Some Aβ seeds die hard. According to two studies from Mathias Jucker’s lab at the German Center for Neurodegenerative Diseases in Tübingen, Germany, Aβ peptides derived from postmortem Alzheimer’s disease brains seeded plaques in mouse brains at sub-attomolar amounts. Even a two-year soak in formaldehyde—a stifling experience for most proteins—won’t stop the seeds. One study, published September 10 in Brain, reported that less than one-billionth of one-billionth of a mole of Aβ from AD brain extracts triggered plaque deposition in mice, whereas peptides derived from cerebrospinal fluid (CSF) failed to seed at much higher concentrations. Published September 6 in Acta Neuropathologica, the second study reported that Aβ peptides derived from formaldehyde-preserved brain tissue retained their toxic seeding ability—an observation that makes Aβ seeds even more eerily reminiscent of prions. Together, the studies further strengthen the view of Aβ as a prion-like molecule. They also highlight questions about what the seeding Aβ species truly is, its role in human disease, and hopes of detecting it in the CSF.
Cortical amyloid aggregates are a hallmark of AD, and researchers have spent decades studying how they form and how they relate to cognitive decline. In vitro studies suggest that aggregates of Aβ serve as seeds that recruit Aβ monomers, which then contribute to growing fibrils. Extracts from human AD brains seed plaques in mice (Kane et al., 2000; Meyer-Luehmann et al., 2006). Jucker’s lab subsequently reported that soluble brain fractions contained the most potent seeds (see Oct 2011 news story). This has prompted researchers to look for seeds in human CSF in hopes of identifying species that cause disease and developing early diagnostic tools (see Feb 2013 news story; Feb 2014 news story; and Mar 2014 news story).
“We believe that the formation of small Aβ aggregates that can act as corruptive seeds is the first step in the disease process,” Jucker told Alzforum. The closer researchers can get to detecting these first seeds, he said, the better.
Straight from the Source. Amyloid plaques line the hippocampus in mice injected with soluble extracts made from postmortem AD brain tissue (left), but not those injected with CSF from AD patients (right). [Image courtesy of Sarah Fritschi.]
To find the smallest amount of Aβ necessary to seed aggregation, co-first authors Sarah Fritschi and Franziska Langer analyzed the soluble fraction of cortical extracts prepared postmortem from AD patients. Mass spectroscopic analysis revealed that these contained an array of Aβ peptides, most notably Aβ1-40, as well as several N-terminally truncated forms starting at the fourth residue. Overall, this composition of Aβ peptides was similar to a previous report (see Portelius et al., 2010). The researchers injected the extracts at different concentrations directly into the hippocampi of young APP23 mice, which are prone to developing plaques. Eight months later, before the mice would have developed amyloid pathology on their own, all of them had amyloid plaques, even those whose injections contained but a single femtogram of Aβ—equivalent to approximately 130,000 monomers or 11,000 dodecamers. “These seeds were incredibly potent,” Jucker said.
The researchers did not inject mice with extracts prepared from normal, non-AD brains, commented Mary Savage of Merck in Rahway, New Jersey. Given reports of Aβ oligomers in the brains of some cognitively normal people, such a comparison would be an interesting next step in implicating disease-associated Aβ species in pathology, she wrote in an email to Alzforum.
The researchers next searched for Aβ seeds in patient CSF. Though the concentration of total Aβ in CSF trumped that in the brain extracts by 10-fold, CSF injected into mouse brain induced no plaques, even after 22 months or when concentrated 15-fold. CSF derived from plaque-ridden, older APP23 mice also failed to seed plaques in younger mice. To rule out factors that might be lurking in CSF and inhibit seeding, the researchers injected mice with brain extract plus CSF. The Aβ in the brain extracts still seeded efficiently. Jucker and colleagues don’t know why CSF from AD patients failed to seed, but noted that the CSF contained no N-terminally truncated Aβ species.
Interestingly, Aβ particles in CSF grew into aggregates in vitro, as measured by elongation when Aβ monomers were added. However, they did so far less potently than did soluble Aβ in brain extracts. Moreover, super resolution fluorescence microscopy revealed that the Aβ particles in CSF were smaller than those in the extracts.
Claudio Soto of the University of Texas in Houston found this unsurprising. “It is common to find prion aggregates in vitro in biological fluids such as blood or urine, which are not able to induce disease in animals,” he said (see full comment below).
Jucker hypothesized that the most potent seeds, perhaps those incorporating N-terminal truncated peptides, are trapped in the brain and never reach the CSF. Alternatively, smaller CSF aggregates may be more vulnerable to degradation when injected into the mice, he added.
Researchers have long noted signs that Aβ species derived from the brain are unlike those from the CSF, but this paper offers the strongest evidence to date of these fundamental differences, wrote David Brody and Michael Gross of Washington University in St. Louis. They co-authored an editorial that will accompany the print version of the paper.
Should the results discourage researchers trying to detect Aβ oligomers in CSF? Not according to Jucker. Aβ species need not have potent seeding capacity to serve as useful diagnostic tools, he said, as long as they distinguish people in the early stages of AD from healthy controls. Karen Ashe of the University of Minnesota in Minneapolis agreed. She has identified an Aβ oligomer, Aβ*56, in CSF. Although it does not possess seeding activity, she said, Aβ*56 correlates with cognitive impairment in humans and may serve as a useful biomarker.
The seeding potency of Aβ peptides derived from brain extracts was on par with what researchers have observed for prion proteins. Researchers in the AD field generally avoid the term prion for Aβ and other amyloidogenic proteins such as α-synuclein and tau, because these peptides are not infectious under everyday circumstances. Some suggest that prions are infectious because they resist degradation and can cross from the gut to the brain. Because prions were initially distinguished from viruses and other infectious agents by their ability to withstand harsh formaldehyde treatment (see Pattison, 1965; Prusiner, 1998), Jucker and collaborator Lary Walker at Emory University, Atlanta, wanted to see how Aβ seeds measured up in this test. If the seeds were as famously hardy as prions, then researchers should be able to use fixed brain-banked samples for seeding.
As reported in the Acta Neuropathologica paper, first authors Fritschi and Amarallys Cintron from the Walker lab found that extracts prepared from postmortem AD brain samples that had been preserved in formaldehyde for two years still seeded plaques in APP23 mice. Extracts from fixed normal, non-AD brains did not induce plaques. To directly compare the seeding efficiency of fixed versus fresh brain samples, the researchers prepared extracts from plaque-laden APPPS1 mouse brains in two different ways: one hemisphere of each mouse brain was simply frozen, while the other was fixed in formaldehyde. The researchers prepared extracts from each side and injected equal amounts into the hippocampi of young APP23 mice. Four months later, both groups of animals harbored plaques throughout the hippocampus, although mice seeded from fixed samples had slightly fewer. Aβ from fixed extracts also seeded plaques in vitro, albeit less efficiently than Aβ from fresh/frozen extracts.
Marc Diamond of Washington University in St. Louis called the study solid in its conclusions, but not a surprise. “Since seeding by PrP has long been known to be resistant to formaldehyde, this result is not unexpected,” he wrote in an email to Alzforum. He added that the relevance of Aβ seeding activity for human disease is not yet clear. “It is not really linked to any clear dysfunction in people, although it is probably a risk factor for tau accumulation,” he commented.
David Westaway of the University of Alberta in Edmonton commented that the formaldehyde resistance of Aβ was reminiscent of prions. Westaway was a postdoc in Stanley Prusiner’s lab at the University of California, San Francisco, at the height of the scientific debate about whether prions contained nucleic acids—i.e., whether they were viruses. Westaway said the study was a nice contribution, but did not necessarily relate to mechanisms involved in the natural pathogenesis of AD. The main implication of the study, he added, is that now researchers may be able to use fixed brain samples in seeding studies. Jucker plans to take advantage of this to compare the seeding capacity of Aβ prepared from different regions of the brain and to potentially link that seeding capacity to different Aβ species. Because Aβ is known to occur in distinct strains that differ between people and regions of the brain, determining which ones cause disease will be important (see Jul 2014 news story; and Sep 2013 news story).
Jucker said his lab has also observed formaldehyde resistance in synuclein seeds, further clustering these various aggregate-prone proteins into the prion-like camp.
Aβ’s seeding persistence in fixed samples is unlikely to be infectious in a laboratory setting, and procedures already in place should be sufficient, researchers commented. However, to be safe, Jucker said his lab now makes an effort to handle fixed samples just as they do fresh ones, especially when making extracts: in the fume hood, and avoiding procedures that generate aerosols. Other researchers noted that these proteins only seed when injected directly into the brain and that the chances of "infection" are minuscule. —Jessica Shugart
- Seeds of Destruction—Prion-like Transmission of Sporadic AD?
- New Assays for Aβ Oligomers—Spinal Fluid a Miss, Brain Awash
- Test Closes in on Oligomers, May Distinguish Alzheimer’s Patients From Controls
- Test Uses 'Seeding' to Detect Aβ Oligomers in Cerebrospinal Fluid
- More Evidence Ties Aβ Strains to Distinct Pathologies
- Does Aβ Come In Strains? Glimpse Into Human Brain Suggests Yes
Research Models Citations
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- Fritschi SK, Langer F, Kaeser SA, Maia LF, Portelius E, Pinotsi D, Kaminski CF, Winkler DT, Maetzler W, Keyvani K, Spitzer P, Wiltfang J, Kaminski Schierle GS, Zetterberg H, Staufenbiel M, Jucker M. Highly potent soluble amyloid-β seeds in human Alzheimer brain but not cerebrospinal fluid. Brain. 2014 Nov;137(Pt 11):2909-15. Epub 2014 Sep 10 PubMed.
- Fritschi SK, Cintron A, Ye L, Mahler J, Bühler A, Baumann F, Neumann M, Nilsson KP, Hammarström P, Walker LC, Jucker M. Aβ seeds resist inactivation by formaldehyde. Acta Neuropathol. 2014 Oct;128(4):477-84. Epub 2014 Sep 6 PubMed.