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A C. Elegans Model of C9orf72-Associated ALS/FTD Uncovers a Conserved Role

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bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 1 A C. elegans model of C9orf72-associated ALS/FTD uncovers a conserved role 2 for eIF2D in RAN translation 3 4 5 Yoshifumi Sonobe 1, 2, 3, Jihad Aburas 1, 3, 4, Priota Islam 5, 6, Tania F. Gendron 7, André E.X. 6 Brown 5, 6, Raymond P. Roos* 1, 2, 3, Paschalis Kratsios* 1, 3, 4 7 8 * These authors contributed equally to this study. 9 10 Correspondence: 11 R.P.R ([email protected]), P.K ([email protected]) 12 13 Affiliations: 14 1 University of Chicago Medical Center, 5841 S. Maryland Ave., Chicago, IL 60637 15 2 Department of Neurology, University of Chicago Medical Center, 5841 S. Maryland Ave., 16 Chicago, IL 60637 17 3 The Grossman Institute for Neuroscience, Quantitative Biology, and Human Behavior, 18 University of Chicago, Chicago, IL, USA 19 4 Department of Neurobiology, University of Chicago, Chicago, IL, USA 20 5 MRC London Institute of Medical Sciences, London, UK 21 6 Institute of Clinical Sciences, Imperial College London, London, UK 22 7 Department of Neuroscience, Mayo Clinic, Jacksonville, FL, USA 23 1 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 24 ABSTRACT (150 words) 25 26 A hexanucleotide repeat expansion GGGGCC in the noncoding region of C9orf72 is the most 27 common cause of inherited amyotrophic lateral sclerosis (ALS) and frontotemporal dementia 28 (FTD). Potentially toxic dipeptide repeats (DPRs) are synthesized from the GGGGCC sequence 29 via repeat associated non-AUG (RAN) translation. We developed C. elegans models that express, 30 either ubiquitously or exclusively in neurons, a transgene with 75 GGGGCC repeats flanked by 31 intronic C9orf72 sequence. The worms generate DPRs (poly-glycine-alanine [poly-GA], poly- 32 glycine-proline [poly-GP]) and display neurodegeneration, locomotor and lifespan defects. 33 Mutation of a non-canonical translation-initiating codon (CUG) upstream of the repeats blocked 34 poly-GA production and ameliorated disease, suggesting poly-GA is pathogenic. Importantly, 35 eukaryotic translation initiation factor 2D (eif-2D/eIF2D) was necessary for RAN translation. 36 Genetic removal of eif-2D increased lifespan in both C. elegans models. In vitro findings in 37 human cells demonstrated a conserved role for eif-2D/eIF2D in RAN translation that could be 38 exploited for ALS and FTD therapy. 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 2 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 54 INTRODUCTION 55 56 The GGGGCC (G4C2) hexanucleotide repeat expansion in the non-coding region of C9orf72 is 57 the most common monogenic cause of inherited amyotrophic lateral sclerosis (ALS) and 58 frontotemporal dementia (FTD)1, 2, and also causes up to 10% of what appears to be sporadic 59 ALS. The G4C2 repeat expands in patients to hundreds or thousands of copies that vary in 60 number in different cells of the same individual. This genetic insult is thought to cause ALS/FTD 61 pathogenesis via three non-mutually exclusive mechanisms: (1) loss of function due to decreased 62 expression of C9ORF72 protein, (2) RNA toxicity from bidirectionally transcribed sense 63 (GGGGCC) and anti-sense (CCCCGG) transcripts, and (3) proteotoxicity from dipeptide repeat 64 (DPR) proteins produced from the expanded nucleotide repeats3. 65 This study focuses on the molecular mechanisms underlying DPR toxicity. Strong 66 evidence suggests that DPRs are toxic in both cell culture and animal models of disease4, 5. 67 Despite the presence of the expanded G4C2 repeat in the non-coding region of the RNA and the 68 absence of an AUG initiating codon, DPRs are translated in all three reading frames from both 69 sense and anti-sense transcripts through a process called repeat-associated non-AUG (RAN) 70 translation4. Poly-glycine-alanine (poly-GA), poly-glycine-proline (poly-GP), and poly-glycine- 71 arginine (poly-GR) are produced from sense transcripts, whereas poly-proline-arginine (poly-PR), 72 poly-proline-alanine (poly-PA), and poly-GP are generated from antisense transcripts6, 7, 8. DPRs 73 are present in neural cells of patients with C9orf72-associated ALS or FTD, indicating that RAN 74 translation occurs in vivo6, 7, 9, 10. A number of potential causes for DPR toxicity have been 75 proposed, including protein aggregation, impaired nucleocytoplasmic transport, and proteasome 76 dysfunction5; however, the relative contribution of each DPR to disease pathogenesis remains 77 unclear. 3 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 78 Recent in vitro and in vivo studies have identified non-canonical translation initiation 79 factors as critical mediators of DPR production. For example, a recent study showed that the 80 ribosomal protein eS25 (RPS25), which is required for efficient translation initiation at the 81 internal ribosome entry site (IRES) of a number of viral and cellular RNAs11, 12, is also required 82 for RAN translation of poly-GP from G4C2 repeats in yeast, fly, and induced pluripotent human 83 stem cell models13. In addition, the eukaryotic translation initiation factor eIF3F, which is 84 thought to control translation of hepatitis C viral (HCV) RNA through binding to the IRES of the 85 viral genome14, regulates poly-GP production from G4C2 repeats in HEK293 cells15. Moreover, 86 a fly study found that eIF4B and eIF4H are important for RAN translation of poly-GR from 87 G4C2 transcripts16. Lastly, knockdown of eIF2A, an unconventional translation initiation factor 88 used by cells under stress conditions17, 18, 19, had a partial role in poly-GA production in HEK293 89 cells and neural cells of the chick embryo20. Collectively, these and other studies21, 22 suggest that 90 multiple factors are involved in RAN translation of DPRs from G4C2 transcripts; yet, their 91 identity and molecular mechanisms of action remain elusive. 92 To identify novel factors involved in RAN translation of DPRs in vivo, we developed 93 two C. elegans models for C9orf72-associated ALS/FTD. C. elegans provides a powerful system 94 for the study of molecular and cellular mechanisms underlying neurodegenerative disorders due 95 to: (1) its short lifespan (~3-4 weeks), (2) its compact nervous system (302 neurons in total), (3) 96 the ability to study neuronal morphology and function with single-cell resolution, (4) the fact that 97 most C. elegans genes have human orthologs, and (5) the ease of genomic engineering and 98 transgenesis, which enables the rapid generation of worms that express human genes, permitting 99 in vivo modeling of neurodegenerative disorders. 100 In this study, we initially developed C. elegans transgenic animals that carry, under the 4 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. 101 control of a ubiquitous promoter, 75 copies of the G4C2 repeat flanked by intronic C9orf72 102 sequences. Compared to controls, these animals produce DPRs, namely poly-glycine-alanine 103 (poly-GA) and poly-glycine-proline (poly-GP), and display evidence of neurodegeneration, as 104 well as locomotor and lifespan defects. A second set of transgenic worms expressing the 75 105 G4C2 repeats exclusively in neurons displayed similar phenotypes, indicating that DPR 106 production in this cell type is sufficient to cause disease phenotypes. These C. elegans models 107 provide an opportunity to study in vivo the molecular mechanisms underlying DPR production. 108 Through a candidate approach, we identified a novel role for the non-canonical translation 109 initiation factor eif-2D (ortholog of human eIF2D) in RAN translation of G4C2 repeats. Genetic 110 removal of eif-2D significantly decreased DPR production, and improved locomotor activity and 111 lifespan in both C. elegans models. Supporting the phylogenetic conservation of our C. elegans 112 findings, knock-down of eIF2D in human cells led to decreased DPR production. Together, these 113 observations suggest that eIF2D plays an important role in RAN translation of DPRs from G4C2 114 repeats, opening new directions for treatment of C9orf72-associated ALS and FTD. 115 116 117 118 119 120 121 122 123 5 bioRxiv preprint doi: https://doi.org/10.1101/2020.06.13.150029; this version posted June 15, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.
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