Biophysical Insights into Biomolecular Condensates of TDP-43

Abstract

Neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), and frontotemporal dementia (FTD) pose significant health challenges worldwide, affecting millions of individuals. A prominent feature of these disorders is the accumulation of pathological protein aggregates that are highly toxic for the cells. Among the proteins implicated in neurodegenerative diseases, TAR DNA-binding protein 43kDa (TDP-43) stands out as a key player, whose aggregates are a characteristic pathological feature of ALS and FTD. TDP-43 possesses an intrinsically disordered domain that plays a critical role in its pathological behavior. This domain has been implicated in the formation of stress granules, dynamic cytoplasmic structures formed in response to cellular stress via the process of liquid-liquid phase separation. Prolonged persistence of these stress granules can lead to the formation of irreversible toxic aggregates, contributing to disease pathology. Emerging evidence also suggests co-existence of TDP-43 aggregates with other pathological proteins exacerbating the severity of the disease. However, the underlying molecular interactions governing these synergistic interactions and phase separation are still elusive. In this work, we investigate the full length TDP-43 and its disease-associated truncations to understand the key molecular interactions driving the liquid- liquid phase separation and liquid-to-solid phase transition of TDP-43 by employing the comprehensive array of biochemical and biophysical techniques. Furthermore, our research delves into investigating the conformational dynamics of the TDP- 43 polypeptide chain within the liquid droplets. By probing the structural dynamics of TDP-43 within these condensates, we seek to elucidate the fundamental mechanisms driving droplet formation and discern aberrant behaviors associated with disease-associated mutants. Our efforts have resulted in the successful recapitulation of TDP-43 droplets in an. To further probe the conformational dynamics of the TDP-43 polypeptide chain within these droplets in an in vitro setting, we create and utilize site- specific labeling of single-cysteine mutants at different locations across the entire sequence of TDP-43. Our study aims at investigating the effects of RNA and other proteins on the material properties of TDP-43 droplets which will help to unravel the complex molecular mechanisms underlying TDP-43 condensation and its transition to pathological aggregates thus broadening our understanding of cellular physiology and pathology.