Acta Neuropathologica https://doi.org/10.1007/s00401-019-01999-w ORIGINAL PAPER ADAR2 mislocalization and widespread RNA editing aberrations in C9orf72‑mediated ALS/FTD Stephen Moore1,2 · Eric Alsop3 · Ileana Lorenzini1 · Alexander Starr1 · Benjamin E. Rabichow1 · Emily Mendez1 · Jennifer L. Levy1 · Camelia Burciu1 · Rebecca Reiman3 · Jeannie Chew4 · Veronique V. Belzil4 · Dennis W. Dickson4 · Janice Robertson5 · Kim A. Staats6 · Justin K. Ichida6 · Leonard Petrucelli4 · Kendall Van Keuren‑Jensen3 · Rita Sattler1 Received: 14 September 2018 / Revised: 28 March 2019 / Accepted: 28 March 2019 © Springer-Verlag GmbH Germany, part of Springer Nature 2019 Abstract The hexanucleotide repeat expansion GGG GCC (G4C2)n in the C9orf72 gene is the most common genetic abnormality asso- ciated with amyotrophic lateral sclerosis (ALS) and frontotemporal dementia (FTD). Recent fndings suggest that dysfunc- tion of nuclear-cytoplasmic trafcking could afect 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 (G 4C2)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 fndings 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. Keywords C9orf72 · ALS · FTD · Nucleocytoplasmic mislocalization · ADAR2 · RNA editing · RNA metabolism · iPSC neurons · RNA-seq · Neurodegeneration · Protein accumulation Electronic supplementary material The online version of this article (https ://doi.org/10.1007/s0040 1-019-01999 -w) contains Introduction supplementary material, which is available to authorized users. Amyotrophic lateral sclerosis (ALS) is a fatal, neurodegen- * Rita Sattler erative disorder caused by progressive loss of both upper [email protected] and lower motor neurons, leading to muscle atrophy and 1 Department of Neurobiology, Barrow Neurological Institute, eventually death due to respiratory failure [65]. A GGG GCC 350 W Thomas Road, Phoenix, AZ 85013, USA (G4C2) hexanucleotide repeat expansion (HRE) in the frst 2 School of Life Sciences, Arizona State University, Tempe, intron of the C9orf72 gene represents the most common AZ, USA genetic abnormality in ALS, as well as in frontotemporal 3 Neurogenomics Division, Translational Genomics Research dementia (FTD), to date [19, 53]. Extensive research on the Institute, Phoenix, AZ, USA role of the (G4C2)n repeat expansion in the C9orf72 gene 4 Department of Neuroscience, Mayo Clinic Jacksonville, has led to the proposal of three alternative-but not mutually Jacksonville, FL, USA exclusive-pathogenic mechanisms: (1) protein loss-of-func- 5 Tanz Centre for Research in Neurodegenerative Diseases, tion [4, 8, 11, 17, 27, 58, 62, 66, 70], (2) toxic RNA gain-of- University of Toronto, Toronto, Canada function [2, 21, 40, 41, 47, 54, 73], and (3) toxicity caused 6 Department of Stem Cell Biology and Regenerative by repeat-associated non-AUG initiated (RAN) translation, Medicine, Eli and Edythe Broad Center for Regenerative which leads to the accumulation of dipeptide repeat proteins Medicine and Stem Cell Biology, Keck School of Medicine, (DPRs) [3, 26, 37, 44, 46, 48, 63, 69, 71, 75, 76]. University of Southern California, Los Angeles, CA, USA Vol.:(0123456789)1 3 Acta Neuropathologica A more recent hypothesis suggests defcits in nucleocy- channel, the large majority of RNA editing events have an toplasmic trafcking of mRNAs and/or proteins are caused unknown impact on cellular function [42, 72]. Nevertheless, by the repeat expansion in the C9orf72 gene [9, 10, 24, 37, dysregulation of crucial post-transcriptional modifcations of 70, 73]. Specifcally, defects in nuclear protein import and editing target genes have been associated with sporadic ALS, nuclear mRNA export were observed in diferent models of Alzheimer’s disease, Huntington’s disease, Parkinson’s dis- C9orf72 disease, spanning human postmortem patient tissue, ease, epilepsy, stroke, as well as many types of cancer [12, human induced pluripotent stem cell-diferentiated neurons 42, 56, 57]. Interestingly, a recent study showed widespread (hiPSC-neurons), drosophila, and yeast [10, 29]. In addition, RNA editing changes in postmortem autopsy brain tissue several nuclear pore complex protein members were identi- from persons with autism spectrum disease (ASD), as well fed as genetic modifers of C9orf72 disease pathogenesis as brains from individuals with Fragile X syndrome, sup- [24, 37], or displayed aggregation at the nuclear membrane porting the broad hypothesis that RNA editing dysregulation [73]. These fndings strengthen the critical role of nucleocy- will uncover novel mechanisms of neurological, neurodegen- toplasmic trafcking in neurodegenerative diseases and may erative and/or neuropsychiatric diseases. explain why altered RNA metabolism is one of the major To thoroughly assess the cytoplasmic mislocalization defcits described in C9orf72 disease pathogenesis. of ADAR2 in C9orf72 ALS/FTD, we examined varying Here, we show the nucleocytoplasmic mislocalization of brain regions of human postmortem C9orf72 patient tissue, the RNA binding protein, adenosine deaminase acting on C9orf72 patient-derived hiPSCs diferentiated into motor RNA 2 (ADAR2), which we hypothesize leads to aberrant neurons (hiPSC-MNs), and wild type (WT) mice express- RNA processing in C9orf72 mediated ALS/FTD. ADAR2 ing (G4C2)149 by means of somatic brain transgenesis using is a member of the ADAR protein family, which deaminates adeno-associated virus (AAV) vectors [16]. Fluorescent adenosine in double-stranded RNA transcripts, thus cata- immunolabeling of ADAR2 protein revealed aberrant lytically converting a single nucleotide from an adenosine ADAR2 staining in the neuronal cytoplasm of C9orf72 to an inosine (A-to-I) [5, 18, 38, 45]. ADAR2 is localized ALS/FTD patient postmortem spinal cord and motor cortex exclusively to the nucleus, and has been proposed to accu- tissue, C9orf72 ALS/FTD hiPSC-MNs, and diferent brain mulate in the nucleus during neuronal development [6]. The regions of AAV9-(G4C2)149-transduced WT mice. Interest- catalytic conversion alters the hydrogen bonding capacity ingly, ADAR2 showed no alterations in gene expression lev- of the edited nucleotide and leads to recognition by cellular els in C9orf72 ALS/FTD, in comparison to what had been machinery as guanosine. ADAR1 and ADAR2 (commonly found in sporadic ALS spinal cord motor neurons [33]. To referred to as ADAR and ADARB1, respectively) are the study the functional consequence of the mislocalization two catalytically active proteins in the ADAR family and are of ADAR2, we utilized RNA sequencing of patient tissue highly regulated. Knockout of these proteins leads to epilep- and diferentiated hiPSC-MNs to analyze the whole tran- tic-like seizures and death in mice, emphasizing its critical scriptome for RNA A to I editing changes. We uncovered roles in normal brain development [34, 35, 67, 68]. ADAR3 widespread RNA editing aberrations (hypo- and hyperedit- (alternate nomenclature is ADARB2), while highly brain ing) in varying brain regions of C9orf72 ALS/FTD patient specifc, is thought to be catalytically inactive and its role tissue as well as C9orf72 patient hiPSC-MNs. These RNA remains mostly unknown [13] (see also Online Resource 1). alterations were assigned to 1,526 genes, including genes Although RNA editing defcits are believed to play an involved in ALS related transcripts and the EIF2 signaling important role in neurodegeneration, there have been rela- pathway. These data provide new insights into the contribu- tively few studies investigating A to I editing in disease. tion of RNA A to I editing in neurodegeneration caused by Previously, studying RNA editing was difcult and relied the C9orf72 repeat expansion. In addition, these data dem- on the serendipitous discovery of A to I editing sites [42, onstrate functional consequences of C9orf72 HRE-mediated 61]. The increasing ease and availability of RNA sequenc- nucleocytoplasmic trafcking defects, supporting the critical ing allow for more robust characterization of A to I RNA role of functional trafcking of RNA processing proteins editing using a more quantitative method. These RNA- between the cytoplasm and nucleus. seq based approaches look for mismatched pairing in the transcriptome to the genome allowing for analysis of every RNA editing site in the transcriptome [61]. Utilizing these Methods methods, there have been millions of A to I RNA editing sites reported, suggesting it may be one of the most com- AAV9‑(G4C2) over expressing mouse tissue mon post-transcriptional