Inhibition of dopamine signaling suppresses cGMP accumulation in rd1 retinal organ cultures

Abstract

The rd1 mouse is a model of retinitis pigmentosa, an inherited photoreceptor neurodegenerative disease. In rd1 retina, early onset rod degeneration is caused by a Pde6b mutation that leads to high levels of intracellular cyclic guanosine monophosphate (cGMP). Cyclic nucleotide-gated ion channels (CNGCs), necessary for phototransduction, are regulated by cGMP. We have previously demonstrated that inhibition of dopamine signaling blocks rd1 photoreceptor degeneration in retinal organ cultures. The mechanism underlying this protection remains unknown. The aim of this study was to determine whether inhibition of dopamine signaling alters cGMP accumulation or CNGC expression. Dopamine depletion from rd1 retinal organ cultures resulted in a significant decrease in cGMP compared with untreated rd1 organ cultures. However, cGMP levels in both treated and untreated rd1 organ cultures significantly exceeded cGMP levels in wild-type (wt) retinal organ cultures. The CNGC expression profile was first determined in vivo. Both channel subunits, Cnga1 and Cngb1, are expressed at low levels by postnatal day 2 (P2), increasing sharply by P6 with a modest increase after P12 in wt retina. A similar pattern is seen in rd1 retina until P12 when expression levels decrease, leading to cell death. No significant difference was observed in the expression of either Cnga1 or Cngb1 in organ cultures from wt, rd1, and dopamine-depleted rd1 retinas. Our results show that dopamine depletion significantly decreases cGMP levels in rd1 retinal organ cultures, but that cGMP accumulation remains high, requiring additional mechanisms for photoreceptor protection. These mechanisms may include activation of protein kinase G-signaling pathways and/or crosstalk with dopamine signaling through cyclic adenosine monophosphate pathways.

Figure 1. cGMP Content in Retinal Organ Cultures

Mean cGMP levels in wt, rd1, and 6-OHDA-treated rd1 retinal organ cultures at P14 as determined by enzyme immunoassay. cGMP levels are expressed as mean concentrations ± SEM in pmol/µg total protein. Sample numbers for each condition were as follows: wt control samples, n = 15; rd1 control samples, n = 20; rd1 6-OHDA-treated samples, n = 22. * p < 2.2e–16; ** p < 0.027.

Figure 2. Expression of CNG channels in rd1 retinas compared to wt

Relative expression of Cnga1 (A) and Cngb1 (B) in rd1 (grey) compared to wt (black) in postnatal retinas between P2 and P21. All time points were normalized to P21 wt. Early time points are shown on an amplified scale (insets). Actb (β-actin) was used as the reference gene. Significant differences between wt and rd1 are indicated (* p < 0.05; ** p < 0.001). The time course of Cngb1 protein expression as determined by Western blot (C) is consistent with mRNA expression. β-actin was used as a loading control.

Figure 3. CNG channel expression in retinal organ cultures

Cnga1 (A) and Cngb1 (B) expression in wt control, rd1 control, and rd1 6-OHDA-treated retinal organ cultures at P10. The ratio of mRNA expression level between wt and treated or untreated rd1 organ cultures was calculated by the 2^−ΔΔCt method after normalization to Actb. For each condition, nine independent samples were tested. No significant difference in expression was observed among the three conditions.