Papers in the March 17 EMBO Molecular Medicine and the April 20 Neuron have reignited a simmering debate about the role of presenilin mutations in familial Alzheimer’s disease. Both studies dispute a claim made previously by Jie Shen, Ray Kelleher, and colleagues that some familial AD PS mutations completely lack γ-secretase activity in vivo. This claim is at the heart of Shen and Kelleher’s presenilin hypothesis of Alzheimer’s disease, which challenges the more widely supported hypothesis that long Aβ species are the central factor in Alzheimer’s disease.
In the EMBO paper, researchers led by Harald Steiner at Ludwig-Maximilians-University, Munich, report that, when expressed in cells, one of these PS variants, L435F, makes Aβ43. This is an aggregating and neurotoxic peptide that only recently started drawing the attention of AD researchers (Saito et al., 2011). Steiner and colleagues also report that Aβ43 accumulates in amyloid plaques in people harboring the L435F presenilin-1 mutation.
In a “Matters Arising” paper in Neuron, researchers led by Bart De Strooper and Lucia Chávez-Gutiérrez at KU Leuven, Belgium, report that L435F—and two other PS mutations purportedly causing loss of function—in fact generate Aβ43 from amyloid precursor protein in vitro, as well.
“This was the missing piece of evidence from the debate, and it clearly supports the amyloid hypothesis,” said Steiner. Together, the papers explain how a small amount of aberrant activity retained by mutant presenilin causes dementia in people who harbor one good and one bad copy of the gene. The good copy, and presumably two copies of presenilin 2, ensure that γ-secretase adequately processes Notch and other substrates that are essential for normal development and biology, while the bad copy over time accrues enough Aβ43 to seed plaques. “Since it has been shown that γ-secretase endopeptidase activity is normal in PS FAD patients, they cannot have major [Notch] signaling defects. However, they have consistent changes in ratios of longer vs. shorter Aβ, and now we find that this happens in [L435F] patients as well,” noted Steiner.
Both Steiner’s and De Strooper’s groups used mouse embryonic fibroblasts (MEFs) to test PS variants for their carboxypeptidase activity. This is the processive chopping of substrate into Aβ peptides of decreasing length, not the endopeptidase activity this enzyme complex also performs. The scientists expressed the protease together with APP harboring the Swedish mutation. This variant accelerates BACE cleavage of APP, giving the researchers a better chance of detecting γ-secretase activity. Both groups used carboxypeptidase null mutations (missing catalytic aspartates) as negative controls, and both measured activity in the absence of wild-type PS1 or PS2 to avoid any ambiguity about what enzyme is doing the catalysis. This is because Shen and colleagues had previously reported a “trans-dominant” effect, whereby PS1 loss-of-function alleles interfere with wild-type PS1 activity in MEFs to modulate activity and generate peptides longer than Aβ40 (see Heilig et al., 2013).
In Munich, joint first authors Benedikt Kretner and Johannes Trambauer found that L435F PS1 made about four times as much Aβ43 as Aβ40. In Leuven, first author Sarah Veugelen found that all three mutants tested (R278I, C410Y, and L435F) reconstitute the γ-secretase complex in PS1/2-negative MEFs, and that they made various forms of Aβ. All three generated very little Aβ40 compared to wild-type PS. R278I and C410Y made about 30 percent less Aβ42 than did wild-type, while L435F made about 90 percent less. However, all three mutants made much more Aβ43 than did normal presenilin. Both R278I and L435F made more Aβ43 than Aβ42.
Responding in the same issue of Neuron, Shen, from Brigham and Women’s Hospital, and Kelleher, from Massachusetts General Hospital, both in Boston, maintain presenilin loss of function is “the most coherent explanation for all existing, reproducible human and mouse data.” First author Dan Xia had reported last year that L435F and C410Y PS knock-ins make no Aβ40/42 in the brain, but he had not checked Aβ43 (see Mar 2015 news). Now he addresses this, reporting three-month-old L435F heterozygous knock-ins made about half as much Aβ43 as did wild-type mice, while finding no Aβ43 in homozygous C410Y embryos.
Xia and colleagues insist that different experimental procedures explain the discrepancies. “It is worth noting the potential for cell type-specific or technical differences in detection of Aβ production between immortalized HEK or MEF clones overexpressing mutant PS1 and APP in culture (Steiner and De Strooper’s work) and the adult brain expressing mutant PS1 at normal endogenous levels (our work),” Kelleher wrote to Alzforum.
De Strooper disagreed. “Our goal was to determine if these variants have any activity,” he told Alzforum. “In my opinion a cell-based assay is a perfectly good way to measure the activity of a membrane protease,” he said, while noting that Veugelen also used knock-in cell models to avoid overexpression. He pointed out that the detergents Xia and colleagues used to make brain extracts may have altered the intra-membrane protease in such a way as to alter its activity and producing artifactual results.
The debate does not hinge on cell data alone. In 2011, researchers led by Takaomi Saido at RIKEN Brain Science Institute, Wako, Japan, reported that the R278I mutation generates Aβ43 in a knock-in mouse model and that this highly amyloidogenic peptide generated extensive plaques (see Jul 2011 news). Last year, Chávez-Gutiérrez and colleagues reported that in brain tissue from 22 familial AD patients who carried various PS1 mutations, γ-secretase’s overall endopeptidase activity was similar between mutant and control tissue, a challenge to the loss-of-function hypothesis (Szaruga et a., 2015). Now, Steiner’s group caps it off by showing that two patients with confirmed L435F genotypes formed extensive Aβ43-containing plaques in the frontal cortex (see image above).
Where does all this leave the presenilin loss-of-function hypothesis? Kelleher insists none of this changes anything. “Our hypothesis does not rely on complete absence of any detectable Aβ production by mutant PS1. Rather, it proposes that loss of presenilin function plays an important role in FAD pathogenesis and provides a more coherent explanation for available data than Aβ oligomerization or deposition alone.”
“That’s a far cry from their earlier claims ‘that FAD mutations in the PSEN genes can cause complete loss of its function and γ-secretase activity in vivo,’” noted De Strooper. “In fact, the partial loss of function, and the possibility that this also contributes to the disease phenotype, has been discussed many times by us and others in the field,” he added. “The point is that the common denominator in Alzheimer’s disease is amyloid production, and loss of function on its own is not sufficient to cause that.”—Tom Fagan
- Mutant Presenilin Knock-in Mice Mimic Knockouts, Stir Old Debate
- What’s Another Amino Acid? Aβ43 Drives Amyloid Pathology
- Saito T, Suemoto T, Brouwers N, Sleegers K, Funamoto S, Mihira N, Matsuba Y, Yamada K, Nilsson P, Takano J, Nishimura M, Iwata N, Van Broeckhoven C, Ihara Y, Saido TC. Potent amyloidogenicity and pathogenicity of Aβ43. Nat Neurosci. 2011 Aug;14(8):1023-32. PubMed.
- Heilig EA, Gutti U, Tai T, Shen J, Kelleher RJ. Trans-dominant negative effects of pathogenic PSEN1 mutations on γ-secretase activity and Aβ production. J Neurosci. 2013 Jul 10;33(28):11606-17. PubMed.
- Szaruga M, Veugelen S, Benurwar M, Lismont S, Sepulveda-Falla D, Lleo A, Ryan NS, Lashley T, Fox NC, Murayama S, Gijsen H, De Strooper B, Chávez-Gutiérrez L. Qualitative changes in human γ-secretase underlie familial Alzheimer's disease. J Exp Med. 2015 Nov 16;212(12):2003-13. Epub 2015 Oct 19 PubMed.
No Available Further Reading
- Kretner B, Trambauer J, Fukumori A, Mielke J, Kuhn PH, Kremmer E, Giese A, Lichtenthaler SF, Haass C, Arzberger T, Steiner H. Generation and deposition of Aβ43 by the virtually inactive presenilin-1 L435F mutant contradicts the presenilin loss-of-function hypothesis of Alzheimer's disease. EMBO Mol Med. 2016 May 2;8(5):458-65. PubMed.
- Veugelen S, Saito T, Saido TC, Chávez-Gutiérrez L, De Strooper B. Familial Alzheimer's Disease Mutations in Presenilin Generate Amyloidogenic Aβ Peptide Seeds. Neuron. 2016 Apr 20;90(2):410-6. PubMed.
- Xia D, Kelleher RJ 3rd, Shen J. Loss of Aβ43 Production Caused by Presenilin-1 Mutations in the Knockin Mouse Brain. Neuron. 2016 Apr 20;90(2):417-22. PubMed.