Direct monitoring of the local dynamic rearrangement of the Alzheimer's peptides secondary structure upon the aggregation
Duration: 2021 - 2025
Research project objectives/Research hypothesis
Neurodegenerative diseases pose one of the most serious health and social problems in the world today. Therefore, the phenomenon of abnormal protein aggregation, responsible for the etiology of these diseases, is of central importance across a wide range of many research disciplines. A better understanding of abnormal protein aggregation can determine strategies for the early treatment of diseases. Prior studies have allowed modeling of only hypothetic aggregation pathways. However, methodological limitations prevented, or largely prohibited direct experimental observations. We are going to prove the scientific hypothesis: "The amyloid-β aregation pathways can be directly monitored through nano-spectroscopic mapping of the secondary structure local rearrangement in individual aggregates". An integral part of the project is a deep investigation into an influence of intrinsic and extrinsic factors such as anti-aggregation agents, and solution conditions on the aggregation scheme. Additionally, cytotoxicity of amyloid-β (aβ) against living neurons will be examined.
Research project methodology
Monitoring of a β aggregation schemes will be achieved by the first ever application of Tip-enhanced Raman (TER) mapping in liquid, to visualize the distribution of β-sheet structure within single protein aggregates. In order to verify the obtained results, Fourier Transform Infrared Spectroscopy at the nanoscale (nanoFTIR) will be applied. Both methods combine the high spatial resolution of Atomic Force Microscopy and chemical selectivity of molecular spectroscopy providing information about the chemical composition of samples at the nanoscale. We postulate that the distribution of the β-sheet structure in peptide aggregates is directly correlated with the aggregation scheme. Therefore, We propose to follow each step of the primary aggregation pathway in single peptide aggregates. The two neurodegenerative peptides will be investigated: aβ(1-42) - the main component of Alzheimer's plaques and the main component of cerebrospinal fluid called aβ(1-40). Moreover, We shall investigate in real-time the influence of the anti-aggregation drug on the molecular structure of aggregates. Various amyloid forms: oligomers, fibrils, and protofibrils of aβ will be continuously mapped upon the introduction of bexarotene into the buffer. Molecular nanospectroscopy has never been carried out in an environment that mimics physiological conditions (on lipid membrane in physiological buffer) and TERS applications in a liquid have also been very limited so far. According to preliminary results, we expect to observe Raman, infrared and TERS markers of amyloid conformational transitions as shifts of spectral positions of the amide I and amide III bands, that are well resolved in the nanoFTIR and TER spectra acquired in liquid. Monitoring of aggregation schemes in bulk samples by confocal Raman, Fourier Transform Infrared Spectroscopy (FTIR) and Circular Dichroism is also planned. A very important aspect of this studies is an application of confocal Raman and FTIR to follow molecular changes induced in living neurons by amyloid treatment. An application of these methods will allow monitoring of proteins, nucleic acids, and lipids damage simultaneously. Acquired spectroscopic data will be treated with multivariate data analysis such as Principal Component Analysis and Hierarchical Cluster Analysis, which is not yet commonly used in the field of nano-spectroscopy. In order to correctly interpret the spectra, theoretical calculations including Density-functional Theory will be performed.
Expected impact of the project on the development of science
This project requires significant development of the methodology, which will be of benefit to the spectroscopic community for a broad range of applications. The methodology (preparation of probes, measurements in physiological buffers, strategy to avoid photo-damage, novel approach to data analysis) will be universal and could be applied in studies of numerous biological systems such as DNA, DNA-proteins mixtures, chromatin, multi-component lipid membranes and many others. This project will extend existing knowledge about the aggregation of Alzheimer's peptides. Understanding the effects of intrinsic and extrinsic factors on the aggregation process at the molecular level is essential for developing effective therapeutic strategies aimed at inhibiting self-assembly.