Makoto Michikawa and colleagues at the National Institute for Longevity Sciences in Aichi-ken, Japan, in collaboration with several other Japanese labs, probed the role of sphingolipids by using myriocin, a potent inhibitor of serine palmitoyltransferase (SPT). SPT is an enzyme essential for sphingolipid biosynthesis.

When first author Naoya Sawamura added myriocin to Chinese hamster ovary (CHO) cells, he found that reducing sphingomyelin synthesis by about threefold affected cleavage of human APP, which had been transfected into the cells. Sawamura found that α cleavage was threefold higher, as determined by the amount of soluble α-secretase product (sAPPα) released by the cells. β cleavage, on the other hand, was unaffected, as was the total amount of expressed APP.

To confirm that these effects were due to changes in sphingolipid biochemistry, the authors examined cleavage of native, rodent APP in CHO cells harboring mutant, inactive forms of SPT. These cells also secreted more sAPPα, but also showed no signs of elevated β-cleavage. In addition, when Sawamura added sphingosine to these cells, or to myriocin-treated cells, the changes in APP cleavage were reversed, indicating that the drug was primarily affecting sphingolipid biochemistry.

Curiously, when the authors measured the amount of Aβ40 and Aβ42 secreted by the cells, they found that myriocin treatment increased Aβ42 release by about 1.5-fold, but had no effect on Aβ40. This suggests that γ-secretase cleavage at the 42 position may be more sensitive to fluctuations of membrane sphingolipids.

How might sphingolipids affect AβPP processing? In the simplest model, they would activate or deactivate the proteolytic secretases directly. But things may be more complex. Sawamura and colleagues found that myriocin also leads to a dramatic increase in phosphorylation of mitogen-activated protein kinase (MAPK). Furthermore, in SPT mutant cells, both elevated sAPPα and phosphorylated MAPK could be returned to normal by not only sphingosine, but also by PD98059, a potent inhibitor of MAPK kinase, or MEK. This result indicates that the effect of reduced sphingolipid on APP processing is mediated by the MAPK pathway, which itself can be activated by protein kinase C (PKC). However, the authors failed to find activation of PKC in myriocin-treated or SPT mutant cells, suggesting MAPK may be activated by a different pathway.

As for rafts, depletion of cholesterol is known to disrupt them and prevent incorporation of APP (see Kojro et al., 2001). Loss of cholesterol has also been shown to increase α cleavage, but it also decreases β cleavage (see ARF related news story). As sphingolipid loss mimics only some of these effects, indications are "that cholesterol and sphingolipids play entirely different roles in determining the properties of lipid rafts," conclude the authors.—Tom Fagan.

Reference:
Sawamura N, Ko M, Yu W, Zou K, Hanada K, Suzuki T, Gong JS, Yanagisawa K, Michikawa M. Modulation of amyloid precursor protein cleavage by cellular sphingolipids. J Biol Chem. 2004 Jan 10 [Epub ahead of print]. Abstract