NRSA in support of Elizabeth Daley: Investigating ETF1 at the Interface of Translation Repression and Nonsense-Mediated Decay in C9-ALS/FTD Neurotoxicity

    Project: Research project

    Project Details


    The purpose of my proposed study is to elucidate how defects in the balance between protein translation and RNA degradation lead to the degeneration of human motor neurons (MNs). The most common genetic cause of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD) is a GGGGCC hexanucleotide repeat expansion in the first intron of the C9orf72 gene (C9). RNA and dipeptide repeat proteins that are transcribed and translated from the C9 expansion respectively, drive neurotoxicity through a number of toxic gain-of-function mechanisms. While several lines of evidence suggest that C9 MNs exhibit defects in nucleocytoplasmic transport, mRNA metabolism and protein translation that are tightly associated with neurotoxicity, the source of these impairments, their interplay, as well as their relative contribution to neurotoxicity remain unclear. In preliminary experiments I performed a proteome-wide nucleocytoplasmic localization screen and discovered that several proteins are redistributed in C9-expressing cells. Among these, is eukaryotic termination factor I (ETF1), a protein which translocates from the cytoplasm to the nucleus in C9 iPSC patient-derived MNs and C9-ALS postmortem tissue. ETF1 associates with the scanning ribosome and mediates the balance of protein translation and RNA degradation. Specifically, it initiates translation termination to release nascent polypeptides; however, in cases of mis-spliced or aberrant mRNA transcripts it triggers their degradation through a highly conserved mRNA surveillance pathway known as nonsense-mediated decay (NMD).
    I hypothesize that the change in subcellular distribution of ETF1 that I found in C9 models elicits an imbalance between protein translation and RNA degradation through nonsense-mediated decay (NMD), and likely represents a critical step in C9-ALS/FTD cellular pathobiology. Here, I will use mutant C9 cellular models, patient-derived MNs, and ALS patient CNS tissue to: a) determine how ETF1 redistribution relates to C9 neurotoxicity, and b) determine the relationship between ETF1, the axis of protein translation and RNA degradation, and C9 neurotoxicity. The proposed experiments aim to elucidate the link between defective nucleocytoplasmic transport, translational repression and aberrant RNA metabolism. Together, my work will shed light on three key pathomechanisms and potentially identify new viable therapeutic targets for a large proportion of ALS and FTD patients.
    Aim 1: Determine the mechanism of ETF1 redistribution and the consequent role of ETF1 in C9 ALS/FTD neurotoxicity. In previous work, I found that ETF1 translocates to the nucleus of C9-expressing cells and was the strongest genetic modifier of C9 neurotoxicity in in vivo Drosophila studies. Here, I will interrogate how ETF1 becomes redistributed and relates to C9 neurotoxicity in iPSC patient derived and isogenic control MNs. Specifically, I will determine whether ETF1 associates with three C9-related pathologies: sequestration by C9-RNA in nuclear foci, sequestration by toxic C9 dipeptide protein products, and/or deposition within nuclear membrane invaginations. I will quantify the colocalization between nuclear ETF1 and each one of these relevant pathological markers and I will correlate the nucleocytoplasmic distribution of ETF1 protein with the magnitude of each pathology at single-cell resolution. Next, I will test the role of ETF1 in C9 disease pathogenesis by manipulating ETF1 expression in patient MNs and measuring neuronal viability, C9-related pathology and neuronal function. These experiments
    Effective start/end date3/1/202/28/21


    • National Institutes of Health (NOT SPECIFIED)

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