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Molecular mechanism of amyloid β aggregation

Final Report Summary - MAMBA (Molecular mechanism of amyloid β aggregation)

The ultimate goal of this project was to understand the molecular events leading to the formation of amyloid fibrils and neuro-toxic species in Alzheimer’s disease, and to quantify how the rates of these events are modulated by extrinsic (solution conditions) and intrinsic (sequence) factors. We have established the experimental framework needed to approach this question from a physico-chemical viewpoint staring with ultra-pure peptide under well-controlled conditions. This has led to an understanding of the molecular mechanism of Amyloid beta peptide aggregation in terms of the underlying microscopic (composite) steps. Importantly, the same steps are prevalent under all conditions and for all sequences examined, but the way the rates are modulated by the conditions differ between the steps. The steps are primary nucleation of monomers in solution, elongation of fibrils by monomer addition, and secondary nucleation of monomers on the fibril surface. The differential modulation means that the dominance of secondary nucleation, the step responsible for generation of the majority of toxic species, may vary between conditions and sequences. For example, we find that the dominance of secondary nucleation is enhanced for several of the disease-associated Amyloid beta peptide variants. Our results a basis for a new approach of kinetics-guided development of therapeutic leads.
Secondary nucleation dominates generation of new aggregates for all variants of Aβ42 examined this far. Secondary nucleation is in many cases a multi-step process. N-terminal extension retard all microscopic steps through a general polymer effect. C-terminal truncation (Aβ40 vs Aβ42) reduces the rate of all microscopic steps, but most significantly primary nucleation. Familial mutants of Aβ42 increase the rate of secondary nucleation, and thereby increase the generation of toxic species.
We have established measure the kinetic barriers of primary nucleation, secondary nucleation and elongation. We found a distinct thermodynamic signature of secondary nucleation. This process has much weaker temperature dependence than the other processes.
Aβ40 and Aβ42 form separate fibrils in binary monomer mixtures. The two peptide cross-react at the level of primary nucleation but not in elongation or secondary nucleation.
We have developed methodology to understand chaperone action down to the level of which microscopic steps they interfere with. We have found that the chaperone domain Brichos inhibits secondary nucleation specifically and significantly reduces oligomer production and the toxicity associated with Aβ42 aggregation. The chaperone DNAJB6 inhibits primary nucleation. We developed a phage display strategy to screen libraries of single-chain antibodies for fibril-specific antibodies which serve as specific inhibitors of secondary nucleation.
We have produced lare quantities of several isotope-enriched (13C,15N) batches of Aβ42 and Aβ40 established a high-resolution structure of Aβ42 fibrils. The high-resolution structure of Aβ40 fibrils is under way.
We have investigated which cellular proteins interact with the products of secondary nucleation, and in an un-biased experiment we found a single hit among ca. 10,000 examined proteins in the form of a kinase that phosphorylates tau, another amyloid-forming protein associated with Alzheimer’s disease. On-pathway oligomers of Aβ(M1-42) are thus found to bind to glycogen synthase kinase 3 (isoforms α and β, previously known as tau kinase-1) and up-regulates its phosphorylation of tau.
We have used protein array screening of interaction partners of on-pathway oligomers formed duing Aβ(M1-42) aggregation, and identified one target as glycogen synthase kinase 3 (isoforms α and β, previously known as tau kinase-1). We found that Aβ42 up-regulates the kinase in its phosphorylation of tau.
We have used dSTORM and cryo-electron microscopy to get high-resolution images of secondary nucleation events along the sides of fibrils.
The aggregation mechanism of alpha-synuclein is modulated by pH. Secondary nucleation occurs at mildly acidic pH. alpha-synuclein aggregation is accelerated by exosomes and membranes with gangliosides and other negatively charged lipids.
We have developed custom-made instruments for liquid handling and simultaneous measurement of several readouts during protein aggregation.