Purification of the neurodegenerative disease associated protein TDP-43 and development of TDP-43 aggregation inhibitors.

Marshall, Toby and Benson, Fiona and Parkin, Edward and Fullwood, Nigel and Allsop, David (2024) Purification of the neurodegenerative disease associated protein TDP-43 and development of TDP-43 aggregation inhibitors. PhD thesis, Lancaster University.

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Abstract

Transactive response DNA-binding protein-43 (TDP-43) is a protein that has been implicated in multiple neurodegenerative diseases, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD). In these diseases, TDP-43 is found aggregated in the cytoplasm of neurones in the brain and spinal cord and it is hypothesised that this aggregation leads to neuronal degeneration. Despite identification of TDP-43 as a constituent of pathological aggregates in 2006, progress in biochemical characterisation of TDP-43 and its aggregation has been limited by an inability to purify sufficient soluble protein to allow characterisation. In this study, a novel method for the purification of TDP-43 has been developed. The resulting purified protein exists in multiple oligomeric states depending on buffer conditions, displays evidence of secondary structural content by circular dichroism spectroscopy and in preliminary studies demonstrates DNA binding activity. A TDP-43 C-terminal fragment was also purified and a fluorescence-based assay developed to monitor its aggregation, with transmission electron microscopy (TEM) used to image the aggregates produced. In this assay, small molecules were tested as aggregation inhibitors. Following minimal success re-purposing generic aggregation inhibitor molecules, a series of targeted peptide-based inhibitors were designed. The third-generation peptide inhibitors, designed with the aid of the artificial intelligence system AlphaFold, reduced the aggregation of the C-terminal fragment, with TEM identifying changes to the morphology of the aggregates produced. Finally, a “druggable” Saccharomyces cerevisiae yeast cell model of TDP-43 proteinopathy was developed, in which molecules with potential as TDP-43 aggregation inhibitors can be tested further.