Involvement of OA1, an intracellular GPCR, and G alpha i3, its binding protein, in melanosomal biogenesis and optic pathway formation

Abstract

Purpose: Ocular albinism type 1 (OA1) is characterized by abnormalities in retinal pigment epithelium (RPE) melanosomes and misrouting of optic axons. The OA1 gene encodes a G-protein-coupled receptor (GPCR) that coimmunoprecipitates with the G alpha i-subunit of heterotrimeric G-proteins from human melanocyte extracts. This study was undertaken to test whether one of the G alpha i proteins, G alpha i3, signals in the same pathway as OA1 to regulate melanosome biogenesis and axonal growth through the optic chiasm.

Methods: Adult G alpha i3(-/-) and Oa1(-/-) mice were compared with their respective control mice (129Sv and B6/NCrl) to study the effects of the loss of G alpha i3 or Oa1 function. Light and electron microscopy were used to analyze the morphology of the retina and the size and density of RPE melanosomes, electroretinograms to study retinal function, and retrograde labeling to investigate the size of the uncrossed optic pathway.

Results: Although G alpha i3(-/-) and Oa1(-/-) photoreceptors were comparable to those of the corresponding control retinas, the density of their RPE melanosomes was significantly lower than in control RPEs. In addition, the RPE cells of G alpha i3(-/-) and Oa1(-/-) mice showed abnormal melanosomes that were far larger than the largest 129Sv and B6/NCrl melanosomes, respectively. Although G alpha i3(-/-) and Oa1(-/-) mice had normal results on electroretinography, retrograde labeling showed a significant reduction from control in the size of their ipsilateral retinofugal projections.

Conclusions: These results indicate that G alpha i3, like Oa1, plays an important role in melanosome biogenesis. Furthermore, they suggest a common Oa1-G alpha i3 signaling pathway that ultimately affects axonal growth through the optic chiasm.

Figure 1

Appearance of photoreceptors and RPE melanosomes in Gαi3^−/−, Oa1^−/−, and corresponding control mice. (a–d) Light micrographs of semithin sections show that the photoreceptors of Gαi3^−/− and Oa1^−/− retinas have normal morphology. Scale bar, 5 μm. (e–h) Electron micrographs of ultrathin sections (scale bar, 0.1 μm) show larger melanosomes in Gαi3^−/− (e) than in 129Sv RPE (f). They are oval and have blebby membranes (e, white arrows). Smooth, round, or oval macromelanosomes in Oa1^−/− (g) are not present in B6/NCrl RPE (h). OS, outer segment; IS, inner segment; ONL, outer nuclear layer.

Figure 2

Density and size distribution of melanosomes in Gαi3^−/− and Oa1^−/− and their respective control mice. (a) Melanosome density in Gαi3^−/− and 129Sv RPEs. (b) Melanosome size distributions for Gαi3^−/− and 129Sv RPEs. (c) Melanosome density in Oa1^−/− and B6/NCrl RPEs. (d) Melanosome size distributions for Oa1^−/− and B6/NCrl RPEs. *Significant differences.

Figure 3

Standardized electroretinography was performed in all experimental and control mice. Amplitudes and latencies of the photopic a-and b waves, flicker function, scotopic b waves, and dark-adapted maximum stimulation a- and b waves were measured. (a) Responses obtained from Gαi3^−/− and control 129Sv mice. (b) Responses obtained from Oa1^−/− and control B6/NCrl mice.

Figure 4

Retrogradely labeled cells in the GCL (a) and INL (b) of the ventrotemporal retina after injection of HRP into the ipsilateral optic pathway. Labeled cells were counted in c2J, B6/J, Oa1^−/−, B6/NCrl, Gαi3^−/−, and 129Sv mice.

Figure 5

(a) Retinal flatmounts showing the distribution of uncrossed RGCs in B6/J and c2J mice. (b) Total number of uncrossed RGCs in retinas of B6/J and c2J mice. (c) Proportion of uncrossed cells in B6/J and c2J displaced to the INL. (d) Proportion of uncrossed cells positioned in the nasal retina of B6/J and c2J mice. (e–h) Retinal distributions and histograms for the 129Sv and Gαi3^−/− mice (conventions as in a–d). (i–l) Retinal distributions and histograms for B6/NCrl and Oa1^−/− mice (conventions as in a–d). *Significant differences.