30 April 2008. At the Alzheimer disease conference held 24-29 March in Keystone, Colorado, both Dennis Selkoe and Michael Wolfe of Brigham and Women’s Hospital, Boston, described in their respective talks joint new research on how the lipid composition of the membrane influences the enzyme activity of γ-secretase. γ-secretase modulation has become a buzzword in the field, and until recently, scientists thought primarily of small molecules (such as flurizan) and protein partners (such as TMP21) when studying γ-secretase modulation. But the enzyme is embedded in the lipid bilayer, after all, so lipid changes in the surrounding membrane environment would seem to be an obvious way of affecting its activity, as well. This may well be relevant to Alzheimer disease, as numerous groups have reported lipid differences between brain samples of AD patients and controls over the past dozen years. Even within a given cell, γ-secretase activity varies from compartment to compartment along with changes in their respective membrane lipid composition.

To study this question, postdoc Pamela Osenkowski, working with both Wolfe and Selkoe, modified a γ-secretase activity assay. Importantly, she removed the detergents that previously were routinely present in in-vitro activity assays as a leftover from the extraction protocol used to obtain γ-secretase in solution. (Besides being artificial and absent from γ-secretase in brain, detergents alter the enzyme’s activity in vitro, Wolfe said.) Specifically, Osenkowski added defined lipids and lipid mixtures to purified human γ-secretase, then removed the detergent. This allowed the lipids and γ-secretase to form proteolipid vesicles, or liposomes. This method creates a more natural environment of lipid bilayers to study the activity of purified γ-secretase, while at the same time giving Osenkowski control over precisely which lipids are present in those bilayers, Wolfe said. It allowed her to systematically characterize γ-secretase activity in different lipids and lipid mixtures.

First off, getting detergents out of the picture doubled the enzyme’s baseline activity, the scientists found. Further, the type of lipid present mattered greatly. Cerebrosides or gangliosides boosted γ-secretase activity over a baseline established with phosphatidylcholine only (this lipid is the most common kind in cell membranes and thought to provide their structural bulk). By contrast, phosphatidylinositol slowed the enzyme down. But a given lipid did not merely boost or crimp γ-secretase activity wholesale; some had quite specific effects. For example, adding cholesterol to phospholipids changed cleavage of APP, APLP1, or Notch-like substrates differentially and dose-dependently.

A more realistic recapitulation of the natural environment had different effects still. Complex lipid mixtures reconstituted γ-secretase in structures resembling lipid rafts, and this environment afforded high γ-secretase activity. Furthermore, brain lipid extracts by far topped liver or heart lipid extracts in terms of revving up γ-secretase activity. Brain lipid extract led to the greatest Aβ40 production of all lipid mixtures tested, whereas E. coli or soybean lipid extract did not support γ-secretase activity at all.

It’s not clear yet whether the lipids affect γ-secretase because they change the general properties of the surrounding membrane, for example, its fluidity and packing, or because certain charged lipids interact directly with γ-secretase. This research is important in part because understanding and tweaking the lipid environment may aid efforts to grow γ-secretase crystals suitable for X-ray crystallography. It may also add small molecules that target lipids to the mostly protein-oriented drug development efforts that predominate today, the scientists note. Finally, interest in the interactions between membrane lipids and APP secretases is growing across the field. Just last Friday, a paper in Science reported that tethering a β-secretase inhibitor to membrane lipids, rather than having it float through soluble compartments, sharply increased its activity (Rajendran et al., 2008). The work of Osenkowski et al. is submitted.—Gabrielle Strobel.

This is Part 2 of a three-part series. See also Part 1 and Part 3.