​​​​Pierre De Rossi​

Using super-resolution microscopy to unravel the physiological and pathological roles of TDP-43 liquid-liquid phase separation

Scientific abstract: Liquid-liquid phase separation (LLPS) is a physical property allowing for oligomerization of proteins by weak non-covalent interactions. LLPS is a newly identified mechanism that regulates several functions in cells, including RNA processing and transport, as well as stress response. In this context, the TAR DNA-binding protein 43 (TDP-43), an RNA binding protein, is under high scrutiny for its central role in cellular physiology. TDP-43 oligomerization is necessary for RNA splicing functions, and destabilization of TDP-43 oligomers leads to aggregation and cellular toxicity. Moreover, aggregation of TDP-43 is a hallmark of two devastating neurodegenerative diseases, amyotrophic lateral sclerosis (ALS), frontotemporal lobar dementia (FTLD) and limbic-predominant age-related TDP-43 encephalopathy (LATE). A central question over the past decade has been how LLPS drives protein aggregation. While the mechanism(s) of this transition remain far from resolved, the field has made great progress in deciphering several key steps and drivers in the process. However, the complexity of TDP-43 function and regulation has been a central problem to understand its role in human disease. One of the limitations stems from the techniques employed to explore these processes. Biochemical approaches do not offer spatial information, and limited resolution of conventional microscopy leads to incomplete answers, and usually requires the use of overexpression of fusion proteins to explore these questions. To overcome these limitations and uncover the physiological and pathological mechanisms involving TDP-43, I will use super resolution microscopy (SRM), in particular direct stochastic optical reconstruction microscopy (dSTORM) and structured illumination microscopy (SIM). The goal of this project is to improve our knowledge on TDP-43 in regard to physiological oligomerization and pathological mechanisms leading to aggregation. I will use SRM to identify the role of physiological TDP-43 oligomers in RNA processing using dSTORM imaging and markers for specific subnuclear compartments. Using live-SIM imaging, I will explore the regulation of physiological TDP-43 oligomers under stress conditions using cell lines and human induced pluripotent stem cell (iPSC)-derived neurons and astrocytes. Finally, I will systematically assess the role of phosphorylation currently exclusively associated with pathological TDP-43 aggregates in the physiological regulation of TDP-43 phase separation and its transition to pathology. Altogether, this study will unravel the molecular machinery controlling TDP-43 compartmentalization and will highlight key steps mediating its dysregulation and aggregation associated with TDP-43 proteinopathies. My results will open new paths for developing efficient drugs to specifically target pathological aggregation of TDP-43 and restore its physiological functions.

​

Lay abstract (english): Liquid-liquid phase separation (LLPS) is a newly identified mechanism that regulates several functions in cells, including RNA metabolism and cellular stress response. In this context, the TAR DNA-binding protein 43 (TDP-43) is under high scrutiny for its central role in cellular physiology and its central role in amyotrophic lateral sclerosis, frontotemporal lobar dementia, and in a subset of Alzeimer's disease cases. The complexity of TDP-43 function and regulation has been a central problem to understand its role in human disease. The goal of this project is to improve our knowledge on TDP-43 and the pathological mechanisms leading to aggregation using advanced super resolution microscopy techniques. Altogether, this study will unravel the molecular process regulating TDP-43 and will highlight key steps leading to the pathology. My results will open new paths for developing efficient drugs to specifically target pathological aggregation of TDP-43 and restore its physiological functions.

​

Lay abstract (french): La séparation de phase liquide-liquide a récemment été identifiée comme mecanisme regulateur de plusieurs fonctions cellulaires, comme la régulation des ARNs ou la réponse cellulaire au stress. Parmis les proteines impliquées dans ce processus, TAR DNA-binding protein 43 (TDP-43) a une place centrale de part son role physiologique et son implication dans la sclérose latérale amyotrophique, la démence fronto-temporale et dans certains cas de la maladie d'Alzheimer. La complexité des fontions de TDP-43 et sa regulation est une question centrale pour comprendre son role dans les maladies neurodégénératives. Le but de mon projet est d'améliorer nos connaissances sur TDP-43 et de comprendre les méchanismes d'aggrégation en utilisant des techniques de microscopie à super-résolution, pour lever le voile sur les etapes clés de l'apparition de la pathologie. Ces résultats ouvriront de nouveaux champs d'étude pour le developpement de traitements qui viseront directement l'aggrégation pathologique de TDP-43 pour restaurer ses fonctions physiologiques.

​