First, we conducted AFM imaging experiments of model membranes as we injected amyloid peptides in the system in order to investigate their action on membranes in real time. We characterized controlled model membranes mimicking the outer or inner side of the neuronal membrane for interaction with extracellular Aβ and intracellular tau, respectively. Our results showed that the amyloid / membrane interaction is sensitive to lipid composition and allowed us to precise the role of specific lipids. We contributed to demonstrate that ganglioside GM1 promotes amyloid / membrane interaction, while cholesterol does not. Yet, when both are present, a fast dissolution of membranes occurred, induced by oligomeric Aβ through a detergent effect, thus highlighting cooperation between cholesterol and GM1. This effect had not been observed with such high temporal resolution before. In the case of Tau, we explored the impact of lipid phase separation, as well as the roles of POPS and PIP2 lipids, which induce aggregation of the peptide in vitro. We report dissolution of the fluid phase of the membrane, and observe aggregation of the peptide when negatively charged lipids are present, with occasional fibrillar morphologies in the case of PIP2. These results would tend to confirm lipid recruitment in the process of tau aggregation.
In parallel, we initiated force spectroscopy experiments with functionalized AFM probes carrying a covalently-attached amyloid peptide, in order to directly probe the amyloid peptide / membrane interaction at the molecular level. We validated our functionalization approach using K18, a fragment of the Tau protein, as a model amyloid peptide, notably by comparing the interaction between a bare AFM probe and a functionalized one toward a specific antibody against Tau. This proof-of-concept of K18 functionalization holds potential for future amyloid molecular studies, and we have started probing model membranes, focusing on the negatively charged lipids identified as targets for the K18 / membrane interaction.
Finally, we worked on the development of an incubator-like chamber with controlled temperature and atmospheric content, with the goal of maintaining environmental conditions close to biological relevance and opening our research to live cell studies. After evaluating the geometrical constraints, we drafted plans using computer-assisted design and identified appropriate materials, as well as key components (Peltier heater). A prototype will soon be fabricated and tested.
The work performed in AMNEsIA has led to 1 published journal article, and two manuscripts in preparation. The results were presented at 2 international and 1 local conferences, as well as 2 public outreach events. So far, 1 article and 1 poster were deposited in open access repositories.
