16 November 2007. Over the past decade, researchers have shifted away from a literal interpretation of Alois Alzheimer’s groundbreaking discovery of plaques and tangles as the likely cause of Alzheimer disease. After years of argument—mostly in the 1990s—about whether plaques or tangles were the culprit, the answer appears to be, “Both and neither.” How can that be true? Scientists have recognized that both constituent proteins of those hallmark pathologies—the amyloid-β (Aβ) peptide and tau—play essential roles in the development of the disease, relegating the Baptist-Tauist divide solidly to the past. That’s the “both” part. But scientists also increasingly agree that the microscopically visible protein deposits are not the worst offenders: hence, the “neither.” Instead, they blame smaller, oligomeric forms of the Aβ peptide that they believe exist in a complex equilibrium with higher-order protofibrils along a path to aggregation. These, they say, damage synapses and interfere with cognitive function. In short, they say plaques are bad, but oligomers are worse. For tau, this story isn’t nearly as far along, but trends suggest that it may well develop along similar lines. (And ditto for α-synuclein.)
The Society for Neuroscience conference, held 3-7 November in San Diego, was a testament to how deeply the science of Aβ oligomers has taken hold in the field. There were some 35 presentations about Aβ species variably called oligomers, ADDLs, AβOs, or protofibrils. Speakers increasingly cited the “Amyloid Oligomer Hypothesis” rather than the “Amyloid Hypothesis” in the introductory slide of their talk. Indeed, a range of presentations from a diverse group of labs reported data largely concurrent with its essential tenet that AD begins with synaptic dysfunction caused by soluble Aβ species. Here are selected highlights.
Perhaps the most direct support came from Ganesh Shankar, an M.D.-Ph.D. student working with a team of colleagues in Dennis Selkoe’s laboratory and Cindy Lemere at Brigham and Women’s Hospital, Boston, Dominic Walsh’s group and Ciaran Regan’s group, both at University College in Dublin, Ireland, and with Bernardo Sabatini at Harvard Medical School. In a sparsely attended slide session on the last afternoon of the conference, Shankar expanded on what a poster presented by Shaomin Li from the same team had foreshadowed days before. The scientists isolated soluble Aβ species from cortex of human AD brain, and report that oligomers as small as a dimer recapitulated the synaptotoxic effect the scientists had previously published for similar small oligomers secreted by cultured cells.
Prior studies from several laboratories have consistently found synaptotoxic effects for various forms on Aβ oligomers (e.g., Walsh et al., 2002—from conditioned media of 7PA2 Chinese hamster ovary cells; Lambert et al., 1998—from synthetic Aβ42; Lesne et al., 2006—from Tg2576 mouse brain). Yet these studies begged the question of how relevant to human Alzheimer disease all this can be until human Aβ oligomers are in hand. To address this question, Shankar and colleagues obtained postmortem cortical tissue from several patients with late-onset AD (one of whom had had no clinical AD but pathological AD upon autopsy). As controls, the scientists used cortex from patients with Lewy body dementia (LBD—they get parkinsonism and dementia at about the same time and are thought to have mixed pathologies), Down syndrome (who have typical AD-type amyloid pathology), and frontotemporal and multi-infarct dementia (who do not). Readily detectable soluble Aβ showed up in cortex from all clinically demented AD patients but not in one cognitively normal person who had the plaque pathology. It also showed up in the Down brain, and to a much smaller extent in the LBD brain. Curiously, soluble extracts from normal control brains appeared to contain very little or no soluble monomeric Aβ by this immunoprecipitation/Western blot assay, even though the brain presumably produces some all the time.
These AD cortical extracts were made merely in TBS buffer without detergent, and they showed primarily monomer at a weight of 4 kDa and dimer at 8 kDa. Extracts made in parallel with detergent also had monomer and dimer in them. Shankar showed experiments suggesting that besides the dimer, soluble Aβ extract from human AD brain also contains complexes having a larger molecular weight—either Aβ aggregated with itself or bound to other proteins—but that these fall apart upon treatment with detergent. This is a technical difference with studies on ADDLs and Aβ*56, both of which are reported to be SDS-stable. Shankar said that his colleagues and he searched for SDS-stable species in the human extracts but so far have been unable to find any that are larger than trimer. Shankar and colleagues used detection by two antibodies that detect the free N- and C-terminus of Aβ, respectively, and also used mass spectrometry, to ascertain that the dimers contained true Aβ, and to exclude any other Aβ-containing APP cleavage fragments that might be contained in the extracts.
Next, the scientists applied their preparations to tests of LTP and spine integrity that they had developed previously. The TBS extracts from AD brains blocked LTP induction, whereas extracts from the other diseases and age-matched controls did not, Shankar reported. (The Down’s extract was not tested.) Immunodepletion of Aβ restored LTP, meaning the effect was specific to Aβ. The effect was potent, acting in the picomolar range. By enriching Aβ through immunoprecipitation, eluting with SDS buffer and then running on size exclusion chromatography, only the fraction enriched for Aβ dimers inhibited LTP significantly; the monomer had no effect. The soluble AD brain extract also facilitated long-term depression, reducing neuronal excitability after a period of stimulation. The main point, Shankar said, is that Aβ dimer extracted from human AD brain is sufficient to disrupt the molecular basis of learning and memory. It is not the only form, but the smallest form that can be toxic.
Various anti-Aβ antibodies are in clinical trials at present, and one debate in the field revolves around which type of antibody might be most potent. Shankar and colleagues indirectly addressed this debate by testing which of the three classes of anti-Aβ antibody used in those trials—N-terminal, mid-region, C-terminal—was best able to rescue the detrimental effect on LTD of the human AD extract. In a subtraction experiment, where the investigators selectively depleted the extract with only one kind of antibody, N-terminal antibodies best protected LTP, Shankar reported. This electrophysiology result concurred with associated biochemistry, in that the N-terminal antibodies also captured the most Aβ from the extract. (Not all immunotherapy clinical trials, however, are based on the premise of directly counteracting Aβ oligomers in brain; some aim to draw down Aβ from the periphery, or target Aβ more generally.)
Beyond LTP and LTD, do these human oligomers really matter to the structure of synapses? There is strong consensus in the field that synapses in AD-relevant brain areas gradually decrease in number early on as people develop cognitive symptoms (Davies et al., 1987; Scheff et al., 2007). At Neuroscience, Shankar showed evidence that the human AD oligomers reduced the density of dendritic spines in cultured brain slices in much the same way as cell-secreted oligomers do (Shankar et al., 2007). Furthermore, Shankar showed data on a rat behavioral test. The human AD extract impaired learning in a passive avoidance paradigm. It did so when infused 3 hours after the rats had initially learned, the period that other studies have identified as the time when synapses undergo remodeling following learning.
Finally, the researchers reported taking a hard crack at the Aβ dimers. Acting on a hunch that the dimers might represent a seed for plaque formation, the team isolated mature, cored plaques and removed as many associated components from them as possible by repeated washes in detergent and TBS buffer. This left behind insoluble, microscopic cores that stained with Congo red. These cores did not inhibit LTP. They were very hardy, but when the scientists blasted them apart with highly concentrated formic acid, Aβ dimers were released, and those did inhibit LTP. Taken together, these investigators interpret their data to mean that soluble Aβ oligomers from typical AD patients, starting with dimers, disrupt synaptic function in humans, and that insoluble cores sequester these species. As to plaques, they represent a reservoir of soluble Aβ in a given brain region, Shankar said. For their part, the dimers would seem to be a relevant substrate for both research into the molecular pathways of synaptic impairment, and also for testing prospective therapeutic agents preclinically, Shankar added.
Other labs need to replicate these findings. When asked whether the recipe for isolating the human oligomers was technically difficult, he replied: “No, it’s pretty much standard biochemistry. But rigorous clinical and histopathological information on the patients should be available before attempting it, so close interaction with a brain bank is key.”—Gabrielle Strobel.
- San Diego: Oligomers Live Up to Bad Reputation, Part 2
- San Diego: Oligomers Live Up to Bad Reputation, Part 3
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