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Interactions of amyloid peptides with the neuronal membrane interface: molecular mechanisms involved in Alzheimer’s disease

Periodic Reporting for period 1 - AMNEsIA (Interactions of amyloid peptides with the neuronal membrane interface: molecular mechanisms involved in Alzheimer’s disease)

Período documentado: 2018-09-01 hasta 2020-08-31

Alzheimer’s disease (AD) is a common neurodegenerative disease leading to loss of cognitive functions and dementia. With an aging population, Europe alone counts over 7 million people suffering from AD, making it a critical public health issue. AD is characterized by the presence of 2 types of amyloid peptides: Aβ, found in extracellular plaques, and Tau, found in intracellular aggregates. During the development of the disease, both self-aggregating proteins are involved in a complex cascade of events leading to neuronal dysfunction and death. The neuronal membrane has been identified as a target of amyloid-induced toxicity, yet the mechanisms involved remain elusive and require investigation at the local scale.

The general objective of the AMNEsIA project was to unravel the deleterious effects of AD-associated amyloid peptides on the neuronal membrane in order to better understand the underlying mechanisms of AD. To that end, we used Atomic Force Microscopy (AFM), a powerful biophysical technique that provided high-resolution information on the amyloid morphology, as well as the amyloid-induced structural changes on the neuronal membrane. We also addressed the amyloid / membrane interaction on a molecular level and investigated the mechanism of interaction.

We exploited the high temporal resolution of AFM imaging, in collaboration with the Laboratory of Research in Nanosciences (University of Reims, France), to assess the effects of an oligomeric toxic variant of Aβ on model membranes of controlled lipid compositions mimicking the external neuronal membrane. Our results revealed a strong impact of the lipid composition and allowed us to propose a mechanism for the oligomeric Aβ / membrane interaction. The simultaneous presence of cholesterol and ganglioside GM1 was required in the membrane to observe its fast amyloid-induced dissolution through detergent effect. In parallel, we investigated the interaction of a Tau fragment with model membranes mimicking the inner neuronal membrane. Our data highlighted a preferential role of negatively charged lipids (POPS, PIP2) in the Tau / membrane interaction, with lipid-induced aggregation of the peptide.

In addition, we have successfully initiated functionalization experiments, where we attach amyloid peptides on AFM tips in order to directly probe the amyloid peptide / membrane interaction by force spectroscopy. We also initiated the development of an incubator-like chamber with controlled environmental parameters, which will allow maintaining experimental conditions close to biological relevance.

In brief, AMNEsIA has contributed to deepening our understanding of AD-associated amyloid peptides, and has laid the foundations to go further in future molecular and live cell studies.
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.
The AMNEsIA project has contributed to deepening our understanding of Alzheimer’s disease associated amyloid peptides, by providing high resolution imaging of the amyloid / membrane interaction with high temporal resolution. Furthermore, the developments performed in AFM tip functionalization and in the design of an incubator-like chamber hold great promise for future molecular and live cell studies, going further in our understanding of these peptides at the molecular and cellular level.

The AMNEsIA project has been a tremendous opportunity for the fellow to learn new skills, collaborate with great scientists and gain visibility and credibility. It has boosted her academic career, and she was recruited as a permanent researcher at the French National Center for Scientific Research (CNRS), working at host institute CBMN (Institute of Chemistry and Biology of Membranes and Nanoobjects, Bordeaux, France).

She has applied for an ERC starting grant, as well as local funding, to build up on AMNEsIA’s results and further her research on amyloid peptides by developing innovative AFM-based imaging and spectroscopy methods.
Illustration of the key biophysical methods used in AMNEsIA