Characterizing actin assembly mechanisms in ALS pathology

Abstract

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease linked to >sixty genes. However, the mechanistic details of onset are unknown and there is no known cure or effective treatment. Disease mechanisms linked to the dysfunction of the neuronal cytoskeleton are arguably the least explored, despite being involved in important cell processes. Eight cytoskeleton associated proteins are genetically linked to ALS onset, including tubulin-4A, spastin, kinesin-5A, dynactin subunit-1, neurofilament, peripherin, alsin, and profilin-1 (PFN1). Most of these genes are linked to microtubule dynamics, yet only PFN1 directly regulates actin assembly. Profilin is essential for many neuronal cell processes mediating the dynamics of lipids, nuclear signals, and the cytoskeleton in neurons. We performed a quantitative biochemical comparison of the impact of the eight ALS-associated profilin variants on actin assembly using single-molecule microscopy assays. The binding affinity of each ALS-related profilin for actin monomers was loosely correlated with the actin filament nucleation strength. The A20T, R136W, Q139L, and C71G variants failed to activate formin-based actin assembly, mostly explained by deficiencies in binding to poly-L-proline stretches in formin. In addition, chemical denaturation experiments suggest that the folding stability of some ALS profilin proteins impacts on actin assembly. Finally, to better understand the connection between the cytoskeleton and ALS we investigated the role of profilin and its direct binding ligand TDP-43 (TAR DNA-binding protein 43) on actin assembly. TDP-43 misregulation is a hallmark of nearly all forms of neurodegeneration, including an estimated 40% of familial ALS. Using advanced microscopy assays, we visualized purified TDP-43 forming biomolecular condensates. Actin was sequestered within TDP-43 condensates tempering total filament assembly. These observations were further exacerbated in the presence of profilin. These results indicate disruptions to actin assembly contribute to ALS and suggest therapeutic interventions targeting actin assembly may be a useful in treating ALS.