3 February 2006. The search is on for factors that interact with the complex protein cutter at the upstream end of the amyloid cascade, that is, γ-secretase, and the enzyme phospholipase D1 (PLD1) could be one of them, according to back-to-back papers released in PNAS online this week. Paul Greengard at Rockefeller University, New York, and colleagues report that the lipase, which hydrolyzes phosphatidylcholine to generate phosphatidic acid, independently affects two processes, the trafficking of amyloid-β precursor protein (AβPP)-loaded vesicles through the trans-Golgi network, and the presenilin-catalyzed cleavage of amyloid-β (Aβ) from AβPP. The findings suggest that the phospholipase could be a target for therapeutics.
Though the work was a collaboration among many labs in the U.S., Rockefeller’s Dongming Cai was lead author on both papers. In the first, Cai and colleagues report how the phospholipase restores AβPP trafficking in neurons harboring presenilin (PS) mutants that cause familial AD. They found that when PLD1 is overexpressed in neuroblastoma cells, it rescues vesicle budding that has been impaired by the expression of PS1ΔE9, a presenilin mutant that causes overproduction of Aβ42 (see related ARF mutation data pages). The rescue seems to depend on the enzymatic activity of the lipase because overexpression of PLD1 increased budding of AβPP vesicles from the tans-Golgi network by over twofold, whereas overexpression of catalytically inactive lipase did not. The authors also found that the PLD1 inhibitor 1-butanol prevents increases in AβPP vesicle budding that occur in PS1 knockout fibroblasts. This not only supports the idea that the catalytic activity of PLD1 is crucial for its effects on budding, it also suggests that this action of PLD1 is independent of presenilin.
Yet there is another side to the story. In the second paper, the researchers report that PLD1 does interact with presenilin, after all: It apparently inhibits the protease, reducing production of Aβ. This action, by contrast, does not depend on the catalytic activity of PLD1. The conclusions are based on the following data. First, Cai used coimmunoprecipitation experiments to show that PLD1 and PS1 interact in embryonic stem cells. The authors narrowed down the site of interaction to the C-terminal end of PS because antibodies to a loop in this region blocked the interactions. Next, the authors determined that overexpression of PLD1 reduces intracellular and secreted Aβ by nearly half. However, unlike in the trafficking experiments, catalytically inactive PLD1 was just as effective here, reducing production of Aβ by similar amounts. The inactive lipase also bound to the C-terminal of PS1.
Taken together, the two papers show that PLD1 has mechanistically distinct actions on vesicle budding and PS1 activity. How the lipase affects the former is unclear, though given the dependence on catalytic activity, it may well be related to levels of phosphatidylcholine or phosphatidic acid. And, while PLD1 can accelerate vesicle budding in PS-negative cells, the lipase’s impact on vesicle trafficking may depend to some degree on the protease, because Cai also found that PLD1 activity was significantly reduced in cells expressing certain PS FAD mutations.
As for the effect of PLD1 on AβPP processing, this seems to be due to binding of the lipase to presenilin, causing disruption of the γ-secretase complex. The authors found that overexpression of PLD1 caused dissociation of the γ-secretase components—PEN2, APH1, and nicastrin—from PS1.
What effect PLD1 might have in vivo, and in particular on the pathology of AD, if any, is uncertain because most of the reported experiments depended on overexpression of PLD1 in cultured cells. When the authors used RNAi to knock down PLD1 protein by around 70 percent, they observed almost a threefold jump in intracellular Aβ, suggesting that relatively modest losses of PLD1 activity could have a significant effect on AβPP processing. In addition, they found that transfecting PLD1 into cortical neurons expressing the M146V PS1 mutation restored neurite outgrowth. When one considers that there may be a vicious cycle brought on by FAD PS mutations—first inactivating PLD1, which then fails to inhibit γ-secretase, which then produces more Aβ—then maintaining PLD1 activity might go some way toward ameliorating AD pathology.—Tom Fagan.
Cai D, Zhong M, Wang R, Netzer WJ, Shields D, Zheng H, Sisodia SS, Foster DA, Gorelick FS, Xu H, Greengard P. Phospholipase D1 corrects impaired betaAPP trafficking and neurite outgrowth in familial Alzheimer’s disease-linked presenilin-1 mutant neurons. PNAS. February 7, 2006;103:1936-1940. Abstract
Cai D, Netzer WJ, Zhong M, Lin Y, Du G, Frohman M, Foster DA, Sisodia SS, Xu H, Gorelick FS, Greengard P. Presenilin-1 uses phospholipase D1 as a negative regulator of beta-amyloid formation. PNAS. February 7, 2006;103:1941-1946. Abstract