Abstract
TAR DNA-binding protein 43 (TDP-43) is a ubiquitously expressed RNA-binding protein of the hnRNP family. It consists of an N-terminal domain (NTD) that mediates its physiological oligomerization, two RNA recognition motifs (RRMs) that are crucial for the roles of TDP-43 in RNA processing, and an intrinsically disordered domain of low complexity (LCD) that is essential for TDP-43 liquid-liquid phase separation and renders it prone to aggregation. In healthy cells, TDP-43 primarily localizes to the nucleus, where it engages in physiological oligomerization through its NTD to fulfill various roles related to RNA metabolism. There it regulates the splicing events of its targets, many of which are essential for neuronal development and health. Under pathological conditions, TDP-43 undergoes cytoplasmic mislocalization, polyubiquitination, hyperphosphorylation, and partial proteolytic cleavage. Both full-length TDP-43 and its C-terminal fragments accumulate in various cytoplasmic (and occasionally nuclear) inclusions within affected neurons leading to cellular toxicity. These TDP-43 proteinaceous aggregates are found in a subset of neurodegenerative proteinopathies, including amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD), and are collectively known as TDP-43 proteinopathies. Although certain hallmarks of TDP-43 inclusion are common among TDP-43 proteinopathies, the resulting aggregates exhibit differences in morphology, subcellular localization, and underlying structural properties, suggesting distinct aggregation mechanisms. RNA binding of TDP-43 through its RRMs has been extensively studied over the years. However, several publications have indicated that other domains may be at play when it comes to its RNA binding ability. The potential interactions of other TDP-43 domains with RNA and their implications in TDP-43 physiology and pathological transition, however, have not been thoroughly investigated. Moreover, the connection between RNA binding by the various TDP-43 domains and its oligomerization, as well as how the loss of such interactions contributes to TDP-43 pathogenesis, remains unclear. It is therefore essential to investigate the interplay between RNA binding and oligomerization and study their impact on TDP-43 to gain a comprehensive understanding of its physiological roles and pathological transitions. This dissertation delves into the molecular mechanisms of TDP-43 physiology and pathology in relation to its RNA binding and the interplay thereof with oligomerization. We demonstrate that all domains of TDP-43 can interact with RNA and that RNA binding and oligomerization are linked. We show that the NTD of TDP-43 interacts with RNA through its oligomerization interface solely in its monomeric state. We demonstrate that this interaction is involved in the regulation of TDP-43’s own mRNA and is essential for full nuclear retention and protection against TDP-43 aggregation, highlighting for the first time the existence and importance of functional monomeric TDP-43. Moreover, we demonstrate that NTD-mediated oligomerization enables high-affinity binding of RNA through its RRMs, implying that oligomerization is required for certain RNA-mediated TDP-43 functions. Finally, we demonstrate that the loss of NTD-mediated oligomerization and RNA binding as well as the loss of RNA binding by the RRMs lead to distinct aggregation mechanisms resulting in pathological aggregates with differences in subcellular localization and morphologies, reminiscent of those found in TDP-43 proteinopathies. Our data broaden the current understanding of TDP-43 physiology on a mechanistic level and provide insights into the pathological transition of TDP-43. Our results furthermore shed light on the potential molecular basis for disease heterogeneity observed across the spectrum of TDP-43 proteinopathies.
Zbinden, Aurélie