What determines if APP will be processed to Aβ? Researchers led by Dora Kovacs, Massachusetts General Hospital, Charlestown, present a potential explanation in the July 3 Journal of Neuroscience. They report, for the first time, that APP binds palmitic acid in the endoplasmic reticulum (ER). This post-translational modification makes APP more hydrophobic and targets it to lipid rafts. There, beta-secretase cleaves the protein, leaving it ripe for g-secretase to generate Aβ. Curbing APP palmitoylation could prevent or treat AD, suggest the authors. “The study provides a clear example that not all APP is equal,” wrote Tobias Hartmann, University of Saarland, Homburg, Germany, to Alzforum in an email (see full comment below). “For some APP molecules, their amyloidogenic fate may already be foretold in the endoplasmic reticulum.”
Palmitoylation renders proteins more lipophilic and likely to associate with cell membranes. Kovacs wondered if APP was palmitoylated when she found that inhibitors of acyl-coenzyme A:cholesterol acyltransferase (ACAT) reduce Aβ production, (see ARF related news story). An ER enzyme, ACAT creates cholesteryl esters from free cholesterol, moving the lipid from the plasma membrane into the cytoplasm. To produce those esters, ACAT attaches palmitate to the steroid.
To investigate the role of palmitoylation in APP processing, lead author Raja Bhattacharyya and colleagues looked in Chinese hamster ovary (CHO) cells that stably expressed the precursor protein. Immunohistochemistry and labeling with a florescent palmitic acid analog revealed that about 10 percent of the APP underwent palmitoylation. The group also detected palmitoylated APP (palAPP) in human neuroglioma cells, rat neuroblastoma cells, and non-transgenic mouse brain extracts. Using an algorithm to predict palmitoylation sites, and N-terminal deletion mutants of APP to map them, the researchers narrowed down the locations of palmitoylation to two cysteine residues—C186 and C187—that protrude into the lumen of the endoplasmic reticulum (ER). When the researchers mutated these cysteines in CHO cells, APP neither left the ER nor generated C-terminal fragments. Aβ42 and Aβ40 levels also dropped by 95 percent in these mutants. These results suggested that palmitoylation, or perhaps disulfide bridges involving the two cysteine residues, are required for exit from the ER and entry into later compartments for APP processing.
Since palmitoylation directs proteins such as BACE1 and γ-secretase to lipid rafts, Bhattacharyya and colleagues wondered if it did the same for APP. They separated raft and non-raft portions from CHO cell membranes and found that 20 percent of the APP in lipid rafts was palmitoylated, versus only 2 percent in non-raft fractions. About the same proportions occurred in membranes isolated from wild-type mouse brains.
Would a 20 percent bump in palmitoylated APP be sufficient to boost Aβ production? Overexpression of palmitoyl acyltransferases in CHO cells drove up palAPP production and doubled Aβ output, while palmitoylation inhibitors such as 2-bromopalmitate (2-BP) and cerulenin lowered both. Interestingly, BACE1 cleaved a larger percentage of palAPP than unmodified APP. In addition, BACE1 inhibitors boosted palAPP levels while an α-secretase inhibitor did not, implying that BACE1 cleaves the modified protein more readily than does α-secretase.
It remains to be seen whether APP palmitoylation drives Aβ pathology in people, but it may increase it in animals. In wild-type mice, PalAPP levels rose nearly two fold between 3- and 18-months. This suggests that with age, palAPP may contribute more to amyloidogenic processing.
To find out if ACAT inhibitors lowered palAPP levels, the researchers applied CI-1011, aka avasimibe (see ARF related news story) to CHO cells. The inhibitor was developed by Pfizer and reached Phase 3 clinical trials for cardiovascular disease but did not improve atherosclerosis. It reduced palAPP, particularly in the lipid raft fraction, and attenuated both β- and α-secretase processing. Likewise, cells that lacked ACAT activity produced very little palAPP. “It looks like we have identified a mechanism by which ACAT inhibitors decrease Aβ generation,” Kovacs told Alzforum.
APP represents the first transmembrane protein found to be palmitoylated in the lumen of the ER. A growing list of secreted proteins, hormones, and receptors are palmitoylated there, wrote the authors. They plan to quantify APP palmitoylation in human brains, including of people with AD, and further probe the mechanism of enhanced cleavage by BACE1. The group is also developing antibodies against palAPP to better visualize it in the cell. While no ACAT inhibitors are currently approved by the Food and Drug Administration, several have been developed against atherosclerosis and hypercholesterolemia and tested in clinical trials (see Farese et al., 2006).
“This is a significant step in a new direction for the APP processing field,” said Gopal Thinakaran, University of Chicago, Illinois. “The authors make a pretty convincing argument that palmitoylated APP is preferentially cleaved by BACE,” he added. Robert Vassar, Northwestern University, Chicago, Illinois, agreed. “The authors present a very thorough, rigorous study that convincingly demonstrates the role of APP palmitoylation in lipid raft localization, BACE1-mediated cleavage of APP, and Aβ generation,” Vassar wrote to Alzforum (see full comment below). However, Thinakaran wondered why ACAT inhibitors lowered both α– and β–secretase cleavage products, and suggested further research would be needed to figure that out.—Gwyneth Dickey Zakaib