The classic neuropathological hallmarks of Alzheimer’s disease are the appearance of amyloid plaques composed primarily of amyloid beta (Aβ) peptides, and neurofibrillary tangles composed mainly of hyperphosphorylated tau protein. For many years, research into treatments for Alzheimer’s disease proceeded on the hypothesis that the plaques were toxic to the surrounding neurons. More recently, however, evidence has shown that soluble Aβ oligomers may be the primary toxic species. A recent paper in Proceedings of the National Academy of Sciences supports this hypothesis by showing that Aβ oligomers isolated from the brains of Alzheimer’s sufferers cause neuronal degradation and improper phosphorylation of tau (1). This paper is open access, so open it upand read along.
Jin et al. isolated dimers of Aβ from homogenates of human brains from Alzheimer’s patients. Dimers were separated from monomers and higher-order oligomers by size-exclusion chromatography in the presence of a strong detergent that typically breaks up folded proteins and repeating aggregates. This separates the dimers and higher oligomers from each other, and also dissociated weakly-interacting peptides (due to the effects of the detergent). As you can see from Fig. 1A, this produced fractions that contained either detergent-stable Aβ dimers (AD-TBS) or normal cortical proteins (cont-TBS) in identical solution conditions. They also created synthetic dimers by mutating Aβ to contain a cysteine that could form a covalent linkage between peptides (Aβ40S26C). They then used these various materials to treat primary cultures of neurons (that is, neurons that were obtained by directly harvesting them from an animal), with the dimers reaching a final concentration 0.5 nM in the growth medium.
Fig 1B establishes that, among the materials studied here, Aβ dimers are uniquely responsible for the appearance of tau “beads” along the neurites of the cultured cells after 3 days (the widespread dots in the final column of images). This effect is quantified in 1C, which shows that the dimer-containing fractions produced a dramatic increase in this clumping. According to the authors, these easily-visible clumps are only one symptom of widespread problems with the cells’ cytoskeletons. This sort of cytoskeletal trouble is expected because tau’s function is to stabilize and assist in the formation of microtubules from tubulin. The upshot of this figure is that continuous exposure to Aβ dimers (Fig 1D establishes that the dimers persist through the treatment period) appears to cause some sort of trouble with tau, which may reflect the incipient formation of the famous tangles.
The natural follow-up question is whether tau is necessary for this cytoskeletal derangement. The fact that the cultured neurons must mature, with a correlated increase in tau expression, for Aβ dimers to have an effect suggests that it must be. To check this, the authors used RNA inhibition to knock down tau levels. Fig 2A demonstrates that tau, but not tubulin, expression was altered using the tau-specific RNAi (but not the scrambled cont-RNAi). The cytoskeletal damage caused by both the natural dimer and the Aβ40S26C synthetic dimer were suppressed by tau-RNAi (Fig. 2B). At least at this timescale, it therefore appears that normal tau expression levels are necessary for this toxic effect of Aβ dimers. However, as tau in neurofibrillary tangles never breaks down, it seems like a longer exposure to Aβ under these conditions should produce similar toxic effects eventually.
The complementary experiment, is shown in Fig. 3, using a hybrid construct where human tau was fused to a fluorescent protein. As you can see from these images, under control conditions (columns labeled EGFP), cells treated with Aβ monomers and dimers have only subtle differences after two days, and beading is only evident after three days of treatment. When tau is overexpressed (columns labeled tau-EYFP), the cytoskeletal issues are obvious a day earlier. The tau-EYFP appears to be distributed in the same places as normal tau (fourth row), so the EYFP tag probably isn’t responsible for this effect, and the normal behavior of monomer-treated cells is reassuring. However, the EYFP tag may make tau more susceptible to some kind of dysregulation. Because this experiment both increases the total amount of tau and introduces the human protein, the reason for the enhanced susceptibility is difficult to determine. A control experiment in which rat tau-EYFP was expressed in the same construct would have been very helpful in clarifying this point.
As I mentioned above the formation of the tangles is associated with tau becoming highly phosphorylated. Jin et al. therefore made an effort to confirm that this was happening in their cultured cells, using antibodies that would recognize some specific sites in the tau protein that receive phosphate tags. Fig. 4 summarizes the results, indicating that human tau expressed in rat neurons becomes highly phosphorylated at serines 202, 205, and 262. For some reason, the endogenous rat tau did not become significantly phosphorylated at S262; this may have something to do with the apparently enhanced toxicity of Aβ dimers in the presence of human tau.
The paper’s final figure tests whether antibodies directed against specific sites in Aβ can prevent the observed cytoskeletal degradation. They found that two antibodies that bound to the N-terminus of Aβ significantly suppressed the effect of the dimers over the three-day timespan (fourth and fifth columns of A). However, an antibody directed towards the C-terminus of Aβ42 did not have much effect. Fig. 5C suggests that this is because this antibody simply didn’t bind to much of the Aβ in solution, either because most of the isoforms are shorter or because the C-terminus is protected in some way.
These results clearly link cytoskeletal disruptions caused by tau to the presence of soluble Aβ dimers, linking the two well-known pathological hallmarks of Alzheimer’s disease. That soluble oligomers, rather than fibrils, were responsible for the effect does not necessarily prove that the plaques aren’t important, for two reasons. The first is that, while these results clearly demonstrate dysregulation and aggregation of tau, true neurofibrillary tangles did not appear, and until we can assemble a full chain of events leading from beads to tangles the case, though strong, is still unproven. Secondly, as I’ve discussed previously, research has shown that the plaques can release soluble oligomers into the surrounding neural tissue and will therefore serve as reservoirs of toxic protein even if the fibrils themselves are completely inert.
That Aβ dimers can derange tau regulation in cultured neurons is not a new finding; similar results were reported last year using synthetic dimers (2). Zempel et al.‘s experiments used Aβ concentrations up to 5 μM, but Jin et al. show that naturally-obtained dimers have toxic effects at much, much lower concentrations. As the Zempel et al. paper suggests (consistent with much previous work), dysregulation of calcium levels caused by Aβ oligomers may be how they cause these effects. It is not presently clear why natural oligomers should be four orders of magnitude more potent than the various kinds of synthetic dimers at causing the effect; an understanding of this difference may be crucial in developing a suite of effective treatments for the disease.
1) Jin, M., Shepardson, N., Yang, T., Chen, G., Walsh, D., & Selkoe, D. (2011). Soluble amyloid β-protein dimers isolated from Alzheimer cortex directly induce Tau hyperphosphorylation and neuritic degeneration. Proceedings of the National Academy of Sciences DOI: 10.1073/pnas.1017033108 OPEN ACCESS
2) Zempel, H., Thies, E., Mandelkow, E., & Mandelkow, E. (2010). Aβ Oligomers Cause Localized Ca2+ Elevation, Missorting of Endogenous Tau into Dendrites, Tau Phosphorylation, and Destruction of Microtubules and Spines. Journal of Neuroscience, 30 (36), 11938-11950 DOI: 10.1523/JNEUROSCI.2357-10.2010