NANOoligo

Amyloid fibrils are involved in many age-related degenerative diseases, including Alzheimer's, Parkinson's, and Huntington's diseases, as well as type II diabetes 1. The study of such biological higher-order structures requires the identification of numerous self-assembled cytotoxic intermediates formed from smaller building blocks. Although different approaches and techniques have been used to obtain kinetic and structural information on these intermediates, they are still difficult to study. This is because the species formed during early stages of amyloid aggregation are heterogeneous comprising a range of aggregation states and they form transiently evolving as a function of time. Developing tools for amyloid analysis at early stage able to discriminate the different population of oligomers and protofibrils in order to evaluate the impact of promoters or inhibitors through amyloid shape analysis is particularly important to understand the external factor on Alzheimer disease. Presently, no dedicated equipment that allows simultaneously a continuous experimental characterization, at the single molecule scale, of the different intermediates emerging during amyloid fibril formation. Our project aims to address these issues by proposing an innovative analytical method based on nanopore technology and using it to solve a biological question. We chose the Aß42 oligomers regading their involvement on Alzheimer's desease. We base our project on our knowledge and recent works proving that single nanopore will be the unique solution to obtain information about the morphology and the size distribution of protein assembly under continuous measurement. To achieve this, we propose a methodology which combines on single nanopore experiments, simulation and biological approaches. The methodology follows in our project is composed by four main steps that are essential to develop a sensor: conception, calibration, application and transfer. The conception will include the single nanopores design and the study the adsorption of amyloid on the nanopore surface. The calibration involves the production the separation a complete investigation of the dynamics of amyloid translocation through the single nanopore in order to assign each current perturbation to the amyloid population. The application will consist to use nanopore sensor to investigate the effect of inhibitor and mutation as well as provide an answer, for the first time, to the impact of pesticides in the Aß assembly. The transfer phase will be the validation our method and its upscale to first prototype of our nanopore sensor inside an easy-to-handle device. Finally we expect from the development of this new analytical method to make significant scientific and technological advances in different areas allowing to (i)Propose a generic nanopore technology transferable to other proteinaceous that are thought to be responsible for the cellular toxicity and the autocatalytic self-propagation of the aberrant fold, involved in various fatal neurodegenerative disorders (ii)Improve our basic knowledge on physics and physical chemistry of protein assembly translocation under confinement. (iii)Improve our understanding on aberrant protein self-assembly that underlies neurodegenerative disorders and specifically the molecular mechanism of Aß fibril formation at early stage. (iv) Provide clues to help identify pesticide exposure as an environmental risk factor in Alzheimer’s disease.