CHARACTERIZATION OF PROTEIN SYNTHESIS DYSREGULATION IN A DROSOPHILA MODEL OF TDP-43 PROTEINOPATHY

Abstract

TAR DNA-binding protein 43 (TDP-43) is a DNA/RNA binding protein whose cytoplasmic aggregation is a pathological hallmark of amyotrophic lateral sclerosis (ALS). How disease-associated TDP-43 aggregates may exert toxicity within motor neurons, which rapidly deteriorate in ALS patients, is not fully understood. Given the important role TDP-43 plays in RNA metabolism within nerve cells and previous findings that TDP-43 regulates the local translation of specific mRNAs, we investigated alterations in global protein synthesis associated with overexpression of wild-type or mutant human TDP-43 in Drosophila motor neurons. We show the translation initiation regulators 4EBP and S6K can alter locomotor phenotypes induced by TDP-43 proteinopathy. In addition, wild-type animals show increased vulnerability to pharmacological inhibition of cap-dependent translation, which suggests a preexisting translational impairment in our ALS models. Using fluorescent noncanonical amino acid tagging (FUNCAT) to label nascent proteins in-vivo we obtained preliminary data showing evidence of reduced newly synthesized proteins at the neuromuscular junction (NMJ) in our Drosophila ALS models. Furthermore, we paired quantitative mass spectrometry with noncanonical amino acid tagging (BONCAT) to identify candidate genes down-translated at the NMJ of ALS flies. Consistent with these observations of broad translational impairment, we found decreased puromycin incorporation into nascent peptides of lymphoblastoid cells derived from an ALS patient harboring a TDP-43 mutation. Taken together, our data point toward compartment specific, cap-dependent protein synthesis deficits, including locally at synaptic boutons, as a key process disrupted by ALS-like pathology.