Apr 122011
 

ResearchBlogging.orgThe 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

Feb 262010
 
ResearchBlogging.orgSpongiform encephalopathies are transmissible diseases that can have a major economic impact on agricultural exports, and pose a significant challenge for surveillance of the food supply. Scientists generally believe that these diseases are transmitted via a self-propagating, aberrant conformation of the prion protein (PrP). This prion hypothesis suggests that PrP alone should be sufficient to cause symptoms or death. If this hypothesis is true, then it should be possible to reproduce the disease using recombinant proteins expressed in yeast or bacteria. In tomorrow’s Science, researchers from Columbus and Shanghai report that they have managed to do this, establishing that PrP alone can account for prion disease transmission.

Previously, other groups had successfully produced recombinant PrP (recPrP) and generated amyloid fibrils that appeared to contain the pathogenic conformation (PrPSc). When injected into mice, however, these amyloids had limited infectivity, which raised doubts as to whether these fibers are the cause of the disease. Wang et al. decided to take a different route, using a technique known as protein misfolding cyclic amplification (PMCA). In this approach, misfolded aggregates of a protein are broken up using sound waves and then incubated with normal, folded protein. If the misfolded protein can cause normal protein to adopt an aberrant conformation (as PrPSc can), then the misfolded protein will be amplified. By performing many cycles of this experiment, one can in principle produce a very large amount of PrPSc from a single misfolded chain.

Wang et al. also added some ingredients to their reactions that they believed would promote prion formation: RNA and a lipid called POPG. Under these conditions, they detected a protease-resistant protein after 17 rounds of PMCA amplification. Under normal circumstances, PrP is cleaved by the protease, like a pair of scissors cutting a string. If PrP has aggregated, however, it becomes much more difficult to cut, as if you were using safety scissors to cut a thick hemp rope. The researchers discovered that the protein from this reaction (which they called rPrP-res) could cause normal protein to also become protease resistant. And, when they treated protein from mouse brains with rPrP-res, they found that it, too, formed protease-resistant aggregates.

To really put the hypothesis to the test, however, tests in live animals were needed. The researchers injected one group of animals with the product of a PMCA seeded with rPrP-res. They also injected three additional groups of animals with control cocktails to prove that neither the non-protein ingredients, nor the unprocessed protein, caused the disease. None of the control animals developed symptoms of encephalopathy during the experience, but all of the mice injected with the PMCA product died in about five months, while displaying clinical signs of prion disease. Their brains were inspected post-mortem using histological and molecular means. The brains clearly showed the formation of the vacuoles that give spongiform encephalopathy its name. Additionally, protease-resistant PrP was detected in homogenates of the brain tissue, indicating that the rPrP-res had propagated in the living mouse brains, causing disease and eventual death. When these homogenates were injected into the brains of other healthy mice, a similar pattern of pathology recurred. This proved that the effects of rPrP-res could be serially propagated, just as prion disease is.

These results leave little room for doubt that misfolded PrP is sufficient to cause prion disease; no other infectious agent was required. The effectiveness of RNA and POPG in promoting the pathogenic conformation may indicate that these or similar molecules play a role in the spontaneous development of prion disease. Aside from adding to our knowledge directly, this research has the potential to significantly increase our ability to investigate prion disease. The ability to produce bona fide infective prion molecules in vitro from recombinant protein opens up new avenues for experiments in structural biology and biochemistry that may enable us to cure or entirely prevent these diseases, rather than just trying to contain them.

Wang, F., Wang, X., Yuan, C., & Ma, J. (2010). Generating a Prion with Bacterially Expressed Recombinant Prion Protein Science, 327 (5969), 1132-1135 DOI: 10.1126/science.1183748

Sep 022009
 
ResearchBlogging.orgOne of the many ways that a living cell does not resemble a test tube is in the degree to which its internal environment is crowded. Cells are crammed full of massive protein complexes, vesicles, organelles, carbohydrates, peptidoglycan, and other assemblies that occupy a great deal of space. The tubes and cuvettes used for biochemical experiments, in contrast, typically contain nothing more than a few proteins and small molecules of interest along with a relatively dilute set of salts and buffering agents. Because many complexes appear to be stabilized by crowding, and excluded volume effects are known to favor compact, folded protein chains, there is considerable interest in estimating how protein behavior changes in the spatially-restricted environment of the cell. In some cases this is approached using NMR to study changes in dynamics and structure inside cells. In a recent issue of Biophysical Journal, groups from Florida State University and Rehovot University, in separate papers, address how crowding affects protein-protein interactions.

Both groups perform biochemical experiments in vitro to examine this question. In order to crowd the solution, they add reagents such as ficoll, polyethylene glycol (PEG), and dextran, and compare the changes in binding and kinetics to solutions that have merely been made more viscous (through the addition of glucose, for instance). Batra et al. study the association of two components of the E. coli DNA polymerase III and find that the presence of crowding agents slightly stabilizes the complex. However, as the size of the crowders is increased, this stabilization is diminished. Batra et al. develop a relatively simple mathematical model that suggests this observation results from the fact that larger particles pack less efficiently, leaving larger “holes” in which the protein complex sees something more like dilute solution.

Phillip et al. study several protein complexes. Similar to Batra’s group, they find that crowding with dextran modestly increases the binding affinity of two of their protein pairs, but that this is not replicated for crowding with PEG. Particularly for PEG-1000, there was a clear decrease in affinity, although a non-crowding viscogen (ethylene glycol) had an even greater effect. Phillip et al. also measured the kinetics of binding, and found that the association rates were significantly lower in crowded solutions, as compared to buffer. However, when the rates were corrected for the effect of viscosity, it appeared that the crowding agents slightly increased the association rate. The authors attribute this to excluded volume effects in the binding transition state. The dissociation rate was also slightly reduced in crowded solutions, which the authors explain by the longer lifetime of the encounter complex (allowing a larger fraction of complexes to fall back to the lower-energy bound state).

Given that crowding appears to have a profound effect on the function of certain complexes, the relatively small effects observed in these studies might seem confusing. Batra et al. argue that although each individual binding interaction is only modestly stabilized, the effect should be cumulative. As a result, multi-subunit complexes will experience a greater effect than small heterodimers. Additionally, the most famous examples of crowding enhancement involve very large complexes — the ribosome, decameric assemblies, hemoglobin polymers, etc. In comparison, the complexes formed in these model studies are quite small. It may be that the stabilizing effect of crowding depends to some degree on the size of the complex to be formed. While similar size is difficult to achieve in strictly heterodimeric systems, it should be possible to monitor the assembly of large complexes like GroES/GroEL under crowded conditions. A study of the relationship between the size or number of components in a complex and crowding stabilization may prove instructive.

Batra, J., Xu, K., Qin, S., & Zhou, H. (2009). Effect of Macromolecular Crowding on Protein Binding Stability: Modest Stabilization and Significant Biological Consequences Biophysical Journal, 97 (3), 906-911 DOI: 10.1016/j.bpj.2009.05.032

Phillip, Y., Sherman, E., Haran, G., & Schreiber, G. (2009). Common Crowding Agents Have Only a Small Effect on Protein-Protein Interactions Biophysical Journal, 97 (3), 875-885 DOI: 10.1016/j.bpj.2009.05.026

Mar 172009
 
ResearchBlogging.orgThe observation of plaques composed primarily of amyloid-β (Aβ) peptides in the brains of Alzheimer’s patients long ago gave rise to a hypothesis that Aβ was the agent that caused the disease. The plaques themselves, composed of long, insoluble fibrils of Aβ, were believed to cause the synapse loss and nerve death characteristic of the disease, and some data supports this model. However, several experiments have suggested an alternative possibility: that the symptoms of Alzheimer’s may be attributed to soluble Aβ oligomers. In this view the fibrillar deposits may be an incidental feature of Alzheimer’s disease, or even a defense mechanism whereby the body tries to get rid of the oligomers by forcing them into insoluble aggregates. In the March 10 edition of PNAS, a team led by researchers at Massachusetts General Hospital claim to have reconciled these two models. Using fluorescence microscopy, they find that amyloid plaques are surrounded by a “halo” of Aβ oligomers that kill the surrounding synapses.

The authors of this studied used fluorescence labeling to identify plaques, oligomers, and synapses in thinly-sliced tissue sections and living brains. They performed their experiments in mice that had been genetically manipulated so as to develop amyloid plaques. When they examined the brains of live mice, Koffie et al. noticed that the fibrillar plaques were surrounded by a cloud of the oligomers, as you can see for yourself in the figure below. On the left you can see the plaque core labeled by a fluorescent dye, and the middle image shows fluorescence associated with an antibody that specifically binds to amyloid oligomers. When these images are merged, the diffuse “halo” of oligomers becomes obvious. The authors see a similar result when they perform a similar experiment using thin slices of brains.

The authors also used a fluorescent-conjugated antibody to identify elements of the post-synaptic density (PSD), so that they can identify healthy synapses in the brain. Experiments in tissue sections demonstrated that the number of healthy synapses was reduced not only right next to the plaque, but also in a region extending up to 50 µm away (a length comparable to the diameter of a human hair). Aβ oligomers are also enriched in this region, and the relative concentration of the oligomer roughly correlates with the loss of synapses. By comparing the pattern of Aβ fluorescence to that of the PSD, the authors determined that oligomers were associated with many synapses, and that interactions between PSD and Aβ oligomers resulted in decreased synapse size. The relationship between Aβ binding and reduced synapse size was also shown to hold in control mice expressing normal levels of native amyloid precursor protein.

The observation that the presence of Aβ oligomers correlates with synapse loss, and the apparent degradation of synapses by Aβ, indicates that the soluble oligomers are a significant cause of Alzheimer’s symptoms, although this study does not rule out the possibility that the plaque itself is also toxic. Even if the plaques have no immediate toxic effect, the authors propose that they serve as reservoirs, releasing synaptotoxic Aβ oligomers into the surrounding neural tissue, increasing the size of the lesions beyond the extent of the plaque itself. In this way Koffie et al. believe they have reconciled the previous models — oligomers are directly toxic, plaques release toxic oligomers, so both can serve as causative agents in Alzheimer’s disease.

If this model is accurate, it implies that Alzheimer’s disease may be quite resilient to attack. Antibodies or drugs that break up the Aβ oligomers will be effective in mitigating the synaptic damage, but as long as the plaques persist they will continue to replenish the pool of oligomers. Treatments that successfully break up the plaques will probably result in worsening symptoms due to the release of toxic oligomers as the fibrils disintegrate. These possibilities reinforce the idea that the most treatment for Alzheimer’s will involve reducing the concentration of amyloidogenic Aβ peptides to prevent them from forming plaques in the first place.

(1) Koffie, R., Meyer-Luehmann, M., Hashimoto, T., Adams, K., Mielke, M., Garcia-Alloza, M., Micheva, K., Smith, S., Kim, M., Lee, V., Hyman, B., & Spires-Jones, T. (2009). Oligomeric amyloid associates with postsynaptic densities and correlates with excitatory synapse loss near senile plaques Proceedings of the National Academy of Sciences, 106 (10), 4012-4017 DOI: 10.1073/pnas.0811698106