S-017 | Single-cell analysis shows that expression of cytoplasmic TDP-43 activates the ATF4 Unfolded Protein Response pathway in neuroblastoma N2a cells

Cellular and Molecular Neurobiology
Author: Mauricio Norman Montenegro | Email: mauricio.montenegro909@gmail.com

Mauricio Montenegro^1°2°, Calén Sansalone^3°, Gabriel Vera Candia^1°2°,  Matías Blaustein^3°, Lionel Muller Igaz^1°2°

^1° Universidad de Buenos Aires, Facultad de Ciencias Médicas, Departamento de Ciencias Fisiológicas. Grupo de Neurociencia de Sistemas. Buenos Aires, Argentina.
^2° CONICET – Universidad de Buenos Aires. Instituto de Fisiología y Biofísica Bernardo Houssay (IFIBIO Houssay). Buenos Aires, Argentina.
^3° Instituto de Biociencias, Biotecnología y Biología traslacional (iB3), Departamento de Fisiología y Biología Molecular y Celular, Facultad de Ciencias Exactas y Naturales, Universidad de Buenos Aires

TAR DNA-binding protein 43 (TDP-43) is a key player in neurodegenerative diseases, notably Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD). While TDP-43 is primarily known for its function in RNA metabolism, emerging evidence highlights its involvement in the Unfolded Protein Response (UPR), a critical cellular mechanism essential for managing endoplasmic reticulum stress and maintaining proteostasis. This study explores the impact of mislocalized, cytoplasmic TDP-43 on UPR signaling at the single cell level. Immunofluorescence staining combined with high-content image analysis of neuroblastoma N2a cells transfected with mutated, cytoplasmic TDP-43 (TDP-43-ΔNLS) revealed a significant increase in endogenous ATF4 expression, indicating activation of the PERK/ATF4 UPR branch. Moreover, co-treatment with the UPR inductor Tunicamycin further increased ATF4 signal. We also examined cell-to-cell correlations between cytoplasmic TDP-43 intensity and ATF4 expression levels. As a novel tool to investigate activation of the three UPR signaling branches, we developed pathway-specific fluorescent reporters and we confirmed ATF4-Scarlet induction in TDP-43-ΔNLS positive cells compared to non-transfected ones. Lastly, we demonstrate successful expression of additional UPR reporters for the ATF6 and IRE1 branches. Our results on the role of TDP-43 in UPR modulation provide insights into its pathological contributions to protein misfolding diseases such as ALS/FTD.