Tau and α‐synuclein protein expression, purification, and their amyloid inhibitor discovery

Abstract

Alzheimer's and Parkinson's diseases (AD/PD) are two major neurodegenerative diseases. AD is characterized by the accumulation in brain of two types of abnormal structures, extracellular amyloid plaques and intraneuronal neurofibrillary tangles. Prevalent evidence now indicates that the respective building blocks of plaques and tangles – oligomers of Aβ peptide and the neuron‐specific, axon‐enriched microtubule‐associated protein, tau, represent the most toxic molecular species contributing to AD pathogenesis. Similarly, alpha‐synuclein protein amyloid is a major constitute of Lewy body, protein aggregates that are the pathological hallmark of Parkinson's disease.Currently there are no effective drugs to prevent or cure AD and PD. Because of protein aggregates, including oligomers, mediate neuronal toxicity in AD/PD, small molecule aggregation inhibitors are attractive drug candidates. Many drug trials aimed at preventing or clearing Aβ pathology have failed in recent years. Investigations on tau yielded innovative targets backed by recent findings about the central role of tau in AD pathogenesis (1,2). Preliminary data from a phase 2 trial with the small molecule, methylene blue chloride, have shown some promise and thereby validate the potential of tau aggregation inhibitors (TAIs), but superior TAIs are needed.In this poster we will present our work on expression and purification of human tau and α‐synuclein proteins and their amyloid formation and inhibitor discovery. Tau and α‐synuclein are expressed as recombinant proteins in E. coli and were purified to homogeneity using multiple chromatographic steps. They are further validated by western blot analysis and mass spectrometry. We have developed multiple biochemical, biophysical, and cell‐based assays to monitor oligomer/amyloid formation and amyloid‐induced cytotoxicity assays in characterizing amyloidogenic proteins (3). We use ThT fluorescence‐based assay for semi‐high throughput library screens. Upon identification of “hit” compounds, we validate them with multiple secondary assays including transmission electron microscopy (TEM), photo‐induced crosslinking of unmodified proteins (PICUP), IC50 measurements, and cell‐based cytotoxicity assays. We will further perform mechanistic studies for these validated inhibitors.Support or Funding InformationThis work is in part supported by Virginia Tech new faculty start‐up funds, Commonwealth Health Research Board (CHRB), Alzheimer's and Related Diseases Research Award Fund (ARDRAF) from Virginia Center on Aging, and Diabetes Action Research and Education Foundation (DAREF) to B.X.