Inhibition of soluble adenylyl cyclase (sAC) rescues defective melanosomal pH and pigmentation in oculocutaneous albinism type 2 (OCA2).

Abstract

Purpose : OCA2 is caused by mutations in an ion channel critical for the maintenance of the pH set-point of the melanosome, the organelle that makes melanin. Melanosomal pH controls pigmentation because tyrosinase is pH sensitive. Currently, there are no treatment options for patients who suffer from OCA2. We discovered that the sAC cAMP pathway regulates melanosomal pH. We hypothesize that inhibition of sAC would correct melanosomal pH and pigmentation in OCA2 melanocytes and mice.

Methods : We measured melanosomal pH in wild type and OCA2 loss of function (LOF) melanocytes using the pH sensitive molecule DAMP and microscopy. We measured melanosomal pH on a per-organelle level and assessed changes across thousands of melanosomes and multiple cells (n=30). We measured tyrosinase activity in live melanocytes using ³H-tyrosine and detecting the accumulation of ³H-H₂0 in the media (n=10). We generated a OCA2^-/-, sAC floxed (f/f), Tyr-CRE-ERT2 mouse model to study the effects of sAC LOF in tyrosinase expressing cells on an OCA2 LOF background. sAC LOF was induced on post-natal days 2-4 by topical application of 4-hydroxytamoxifen to the skin and eyes. Pharmacological inhibition of sAC was conducted by daily intraperitoneal (IP) injection of sAC inhibitors (36mg/kg) for one month.

Results : sAC inhibition restored melanosomal pH and tyrosinase activity to normal levels in OCA2 melanocytes. Oca2^-/-;sAC^-/- (sACKO) mice (n=20) had darker hair color at P28 as compared to Oca2^-/-;sAC^f/f (sACWT) littermates (n=9) . HPLC analysis of hair showed a significant increase in total melanin in sACKO mice. In addition, iris and choroid pigmentation was significantly increased in sACKO mice as measured by slit lamp (n=23) and Fontana-Masson staining (n=5) as compared to sACWT mice (n=14 and n=4, respectively). Electromicroscopy of sACKO eyes (n=5) revealed an increase in mature melanosomes in both melanocytes and RPE cells as compared to sACWT littermates (n=4). Furthermore, IP injection of sAC inhibitors increased pigmentation of Oca2^-/- eyes as compared to vehicle (n=4).

Conclusions : Our data reveals that restoration of normal melanosomal pH by inhibition of sAC can improve the pigmentation of cells and mice with OCA2 LOF. We propose that therapeutics designed to restore melanosomal pH may provide treatment opportunities for patients with OCA2 and other forms of albinism.

This is a 2021 ARVO Annual Meeting abstract.