Understanding Alzheimer’s Disease through Biophysics Research

September 21 is World Alzheimer’s Day. Alzheimer’s disease is the leading cause of dementia, from which 47 million people worldwide suffer. It affects memory, thinking, orientation, comprehension, calculation, learning capacity, language, and judgement. 

In recognition of World Alzheimer’s Day, we spoke with two Biophysical Society members whose research aims to improve understanding of the mechanisms behind Alzheimer’s and other neurodegenerative diseases.


Liz Rhoades, University of Pennsylvania

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What is the connection between your research and Alzheimer’s disease?

We study the protein tau, which is a microtubule associated protein.  In Alzheimer’s and other neurodegenerative disorders, tau forms fibrillar aggregates that are deposited in brain tissue.  There are six isoforms of tau found in adult humans and alteration in the amounts of the isoforms are linked to disease development. Interactions of tau with microtubules are normally regulated by phosphorylation and tau aggregates derived from patient tissues are hyperphosphorylated, providing a link between loss of native tau function and disease as well.

Why is your research important to those concerned about Alzheimer’s?

We are working to understand basic aspects of tau function because we of the insight it provides to loss of function in disease.  Despite rather intensive study, molecular details of tau function are still lacking.  This is at least in part due to the fact that tau is large intrinsically disordered protein and thus it is challenging to characterize its structural features, particularly when associated with tubulin (the soluble building blocks of microtubules) and microtubules. For example, a few years ago, we observed that tau binds to soluble tubulin, a feature that had not previously received much attention. Our results suggests that it binds with similar affinity to tubulin as it does to microtubules which suggests that understanding how mutation impacts its interactions with tubulin is as important as characterizing how It interacts with microtubules. This is important because therapeutic strategies may very well involve targeting interactions between tau and its functional binding partners – we need to know who those partners are and the relevant features of the interaction!

Rhoades

The image here is from a paper that was published in PNAS last fall. It shows tau binding to two soluble tubulin dimers, and is based on our single molecule FRET measurements. It highlights how tau retains a primarily disordered state while binding and initiating tubulin polymerization. 

How did you get into this area of research?

We had been looking at tau aggregation in the lab for a couple of years, and then I had a few students – two graduate students and an undergraduate –  who were very interested in working with tubulin.  They were they ones who really pushed to get things up and running.  Anyone who has ever done a tubulin purification in their lab knows that this is not a trivial undertaking!

How long have you been working on it?

6 or 7 years

Do you receive public funding for this work? If so, from what agency?

In the past we received funding from NSF (MCB)  and currently we receive funding from NIH (NIA).

Have you had any surprise findings thus far?

I think most findings I get excited about are surprises, but there are three that I can think of as particularly surprising.  The first was that tau binding to soluble tubulin had been largely overlooked in previous studies. We started working with soluble tubulin because it was easier for us to use in single molecule experiments (one of our primary tools) and only as we began to get interested results, do we recognize that there really was not a deep literature on tau-tubulin. The second was the tau point mutants linked to different neurodegenerative disorders bound more tightly to tubulin than the wild-type tau.  Our expectation based on the tau-microtubule literature was the mutation should decrease the binding affinity.  We are still working to understand the impact of this on tau function.  The third is that a region which flank the microtubule binding region has a high affinity for tubulin and allows for tau to bind to multiple tubulin dimers simultaneous to form a ‘fuzzy complex’.  This work was published in Biophysical Journal this summer.

What is particularly interesting about the work from the perspective of other researchers?

I think some of it is probably methodology – we are using single molecule FRET and FCS to investigate tau-tubulin and working to make useful measurements in relatively complex, heterogeneous systems.

What is particularly interesting about the work from the perspective of the public?

Understanding tau’s interactions with native binding partners may provide new targets for therapeutics.  I think anyone who has a family member or loved one who suffers from Alzheimer’s or another neurodegenerative disorder would find that interesting.


Dieter Willbold, Heinrich-Heine-Universität Düsseldorf and Forschungszentrum Jülich

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What is the connection between your research and Alzheimer’s disease?

My interest is focused on three-dimensional structures and dynamics of medically relevant proteins at atomic resolution and their interactions with native and artificial ligands. Autophagy and neurodegenerative diseases, which by the way do have a clear connection with each other, fall within my main interest areas. I want to understand protein aggregation in time and space at high resolution. And, I want to develop strategies and compounds that allow intervention and prevention. And the protein I am working on now for a very long time as a researcher is the Alzheimer’s disease (AD) related amyloid-beta protein (Aβ).

Why is your research important to those concerned about Alzheimer’s?

We do a lot of in-depth basic science on aggregation of Aβ, tau, alpha-synuclein, and many other proteins. We also design compounds for novel disease intervention strategies, and develop novel biomarkers and assays to measure them appropriately and most sensitively. All three, basic science, drug design and biomarker development, are based on biophysical principles and physico-chemical “thinking” and heavily rely on respective methods, such as NMR, x-ray crystallography, cryo-electron microscopy, ultracentrifugation, surface plasmon resonance, micro-calorimetry, TIRF microscopy, AFM, micro-thermophoresis, biolayer interference, and all kinds of spectroscopies.

Willbold

Figure 1: Cross section through the Aβ fibril illustrating the stepwise overlapping arrangement of the Aβ proteins. (Copyright: Forschungszentrum Jülich / HHU Düsseldorf / Gunnar Schröder). See also: http://www.fz-juelich.de/SharedDocs/Pressemitteilungen/UK/EN/2017/17-09-08-alzheimer-fibrillen.html .

How did you get into this area of research?

Already during my PhD project, which was mainly on the 3D structure determination of the transactivator protein (Tat) of the equine homologue of the HIV virus, I was engaged in structural studies of the amyloid-beta protein (Aβ) by NMR spectroscopy with some of the results published in 1995 with Paul Rösch being my supervisor and mentor. Ever since then, I was thinking of potential therapeutic intervention strategies. Since 1999, when I was heading my own junior research group in Jena, I had the necessary resources to at least start research on intervention strategies.

Soon after, I became involved in projects on prion diseases and prion protein (PrP) aggregation, when I accepted my first professorship at the Heinrich Heine University Düsseldorf in very close collaboration with Detlev Riesner. The common themes in these protein misfolding or protein aggregation diseases became quite clear. In my view, any intervention strategy – rather than a prevention strategy – needed to target toxic aggregates and get rid of them, rather than to reduce the formation of the monomeric species. As a biophysicist, I thought it would be a good idea to shift equilibria between monomers and aggregates away from the toxic aggregates (or oligomers as they are called today). To do this, we looked for compounds that bind to Aβ monomers with the free binding energy being used to lower the free energy level of monomers thus shifting the thermodynamic equilibrium towards Aβ monomers. The wording we use nowadays is that such a compound stabilizes Aβ in an aggregation-incompetent conformation. Because this is also happening with Aβ monomer units within Aβ oligomers, such a compound is also able to damage and destroy already pre-formed Aβ oligomers leading ultimately to their elimination. To identify a useful lead compound, we used mirror image phage display selection, a tool that allows selection of a compound from huge peptide libraries, but yielding a fully D-enantiomeric peptide, that does not have the disadvantages of normal L-peptides, which are very easily degraded and immunogenic. Our lead compound with the name “D3” (D-peptide from the third selection trial) showed really nice properties in vitro and in vivo. When we then wanted to elucidate the mechanism of action, it was essential to establish a whole zoo of methods and assays, which brought me even deeper into the field of protein aggregation in general and Alzheimer’s in particular. I just wanted to elucidate and pinpoint the mechanism of action and to reveal structural details of any interaction of Aβ with itself and with ligands.

Do you receive public funding for this work? If so, from what agency?

Yes, now we do. In the beginning, I was not successful at securing additional money from funding agencies, e.g., the DFG. The project received great review reports about the underlying idea, but the panels ultimately decided that the project was too risky. Therefore, I used most of the institutional resources (which was not much in those years) for the project. Only in 2007, the Volkswagen-Stiftung funded a side project. Since 2013, I did and still do receive significant funding from the Helmholtz-Gemeinschaft, the federal ministry BMBF, the EU, and also from the Michael J Fox Foundation, the Weston Brain Institute, Alzheimer’s Research UK, as well as the Alzheimer’s Association. We have also been part of a JPND network.

Have you had any surprise findings thus far?

Yes indeed — many! Just to describe some: our lead compound D3 worked effectively not only in vitro, but also in several animal models. This has been a successful long-term collaboration with my dear colleagues Thomas van Groen, Inga Kadish and Antje Willuweit. D3 improved cognition in several models and decelerated neurodegeneration in an additional animal model that we have received from my dear collaborator Uli Demuth. We established an assay called QIAD that allows us to quantify Aβ oligomer elimination efficiency (https://www.ncbi.nlm.nih.gov/pubmed/26394756). We found that D3 efficiently eliminates Aβ oligomers, but many compounds that have already been in the clinics and failed are not able to do this. By following aggregation of N-terminally truncated and pyro-glutamate-modified Aβ (pEAβ) by NMR and CD spectroscopy, we found intermediates with helical secondary structure during aggregation.

When we tried to follow Aβ aggregation by SANS and analytical ultracentrifugation (AUC), we did not find any intermediates between monomers and penta- or hexamers (https://www.ncbi.nlm.nih.gov/pubmed/28559586). Thus, Aβ seed formation may be a reaction of very high order. In our recent research, (7th Sep 2017, https://www.ncbi.nlm.nih.gov/pubmed/28882996) we published a high resolution cryo-EM structure of Aβ fibrils. This structure provided many surprising findings in one hit including: all 42 residues of Aβ(1-42) are part of the fibril structure, there is no C2 symmetry between the two proto-filaments of the fibril, both ends of the fibril are different, each Aβ monomer subunit contacts many other subunits and six Aβ monomers form the minimal fibril unit. See also the respective report in alzforum.org: http://www.alzforum.org/news/research-news/amyloid-v-fibril-structure-bares-all .

What is particularly interesting about the work from the perspective of the public?

I think that from the perspective of the public, two questions are relevant and interesting: Can we visualize highly complex things? Especially as structural biologists, we indeed can. Just look at the beautiful picture of the Aβ fibril at atomic resolution below. The second question is, of course: Can we contribute to efforts for improving the quality of life, for example by developing therapeutic strategies and drug candidates? Yes, we can also do (or at least try to do) this. It is, however, a huge undertaking that needs substantial funding and teamwork with many experts and specialists that you would not contact for basic science.

Just today (18th Sep 2017), we founded a company named Priavoid that will take an optimized derivative of D3 into clinical studies and hopefully to the market someday. In parallel, because Aβ oligomer elimination is our most favored mechanism of action, we have developed a technology called sFIDA (surface-based fluorescence intensity distribution analysis), which is able to quantify Aβ oligomers in body liquids like CSF and blood at single particle sensitivity (https://www.ncbi.nlm.nih.gov/pubmed/27823959). The development of this technology was and is important to ultimately show target engagement of our Aβ oligomer eliminating compounds. sFIDA will also be useful for early diagnosis of any protein misfolding disease, to recruit the “right” patients for clinical studies and to follow treatment success, if there is one. Thus, all in all, I think we have developed interesting results for the public domain.

Is there anything else you would like to add?

During the initial stages of the above described project to develop a novel therapeutic strategy for AD and to identify suitable compounds, there was only myself and my PhD student, Katja Wiesehan. Currently, there are many colleagues and coworkers that are the most experienced experts in their fields. It is such a beautiful experience to work and think with all of them and all the junior researchers, and to finally get things done. Please, have look at them and their groups in Düsseldorf (http://www.ipb.hhu.de/en.html) and Jülich (http://www.fz-juelich.de/ics/ics-6/EN/) with outstations in Grenoble and Hamburg. Finally, I shall not forget the many, many collaborators, of which I named only a few above.

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