Abstract
Purpose::
The retinal pigment epithelium (RPE) produces melanosomes similar to those found in melanocytes of the skin and choroid. The majority of the melanosomes in mammalian RPE appears to be formed during embryonic life, contrary to skin melanocytes. However, the timing and extent of RPE melanogenesis remains controversial. Recently, Rab38 and Rab32 were implicated in the transport of tyrosinase to premelanosomes in skin melanocytes, as deduced from skin melanocyte studies in Rab38-deficient chocolate (cht) mice and siRNA-mediated Rab32 downregulation.
Methods::
Conventional and cryo-IEM were performed in wild-type, cht, and albino (tyrosinase-negative) mice, at different ages (E14.5, P1, P8 and 4 months old). The number of stage II, III and IV melanosomes in RPE was determined by quantitative analysis, and RPE sections were stained for Pmel17, tyrosinase, Rab38 and Rab32.
Results::
RPE of adult homozygous cht mice exhibit a severe reduction in the number of RPE melanosomes, when compared with wild-type controls, while heterozygous cht mice have an intermediate phenotype. Choroidal melanosomes also exhibited similar trend, although the defect was less pronounced. All stages of melanosome biogenesis could be observed in the RPE at E14.5. At this stage, melanosomes exhibited strong staining for melanin-synthesising enzymes, which was reduced at P1 but barely detectable at P8. At P1, 60% of all melanosomes are stage II in cht RPE versus approx. 20% in heterozygous controls. Double labelling of E14.5 sections with Pmel17 and tyrosinase suggested a defect in delivery of tyrosinase to immature melanosomes in cht RPE, despite the expression of the redundant Rab32 protein. No premelanosomes were observed in adult mice, in both wild type and cht RPE. Interestingly, similar observations were obtained in albino RPE, ie, presence of stage II and III melanosomes at P1, but not in adult mice.
Conclusions::
Loss of functional Rab38 causes a dramatic loss of mature melanosomes due to a major reduction in the delivery of tyrosinase to premelanosomes. In the absence of Rab38 in the RPE, premelanosomes are synthesised but fail to deposit melanin, and are short lived. We suggest that the RPE pigmentation defect observed in cht mice is due to the short time frame in which melanogenesis occurs in the RPE, compared with skin and choroidal melanocytes where the process continues throughout life. This short window is insufficient for Rab32 to compensate for the loss of Rab38 activity.
Keywords: retinal pigment epithelium • melanocytes