Raman Spectroscopic Studies of Amyloids

National Heart, Lung, And Blood Institute

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Abstract

We have performed foundational studies by Raman spectral imaging (RSI) towards the goal of studying amyloid formation in cells. This method couples the chemical specificity of vibrational spectroscopy and the spatial resolution of a microscope, so that polypeptide conformational changes can be evaluated and pinpointed. Because intrinsic molecular vibrations are measured, protein secondary structure and cellular biomolecules (e.g. nucleotides and lipids) can be simultaneously observed, offering information unattainable by standard imaging modalities. By leveraging chemical biological approaches, unique vibrational probes have been installed in alpha-synuclein for in vitro and in cellulo studies. We want to understand under what circumstances the protein transforms from a functional to a pathological state and in which cellular compartment. The discovery of initiating sites for alpha-synuclein accumulation would inform on interventional approaches. These studies will provide insights into the biological function of alpha-synuclein and the mechanisms by which alpha-synuclein and its molecular interactions contribute to neurodegeneration. In recent work, we have expanded our RSI studies towards studies of liquid-liquid phase separation (LLPS) of transactive response (TAR) DNA-binding protein of 43kD (TDP-43), the primary component of the inclusions in both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration with ubiquitin (now known as FTLD-TDP). Here, we aim to delineate the relationship between LLPS and aggregation of TDP-43. We are testing the prevailing hypothesis that LLPS serves as an amyloid intermediate by directly measuring polypeptide conformational state(s) inside protein droplets and monitoring how they evolve over time through RSI. The goal is to gain mechanistic understanding of the processes of TDP-43 phase separation and aggregation and to determine their roles in disease etiology.