CYTOPLASMIC EXPRESSION OF TDP-43 DECREASES GLOBAL TRANSLATION BOTH IN VITRO AND IN VIVO

VILA, A; LUCHELLI, L.; MULLER IGAZ, L.; CHARIF, S. E.; BLAUSTEIN, M.

Congreso; The Alzheimer?s Association International Conference (2020) Virtual Event; 2020

The Alzheimer?s Association

Background: TDP-43 is a major component of cytoplasmic inclusions observed in neurodegenerative diseases like frontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS). To further understand the role of this predominantly nuclear RNA-binding protein in disease-related mRNA/protein metabolism and proteostasis, we used a combined approach with cellular and animal models that overexpress a cytoplasmic form of human TDP-43 (TDP-43-ΔNLS), Recapitulating ALS/FTD features. Method: HEK293 cells were transfected with empty vector or hTDP-43-ΔNLS followed by a method for labeling de novo translation, surface sensing of translation (SUnSET), based on puromycin (PURO) incorporation. We also assessed whether cytoplasmic TDP-43 regulates global translation in vivo using a transgenic TDP-43-ΔNLS mouse model. Brain cortices from control and transgenic mice were subjected to polysome profiling and western blotting. Lastly, cellular level analysis of ongoing protein synthesis was performed by applying the SUnSET method in acute thick brain slices from control and TDP-43-ΔNLS mice. Immunofluorescence for TDP-43 and PURO was performed and signal intensity was measured.Result: Our in vitro approach showed that, while control cells displayed robust puromycilation, TDP-43-ΔNLS positive cells exhibited reduced ongoing protein synthesis when compared to control groups. In vivo results from polysome profiling revealed a shift towards light (non-polysomal) fractions as compared to wild-type littermates, indicating a decrease in global mRNA translation. Cellular level analysis of ongoing protein synthesis showed that slices from control mice incubated with PURO exhibited robust cytoplasmic PURO signal in layer 5 neurons from motor cortex, and normal nuclear TDP-43 levels similar to control (-PURO) neurons. Slices from TDP-43-ΔNLS mice incubated with PURO exhibited high cytoplasmic expression of TDP-43 and a reduction in puromycilation respect to control mice. Quantification of neuronal staining intensity revealed a significant decrease in PURO incorporation in transgenic vs. control mice. Conclusion: In vitro and in vivo results indicate that cytoplasmic TDP-43 can decrease global translation and potentially cause functional/cytotoxic effects as observed in ALS/FTD. We provide in vivo evidence (by two independent and complementary methods) for a role of mislocalized TDP-43 in the regulation of global protein synthesis, with implications for understanding the molecular changes underlying the clinicpathological manifestations of TDP-43 proteinopathies.