Targeting oligomer dynamics provides hope for new Alzheimer’s therapies

Researchers have known for some time that the aggregation of proteins into amyloid fibrils and plaques are the factors that lay behind neurodegenerative disorders such as Alzheimer’s disease, Parkinson’s disease, Huntington’s disease and amyotrophic lateral sclerosis (motor neuron disease).

More recently scientists have become aware that specific types of aggregates known as oligomers, which are small and soluble, play a major role in cell and tissue toxicity in these diseases.

“Oligomers are key toxic species in many of the neurodegenerative disorders,” said Professor Tuomas Knowles, co-director of the Department’s Centre for Misfolding Diseases.  “They trigger the aberrant biology that leads to the disease.”

And it has become increasingly clear that these oligomers can aggregate further into amyloid fibrils, leading to the tell-tale protein deposits in the brains of disease sufferers. However, until now the fundamental molecular pathways that control oligomer formation during this amyloid aggregation cascade have remained unclear. “It has been very challenging to study oligomeric dynamics because these assemblies are so transient and dynamic,” said Knowles.

But in a paper published in Nature Chemistry today, Knowles and his colleagues in the Centre for Misfolding diseases and Lund University in Sweden, reveal how they developed and applied a powerful chemical kinetic framework to measure these oligomer reactions for the first time, thus opening the way for new therapeutic interventions to combat protein misfolding diseases.

“Before this, we knew the concentration of the monomers and the final aggregates, which are pretty benign,” said co-author Professor Sara Linse from Lund University.  “But this research is important because what we have been able to do here is measure the concentration of the most toxic species for the first time.”

The researchers combined theory, experiment and simulation to reveal in molecular detail the dynamics of oligomers during amyloid fibril formation.

“One aspect of this work I’m personally very excited about is the interdisciplinarity, and how exciting it is to see people from different scientific backgrounds combining their different strengths to understand something really fundamental and important,” said first author Dr Thomas Michaels, a Junior Research Fellow at Peterhouse and researcher in the Centre for Misfolding Diseases.

“By combining our mathematical modelling with the ground-breaking experiments designed by Sara Linse, we have been able to develop a powerful chemical kinetic model,” he said.

Linse’s group has pioneered the experiments to measure the oligomers as they aggregate, which is something that researchers have not previously been able to do. 

Working closely with colleague Dr Katja Bernfur, she obtained reproducible and quantitative measurements of the oligomer concentrations which formed during aggregation reactions.  Centrifuges were used to remove the fibrils, and they employed techniques such as size-exclusion chromatography and liquid scintillation counting to identify the resulting oligomer fractions without disturbing their aggregation behaviour.  

Researchers in the Centre for Misfolding Diseases then compared a series of mechanistic scenarios with these direct measurements, followed by mathematical analysis, which enabled them to develop a detailed understanding of how the oligomers were interacting with monomers, fibrils and aggregates.

To provide a structural interpretation of their results, Dr Andela Šarić at UCL performed computer simulations, which enabled them to shed light on some of the key parameters determining the mechanism, such as oligomer formation times, while retaining molecular-level resolution.

 “Using this framework, we were able to respond to questions about what happens to the oligomers once they are formed,” said Michaels. “These are questions to which previously no one knew the answer.”

The researchers discovered that even though all mature amyloid fibrils originate as oligomers, the oligomers themselves are structurally distinct from the fibrillar aggregates.

This implies that at least some oligomers must undergo a structural conversion to become these faster, elongating fibrils.

They also found that most oligomers do not form fibrils, but instead dissociate back into nontoxic monomers. “Even though oligomers are the key source of fibrils, we found that less than 10% of oligomers successfully converted into fibrillar species, whereas the remaining 90% of the oligomers dissociated back to monomers,” noted Michaels.

This means the initial oligomer formation step is followed by a large barrier for oligomer conversion. “We now know that only a few of the oligomers form fibrils. All of the fibrils start as oligomers, but not all oligomers go on to form fibrils,” added Knowles.   

They concluded from the data that there is a two-step mechanism that involves oligomers as a necessary intermediate step in the formation of fibrils. In this two-step nucleation process, there is a competing process of oligomers converting either to fibrils or dissociating back to monomers.

 “After discovering that the secondary nucleation mechanism is the main driver of fibril formation,” said Michaels “it has now become clear that the oligomers need to undergo a structural reorganisation step while in contact with fibril surfaces. 

“It’s similar to water droplets forming on a shower door – having the surface of the door makes it much more favourable as a place for the droplets to form. The oligomers use the fibril surface to form.“

As more fibrils are formed, this creates more fibril surfaces on which fibrillar oligomers can be formed, in an exponential process. In a nod to previous research they concluded that the fibril surfaces serve as an oligomer “breeding factory.” 

The oligomers produced via this secondary nucleation persist for a significant amount of time in solution before they convert into fibrils, and both oligomer formation and conversion are slow steps in the reaction.  Most of the oligomers dissociate back to monomers, and multiple oligomers typically form and dissociate before one successful conversion event into a fibril occurs.  “Because of their low stability the oligomers dissolve but the monomers keep forming into oligomers, so there is constant recycling,” said Linse.

These results mean that scientists can now break down the cycle of fibril self-replication into a series of elementary steps, and quantify the importance of each step. “Using this chemical kinetics model, we can now predict concentrations of the oligomers when the model parameters are varied, and then compare them with direct measurements to test our theory,” said Michaels.  

“This is very exciting because there are new steps we didn’t know existed before, and so using this framework we can systematically screen different compounds and find the ones that are most effective, and also target different stages of the process.

The discovery that the majority of oligomers dissolve before they can convert into amyloid fibril precursors has revealed an unexpected vulnerability in the protein aggregation process. “It is a vulnerability that we are now very keen to exploit with new drugs that target it,” added Professor Michele Vendruscolo, co-director of the Centre for Misfolding Diseases.

"Chemical kinetics is a gold standard tool to discover and test molecular mechanisms,” said Knowles. “Now by using this powerful framework to focus on oligomeric species, we have been able to show that it is equally important in studying amyloid aggregation.”

Linse said that the team now plans to look at inhibitors of oligomer formation, and quantify how different inhibitors affect the oligomers.

 “The obvious next step is going to be to use this framework to better understand how we could design therapies,” agreed Michaels. “There is already a very strong drug discovery programme in Cambridge which relies on this iteration between experiments and theories,” he said.  “What I love most is the fact that we have been able to use mathematical tools to solve a very clear problem in the fight against Alzheimer’s.”