Scientists and clinicians gathered at the University of Southern California in Los Angeles on April 15, 2016, for a day jam-packed with a dozen presentations. It was the third annual Zilkha Symposium on Alzheimer Disease & Related Disorders, co-organized by USC’s Berislav Zlokovic, David Holtzman of Washington University, St. Louis, and Rudy Tanzi of Massachusetts General Hospital in Boston. In talks that stretched from genetics to clinical symptoms to therapeutic targets, the overarching theme was coming to grips with complexity and heterogeneity. Scientists agreed they need to better understand not only Alzheimer’s central pathways of Aβ and tau, but also the many additional processes that influence its pathogenesis in important ways, such as neuroimmune and neurovascular regulation. On the Aβ topic, speaker David Brody of Washington University in St. Louis offered preliminary results that might open a new door to the study of oligomers. Brody purified oligomers from human brain and came up with a set that look nothing like the oligomers and fibrils previously obtained by scientists working with mouse models or synthesizing oligomers in vitro.
Scientists know that while the distribution of amyloid plaques does not correlate tightly with dementia in people, oligomeric Aβ does suppress learning and memory, and kills neurons in various experimental systems. However, for years, the individual labs studying Aβ oligomers have seemed to find, or make, slightly different types of oligomer. Scientists have isolated dimers from human cerebrospinal fluid and cultured cell lines, collected dodecamers from the brains of AD models, or synthesized their own artificial versions in vitro. Only some oligomers are toxic at low concentrations, and labs have not broadly reproduced each other’s methods and findings, leaving the field at an impasse (see Oct 2011 webinar). What’s more, scientists have cautioned that studying oligomers in the lab may alter their properties. Detergents used in purification protocols or gel electrophoresis buffers can induce oligomerization of monomers within minutes (Hepler et al., 2006; Watt et al., 2013) Roughly douncing or otherwise homogenizing tissues might break up larger aggregates, Brody speculated. “It is not clear what exactly is going on with amyloid-β oligomers, and which ones are most toxic,” Brody said.
Zlokovic called Brody’s work “the most detailed and comprehensive analysis of oligomers from brain tissue that I saw so far.” However, Brody noted his results are still preliminary. “I do not think we have in any way solved this problem; what we are doing is pushing the technical envelope a little bit.”
Brody, and Thomas Esparza in his lab, set out to purify native Aβ oligomers from human brain tissue of people who died of AD, despite warnings from colleagues. “Many people tried and gave up,” Brody said. “I have been told a dozen times not to do this, that this was impossible.” Part of the challenge is that oligomers are thought to occur in minute concentrations in the brain; another was the lack of a good assay to quantify the presence of oligomers throughout the steps of a purification protocol.
Brody and Esparza thought they could succeed because they had previously developed a quantitative ELISA for oligomers (see Feb 2013 news). Another advantage, Brody told Alzforum, was the availability of relatively large quantities of AD brain tissue from the Washington University Alzheimer’s Disease Research Center. Other scientists trying to develop protocols for purifying amyloid oligomers have had to rely on non-human sources, Brody said, such as synthetic oligomers that might differ from the natural versions. In contrast, Esparza was able to use human tissue for all his experiments, over years spent developing the protocol.
The researchers started with amyloid-loaded brain tissue from people who had died of autopsy-confirmed Alzheimer’s. Esparza minced cortical tissue, then homogenized it in buffer with CHAPS, a detergent he found does not facilitate Aβ oligomerization. He spun the homogenate in a centrifuge to pellet out large debris, including plaques. Then he took that supernatant, which included a mix of monomers and oligomers, and spun it again at 475,000 x g. Esparza had to use a special ultracentrifuge to reach this enormous speed. He included a sucrose cushion at the bottom of his tubes to catch the oligomers; otherwise, they would become irretrievably stuck to the tube walls, Brody said. Monomers remained in the supernatant.
Next, Esparza limited his preparation to oligomer size with size-exclusion chromatography, and immunoprecipitated the oligomers with two different antibodies to Aβ. All the while, he checked his intermediate preps to make sure the oligomers were still present. One important trick, Brody said, was to coat every pipette tip, test tube, or other apparatus with albumin. Otherwise, the Aβ sticks to the equipment and a bit gets left behind at every step.
As it turned out, it was not impossible to isolate oligomers from brain—just “very, very difficult,” Brody said. Overall, Esparza lost less than 30 percent of the amyloid he had in the original homogenate, and he concentrated the protein by 10,000 times. The oligomers’ structures remained stable throughout the procedure, Esparza believes, because they had the same size according to size-exclusion chromatography in the initial lysates and final purified preparation. No new oligomers formed; Esparza confirmed this by spiking in monomers, which stayed monomers.
What kinds of oligomer were in the AD brains? Some were large, more than 500 kilodaltons according to size-exclusion chromatography. Using an electron microscope, Esparza spied clusters of spheres, each 10-20 nanometers across. Some clusters contained just three or four spheres; others grouped together dozens. They were decidedly non-fibrillar. “What we see does not look like anything that has been previously reported,” said Brody. He suspects each sphere contains one or more Aβ molecules, plus associated proteins the amyloid may bind.
Norelle Wildburger in Brody’s lab used mass spectrometry to identify the specific Aβ species in his oligomers. She saw an assortment. Besides those that start with Aβ’s first amino acid, there were others truncated at various sites up to position 11. Measuring Aβ’s other end, she saw peptides ending between positions 34 and 43. The molecules were sometimes modified, for example having lost amine groups or gained cyclized pyroglutamate.
Brody suspects, and other scientists at the Zilkha Symposium agreed, that some previous studies of Aβ oligomers may have been confounded by their protocols. Maria Carrillo of the Alzheimer’s Association said it was intriguing to consider oligomers might be larger than previously thought, and that some other oligomer studies might have focused on artefacts. However, Brody cautioned that his own prep still might be subject to other types of artefact. For example, oligomers might form postmortem as tissues cool from body to room temperature, before the medical examiner arrives.
Ideally, one would like to detect oligomers in the living brain, perhaps with imaging agents specific to oligomer epitopes. Alas, Brody said such tracers are nowhere near ready (Feb 2016 news; Dec 2014 news). Scientists are also making progress with methods to detect oligomers in cerebrospinal fluid, but still wish for more sensitivity in those assays (see Jul 2012 conference news; Feb 2014 news).
What does seeing a zoo of larger Aβ oligomer species in human AD brain mean for the field? “It is, in one sense, a giant step backwards, because a lot of what we thought we knew about Aβ oligomers may not be that relevant to the human brain,” Brody said. Potentially, therapeutics that target synthetic oligomers might not work in people, he speculated. However, he added, his work also offers ways to take oligomer research forward. “It is, perhaps, an opportunity to look more broadly at the other forms of amyloid-β oligomers that might exist in human brain,” Brody told Alzforum. Once published, Brody will make the detailed protocol publicly available.
Many questions arise from this ongoing work, Brody noted. For one, he is interested in comparing the various Aβ constituents and their proportions between human and mouse brains. For another, he wants to test how the oligomers interact with plaques, and if plaques from the brains of donors who died without dementia are better at buffering loose oligomers. This might explain in part why people with brain amyloid were protected from dementia.
Sangram Sisodia of the University of Chicago called Brody’s presentation “the coolest stuff I saw.” He said it would be key to show which species are toxic. Brody plans to inject mouse brains and check for synaptic degeneration or loss. He also wants to expose hippocampal slices to the oligomers and measure effects on long-term potentiation. Washington University’s Holtzman said the work took a different direction from the rest of the field, and wondered if these oligomers might be the long-sought connection between Aβ and tau—in other words, if their presence drives the emergence of tauopathy.—Amber Dance
- New Assays for Aβ Oligomers—Spinal Fluid a Miss, Brain Awash
- Can Antibody-Based PET Scans Pinpoint Aβ Oligomers in the Brain?
- Antibody Probe Seeks Aβ Oligomers in MRI
- New Assays for Aβ Oligomers in CSF Claim Femtogram Sensitivity
- Test Closes in on Oligomers, May Distinguish Alzheimer’s Patients From Controls
- Hepler RW, Grimm KM, Nahas DD, Breese R, Dodson EC, Acton P, Keller PM, Yeager M, Wang H, Shughrue P, Kinney G, Joyce JG. Solution state characterization of amyloid beta-derived diffusible ligands. Biochemistry. 2006 Dec 26;45(51):15157-67. PubMed.
- Watt AD, Perez KA, Rembach A, Sherrat NA, Hung LW, Johanssen T, McLean CA, Kok WM, Hutton CA, Fodero-Tavoletti M, Masters CL, Villemagne VL, Barnham KJ. Oligomers, fact or artefact? SDS-PAGE induces dimerization of β-amyloid in human brain samples. Acta Neuropathol. 2013 Apr;125(4):549-64. PubMed.
No Available Further Reading