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ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD

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A Correction to this article was published on 26 September 2019

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Abstract

The hexanucleotide repeat expansion GGGGCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality associated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent findings suggest that dysfunction of nuclear-cytoplasmic trafficking could affect the transport of RNA binding proteins in C9orf72 ALS/FTD. Here, we provide evidence that the RNA editing enzyme adenosine deaminase acting on RNA 2 (ADAR2) is mislocalized in C9orf72 repeat expansion mediated ALS/FTD. ADAR2 is responsible for adenosine (A) to inosine (I) editing of double-stranded RNA, and its function has been shown to be essential for survival. Here we show the mislocalization of ADAR2 in human induced pluripotent stem cell-derived motor neurons (hiPSC-MNs) from C9orf72 patients, in mice expressing (G4C2)149, and in C9orf72 ALS/FTD patient postmortem tissue. As a consequence of this mislocalization we observe alterations in RNA editing in our model systems and across multiple brain regions. Analysis of editing at 408,580 known RNA editing sites indicates that there are vast RNA A to I editing aberrations in C9orf72-mediated ALS/FTD. These RNA editing aberrations are found in many cellular pathways, such as the ALS pathway and the crucial EIF2 signaling pathway. Our findings suggest that the mislocalization of ADAR2 in C9orf72 mediated ALS/FTD is responsible for the alteration of RNA processing events that may impact vast cellular functions, including the integrated stress response (ISR) and protein translation.

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  • 26 September 2019

    The original article was published erroneously without mentioning the support of the U.S.

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Acknowledgements

We would like to thank the Sattler Laboratory for suggestions and comments towards the manuscript. We would also like to thank all ALS patients and families that have contributed to this research via postmortem brain tissue donations. Specifically, we would like to thank Doug Clough for assistance with data analysis and insightful discussions. We further thank the Target ALS Human Postmortem Tissue Core, New York Genome Center for Genomics of Neurodegenerative Disease, Amyotrophic Lateral Sclerosis Association and TOW Foundation for providing access to their postmortem patient tissue samples collection. We thank both the Target ALS Consortium and the New York Genome Center for access to their RNA sequencing database. In particular, we would like to thank Drs. Lyle Ostrow, Hemali Phatnani and Robert Bowser. We would also like to thank Drs. Sylvia Perez and Elliott Mufson for generously providing us with AD patient postmortem tissue samples. Further thanks go to Dr. Stella Dracheva for helpful discussions throughout this project. This work was support by the National Institute of Neurological Disorders and Stroke, NIH RO1NS085207 (RS); the Muscular Dystrophy Association (RS); the ALS Association (RS); the Robert Packard Center for ALS Research (RS); and the Barrow Neurological Foundation (RS). Part of this work was also made possible by NIH Grant R01NS097850 (JKI), US Department of Defense Grant W81XWH-15-1-0187 (JI), and grants from the Donald E. and Delia B. Baxter Foundation (JKI), the Alzheimer’s Drug Discovery Foundation (JKI) and the Association for Frontotemporal Degeneration (JKI), the Harrington Discovery Institute (JKI), the Tau Consortium (JKI), the Pape Adams Foundation (JKI), the Frick Foundation for ALS Research (JKI), the Muscular Dystrophy Association (JKI), the New York Stem Cell Foundation (JKI), the USC Keck School of Medicine Regenerative Medicine Initiative (JKI), the USC Broad Innovation Award (JKI), and the Southern California Clinical and Translational Science Institute to JKI. JKI is a New York Stem Cell Foundation-Robertson Investigator and a Richard N. Merkin Scholar. We would additionally like to thank the National Institutes of Health/National Institute of Neurological Disorders and Stroke [R35NS097273 (L.P.); P01NS084974 (L.P.); P01NS099114 (L.P.); R01NS088689 (L.P.)]; the Mayo Clinic Foundation (L.P.); the Amyotrophic Lateral Sclerosis Association (L.P.), the Robert Packard Center for ALS Research at Johns Hopkins (L.P.), the Target ALS Foundation (L.P.), and the James Hunter Family ALS Initiative (JR).

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Authors and Affiliations

  1. Department of Neurobiology, Barrow Neurological Institute, 350 W Thomas Road, Phoenix, AZ, 85013, USA

    Stephen Moore, Ileana Lorenzini, Alexander Starr, Benjamin E. Rabichow, Emily Mendez, Jennifer L. Levy, Camelia Burciu & Rita Sattler

  2. School of Life Sciences, Arizona State University, Tempe, AZ, USA

    Stephen Moore

  3. Neurogenomics Division, Translational Genomics Research Institute, Phoenix, AZ, USA

    Eric Alsop, Rebecca Reiman & Kendall Van Keuren-Jensen

  4. Department of Neuroscience, Mayo Clinic Jacksonville, Jacksonville, FL, USA

    Jeannie Chew, Veronique V. Belzil, Dennis W. Dickson & Leonard Petrucelli

  5. Tanz Centre for Research in Neurodegenerative Diseases, University of Toronto, Toronto, Canada

    Janice Robertson

  6. Department of Stem Cell Biology and Regenerative Medicine, Eli and Edythe Broad Center for Regenerative Medicine and Stem Cell Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA, USA

    Kim A. Staats & Justin K. Ichida

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  1. Stephen Moore
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  2. Eric Alsop
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  3. Ileana Lorenzini
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  4. Alexander Starr
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Electronic supplementary material

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Online Resource 1. Structure and Domains of ADAR1, ADAR2, and ADAR3.

(a) ADAR1 can commonly be referred to ADAR, contains a nuclear export sequence, 2 Z-DNA binding domains, 3 double-stranded RNA binding domains, a nuclear localization sequence and a catalytically active deaminase domain. (b) ADAR2 sometimes referred to as ADARb1 only contains a nuclear localization signal and is confined to the nucleus, it has two double-stranded RNA binding domains and a catalytically active deaminase domain. (c) ADAR3 sometimes referred to ADARb2 is the only member of the ADAR family thought to be catalytically inactive. (TIFF 3488 kb)

Online Resource 2. Demographics of Patient Postmortem Tissue, hiPSC Lines, and RNA Samples from the NYGC.

We received C9orf72 ALS/FTD and non-ALS postmortem spinal cord, motor cortex, and cerebellum for immunohistochemistry from both Dr. Janice Robertson as well as Target ALS. hiPSC motor neurons were differentiated in Dr. Rita Sattler’s lab and Dr. Justin Ichida’s lab, these neurons were used for immunocytochemistry, RNA sequencing, and RNA editing analysis. We utilized RNA sequencing of C9orf72 ALS/FTD spinal cord, motor cortex, frontal cortex, and cerebellum performed by the NYGC to analyze and characterize RNA editing in postmortem C9orf72 ALS/FTD patient tissue. (XLSX 19 kb)

Online Resource 3. RNA A to I Editing Sites Analyzed for Editing Aberration.

Complete list of sites determined to display A to I RNA editing that satisfied our requirements of a coverage of 20x across half the non-ALS controls and half the C9orf72 ALS/FTD analyzed. Also included are all differentially edited A to I editing sites and genes in C9orf72 ALS/FTD tissue, hiPSC motor neurons, siRNA treated hiPSC motor neurons and HEK293 cells expressing nuclear or cytoplasmic ADAR2. (XLSX 8938 kb)

Online Resource 4. ADAR2 Mislocalization in Patient Postmortem Spinal Cord Tissue.

(a) Four additional MAP2 positive neurons as examples of normal ADAR2 mislocalization in non-ALS patient postmortem spinal cord. (b) Four additional MAP2 positive neurons as examples of cytoplasmic accumulations of ADAR2, these images display different types of cytoplasmic accumulations that have been observed in patient postmortem tissue. (c-e) ADAR2 accumulations in the dendrite of a spinal motor neuron of a C9orf72 ALS/FTD patient. (f-j) No primary antibody negative control. (TIFF 37158 kb)

Online Resource 5. Co-pathology of ADAR2 and TDP43.

(a-h) two examples of spinal motor neurons that have dual TDP-43 pathology and cytoplasmic ADAR2 accumulation. Quantification is provided as the percentage of neurons that display both ADAR2 and TDP-43 cytoplasmic accumulation (* p < 0.05). (TIFF 9510 kb)

Online Resource 6. ADAR2 Mislocalization in Patient Postmortem Motor Cortex Tissue.

(a) four additional examples of ADAR2 localization in non-ALS control motor cortex. (b) four additional examples of MAP2 positive neurons in C9orf72 ALS/FTD motor cortex, four neurons displaying ADAR2 cytoplasmic accumulations and one neuron displaying typical nuclear ADAR2 distribution. (Bottom) No primary antibody negative control. (TIFF 19370 kb)

Online Resource 7. ADAR2 is Not Significantly Mislocalized in C9orf72 Frontal Cortex.

(a) 3 neurons from non-ALS control patient postmortem tissue displaying typical nuclear ADAR2 staining. (b) 3 neurons from C9orf72 ALS/FTD patient postmortem tissue displaying cytoplasmic accumulations of ADAR2. (c) No significant difference in the percentage of neurons that display cytoplasmic accumulation of ADAR2. (p < 0.10, t-test). (d – h) No Primary Antibody negative control. (TIFF 18119 kb)

Online Resource 8. ADAR2 Mislocalization in hiPSC Derived Motor Neurons.

(a) 10 motor neurons stained for ADAR2 in healthy control hiPSC-MNs displaying typical nuclear localization. (b) 10 C9orf72 ALS/FTD hiPSC differentiated motor neurons displaying cytoplasmic accumulation of ADAR2. (TIFF 35055 kb)

Online Resource 9. ADAR2 Mislocalization in Severe AD Frontal Cortex.

(a-d) cytoplasmic accumulation of ADAR2 in a patient with mild cognitive impairment. (e–h) patients with mild AD present with limited ADAR2 mislocalization. (i-l) Cytoplasmic accumulation of ADAR2 in a patient with severe AD. (m) Quantification of MAP2 positive neurons in the frontal cortex of MCI, mild AD, and severe AD reveal 20.3% (p = 0.49) of neurons in patients with mild cognitive impairment, 21.5% (p = 0.43) of neurons in patients with mild AD and 39.66%(p = 0.04, ANOVA) of neurons in tissue from severe AD patients. (TIFF 8341 kb)

Online Resource 10. Gene Expression of AMPA Receptor Subunits (

GluA1-4) andADAR 1-3in C9orf72 ALS/FTD Human Postmortem Tissue and hiPSC Motor Neurons. Gene expression from RNA sequencing performed by the NYGC on C9orf72 ALS/FTD human postmortem tissue was compared to control human postmortem tissue and gene expression of C9orf72 hiPSC motor neurons was compared to control hiPSC neurons. Fold change gene expression of the AMPA receptor and ADAR family collected from RNA sequencing of C9orf72 ALS/FTD tissues and hiPSC derived motor neurons compared to controls. (**, p = 0.01, t-test) (TIFF 4797 kb)

Online Resource 11. Volcano Plots of RNA Editing Aberrations in Other Human Tissues.

(a-c) Volcano plots showing the spread of all RNA editing aberrations in (a) motor cortex, (b) frontal cortex, and (c) cerebellum. (TIFF 14962 kb)

Online Resource 12. RNA Editing Aberrations in C9orf72 ALS/FTD derived hiPSC Motor Neurons.

RNA sequencing was performed on C9orf72 ALS/FTD hiPSC derived motor neurons by Kim Staats at USC (a) Volcano plot depicting the spread of all RNA editing aberrations in C9orf72 ALS/FTD hiPSC derived neurons. (b) Top hits of all RNA editing aberrations detected in C9orf72 ALS/FTD hiPSC motor neurons. (TIFF 10366 kb)

Online Resource 13. Classification of all Detected RNA Editing Aberrations.

Genomic alignment of RNA A to I editing sites detected in our analysis reveals that the majority of sites are contained in introns, 3′ UTR, non-coding transcripts, relatively few sites are in coding regions that lead to missense variations (1%). (TIFF 5977 kb)

Online Resource 14. siRNA Knockdown of ADAR 1 and 2 Disrupts RNA Editing.

(a) hiPSC motor neurons show successful knockdown after treatment with ADAR1 siRNA in isolation and multiplexed with ADAR2 siRNA. (b) hiPSC motor neurons show successful knockdown after treatment with ADAR2 siRNA in isolation and multiplexed with ADAR1 siRNA. (c) Knockdown of ADAR1 leads to primarily hypo-editing. (d) Knockdown of ADAR2 leads to hyper- and hypo-editing. (e) Knockdown of both ADAR1 and ADAR2 leads to hypo-editing. (TIFF 24682 kb)

Online Resource 15. Tissue Specific and Overlapping RNA Editing Aberrations in C9orf72 ALS/FTD.

Each tab displays a list of genes that are differentially edited in C9orf72 ALS/FTD in each specific tissue and overlapping areas, corresponding to the Venn diagram in Fig. 7a. (XLSX 130 kb)

Online Resource 16. The GluA2 Q/R Site is Mis-Edited in C9orf72 ALS/FTD Postmortem Spinal Cord Tissue.

RNA sequencing analysis of the GluA2 Q/R site reveals modest, yet significant A to I editing differences in C9orf72 ALS/FTD spinal cord and motor cortex, but no differences in frontal cortex and cerebellum. (TIFF 4392 kb)

Online Resource 17. Direct Comparison of Genes with RNA editing Aberrations in Different Models of RNA Editing Dysregulation.

(a) Venn Diagram displaying the common and unique genes which exhibit RNA editing alterations in HEK293T cells overexpressing ΔNLS-ADAR2, C9orf72 ALS/FTD, C9orf72 ALS/FTD hiPSC MNs, and hiPSC MNs treated with ADAR2 siRNA. (b) Genes of interest that are misedited in all different models of abnormal RNA editing. (TIFF 16873 kb)

Online Resource 18. Tissue and Cell Model Specific and Overlapping RNA Editing Aberrations.

Each tab displays a list of genes that are differentially edited in each specific tissue and cell model as well as overlapping areas, corresponding to the Venn diagram in Online Resource 17. (XLSX 203 kb)

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Moore, S., Alsop, E., Lorenzini, I. et al. ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD. Acta Neuropathol 138, 49–65 (2019). https://doi.org/10.1007/s00401-019-01999-w

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