Abstract
PurposeOcular Albinism type 1 (OA1) is a disease caused by mutations in the OA1 gene and characterized by the presence of macromelanosomes in the retinal pigment epithelium (RPE) as well as abnormal crossing of the optic axons at the optic chiasm. We showed in our previous studies in mice that Oa1 activates specifically Gαi3 in its signaling pathway and thus, hypothesized that a constitutively active Gαi3 in the RPE of Oa1−/− mice might keep on the Oa1 signaling cascade and prevent the formation of macromelanosomes. To test this hypothesis, we have generated transgenic mice that carry the constitutively active Gαi3 (Q204L) protein in the RPE of Oa1−/− mice and are now reporting the effects that the transgene produced on the Oa1−/− RPE phenotype.MethodsTransgenic mice carrying RPE-specific expression of the constitutively active Gαi3 (Q204L) were generated by injecting fertilized eggs of Oa1−/− females with a lentivirus containing the Gαi3 (Q204L) cDNA. PCR, Southern blots, Western blots and confocal microscopy were used to confirm the presence of the transgene in the RPE of positive transgenic mice. Morphometrical analyses were performed using electron microscopy to compare the size and number of melanosomes per RPE area in putative Oa1−/−, Gαi3 (Q204L) transgenic mice with those of wild-type NCrl and Oa1−/− mice.ResultsWe found a correlation between the presence of the constitutively active Gαi3 (Q204L) transgene and the rescue of the normal phenotype of RPE melanosomes in Oa1−/−, Gαi3 (Q204L) mice. These mice have higher density of melanosomes per RPE area and a larger number of small melanosomes than Oa1−/− mice, and their RPE phenotype is similar to that of wild-type mice.ConclusionsOur results show that a constitutively active Gαi3 protein can by-pass the lack of Oa1 protein in Oa1−/− mice and consequently rescue the RPE melanosomal phenotype.
Highlights
X-linked ocular albinism is a disorder of melanosome biogenesis leading to congenital visual impairment in males [1]
While the precise function of GTPbinding protein alpha subunit polypeptide 3 (Gai3) in retinal pigment epithelium (RPE) melanosome biogenesis remains to be delineated, our studies suggest that this protein plays an important role in the control of the size and density of RPE melanosomes and in the regulation of RPE pigmentation [10,20]
It has been shown that transgenic mice can be generated efficiently by microinjection of lentivirus into the perivitelline space of single-cell mouse embryos, and that injecting a high dose of virus can produce near 100% transgenesis with variable copies of transgene in transgenic lines [21]
Summary
X-linked ocular albinism is a disorder of melanosome biogenesis leading to congenital visual impairment in males [1]. OA1 patients (as well as Oa1 knockout mice) have a reduction of ipsilateral retinal ganglion axons at the optic chiasm. These are the two main phenotypic characteristic of ocular albinism [3]. More than 60 mutations (missense, nonsense, frameshift or splice-site mutations) have been identified in affected individuals [4,5] These changes result in a nonfunctional OA1 protein and often prevent it from reaching its normal location at melanosomal membranes [6,7,8] or from interacting with other molecules of its signaling pathway [9]. Without a functional GPR143 protein, melanosomes in the RPE and melanocytes of the skin become abnormally large, but it is unclear how these macromelanosomes are related to vision abnormalities in patients with ocular albinism
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