Aggregated proteins lie at the heart of many neurodegenerative diseases, but researchers have struggled to pin down the precise conformations that trigger pathology. For amyloid-β (Aβ), the Alzheimer disease protein, it appears that small, structured oligomers are the evil players, but how they get their shape, and why they are so toxic, remain unsolved mysteries.
A pair of recent papers features in silico, in vitro, and in vivo approaches to address those questions. In one report, Christopher Dobson, Damian Crowther, David Lomas, and colleagues at the University of Cambridge in England provide evidence that the tendency of Aβ peptides to aggregate into small, oligomeric protofibrils, as predicted from primary amino acid sequence, predicts pathogenicity in a fly model of Alzheimer disease. The investigators calculated the propensity of different mutated forms of Aβ42 to form oligomers using a novel algorithm, and backed up their calculations by direct measurements in the test tube. The paper, out October 30 in PLoS Biology, raises the possibility that researchers might be able to assess the potential toxicity of any protein based on its amino acid sequence.
Looking at how oligomers come to be, an earlier publication from other Cambridge researchers Giorgio Favrin, Michele Vendruscolo, and again Dobson suggests that the generation of protofibrils occurs via a two-step process and is heavily dependent on the hydrophobicity of the starting peptide. These scientists used computer modeling to simulate the aggregation and folding of two different peptides derived from Aβ42. They found that the first stage of oligomerization occurs when peptides cluster through hydrophobic interactions. Then, the aggregated peptides reorganize into β-sheet-containing amyloid structures. This second step re-exposes hydrophobic residues to solvent, an event that may explain the toxicity of the oligomers, the authors speculate. The work was published in the September issue of PLoS Computational Biology.
In recent years, it has been recognized that the ability to aggregate and form amyloid fibrils does not depend on a given protein’s primary sequence, but may be a common property of proteins with widely varying amino acid sequence. Not all proteins form toxic oligomers, however, and it has been difficult to figure out precisely what determines toxicity in vivo. In the first paper, lead author Leila Luheshi and coworkers use an in-vivo measure of Aβ toxicity, the Drosophila model of AD. In this model, expression of Aβ42 peptide in the fly nervous system leads to early death and motor problems due to neuronal toxicity (Crowther et al., 2005). Using a computer algorithm to predict the propensity of peptides to aggregate based on their amino acid sequence (Chiti et al., 2003), the investigators assigned aggregation scores to all of the 798 possible point mutants of Aβ42. They then chose 15 of these with varying aggregation tendencies, expressed them in Drosophila, and assessed toxicity by changes in lifespan and motor activity.
From this analysis, the scientists found that the predicted tendency of peptides to aggregate correlated with shorter lifespan and greater decrements in motor ability. The correlation with aggregation was confirmed by measuring aggregation in vitro for five of the mutant peptides. For example, the F20E mutant had a low aggregation score and displayed minimal neurotoxicity. In vitro, the protein aggregated slowly compared to Aβ42, and in vivo, it formed no deposits in the fly brain under the same conditions where Aβ42 produced abundant accumulation. The researchers speculate that because the F20E peptide aggregates slowly, natural clearance mechanisms have a chance to rid the brain of it before it forms deposits.
Some peptides did not fit the pattern, however. One particular double mutation (I31E/E22G) stood out because its primary sequence predicted it would aggregate as avidly as the highly toxic E22G Aβ42, yet the double mutant was not toxic in the flies. In vitro, the two peptides aggregated at similar rates, and flies showed similar levels of deposition, suggesting that the computer model accurately predicted aggregation, but for some reason aggregation did not predict toxicity in this case.
This stimulated the researchers to look at another parameter, which was the tendency of peptides to form protofibrils. These small but highly organized oligomeric precursors to amyloid fibrils are the leading candidates for causing Aβ neurotoxicity (see ARF related news story and ARF news story). The investigators developed a new algorithm to calculate the tendency of mutant peptides to form protofibrils, based on experiments with other protofibril-forming peptides (Pawar et al., 2005). The scores from this calculation correlated even better with toxicity than the aggregation scores. In a separate experiment, the researchers report that analysis of the E22G multimers by electron microscopy revealed many protofibrils, while the I31E/E22G protein produced only fibrils, further strengthening the association between protofibrils and toxicity.
“These results provide compelling evidence that, despite the presence within the cell of multiple regulator mechanisms such as molecular chaperones and degradation systems, it is the intrinsic, sequence-dependent propensity of the Aβ42 peptide to form protofibril aggregates that is the primary determinant of its pathological behavior in living systems,” the authors write. Their fly system provides a way to measure the relationship between neuronal dysfunction in a complex organism and the fundamental factors that determine whether Aβ42 peptides will form protofibrils. The same principles may apply to other aggregation diseases as well, they conclude.
In the second paper, the Cambridge researchers take an in-silico approach to ask how protofibrils form, and whether the process reveals anything about their toxicity. Because of their amorphous and transient nature, the structure of early-stage aggregates of Aβ has been hard to determine. To get a virtual view of the process, first author Mookyung Cheon, who is also affiliated with the Pusan National University in Pusan, Korea, carried out a computer simulation of the folding and aggregation of two Aβ fragments, covering residues 16-22 and 25-35.
According to the simulations, the formation of oligomers followed a two-step pathway that depended on the degree of hydrophobicity of the peptide. The first step, which Cheon readily detected with the more hydrophobic 16-22 fragment, involved the aggregation of peptides into large disordered molten oligomers, driven by hydrophobic interactions. Then, over time, these amorphous blobs with greasy centers reorganized into β-sheet structures by forming interchain hydrogen bonds. If a peptide is not very hydrophobic, it may bypass the first step, as the authors observed with the 25-35 peptide, which proceeded straight to the hydrogen-bond interactions.
As a net result of the formation of the β-sheet, hydrophobic residues become exposed to the solvent. Therein could lie the toxicity of oligomers, the authors speculate, as the sticky protofibrils might interact with any number of proteins in cells. Further, Cheon and colleagues found that the extent of hydrophobic surface exposed depends on the size of Aβ(17-21) oligomers. Because smaller assemblies have weaker total hydrophobic interactions, they expose a larger fraction of their hydrophobic residues. As the oligomers grow larger, the number of exposed hydrophobic residues decreases because of decreasing surface-to-volume ratio and a reduction in the total number of oligomers. This model leads to the prediction that there will be an optimal aggregate size where toxicity is greatest, a situation borne out by experimental results (see ARF related news story and Baglioni et al., 2006).
This scenario of oligomer formation pits the tendency of peptides to make hydrophobic interactions against the propensity to form ordered, hydrogen-bonded structures. In this competition, the hydrophobicity of the peptide sequence balances with the generic, sequence-independent tendency to form interchain hydrogen bonds. It must be noted that the simulations were carried out with Aβ fragments, and thus may not reflect the behavior of the entire protein. However, the fragments used are known to be important to lend Aβ its toxicity. It remains to be seen if the process outlined with these small peptides might supply a common mechanism for the toxicity of oligomers derived from a variety of proteins, as occurs in other neurodegenerative diseases.—Pat McCaffrey