlo, a single-step chemiluminescent reagent. In the differentiation assay, cells are isolated from the rhodopsin-GFP knock-in mouse and PR differentiation is assessed by fixing cells after 21 days in culture and imaging with the Acumen plate-based laser cytometer (TTP Labtech) to determine number and intensity of GFP-expressing cells. Positive wells are re-imaged at higher resolution with an INCell2000 automated microscope (GE). Concentration-response curves are generated to pharmacologically profile each compound and hits identified by xx.
Results: We have developed PR differentiation and neuroprotection assays with a signal to background (S/B) ratios of 11 and 3, and a coefficient of variation (CV) of 20 and 9 %, suitable for chemical screening. Staurosporine has been shown in our differentiation assay to simultaneously increase the number of rhodopsin positive objects while decreasing the mean rhodopsin intensity and punctate rhodopsin fluorescent objects.
Conclusions: Using primary murine retinal cells, we developed high throughput assays to identify small molecules that influence PR development and survival. By screening multiple compound concentrations, dose-response curves can be generated, and the false negative rate minimized. It is hoped that this work will identify both potential preclinical candidates as well as molecular probes that will be useful for analysis of the molecular mechanisms that promote PR differentiation and survival.