Fans of Douglas Adams’s The Hitchhiker's Guide to the Galaxy may remember that 42 is the answer to the “ultimate question of life, the universe, and everything.” Amyloid-β42 is almost certainly not the ultimate answer to the question of what causes Alzheimer’s disease. Case in point, Aβ43. A paper in today’s Nature Neuroscience contends that the longer peptide can play a major role in pathology. Researchers led by Takaomi Saido at the RIKEN Brain Science Institute, Wako, Japan, engineered a mouse that overproduces Aβ43. The animals have increased amyloid pathology and severe learning and memory deficits. The work may have ramifications for the AD field, suggesting that new looks at pathology, biomarkers, and treatment options might be warranted. “Aβ43 may turn out to be a much bigger player than we have been led to believe,” suggested Michael Wolfe, Brigham and Women’s Hospital, Boston. Wolfe was not involved in the study.

Saido became interested in Aβ43 when he saw a poster presentation indicating that a mutation in presenilin-1, the catalytic component of the γ-secretase that extricates Aβ from its precursor, shifts Aβ cleavage toward the 43 position (see subsequent paper by Nakaya et al., 2005). Shortly afterward, researchers led by Martin Rossor at University College, London, reported language impairment in people carrying one of the same genetic variants, R278I (see Godbolt et al., 2004), suggesting the mutation, and possibly Aβ43, are pathogenic. One of those carriers has since gone on to develop AD-like learning problems.

To test this idea, joint first authors Takashi Saito, Takahiro Suemoto, and colleagues developed a knock-in model, replacing the endogenous mouse presenilin (PS1) gene with one containing the R278I mutation. Mice heterozygous for the mutant PS1 appeared normal, but homozygous animals died in the womb and had severe developmental defects. Though γ-secretase, a complex of presenilin and three other proteins, appeared to form normally in the homozygotes, it did not properly process substrates, including APP and Notch, which are essential for development.

To test if a single copy of the mutant PS1 is sufficient to alter Aβ levels in the heterozygotes, Saito and colleagues developed enzyme-linked immunosorbent assays (ELISAs) to specifically identify Aβ40, 42, and 43. The ELISAs detected more guanidine hydrochloride soluble Aβ43 in the cortex of 24-month-old heterozygous mice than controls. On the other hand, the mutant mice produced less Aβ40. The pattern is in keeping with coauthor Yasuo Ihara’s finding that γ-secretase sequentially cleaves APP in three-amino-acid steps (see Takami et al., 2009 and ARF related news story), and suggests that the mutation retards the cleavage of Aβ43 to Aβ40.

This could have important implications. The ratio of Aβ42 to Aβ40 is commonly used as a measure of toxicity, but Saido believes an increase in that ratio could equally reflect an increase in Aβ43 and a concomitant decrease in Aβ40. Indeed, in the PS1 mutant heterozygotes, the Aβ42/40 ratio is higher than in controls, while Aβ42 levels are unchanged.

The researchers found that the R278I PS1 caused rampant elevations of Aβ43 in cells overexpressing wild-type amyloid precursor protein (APP). To see what effect this might have in vivo, Saito and colleagues crossed the R278I PS1 mutant mice with transgenic cousins overexpressing human APP. Three- and nine-month-old crosses produced more Aβ42 and 40 than controls, but even more Aβ43, such that the ratio of solubilized Aβ42 to 40 appeared normal, but Aβ43/40 was elevated. In the R278I PSs x APP crosses, amyloid deposits began to appear by six months and extensively decorated the hippocampus by nine months. The plaques had more Aβ43 than Aβ42, in contrast to plaques in control mice that overexpressed wild-type human APP with a M146V mutant presenilin. In the latter, plaques were predominantly made up of Aβ42. In R278I PS1 x APP mice, pathological changes were preceded by behavioral dysfunction. At four months old, the mice performed poorly in a Y-maze test of learning and memory.

All told, the evidence suggests that the R278I presenilin mutation shifts APP processing toward Aβ43, which then drives plaque pathology and learning and memory deficits. Could that be relevant to human AD? Researchers led by Lars Tjernberg at the Karolinska Institute in Stockholm found Aβ43 in amyloid plaque cores taken from both sporadic and familial AD cases (see Welander et al., 2009). Quantification by mass spectrometry suggested that Aβ43 represents only about 5 percent of the total; however, the peptide is more difficult to ionize for mass spectrometry than Aβ40 or 42, suggested Saido. “We must be cautious interpreting mass spec data,” he told ARF. Even if Aβ43 makes up a small amount of the total Aβ, it could still be influential. Saito and colleagues found that in a test tube, it aggregates faster, seeds aggregation more efficiently than either Aβ40 or Aβ42, and is more toxic to cells. Clinical evidence points to a role as well. The researchers found a correlation between age of onset, PS mutations, and Aβ43. Those PS mutations that produce the most Aβ43 when expressed in cells cause the earliest onset of AD. The same is true for PS mutations and Aβ42, but the correlation for Aβ43 was tighter.

Though Wolfe noted evidence pointing to poor removal of Aβ as the major problem in Alzheimer’s (see ARF related news story and upcoming Webinar on the role of ApoE isoforms in Aβ clearance), he said that it is an oversimplification to talk about Aβ40 and 42. There is a whole range of different Aβ peptides that are understudied, he said. As Bart De Strooper and Iryna Benilova, KU Leuven, Belgium, point out in an upcoming Nature News & Views, there are two major paths for Aβ production, both yielding peptides that are successively shorter by three amino acids. The Aβ40 line starts with Aβ49, which is chopped down into Aβ46, and Aβ43. Aβ42 originates from Aβ45 and Aβ48. Shifting γ-secretase to favor the former pathway, and, in particular, to have it go to completion, could be a potential therapeutic strategy. Saido said that Ihara already identified some compounds that facilitate processing of Aβ43 to Aβ40. Saido is also launching a screen to search chemical libraries for compounds that can do the same. Aβ43 might also be a useful biomarker. “Aβ42 is reduced in the CSF because it is trapped in the brain. Since Aβ43 is more prone to aggregate than Aβ42, I would expect that the CSF Aβ43 would be a more sensitive marker—if we can measure it,” said Saido. Tjernberg agrees. “My prediction is you would find an increase in Aβ43 in familial cases that have high Aβ42/40 ratios,” he told ARF. The problem is that no one ever measures Aβ43, and, in fact, some of the antibodies, such as BCO5, that were used in earlier studies of Aβ42, also recognize the longer peptide. Both Tjernberg and Saido are working on ELISAs that could be used to measure Aβ43 in CSF and other biological samples. However, in an e-mail to ARF, Erik Portelius, University of Gothenburg, Sweden, added words of caution, noting that the amount of Aβ43 in the brain is very small compared to Aβ42. “Whether Aβ43 can be used as a biochemical marker in cerebrospinal fluid, if present, remains to be shown,” he wrote (see full comment below).

What is clear is that these new mouse models raise many questions about Aβ43 and its role in AD. Ironically, Adams’s followers know that questions are as important as answers. The biological supercomputer that arrived at the ultimate answer to the ultimate question was powered by none other than sagacious mice. Unfortunately, it took them 7.5 million years to arrive at the answer, by which time the original question had long been forgotten. Hopefully, it will take much less time for mouse models to help answer whether Aβ43 is important in Alzheimer’s disease.—Tom Fagan


  1. Saito and colleagues have generated knock-in mice that cause overproduction of Aβ43. They show that this isoform of Aβ has a higher propensity to aggregate and is more neurotoxic than Aβ42. The conclusion is that Aβ43 in cerebrospinal fluid potentially can be used as a biomarker for presymptomatic Alzheimer's disease. The data provided from the knock-in mice are convincing and aid in understanding the complex nature of Aβ isoforms and the processing of amyloid precursor protein.

    However, many studies have shown that Aβ43 in the brain is only a very minor species compared to Aβ42 and N-terminal truncations of the same, and, so far, Aβ43 has not been identified and verified in human cerebrospinal fluid. Whether Aβ43 can be used as a biochemical marker in cerebrospinal fluid, if present, remains to be shown.

    If this Aβ isoform can be identified by highly sensitive immunoassays in CSF, it has a potential to serve as a novel biomarker for AD.

  2. In this important paper, Takashi Saito and colleagues explore the pathogenic role of Aβ43 in Alzheimer’s disease (AD) by generating knock-in mice with a presenilin-1 (PSEN1) gene mutation, R278I, which selectively overproduces Aβ43 (1). Aβ42 has been the major focus of interest in amyloidogenesis in AD, and yet various longer species of Aβ, such as Aβ43, Aβ45, Aβ48, Aβ49, and Aβ50 have all been found in AD brains. Saito et al. demonstrate that the R278I mutation causes loss of γ-secretase activity in a recessive manner. Specifically, it appears to inhibit conversion of Aβ43 to Aβ40 by γ-secretase, leading to an increased ratio of Aβ43:Aβ40 and Aβ42:Aβ40, without altering the level of Aβ42. By cross-breeding heterozygous R278I with APP mice, they go on to show that the R278I mutation leads to accelerated Aβ pathology with an accompanying inflammatory response and cognitive impairment, which precedes plaque formation.

    Aβ43 was found to show higher neural toxicity and to contribute more readily to the formation of the thioflavin T-positive β-sheeted structure than either Aβ40 or Aβ42. They also examined brain sections from patients with sporadic AD and found that Aβ43 accumulated more frequently than Aβ40. Interestingly, homozygous PSEN1 R278I knock-in mice were found to have an embryonic lethal phenotype. This was thought to be due to impaired processing of Notch1, another of the substrates of γ-secretase. It would be of great interest to know whether interaction of PSEN1 with other substrates including Notch1 is in any way affected by the heterozygous R278I mutation.

    The clinical phenotype of the R278I PSEN1 mutation has proved to be equally as fascinating. The mutation was originally identified in two individuals with an atypical AD phenotype, who presented with symptoms of language impairment (2). However, we have subsequently studied individuals from another branch of this family who have had either behavioral or typical amnestic presentations of familial AD (FAD). Marked heterogeneity may be witnessed in the clinical phenotype of FAD, between different mutations and even within single families affected by the same mutation (3). Investigating the mechanisms underlying this heterogeneity will be an important direction for further work in the field. As Saito et al. point out, although the majority of PSEN1 mutations are associated with an increased ratio of Aβ42:Aβ40, they have varying effects on the absolute levels of Aβ40 and Aβ42, and future studies should also consider how Aβ43 levels are affected. Investigation of the additional genetic and epigenetic factors that may modify these molecular mechanisms, or their pathological consequences, in different individuals will be particularly challenging. Technological developments including the generation of stem cells from skin fibroblasts of patients with these mutations may provide new opportunities for exploring these issues. In collaboration with John Hardy and Selina Wray, fibroblast cell lines from one of our patients with the R278I PSEN1 mutation have recently been generated. These will be deposited in the Coriell cell repository so that any research group with an interest in the mutation may benefit from open access to this valuable resource. The up-to-date list of lines available upon request from the NINDS repository can be found here.

    Saito et al. conclude their paper by proposing two future lines of investigation into the potential clinical implications of their findings. Firstly, they suggest that measurement of cerebrospinal fluid Aβ43 levels may have value as a potential biomarker for presymptomatic AD. Secondly, they hypothesize that inhibition of Aβ43 production by facilitating conversion of Aβ43 to Aβ40 by the γ-secretase complex may prevent Aβ amyloidosis. With international collaborative initiatives like the Dominantly Inherited Alzheimer Network (DIAN) now studying presymptomatic biomarker changes in FAD mutation carriers and contemplating the design of prevention trials for these individuals (4), interest in such avenues of research could not be more timely.


    . Random mutagenesis of presenilin-1 identifies novel mutants exclusively generating long amyloid beta-peptides. J Biol Chem. 2005 May 13;280(19):19070-7. PubMed.

    . A presenilin 1 R278I mutation presenting with language impairment. Neurology. 2004 Nov 9;63(9):1702-4. PubMed.

    . Correlating familial Alzheimer's disease gene mutations with clinical phenotype. Biomark Med. 2010 Feb;4(1):99-112. PubMed.

    . Autosomal-dominant Alzheimer's disease: a review and proposal for the prevention of Alzheimer's disease. Alzheimers Res Ther. 2011;3(1):1. PubMed.

    View all comments by Martin Rossor
  3. In this interesting and important paper, Saito and colleagues have engineered knock-in mice that cause overproduction of Aβ43. They showed that Aβ43, an overlooked species, was potently amyloidogenic, neurotoxic, and abundant in vivo. Aβ43 may be a new biomarker for AD.

    But I wonder about some of the data. Aβ43 production in R278I/R278I MEF cells or in R278I/+ MEF cells was markedly reduced in a gene dose-dependent manner as shown in supplementary Fig 10bc, suggesting that γ-secretase complex including PS1-R278I clearly impaired Aβ43 production activity as well as Aβ38, Aβ40, and Aβ42. This unambiguously indicates that the R278 mutation is a loss of γ-secretase function. In this figure, an increase in Aβ43 production via the inhibition of Aβ43-to-40 conversion process cannot be observed. As shown in Figure 2k-n, however, a large amount of Aβ43 was detected in conditioned medium from R278I/R278I MEF cells or from R278I/- MEF cells in the ELISA system. Therefore, Aβ43 in conditioned medium may be produced via a PS1-independent processing pathway, not via the inhibition of the Aβ43-to-40 conversion process in the γ-secretase complex.


    . Distinct presenilin-dependent and presenilin-independent gamma-secretases are responsible for total cellular Abeta production. J Neurosci Res. 2003 Nov 1;74(3):361-9. PubMed.

    . A presenilin-independent aspartyl protease prefers the gamma-42 site cleavage. J Neurochem. 2006 Jan;96(1):118-25. PubMed.

    . Pharmacological evidences for DFK167-sensitive presenilin-independent gamma-secretase-like activity. J Neurochem. 2009 Jul;110(1):275-83. PubMed.

    View all comments by Fuyuki Kametani

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News Citations

  1. Research Brief: Presenilin Simplicity—Evidence for Autoproteolysis
  2. Honolulu: Wake-Up Call—Aβ Clearance, Not Production, Awry in AD

Webinar Citations

  1. Slow Aβ; Clearance Is ApoE4’s Modus Operandi in Late-Onset AD

Paper Citations

  1. . Random mutagenesis of presenilin-1 identifies novel mutants exclusively generating long amyloid beta-peptides. J Biol Chem. 2005 May 13;280(19):19070-7. PubMed.
  2. . A presenilin 1 R278I mutation presenting with language impairment. Neurology. 2004 Nov 9;63(9):1702-4. PubMed.
  3. . 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.
  4. . Abeta43 is more frequent than Abeta40 in amyloid plaque cores from Alzheimer disease brains. J Neurochem. 2009 Jul;110(2):697-706. Epub 2009 May 15 PubMed.

External Citations

  1. The Hitchhiker's Guide to the Galaxy

Further Reading

Primary Papers

  1. . Potent amyloidogenicity and pathogenicity of Aβ43. Nat Neurosci. 2011 Aug;14(8):1023-32. PubMed.