Cytoplasmic expression of the ALS/FTD-related protein TDP-43 decreases global translation both in vitro and in vivo.

CHARIF S; BLAUSTEIN M; VILA A; LUCHELLI L; IGAZ LM

Conferencia; AAIC 2020(Alzheimer´s Association International Conference); 2020

Alzheimer´s Association

Background: TDP-43 is a majorcomponent of cytoplasmic inclusions observed in neurodegenerative diseases likefrontotemporal dementia (FTD) and amyotrophic lateral sclerosis (ALS).  To further understand therole of this predominantly nuclear RNA-binding protein in disease-related mRNA/proteinmetabolism and proteostasis, we used a combined approach with cellular andanimal models that overexpress a cytoplasmic form of human TDP-43(TDP-43-ΔNLS), recapitulating ALS/FTD features.  Method: HEK293 cells were transfected with emptyvector 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 whethercytoplasmic TDP-43 regulates global translation in vivo using a transgenic TDP-43-ΔNLS mouse model. Brain corticesfrom control and transgenic mice were subjected to polysome profiling andwestern blotting. Lastly, cellular level analysis of ongoing protein synthesiswas performed by applying the SUnSET method in acute thick brain slices fromcontrol and TDP-43-ΔNLS mice. Immunofluorescence for TDP-43 and PURO wasperformed and signal intensity was measured. Result: Our in vitro approach showed that,while control cells displayed robust puromycilation, TDP-43-ΔNLS positive cellsexhibited reduced ongoing protein synthesis when compared to control groups. In vivo results from polysome profilingrevealed a shift towards light (non-polysomal) fractions as compared towild-type littermates, indicating a decrease in global mRNA translation.Cellular level analysis of ongoing protein synthesis showed that slices fromcontrol mice incubated with PURO exhibited robust cytoplasmic PURO signal inlayer 5 neurons from motor cortex, and normal nuclear TDP-43 levels similar tocontrol (-PURO) neurons. Slices from TDP-43-ΔNLS mice incubated with PUROexhibited high cytoplasmic expression of TDP-43 and a reduction inpuromycilation respect to control mice. Quantification of neuronal stainingintensity revealed a significant decrease in PURO incorporation in transgenicvs. control mice.Conclusion: In vitro and invivo results indicate that cytoplasmic TDP-43 can decrease globaltranslation and potentially cause functional/cytotoxic effects as observed inALS/FTD. We provide in vivo evidence (by two independent andcomplementary methods) for a role of mislocalized TDP-43 in the regulation ofglobal protein synthesis, with implications for understanding the molecularchanges underlying the clinic-pathological manifestations of TDP-43proteinopathies.