Study of Cyclic Guanosine Monophosphate in the Retinal Dystrophy Mouse

Retinitis Pigmentosa is a hereditary group of diseases that leads to blindness. Although there is no known treatment as of now, there is a considerable amount of research on the retina.

When light enters the eye, a phototransduction cascade occurs. A rhodopsin molecule absorbs a photon, which in turn activates the trimeric protein transducin. The next stage involves cGMP-phophodiesterase (PDE), which consists of four parts: a and ß catalytic subunits and two g inhibitory subunits. The activated transducin causes the g subunits to bind, which frees the catalytic subunits. Finally, we reach cyclic guanosine monophosphate (cGMP). cGMP allows the cGMP-gated cation channels on the plasma membrane to remain open in the dark, allowing calcium and sodium to freely enter. Activation of the PDE causes a decrease in the cGMP levels, which in turn, shuts the channels, stopping sodium and calcium from entering. It is uncertain how mutations in this process could result in photoreceptor death.

There are different genetic models that may allow us to learn more about Retinitis Pigmentosa. Our focus has been on the retinal dystrophy or RD mouse. In both humans and mice, mutations in the PDE gene can cause Retinitis Pigmentosa. As a result of a mutation in the ß subunit of the cGMP phosphodiesterase gene, the mice undergo a rapid loss of rod photoreceptors in the three weeks after birth. The Ogilvie lab is focusing on cGMP levels in the mouse retina; the PDE mutation results in a high concentration of cGMP before the photoreceptors degenerate. When the mice reach adult age, photoreceptors have died and levels of cGMP are very low.

To examine the retinas, organ cultures are created to, as much as possible, eliminate any variables. The cultures may be grown in control media or treated with growth factors in hope of rescuing the photoreceptor cells. In one test, Dr. Ogilvie added dopamine antagonists in the control group to test if the growth factors were working on dopaminergic cells, which in turn would work on the photoreceptors. However, Dr. Ogilvie discovered that the addition of dopamine antagonists resulted in complete survival of the photoreceptors. With these findings, a question arose: are cGMP levels stable as well? With this question comes my project. Using an assay kit measuring cGMP, I was to test the kit and make sure the results were accurate and believable. When the assay began to yield reliable results, I was to measure cGMP in RD retinal organ cultures of different ages to determine the relationship between cGMP and age in vitro. Initially, this could be compared to previously published in vivo results. Subsequently, this method could be used to determine cGMP levels in organ cultures treated with the dopamine antagonists.

A graph showing cGMP levels every one to two days from postnatal day 10 to 26 is almost complete. The published graph of in vivo data begins with low levels of cGMP, peaks, and then declines. Preliminary results from our work have shown similar results. However, the peak is at a later date, because in vitro photoreceptors tend to live longer than in vivo photoreceptors. In the future, when the graph is complete, we will be able to analyze the dopamine cGMP levels. By doing so, we can find out if cGMP levels are high, despite recovery of the photoreceptors, or if the dopamine antagonists were able to control the cGMP levels as well.