Peer Pressure? "Pal" Leads APP Down Amyloid Path
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
- ACAT and Mouse—Inhibiting Former Prevents AD-like Pathology in Latter
- Salzburg: The Pony in There—Can ACAT Inhibition Work for AD?
- Vetrivel KS, Meckler X, Chen Y, Nguyen PD, Seidah NG, Vassar R, Wong PC, Fukata M, Kounnas MZ, Thinakaran G. Alzheimer disease Abeta production in the absence of S-palmitoylation-dependent targeting of BACE1 to lipid rafts. J Biol Chem. 2009 Feb 6;284(6):3793-803. PubMed.
- Cheng H, Vetrivel KS, Drisdel RC, Meckler X, Gong P, Leem JY, Li T, Carter M, Chen Y, Nguyen P, Iwatsubo T, Tomita T, Wong PC, Green WN, Kounnas MZ, Thinakaran G. S-palmitoylation of gamma-secretase subunits nicastrin and APH-1. J Biol Chem. 2009 Jan 16;284(3):1373-84. PubMed.
- Bhattacharyya R, Kovacs DM. ACAT inhibition and amyloid beta reduction. Biochim Biophys Acta. 2010 Aug;1801(8):960-5. PubMed.
- Motoki K, Kume H, Oda A, Tamaoka A, Hosaka A, Kametani F, Araki W. Neuronal β-amyloid generation is independent of lipid raft association of β-secretase BACE1: analysis with a palmitoylation-deficient mutant. Brain Behav. 2012 May;2(3):270-82. PubMed.
- Huttunen HJ, Kovacs DM. ACAT as a drug target for Alzheimer's disease. Neurodegener Dis. 2008;5(3-4):212-4. PubMed.
- Hutter-Paier B, Huttunen HJ, Puglielli L, Eckman CB, Kim DY, Hofmeister A, Moir RD, Domnitz SB, Frosch MP, Windisch M, Kovacs DM. The ACAT inhibitor CP-113,818 markedly reduces amyloid pathology in a mouse model of Alzheimer's disease. Neuron. 2004 Oct 14;44(2):227-38. PubMed.
- Huttunen HJ, Peach C, Bhattacharyya R, Barren C, Pettingell W, Hutter-Paier B, Windisch M, Berezovska O, Kovacs DM. Inhibition of acyl-coenzyme A: cholesterol acyl transferase modulates amyloid precursor protein trafficking in the early secretory pathway. FASEB J. 2009 Nov;23(11):3819-28. PubMed.
- Wave of New BACE Inhibitors Heading to Phase 2
- BACE Goes Rafting after APP
- Cellular Compartments Provide New Wrinkle—and a New Target?—in Cholesterol
- Salzburg: The Pony in There—Can ACAT Inhibition Work for AD?
- ACAT and Mouse—Inhibiting Former Prevents AD-like Pathology in Latter
- Merck Launches Largest Trial of BACE Inhibitor in AD
- Fats, Aβ, Oxidative Stress: Feeding Forward and Backward, Killing Neurons?
- BACE Inhibitors Barrel Forward—Next Hurdles: Safety, Efficacy
- Bhattacharyya R, Barren C, Kovacs DM. Palmitoylation of amyloid precursor protein regulates amyloidogenic processing in lipid rafts. J Neurosci. 2013 Jul 3;33(27):11169-83. PubMed.
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University of Wisconsin-Madison
This paper presents data indicating that palmitoylation of APP in the early secretory pathway regulates its processing by BACE1. The findings are novel and offer a previously unknown aspect of APP biology that could be targeted for therapeutic purposes. The conclusions are supported by a combination of biochemical experiments in both cellular and animal models of the disease. The findings are intriguing and open a new area of research. Indeed, a few important points will require further evaluation:
1) The formation of disulfide bonds seems to reduce the palmitoylation of APP. The basic information for the folding of a protein is contained in the primary amino acid sequence. However, co- and post-translational modifications can assist and improve the efficiency of folding. The formation of disulfide bonds is a co-translational event. It occurs during translation and helps the folding of the nascent polypeptide. In contrast, palmitoylation is a post-translational event. It occurs after the folding of the nascent polypeptide. Perhaps palmitoylation is being used here to “mark” unfolded/misfolded APP. In fact, a possible scenario could be that correctly folded APP (with disulfide bonds) is not palmitoylated while unfolded/misfolded APP (without disulfide bonds) is palmitoylated. If this is the case, then the preferential β cleavage of the palmitoylated (and still immature) version of APP in the early secretory pathway could be envisioned as an attempt to remove unfolded/misfolded APP.
2) Ceramide and cholesterol esters (CEs) have been previously associated with the pathogenesis of AD. Both are synthesized at the ER and use palmitoyl-CoA as one of the substrates. In the case of ceramide, palmitoyl-CoA is fused to serine to generate sphingosine, which then is used to synthesize ceramide. In the case of CEs, palmitate is one of the fatty acids that can be attached to the cholesterol moiety. It would be interesting to determine whether there is cross-talk between the above lipid biosynthetic pathways and the palmitoylation of a polypeptide.
Northwestern University Feinberg School of Medicine
The paper by Kovacs and colleagues reports the discovery that a significant proportion of APP in the cell is palmitoylated and enriched in lipid rafts. APP palmitoylation increased the colocalization of APP with BACE1, the β-secretase. Consequently, BACE1 cleavage of APP was increased, as was the generation of Aβ. Using both genetic and pharmacologic approaches, they show a direct relationship between APP palmitoylation and Aβ production. Moreover, the group discovered that ACAT inhibition also reduced APP palmitoylation, lipid raft localization, and Aβ generation.
The authors present a very thorough, rigorous study that convincingly demonstrates the role of APP palmitoylation in lipid raft localization and BACE1-mediated cleavage of APP and Aβ generation. Further, their observations strongly suggest that inhibition of either palmitoylation or ACAT should prove effective for lowering cerebral Aβ levels in AD. Kovacs and colleagues present an intriguing therapeutic alternative to direct inhibition of BACE1, which could be associated with mechanism based toxicities. Thus, further investigation of inhibition of APP palmitoylation as a therapeutic approach for AD is clearly warranted.
University of the Saarland
For many years, evidence accumulated that AD has extensive links to lipid metabolism. Dora Kovacs' group was one of the first to identify such a link on the molecular level with their ACAT targeted experiments. There is also extensive evidence suggesting APP processing, especially Aβ generation, is very sensitive to neuronal cholesterol and other lipids. APP may even serve as a receptor for some lipidated lipoproteins and function directly or indirectly as a sensor for lipid levels. Now, the Kovacs group has identified an even more direct link—APP is covalently bound to palmitoyl at the cysteine residues 186 and 187. Palmitoylation is one way to target proteins into lipid rafts, a specific membrane domain that is strongly implicated with amyloidogenic APP processing. Only a small fraction of APP was found to be palmitoylated, which is in good agreement that only a small fraction of total APP is found in lipid rafts. Without palmitoylation (or disulfide bridging), APP cannot be effectively exported from the ER. Interestingly, this appears to provide some explanation for the original ACAT results, because ACAT inhibition reduces APP palmitoylation and therefore impaired APP processing eventually resulting in decreased Aβ generation. Luminal S-palmitoylation for transmembrane proteins, as it is the case for APP, is thus far unprecedented (maybe a reason why it took so long to be discovered) and clearly offers a whole new range of potential ways to interfere with this modification and possibly some therapeutic options. Moreover, it is a clear example that not all APP is equal. Rather, the amyloidogenic fate of some APP molecules may already be foretold in the endoplasmic reticulum.
National Institute of Neuroscience, NCNP
This paper reported intriguing findings that amyloid precursor protein (APP) is palmitoylated at Cys186 and Cys187, and this palmitoylated form is cleaved by BACE1 in lipid rafts. Palmitoylation of transmembrane proteins usually occurs inside or close to the transmembrane domain or in the cytoplasmic domain (Charollais and Van der Goot, 2009), but in the case of APP, the palmitoylation sites are located in the N-terminal luminal domain. The authors showed that APP mutants with Cys-Ser or Cys-Ala substitutions at these sites do not undergo normal maturation and are retained in the ER. Because these cysteine residues are important for disulfide bond formation and only a small part of APP is palmitoylated, the ER retention of the APP mutants can be explained by improper folding of APP, but not by the lack of palmitoylation. It can be assumed that when APP is palmitoylated at these Cys residues, disulfide bonds will not be formed, which could affect APP maturation. In this regard, it seems that the property of palmitoylated APP differs from APP without palmitoylation and palmitoylated APP might be more unstable, as suggested in Dr. Puglielli’s comments.
In addition, APP is mostly distributed in non-raft membrane domains, as is BACE1. We have previously published experimental data suggesting that BACE1 cleaves APP mainly outside lipid rafts in neurons (Motoki et al., 2012).
Although this study revealed an interesting aspect of APP, further characterization of palmitoylated APP appears necessary to clarify its role in amyloidogenic processing.
Charollais J, Van Der Goot FG. Palmitoylation of membrane proteins (Review). Mol Membr Biol. 2009 Jan;26(1):55-66. PubMed.
Motoki K, Kume H, Oda A, Tamaoka A, Hosaka A, Kametani F, Araki W. Neuronal β-amyloid generation is independent of lipid raft association of β-secretase BACE1: analysis with a palmitoylation-deficient mutant. Brain Behav. 2012 May;2(3):270-82. PubMed.
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