ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72-mediated ALS/FTD
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.
KeywordsC9orf72 ALS FTD Nucleocytoplasmic mislocalization ADAR2 RNA editing RNA metabolism iPSC neurons RNA-seq Neurodegeneration Protein accumulation
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).
- 3.Ash Peter EA, Bieniek Kevin F, Gendron Tania F, Caulfield T, Lin W-L, DeJesus-Hernandez M et al (2013) Unconventional translation of C9ORF72 GGGGCC expansion generates insoluble polypeptides specific to c9FTD/ALS. Neuron 77:639–646. https://doi.org/10.1016/j.neuron.2013.02.004 CrossRefGoogle Scholar
- 19.DeJesus-Hernandez M, Mackenzie Ian R, Boeve Bradley F, Boxer Adam L, Baker M, Rutherford Nicola J et al (2011) Expanded GGGGCC hexanucleotide repeat in noncoding region of C9ORF72 causes chromosome 9p-linked FTD and ALS. Neuron 72:245–256. https://doi.org/10.1016/j.neuron.2011.09.011 CrossRefGoogle Scholar
- 20.Devi L, Ohno M (2014) PERK mediates eIF2α phosphorylation responsible for BACE1 elevation, CREB dysfunction and neurodegeneration in a mouse model of Alzheimer’s disease. Neurobiol Aging 35:2272–2281. https://doi.org/10.1016/j.neurobiolaging.2014.04.031 CrossRefGoogle Scholar
- 26.Gendron TF, van Blitterswijk M, Bieniek KF, Daughrity LM, Jiang J, Rush BK et al (2015) Cerebellar c9RAN proteins associate with clinical and neuropathological characteristics of C9ORF72 repeat expansion carriers. Acta Neuropathol 130:559–573. https://doi.org/10.1007/s00401-015-1474-4 CrossRefGoogle Scholar
- 27.Gijselinck I, Van Langenhove T, van der Zee J, Sleegers K, Philtjens S, Kleinberger G et al (2012) A C9orf72 promoter repeat expansion in a Flanders-Belgian cohort with disorders of the frontotemporal lobar degeneration-amyotrophic lateral sclerosis spectrum: a gene identification study. Lancet Neurol 11:54–65. https://doi.org/10.1016/S1474-4422(11)70261-7 CrossRefGoogle Scholar
- 52.Reed SE, Staley EM, Mayginnes JP, Pintel DJ, Tullis GE (2006) Transfection of mammalian cells using linear polyethylenimine is a simple and effective means of producing recombinant adeno-associated virus vectors. J Virol Methods 138:85–98. https://doi.org/10.1016/j.jviromet.2006.07.024 CrossRefGoogle Scholar
- 66.Waite AJ, Bäumer D, East S, Neal J, Morris HR, Ansorge O et al (2014) Reduced C9orf72 protein levels in frontal cortex of amyotrophic lateral sclerosis and frontotemporal degeneration brain with the C9ORF72 hexanucleotide repeat expansion. Neurobiol Aging 35:1779.e5–1779.e13. https://doi.org/10.1016/j.neurobiolaging.2014.01.016 CrossRefGoogle Scholar
- 69.Wen X, Tan W, Westergard T, Krishnamurthy K, Markandaiah SS, Shi Y et al (2014) Antisense proline-arginine RAN dipeptides linked to C9ORF72-ALS/FTD form toxic nuclear aggregates that initiate in vitro and in vivo neuronal death. Neuron 84:1213–1225. https://doi.org/10.1016/j.neuron.2014.12.010 CrossRefGoogle Scholar
- 72.Yu W, Xu H, Xue Y, An D, Li H, Chen W et al (2018) 5-HT2CR antagonist/5-HT2CR inverse agonist recovered the increased isolation-induced aggressive behavior of BALB/c mice mediated by ADAR1 (p110) expression and Htr2c RNA editing. Brain Behav 8:e00929. https://doi.org/10.1002/brb3.929 CrossRefGoogle Scholar
- 74.Zhang Y-J, Gendron TF, Ebbert MTW, O’Raw AD, Yue M, Jansen-West K et al (2018) Poly(GR) impairs protein translation and stress granule dynamics in C9orf72-associated frontotemporal dementia and amyotrophic lateral sclerosis. Nat Med 24:1136–1142. https://doi.org/10.1038/s41591-018-0071-1 CrossRefGoogle Scholar