Like calling a spade a spade? What about an Aβ oligomer? The study of this peptide’s oligomers has taken off in the last decade. Many labs have developed their own favorite means of making, purifying, and studying them. Researchers have isolated dimers, trimers, and on up to dodecamers and higher molecular mass species. Do any exist in the brain? Do all? What are the most physiologically relevant forms? Researchers are debating that question, but the field has become confusing as labs tend to use their own preparations more than reproduce one another’s, and standards on how to prepare and what to call oligomers have not yet emerged. Is it even clear what the necessary tools are to provide the answers?
On Thursday, 13 October 2011, the Alzforum held a Webinar discussion led by Dominic Walsh. Bart De Strooper, Hilal Lashuel, Sylvain Lesne, and David Teplow joined Walsh to discuss how to come to some common terminology and protocols in the field of Aβ oligomers.
Listen to the Webinar
Dominic Walsh's Presentation
Sylvain Lesne's Presentation
Hilal Lashuel's Presentation
Bart De Strooper's Presentation
View Comments By: Shaohua Xu — Posted 1 February 2012
By Tom Fagan
In recent years, Aβ oligomers have grabbed the research limelight. Bigger than monomers, but not full-fledged fibrils, Aβ oligomers are touted as the most toxic forms of the peptide. But do they really exist in the brain? If so, what form do they take?
Oligomers burst onto the scene over 15 years ago, when researchers in Dennis Selkoe’s lab at Brigham and Women’s Hospital, Boston, found that Chinese hamster ovary (CHO) cells transfected with the amyloid-β precursor protein gene secreted Aβ oligomers (see Podlisny et al., 1995). Working in the lab, Dominic Walsh discovered that these natural oligomers were toxic to neurons. CHO-derived monomers were innocuous, but dimers, trimers, and larger species blocked synaptic function when injected into rat brain (see ARF related news story). Collaborating with Walsh and Selkoe, Karen Ashe’s group at the University of Minneapolis, Minnesota, subsequently found that low doses of these oligomers acutely suppress learning and memory when injected into rat cerebral ventricle (see ARF related news story). About the same time, researchers at Bill Klein’s lab at Northwestern University, Chicago, reported that ADDLs, or Aβ-derived diffusible ligands, are toxic to cells as well. ADDLs are non-fibrillar soluble oligomers formed in the test tube. They were reported to inhibit long-term potentiation (LTP) in neuronal cultures and kill neurons at higher concentrations (see ARF related news story and Lambert et al., 1998). Soon, numerous other labs fingered Aβ oligomers as potentially the most toxic form of the peptide (see, e.g., Dahlgren et al., 2002; Wang et al., 2002).
But do oligomers form in the brain? Early hints that they do came from Alex Roher and colleagues at Sun Health Research Institute, Sun City, Arizona. These researchers isolated water-soluble oligomers from human brain tissue, including dimers that killed neurons in the presence of microglia (see Roher et al., 1996). Walsh isolated dimers from human cerebrospinal fluid, and reported that primary neuronal cells from human brain make oligomers intracellularly (see Walsh et al., 2000), supporting the idea that Aβ multimers form in vivo. Klein’s group used antibodies raised against ADDLs to identify a kind of oligomer in human brain (see ARF related news story
on Gong et al., 2003). Recently, Selkoe’s group isolated AD patient-derived oligomers and showed they are toxic to cells. The majority of the oligomers in this preparation were dimers, reinforcing the idea that these are a particularly nasty form of the peptide (see ARF related news story).
Oligomers have been isolated from mice, too, most notably the 12-mer Aβ*56 identified by Sylvain Lesne and colleagues in Ashe’s lab. This 56 kDa species surfaces in Tg2576 transgenic mice just as their learning and memory problems begin, and levels in the brain correlate with the degree of cognitive dysfunction (see ARF related news story). Last July, researchers at Karen Gylys’ lab at the University of California, Los Angeles, identified a 56 kDa form of Aβ in synaptosomes from human AD patients (see Sokolow et al., 2011). Whether the human and mouse 56 kDa forms are structurally similar is unclear.
Indeed, the variety of different structures assumed by Aβ stands opposite a myriad of properties and toxicities, few of which, it seems, are robustly reproduced by other labs. CHO-derived oligomers were reported to induce synapse loss by acting on NMDA-type glutamate receptors to cause long-term depression (see ARF news on Shankar et al., 2007). ADDLs were reported to bind to and ablate NMDA receptors (see ARF related news story on Lacor et al., 2007). When applied to rat hippocampal neurons, patient-derived oligomers apparently cause hyperphosphorylation of tau and degeneration of neurites (see ARF related news story on Jin et al., 2011), but application of ADDLs also was reported to lead to tau phosphorylation, a breakdown of microtubules, and synaptic degradation (see ARF related news story on Zempel et al., 2010). One problem is that different labs use widely different oligomer concentrations in cell culture experiments (e.g., ADDLs at 500 nM vs. patient-derived oligomers at 0.5 nM).
So what are the physiologically relevant forms of Aβ? While different groups have worked on their own particular oligomer of choice, others have tried and failed to find oligomers or to reproduce their toxicities. This lack of consensus is sowing some seeds of doubt that oligomers truly are crucial to AD.
Researchers are calling for clarification, for some standardization of nomenclature and protocols, and for the sharing of material. As stated in a recent Nature editorial, “it is imperative that all papers reporting the effects of aggregated proteins clearly state the exact source of the protein, its state of aggregation when added to the system, and discuss the potential caveats of the approach.” While this may help the field reach consensus on the properties of certain Aβ oligomers, the Nature editors also acknowledge that oligomers are inherently metastable. In other words, what a scientist thinks (s)he is giving to the cells is not always what the cells are getting, since oligomers can dissociate or associate with other oligomers, monomers, or other entirely different proteins. Walsh suggests going one step further and characterizing oligomers not just at the beginning, but at the end of a given experiment as well.
Pinpointing physiologically relevant oligomers may prove even more challenging. The field grapples with the oligomer version of Heisenberg’s uncertainty principle in that the very act of observing these oligomers may change them. For example, detergents used in gel electrophoresis experiments may metamorphose large aggregates into low-molecular-weight soluble oligomers (see Hepler et al., 2006). How do brain oligomers change during a lengthy and complex purification? Dimers isolated from the brain might not, in fact, have any toxicity themselves, but rapidly assume larger structures that do (see ARF related news story on O’Nuallain et al., 2010). On top of this, Aβ is well known to exist in different lengths and modified forms that may not all oligomerize the same way, or have the same properties even if they do. Bart De Strooper’s group at KU Leuven in Belgium and Hilal Lashuel's group at the Swiss Federal Institute of Technology in Lausanne, Switzerland, showed that Aβ oligomerization dynamics and toxicity can be vastly altered by subtle changes in the ratio of Aβ42 to Aβ40 (see Kuperstein et al., 2010 and Jan et al., 2008), and a report on Aβ43 has further opened up the range of possible forms (see ARF related news story on Saito et al., 2011). Other researchers, including David Teplow and Hilal Lashuel, believe that it is not any one oligomer that is going to be important, but a population of different assemblies (see Jan et al., 2011). Probability theory might then be needed to predict which entities are the prominent/important in the brain.
Researchers agree that the field needs better tools to study these proteins in solution. Some help may come from biophysical and modeling approaches. Teplow, working with Michael Bowers at the University of California, Santa Barbara, has used crosslinking to capture different oligomeric states (see Ono et al., 2009) and modified mass spectrometry to analyze Aβ oligomer assembly. He found that Aβ42 seems to prefer tetrameric and dodecameric conformations (see ARF related news story on Bernstein et al., 2009). Teplow’s group also used molecular models of aggregation to predict that Aβ42 forms pentameric and hexameric assemblies before forming larger aggregates (see Urbanc et al., 2010). Biophysical techniques can characterize the sizes and shapes of molecules in solution, but they tend to require highly pure samples (dynamic light scattering) or are expensive (fluorescence correlation spectroscopy).
In summary, researchers have identified myriad Aβ oligomer species, but not how they relate to each other and to Alzheimer’s disease. Researchers studying tau and α-synuclein may confront similar challenges before too long. What technical and experimental standards are needed to find consensus? What biophysical advances will allow researchers to identify and characterize oligomeric species in vivo? Would a collaborative effort at a round-robin comparison of protocols among the leading labs be useful, as has been done in CSF Aβ/tau measurement and in AD neuropathology?
"Oligomer" is a misnomer here. An oligomer by definition is a molecule that consists of a relatively small and specifiable number of monomers—usually less than five (see the free dictionary).
1. Aβ aggregation species are likely to be larger than assemblies of five monomers.
2. No evidence supports a defined number of monomers in these aggregates. On the contrary, more evidence supports a diverse number of monomers in aggregates of intermediate size.
For tau and Sup35, spherical aggregates that are larger than a monomer but smaller than fibers are estimated to consist of a few dozen monomers.
References: Xu S, Bevis B, Arnsdorf MF. The assembly of amyloidogenic yeast sup35 as assessed by scanning (atomic) force microscopy: an analogy to linear colloidal aggregation? Biophys J. 2001 Jul;81(1):446-54. Abstract
Xu S. Cross-β-sheet structure in amyloid fiber formation. J Phys Chem B. 2009 Sep 17;113(37):12447-55. Abstract
Xu S. Aggregation drives "misfolding" in protein amyloid fiber formation. Amyloid. 2007 Jun;14(2):119-31. Abstract
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