The initial clue linking presenilins to melanin production has been staring researchers in the face for several years. In previous work, Zheng and colleagues attempted to rescue the embryonic lethal phenotype of PS1/PS2 double knockout mice by breeding in a human PS1 transgene whose expression was restricted to the central nervous system. The approach was partially successful, and rather than perishing as embryos, the animals at least survived to birth. They expired soon after, however, and displayed massively abnormal kidney development due to compromised Notch signaling in non-neural tissues (Wang et al., 2003).

In those experiments, the researchers found they could distinguish the rescued mice from their control littermates at a glance—the transgene recipients all had white eyes. In the new work, chasing the white eye phenotype led first author Runsheng Wang and colleagues directly to the melanin synthesis pathway. This black pigment is produced inside melanosomes, membrane-bound organelles that contain the enzyme tyrosinase, which catalyzes the rate-limiting step in the transformation of tyrosine into melanin. [If this sounds familiar, it is. Melanosomes recently grabbed the attention of AD researchers, who found them to be filled with an amyloid of the Pmel17 protein, the first physiological amyloid found in higher organisms (see ARF related news story)].

With no PS1 expression, the eyes of the rescue mice were morphologically normal, but lacked pigment granules in the retinal pigmented epithelium (RPE) cells. The researchers showed that the mice had normal levels of the tyrosinase and two associated proteins, but the tyrosinase was mislocalized. PS+ cells contained a small number of tyrosinase-containing vesicles located near the trans-Golgi network. In normal eye cells, these vesicles appear to shuttle tyrosinase from where it is made in the secretory pathway to nearby early-stage melanosomes. But in the PS-null cells, these small, tyrosinase-loaded vesicles accumulated, and could be seen all over the cell body. The same cells contained only early-stage melanosomes that failed to mature or produce melanin.

Successful melanin synthesis depends on PS in skin cells, as well. Blocking γ-secretase activity with the inhibitor DAPT caused skin melanocytes to behave like the PS-null cells: Melanin synthesis was blocked and melanosome maturation stalled.

For many γ-secretase substrates, inactivation of PS leads to accumulation of a C-terminal cleavage fragment. Tyrosinase, as a type I membrane protein, was a candidate substrate, and the researchers detected a 7 kDa C-terminal fragment in either in PS 1 -/- cells, or in cells treated with DAPT. A CTF was also detected for two other tyrosinase-associated, melanosome-targeted proteins after DAPT treatment, suggesting that all three proteins could be PS substrates.

But what about the effect of FAD mutations on melanin production? From previous work (Wang et al., 2004), Zheng and colleagues knew that animals carrying the M146V FAD allele displayed learning and neurogenesis phenotypes, but only when the wild-type PS1 allele was absent. To look at effects on pigment in vivo, they bred heterozygous PS1 knockouts with knock-ins bearing the M146V FAD mutation in the endogenous PS1 gene, all on a PS2 null background. The mice carrying only the FAD mutant gene (PS2-/-, PS1 M146V/-), had a distinctly lighter coat color than did mice with a wild-type PS1 gene (PS2-/-, PS1+/-). PS1 gene dosage also appeared to affect pigmentation, although to a lesser extent than the mutation. Thus, while there was no noticeable difference in coat color between wild-type mice (PS1+/+) and PS1 knockout heterozygotes (PS1+/-), visual inspection of tail skin pigmentation and biochemical measurements of melanin content revealed that the PS1+/- produced less melanin than did the PS1 wild-type, and the PS1 M146V/- produced the least.

The results of Wang et al. establish a new role for PS in protein trafficking to melanosomes, but the mechanistic details of this regulation remain to be worked out. The physiological significance of the cleavage of tyrosinase cleavage and related proteins by γ-secretase is likewise totally unknown. The systems developed by the investigators provide a good opportunity for future studies to untangle these issues and the role of FAD mutations in presenilin function.

With this new data, melanosome biosynthesis is now known to involve not only a novel amyloid, but also a presenilin. A coincidence? one might ask. Or is there a link between PS and the Pmel17 protein, the precursor to melanosome amyloid? According to Michael Marks of the University of Pennsylvania in Philadelphia, it is unlikely that PS plays a role in the processing of Pmel17. Zheng’s data showed that Pmel17 protein levels and immunostaining patterns were unaffected by PS deficiency. In addition, the PS-deficient cells generate relatively intact early-stage melanosomes, which also argues against an obligatory role of PS in Pmel17 amyloid generation, Marks notes.

In the end, getting out of melanosomes and back to AD, Wang et al. conclude, “These findings raise the intriguing possibility that a compromised post-Golgi vesicle transport may contribute to Alzheimer’s disease pathogenesis.”—Pat McCaffrey.

Reference:
Wang R, Tang P, Wang P, Boissy RE, Zheng H. Regulation of tyrosinase trafficking and processing by presenilins: Partial loss of function by familial Alzheimer's disease mutation. Proc Natl Acad Sci U S A. 2005 Dec 29; [Epub ahead of print] Abstract