Investigating ETF1 at the Interface of Translation Repression and Nonsense-Mediated Decay in C9-ALS/FTD Neurotoxicity
Daley, Elizabeth
Northwestern University at Chicago, Chicago, IL, United States
Search grants from Elizabeth Daley
Search grants from Northwestern University at Chicago
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Amyotrophic Lateral Sclerosis (ALS) and Frontotemporal Dementia (FTD) are devastating neurodegenerative diseases that represent two ends of a single disease spectrum. A GGGGCC hexanucleotide repeat expansion in the first intron of the C9orf72 (C9) gene is the most common genetic cause of ALS/FTD, wherein 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 motor neurons (MNs) exhibit defects in nucleocytoplasmic transport, mRNA metabolism and protein translation that are tightly associated with neurotoxicity, how these defects contribute to neurotoxicity remains unclear. In a previous proteome-wide nucleocytoplasmic localization screen, I discovered that several proteins are redistributed in C9-expressing cells, enriched for function in protein translation and RNA metabolism. Among these, is eukaryotic termination factor I (ETF1), 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 either initiates translation termination to release a nascent polypeptide or initiates nonsense-mediated decay (NMD) to degrade the transcript, depending on mRNA context. 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 NMD, and likely represents a critical step in C9-ALS/FTD cellular pathobiology. To test my hypothesis, the proposed experiments address two specific aims. In the first aim, I will interrogate how ETF1 becomes redistributed and relates to C9 neurotoxicity in iPSC patient derived and isogenic control MNs. 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. 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. In my second aim, I will examine upstream and downstream cellular mechanisms related to the balance of protein translation and mRNA degradation through NMD in iPSC patient derived and isogenic control MNs. I will test the role of ETF1 in mediating the balance between protein translation and mRNA degradation by manipulating ETF1 expression in patient MNs and measuring the efficiency of each pathway. Next, I will test for ETF1-dependent transcriptomic events that may precede neurotoxicity by performing RNA-Seq analysis on MNs with and without NMD ablation. I will examine differentially expressed transcripts between control and patient MNs and differential NMD targets in control and patient MNs. The proposed experiments will elucidate the link between defective nucleocytoplasmic transport, translational repression and aberrant RNA metabolism, shedding light on three key pathomechanisms and potentially identifying new viable therapeutic targets for a large proportion of ALS and FTD patients.
The most common genetic cause of ALS and FTD is a GGGGCC hexanucleotide repeat expansion in the first intron of the C9orf72 gene (C9). In a previous nucleocytoplasmic localization proteomics screen, I identified several redistributed proteins in C9-expressing cells that converge on the pathways of protein translation and RNA metabolism, including ETF1, which is essential to both pathways. While several lines of evidence suggest that C9 motor neurons 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. Completing the proposed experimental aims will elucidate the link between three key pathomechanisms associated with ALS/FTD by investigating the role of ETF1 in C9- related neurotoxicity, highlighting novel points of therapeutic intervention.
National Institute of Neurological Disorders and Stroke (NINDS)
Predoctoral Individual National Research Service Award (F31)
1F31NS117084-01
Special Emphasis Panel (ZRG1)
Gubitz, Amelie
Northwestern University at Chicago
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