Direct and Indirect Modulators of TDP-43 Aggregation in vitro

Abstract

Amyotrophic lateral sclerosis is a devastating neurodegenerative disease that predominately affects upper, bulbar, and spinal motor neurons. Prognosis remains poor, with most patients succumbing to respiratory failure within five years of diagnosis. Although ALS has been clinically recognized and diagnosed for over 150 years, no therapeutics have been affectively developed to target its pathognomonic sign of TAR-DNA Binding Protein 43 kDa (TDP-43) mislocalization and aggregation. G-Quadruplexes (G4s) are guanine-rich nucleic acid structures that form stacked tetrad and can influence protein folding, phase change, and aggregation. Here, we demonstrate the capacity of G4s to bind and modulate TDP-43 condensation under chemically induced stress conditions. This identified a framework of direct endogenous molecular intervention in TDP-43 pathology. These findings provided a basis for synthetically targeting the nucleic acid binding domains of TDP-43 with small molecule protein mimetics. Oligopyridylamides (OPs) are synthetic compounds with tunable side-chains that have demonstrated promise in attenuating aberrant neurodegenerative protein interactions. We present two lead compounds that facilitate direct binding and amelioration of TDP-43 proteinopathy through biophysical and cellular analysis. Proteomic profiling of reporter cells treated with OPs recover from ALS-associated protein signatures after exposure to pathogenic ALS patient neuron-derived exosomes (NDEs). We also report that these NDEs pathogenically differ in patient-matched analyses after a 24-week Phase II clinical trial of oral fasudil, a Rho-kinase (ROCK) inhibitor, performed by Woolsey Pharmaceuticals (New York, NY). Additionally, proteomic analysis of reporter cells exposed to ALS-NDEs identify dysregulated bioenergetic, nuclear pore complex, and autophagic protein files that are recovered after ROCK inhibition. Collectively, our findings present a clear framework for modulating TDP-43 proteinopathy. G4s reveal endogenous nucleic acid chaperones of TDP-43 condensation; OPs demonstrate that synthetic small molecules can directly bind TDP-43 and suppress aggregation; and patient-derived NDEs establish a biologically relevant model of extracellular TDP-43 pathology that can be altered by systemic ROCK inhibition. This work provides a mechanistic insight into TDP-43 regulation and identifies therapeutic strategies aimed at both direct aggregation control and reduced extracellular propagation of ALS-associated pathology.