Mutations in the transmembrane domain of Aβ-protein precursor cause early onset familial Alzheimer’s disease. They can increase the ratio of the amyloidogenic Aβ42 to the less-sticky Aβ40, but how exactly do these FAD mutations alter the processing of APP? To address this, researchers led by Michael Wolfe at the University of Kansas, Lawrence, measured all the peptides, including tri- and tetrapeptides, that got released when the γ-secretase enzyme processed 14 different mutant forms of APP. The study, published January 12 in the Journal of Biological Chemistry, found that not all APP mutations increase the Aβ42 to Aβ40 ratio. However, all of them did lead to a rise in Aβ peptides of 45 or more amino acids, suggesting that these longer forms may contribute to the disease.
- Aβ42 is considered the more pathogenic.
- However, not all APP mutations increase the Aβ42/Aβ40 ratio.
- Fourteen mutations tested generated Aβ peptides of 45 or more amino acids.
“It is interesting to see that FAD-linked APP variants promote the production of longer (≥45 aa) Aβ peptides,” Lucía Chávez Gutiérrez, KU Leuven, Belgium, wrote to Alzforum. “The findings are in line with our observations. Whether these longer peptides are indeed generated in the FAD brain and are disease-causing remains to be clarified,” she wrote. Chávez Gutiérrez and colleagues found that pathogenic presenilin and APP mutations destabilize γ-secretase, thereby promoting the making of longer Aβ peptides (Szaruga et al., 2017; Chávez-Gutiérrez et al., 2012).
To examine the effect of APP mutations in detail, Wolfe and colleagues used ELISAs to measure Aβ42 and Aβ40 produced in cells and cell-free assays using purified γ-secretase. They found that the ratio of Aβ42/Aβ40 did not increase in two out of the 14 substrates, suggesting that an increase in Aβ42/Aβ40 is not necessary for the pathogenesis of FAD. The paper does not address the pathogenicity of many FAD APP mutations that lie outside the transmembrane domain.
What other pathogenic products might be generated from APP? The γ-secretase complex processes the C-terminal fragment (β-CTF) left in the cell membrane after β-secretase has cut the precursor protein. First, γ-secretase cuts β-CTF in half, liberating the APP intracellular domain (AICD) into the cytosol and leaving a membrane-bound fragment of 48 or 49 amino acids (see image at right). After this endoprotease step, the secretase’s carboxypeptidase activity kicks in, sequentially lopping off tripeptides until Aβ escapes its clutches and the membrane.
To quantify those small tripeptides, first author Sujan Devkota and colleagues modified a liquid chromatography tandem mass spectrometry method (Takami et al., 2009). They discovered that γ-secretase processing of APP carrying any of 14 FAD mutations led to an increase in one or more Aβ peptides that were 45 to 49 amino acids long. “Nobody has looked at the pathological role of these long Aβ peptides, or really quantified them, because they are so hard to detect through mass spectrometry,” said Wolfe, adding “This is the first systematic, rigorous determination of the effects of any FAD mutation on all Aβ peptide products.”
Curiously, these peptides of 45 amino acids and more remained anchored to the membrane, suggesting that γ-secretase is unable to process them effectively. Even the L52P mutation, which sits in the AICD domain and is therefore released after the initial endoprotease cleavage, stalled the subsequent carboxypeptidase processivity. “That really surprised us,” Wolfe told Alzforum. “One possibility is that L52P-mutant AICD does not dissociate from the enzyme very easily,” he said.
Whether the membrane-anchored amyloids are linked to FAD pathogenesis is unclear. Wolfe and colleagues speculate that they could form oligomers, form pores in the membrane, or diffuse through the membrane and cause mayhem with other components.
Interestingly, a separate study of 138 mutations in PSEN1—the catalytic component of γ-secretase, not the APP substrate—reported that not only did 10 percent of them not increase the Aβ42/Aβ40 ratio, they decreased it (Sun et al., 2017). Again, this hints that an increase in the ratio is not necessary for pathogenesis. Whether those PS1 mutations cause longer peptides to accumulate remains to be seen.
Moving forward, Wolfe would like to conduct an analogous study on the effect of PSEN mutations on all Aβ species, and test if the long species are neurotoxic. “They’re hard to see, but they may have profound effects on disease progression,” said Wolfe.—Helen Santoro
Mutation Interactive Images Citations
- Szaruga M, Munteanu B, Lismont S, Veugelen S, Horré K, Mercken M, Saido TC, Ryan NS, De Vos T, Savvides SN, Gallardo R, Schymkowitz J, Rousseau F, Fox NC, Hopf C, De Strooper B, Chávez-Gutiérrez L. Alzheimer's-Causing Mutations Shift Aβ Length by Destabilizing γ-Secretase-Aβn Interactions. Cell. 2017 Jul 27;170(3):443-456.e14. PubMed. Correction.
- Chávez-Gutiérrez L, Bammens L, Benilova I, Vandersteen A, Benurwar M, Borgers M, Lismont S, Zhou L, Van Cleynenbreugel S, Esselmann H, Wiltfang J, Serneels L, Karran E, Gijsen H, Schymkowitz J, Rousseau F, Broersen K, De Strooper B. The mechanism of γ-Secretase dysfunction in familial Alzheimer disease. EMBO J. 2012 May 16;31(10):2261-74. Epub 2012 Apr 13 PubMed.
- Takami M, Nagashima Y, Sano Y, Ishihara S, Morishima-Kawashima M, Funamoto S, Ihara Y. gamma-Secretase: successive tripeptide and tetrapeptide release from the transmembrane domain of beta-carboxyl terminal fragment. J Neurosci. 2009 Oct 14;29(41):13042-52. PubMed.
- Sun L, Zhou R, Yang G, Shi Y. Analysis of 138 pathogenic mutations in presenilin-1 on the in vitro production of Aβ42 and Aβ40 peptides by γ-secretase. Proc Natl Acad Sci U S A. 2017 Jan 24;114(4):E476-E485. Epub 2016 Dec 5 PubMed.
No Available Further Reading
- Devkota S, Williams TD, Wolfe MS. Familial Alzheimer's disease mutations in amyloid protein precursor alter proteolysis by γ-secretase to increase amyloid β-peptides of >45 residues. J Biol Chem. 2021 Jan 12;:100281. PubMed.