Abstract
Purpose::
Following rod cell death in retinitis pigmentosa (RP), cone cells die by an uncharacterized secondary pathway which may be due to direct rod-cone interactions, or indirect effects of rod depletion. Understanding the mechanism of this secondary ablation is critical to the development of effective treatments to prevent it. By controlling the conditions of rod cell death, and monitoring the effect on surrounding cells, we can gain a better understanding of the processes that take place in the retina following rod cell death that eventually lead to blindness.
Methods::
We generated transgenic Xenopus laevis that express an inducible form of caspase 9 exclusively in rods. The inducible caspase 9 has binding domains specific for the compound AP20187 (Ariad Pharmaceuticals) which induces its dimerization and auto-activation. When expressed, this exogenous protein sits dormant in rod cells until it interacts with the compound, at which point apoptosis is initiated. We raised three primary transgenic animals past morphogenesis before administering a dorsal sac injection of AP20187. We employed electroretinography to assay retinal function longitudinally.
Results::
We demonstrated that transgenic animals injected with AP20187 show a dramatic reduction in scotopic electroretinograph response. We analyzed a- and b-wave amplitude, projected a-wave asymptote (Rmp3) derived from the Shady et al. modification of the Lamb and Pugh equation, as well as Vmax and K derived from fitting trough-to-peak amplitude at increasing stimulus intensity to the Naka Rushton equation. All measurements were dramatically reduced except K. Waveforms generated under photopic conditions designed to isolate cone function did not deviate from pre-drug measures initially, but a reduction in trough-to-peak amplitude was pronounced three months after the initial AP20187 injection.
Conclusions::
These results indicate that cone function was severely compromised as a consequence of rod cell death in Xenopus laevis, as seen in humans and other model systems. We will corroborate this information with other assays of cone cell function and viability. Using this model, we can compare secondary effects observed in the retina when rod death is initiated at different times or by different mechanisms.
Keywords: retinal degenerations: cell biology • photoreceptors • cell-cell communication