Biophysical methodologies used to assess the aggregation, phosphorylation, and antibody inhibition mechanisms of ALS-associated protein, TDP-43

Josephine Esposto¹, Robert J. Huber³, Sanela Martic^1,2

¹Department of Forensic Sciences, Trent University, Peterborough, ON

²Department of Environmental and Life Sciences, Trent University, Peterborough, ON

³Department of Biology, Trent University, Peterborough, ON

 

In several neurological diseases, the onset and progression of degeneration has been linked to TDP-43 proteinopathy, specifically protein aggregation and post-translational modifications. Hyperphosphorylation and aggregation of the protein have been shown to contribute to the pathology in vivo, suggesting that the inhibition of these mechanisms could be a viable therapeutic target. Pertinent research has only discovered a superficial understanding of the aggregation mechanisms of TDP-43, thus providing us with a critical knowledge gap in the functionality and structural composition of the protein. Using various biochemical and biophysical techniques to analyze the aggregates formed in vitro, phosphorylated, full-length TDP-43 was shown to aggregate under agitated, temperature-controlled environments that resulted in protein fibrils. The proteins were analyzed via Thioflavin T (ThT) fluorescence assays to monitor β-sheet formation, transmission electron microscopy (TEM) for visualization of the structural morphologies of TDP-43 aggregates, and turbidity measurements. When incubated with antibodies specific to epitopes (RRM2-CTD domain), a significant reduction in the formation of insoluble aggregates was observed at a low antibody. The outcomes of the inhibition were highly dependent on the type of protein and antibodies used when compared to negative controls of anti-SOD and anti-tau antibodies that demonstrated no binding to TDP-43. Further studies are necessary to confirm the exact binding mechanism of the polyclonal antibodies but provides a preliminary pathway for immunotherapies as a viable treatment for TDP-43 proteinopathies.