1 Title: Dysregulation of RNA editing may help explain pathogenicity mechanisms of 2 congenital Zika syndrome and Guillain-Barre syndrome 3 4 Helen Piontkivska 1, 2, *, Noel-Marie Plonski 2, Michael M. Miyamoto 3, and Marta L. 5 Wayne 3, 4 6 7 1 Department of Biological Sciences and 2 School of Biomedical Sciences, Kent State University, 8 Kent, OH 44242, USA 9 3 Department of Biology, University of Florida, Gainesville, FL 32611, USA 10 4 Emerging Pathogens Institute, University of Florida, Gainesville, FL 32611, USA 11 12 * Corresponding Author 13 Helen Piontkivska 14 Department of Biological Sciences 15 Kent State University 16 Kent, OH 44242, USA 17 Telephone: (330) 672-3620 18 Fax: (330) 672-3713 19 E-mail: [email protected] 20

1

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 21 Abbreviations: 22 ADAR, Adenosine Deaminases Acting on RNA 23 AGS6, Aicardi-Goutieres syndrome 6 24 ALS, amyotrophic lateral sclerosis 25 CDC, Centers for Disease Control and Prevention 26 CDI, inhibitory Ca2+-feedback 27 CMV, cytomegalovirus 28 CZS, Congenital Zika syndrome 29 GBS, Guillain-Barre syndrome 30 IFN, interferon 31 ISGs, interferon-stimulated genes 32 ISREs, interferon-sensitive response elements 33 WNV, West Nile virus 34 Zika virus (ZIKV) 35

2

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 36 Abstract: 37 Many Zika virus (ZIKV) pathogenesis-related studies have focused primarily on virus-driven 38 pathology and neurotoxicity, instead of considering the possibility of pathogenesis as an 39 (unintended) consequence of host innate immunity: specifically, as the side-effect of an 40 otherwise well-functioning machine. The hypothesis presented here suggests a new way of 41 thinking about the role of host immune mechanisms in disease pathogenesis, focusing on 42 dysregulation of post-transcriptional RNA editing as a candidate driver of a broad range of 43 observed neurodevelopmental defects and neurodegenerative clinical symptoms in both infants 44 and adults linked with ZIKV infections. We collect and synthesize existing evidence of ZIKV- 45 mediated changes in expression of adenosine deaminases that act on RNA (ADARs), known 46 links between abnormal RNA editing and pathogenesis, as well as ideas for potential 47 translational applications, including genomic profile-based molecular diagnostic tools and/or 48 treatment strategies. 49 50

3

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 51 Introduction: ZIKV as a causative agent for an emergent public health crisis 52 Following discovery of a link between Zika Virus (ZIKV, a flavivirus) infection in pregnancy 53 and microcephaly in infants in 2015 [1], this public health crisis continues to present a significant 54 hazard to human health. The Global Disease Burden study estimated that ~7·60 million ZIKV 55 infections occurred in 2016, most of which were in Latin America and the Caribbean [2]. While 56 the vast majority of these cases were asymptomatic infections, with only a small portion 57 resulting in Zika-attributable Guillain-Barré syndrome (GBS) and congenital Zika syndrome 58 (CZS) births, infants born with the CZS will face life-long profound disability [3] and their 59 families face extreme economic burdens as well. Already over 6900 pregnancies with ZIKV 60 infected mothers have been recorded in the US and the US territories as of July 17, 2018 (CDC), 61 from which over 300 infants were born with CZS. Nor are adults immune: known consequences 62 of ZIKV infection in adults include GBS [4, 5], a debilitating peripheral neuropathy that is 63 potentially life-threatening and costly to treat [6]. However, there is currently no available vaccine 64 for ZIKV, nor antiviral treatments. The efficacy of prevention strategies, such as protecting 65 oneself from mosquito bites or sexual abstinence during confirmed infection, is compromised by 66 the frequently asymptomatic nature of ZIKV infection [7] as well as the wiliness of mosquitoes. 67 In the absence of effective prevention, insights into the mechanisms behind ZIKV-associated 68 pathogenesis are critical to the development of treatments that can ameliorate potential ZIKV 69 sequelae. 70 71 What is congenital Zika syndrome (CZS)? 72 The link between ZIKV infection in pregnancy and congenital Zika syndrome (CZS) is a 73 particularly pressing concern, in part because we do not yet fully understand the extent of 74 neurodevelopmental defects – particularly those not obvious at birth - associated with it [8]. CZS 75 includes a broad range of neurodevelopmental defects beyond microcephaly [9, 10], including 76 ocular [11] and auditory defects [12]. The overall risk of ZIKV-linked birth defects has been 77 estimated at up to ~15% for infections in the 1st trimester [13], potentially impacting thousands of 78 infants in the US and US territories alone. However, the risk extends into infections that occur 79 later in pregnancy [14], and central nervous system defects may be present without microcephaly 80 [15, 16]. A recent CDC report showed an increase in prevalence of birth defects potentially 81 consistent with CZS in US areas with documented local ZIKV transmission [8]. Flash flooding

4

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 82 accompanying major hurricanes (such as those associated with 2017 hurricanes Harvey, Irma 83 and Maria in TX, FL and PR, respectively) and the diverse set of ZIKV transmission routes, 84 including non-vector-borne transmission via sexual contact and bodily fluids, such as sweat, 85 saliva or tears [17], likely expand the portion of ZIKV-exposed US population, similar to the 86 observed expansion of West Nile virus (WNV) infections following hurricane Katrina in 2005 87 [18]. 88 89 ZIKV-mediated pathogenesis? 90 Despite major efforts focused on ZIKV research over the last three years, our understanding of 91 ZIKV-mediated pathogenesis remains limited, in part because the symptoms and sequelae of 92 ZIKV infection span from asymptomatic to major birth defects in infants, as well as severe 93 adverse outcomes in some adults. Below we highlight a broad variety of clinical symptoms and 94 outcomes associated with ZIKV infection, with the explicit purpose of identifying connectedness 95 of ZIKV sequelae across different life stages. For in-depth comprehensive surveys focused on 96 fetal or adult outcomes, we refer the reader to the following studies [9, 19, 20]. 97 98 What do we know so far? On the one hand, most ZIKV infections in adults are benign and self- 99 limiting [21], with as many as 80% having asymptomatic infections [7]. It is worth noting that 100 these asymptomatic carriers are still capable of infecting others [19]. Moreover, the risk of 101 neurodevelopmental fetal defects appears to be similar between symptomatic and asymptomatic 102 pregnant women [19]. While relatively few pediatric-focused studies of ZIKV infections are 103 available, the available evidence suggests that the majority of acute ZIKV infections in children 104 are similarly mild in nature to adult ones [22], although recent reports have begun to associate 105 ZIKV with other clinical issues, such as acute abdominal symptoms [23] or post-infection 106 vasculitis resulting in pediatric stroke [24]. 107 108 During pregnancy, ZIKV poses a substantial risk of fetal neurodevelopmental defects, resulting 109 in profound long-term disability and immense costs of future care, influencing individuals and 110 communities [21, 25]. The full spectrum of CZS is still being elucidated, coupled with concerns 111 that ZIKV-related outcomes are underreported, particularly when ZIKV infections result in later 112 onset of developmental delays and learning disabilities without visible microcephaly [26], or

5

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 113 includes other systemic abnormalities that manifest postnatally, such as dysphagia [27], seizures 114 [28], and hearing loss [29]. For example, recent evidence suggests that ZIKV infection in 115 pregnancy may have been linked with microcephaly and other neurodevelopmental defects in the 116 US (Hawaii) as early as 2007, with several (likely travel-related) births in 2009 and 2011 being 117 definitively linked to maternal ZIKV-positive antibodies [30]. In part due to lack of long term, 118 longitudinal studies, the long-term consequences of an in utero ZIKV infection are poorly 119 understood, particularly in infants lacking visible symptoms of CZS and/or from asymptomatic 120 infections; however, there are reported cases of CZS being diagnosed post-birth because the 121 infants showed neurodevelopmental delays and developed postnatal microcephaly [31, 32]. Recent 122 abnormal imaging from normocephalic infants from ZIKV-positive pregnancies [33] and pregnant 123 pigtail macaques [16] further underscores challenges associated with detection of ZIKV-mediated 124 brain injury that may appear subtle, yet could have profound neurocognitive and psychiatric 125 consequences later. 126 127 In addition to causing severe neurodevelopmental defects in prenatal infections, ZIKV infections 128 have also been linked to severe neurotoxicity in some adults, including Guillain-Barre Syndrome 129 (GBS) [4, 5, 34]. GBS is an acute paralytic neuropathy, an inflammatory disorder of the peripheral 130 nervous system, and is one of the leading causes of acute flaccid paralysis worldwide [35, 36]. 131 Despite recent progress in immunotherapy [37], GBS remains a life-threatening condition, often 132 requiring complex and expensive care [38]. Up to 20% of GBS cases result in severe disability, or 133 even death [36], while many other patients experience ongoing motor or sensory issues, fatigue 134 and pain [39]. Compared to other morbidities, ZIKV-triggered GBS represents a very small 135 fraction of the global disease burden [3]. Nonetheless, GBS is associated with substantial risks 136 (including death) as well as financial costs for affected individuals [21]. Moreover, there are 137 challenges with early GBS diagnosis, particular in atypical presentations and/or pediatric patients 138 [38]. For example, cranial nerve enhancement - thought to be a part of GBS in pediatric patients 139 [40] - was recently described from an imaging study of seemingly normal infant born after ZIKV- 140 positive pregnancy. However, not all infants from ZIKV-positive pregnancies receive the 141 recommended postnatal imaging [13, 41]. Moreover, the molecular mechanisms behind GBS 142 remain poorly understood [5, 42]. In addition to its association with GBS, the list of neurological

6

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 143 and other consequences of ZIKV infection in adults and children – including 144 meningoencephalitis, paresis, and vasculitis - keeps growing [11, 24, 43]. 145 146 In other words, ZIKV infection appears to be associated with a broad and disparate range of 147 clinical and subclinical symptoms and outcomes. We would like to propose a novel hypothesis 148 that can help explain the multitude of diverse ZIKV infection outcomes both pre- and post- 149 natally. While it requires experimental validation, this hypothesis is firmly grounded in evidence 150 from multiple areas of study, and allows us to make connections between previously un- 151 connectable observations. It also enables us to generate new, testable hypotheses that may enable 152 development of effective treatment and prevention strategies. 153 154 Hypothesis: Connecting the dots 155 We now build the case that ZIKV-mediated pathogenicity – both fetal and adult - is due to 156 dysregulation of the activity of the RNA-editing enzymes, ADARs (Adenosine Deaminases 157 Acting on RNA), which in turn leads to inappropriate levels of post-transcriptional editing of 158 key proteins involved in metabolism of calcium and other ions, and homeostasis in neural 159 cells, thereby triggering a cascade of diverse neurological effects (Figure 1). We propose that 160 viral-triggered dysregulation of ADAR editing results in a handful of amino acid changes in key 161 neural proteins involved in ion transport and neurotransmitter metabolism. Either an excess or a 162 paucity of developmentally-appropriate edits lead to changes in ion permeability and excitability, 163 ultimately resulting in excitotoxicity and neuronal demise [44-46]. 164 165 What are ADARs? 166 ADARs (adenosine deaminases that act on RNA) are RNA editing enzymes that deaminate 167 adenosines (A) to produce inosines (I) within double-stranded RNAs [47]. These A-to-I changes 168 are translated as A-to-G substitutions in mRNA transcripts, thereby modulating proteome 169 diversity and expression by introducing nonsynonymous (amino acid changing) substitutions or 170 splice site changes, and serving as a mechanism of dynamic and nuanced post-transcriptional 171 regulation of gene expression [48]. There are three mammalian ADAR loci - ADAR, ADARB1 172 and ADARB2 [49]. Only ADAR and ADARB1 have proven deaminase activity [50], while 173 ADARB2 is thought to play a regulatory role through competition with other ADARs for

7

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 174 substrate [51]. It should be noted that expression and editing activities of one family member can 175 be influenced by expression and editing activities of the other ADARs [52, 53]. ADARs play a 176 particularly prominent role in the nervous system as the majority of ADAR editing target genes 177 are expressed in the nervous system, including brain [49]. 178 179 ADAR expression is elevated in ZIKV-infected cells that exhibit cytopathic effects 180 This piece of evidence comes from a recent study that examined consequences of ZIKV infection 181 in three cell lines of primary human neural stem cells originally derived from three individual 182 fetal brains [54]. Two lines (K048 and K054) exhibited signs of reduced neuronal differentiation 183 (which is consistent with other ZIKV infected brain studies, including in vivo), while such signs 184 were not noticed in the third line, G010 [54]. Differential gene expression analysis showed global 185 similarities in transcriptome changes in K048 and K054, which were not shared with G010, 186 particularly among genes involved in innate immune pathways [54]. Their supplementary list of 187 differentially expressed genes reported that ADAR gene expression increased significantly in 188 K048 and K054 cells in response to ZIKV infection, while remained the same in G010 cells 189 (with 1.63 and 1.1-fold change, with the false discovery rate P values < 0.05 in K048 and K054, 190 but not in G010, respectively). In other words, only the cells with the observable cytopathic 191 effects exhibited an increase in ADAR expression (of course, the relationship between 192 expression and editing may not necessarily be linear [55]). While no significant change in 193 expression of ADARB1 and ADARB2 was reported, it should be noted that changes in the 194 ADAR gene expression can interfere with specific editing activity of the other ADARB1 enzyme 195 [52, 56]. 196 197 Our re-analysis of the McGrath et al. (2017) transcriptome data [54] shows that ADAR editing 198 activity per se, not merely expression of ADAR, appears to be higher in the cells with more 199 prominent cytopathic effects. Using the SnpEff variant annotation tool implemented in GATK, 200 we tallied nucleotide changes between mock and ZIKV-infected cells and found that ZIKV- 201 infected samples harbor significantly higher numbers of A-to-G-changes (i.e., changes that can 202 be attributed to ADAR activity) in K048 cells compared to mock-infected cells (Mood’s median 203 test, P = 0.014). However, no significant differences were observed in K054 cells, where 204 cytopathic effects were not as prominent as in K048, and in G010 cells (P > 0.05). An alternative

8

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 205 variant calling was performed using GATK Haplotype caller, and the distribution of missense A- 206 to-G variants was examined in K048 cells, which had the highest read depth. Of the genes with 207 variants not shared between mock and ZIKV-infected cells (in other words, those less likely to 208 be genetic polymorphisms), there was a significantly higher fraction of genes with variants in at 209 least 2 samples in ZIKV-infected cells compared to mock-infected cells (10 versus 70 in mock 210 cell, and 59 versus 127 in ZIKV-infected cells), including well-known editing target GRIA3. 211 However, these preliminary analyses have significant limitations, one of which is the significant 212 difference in sequencing depth between samples (K048 had twice as many reads as the other two 213 cell lines) Thus, these preliminary findings offer only an initial glimpse into the potential effects 214 of ADAR editing in ZIKV infection, and require further experimental confirmation, including 215 careful validation of editing changes (if any) of specific key neural genes and/or whether 216 potential differences of normal editing patterns among individuals may be contributing to 217 pathogenesis mechanisms. Nonetheless, these preliminary findings are consistent with the 218 proposed hypothesis about the role of dysregulated editing changes in ZIKV infections; although 219 it remains to be determined whether these changes result from the redirection of ADAR p150 220 editing activity away from the host editome to ZIKV, or through changes in normal host editing 221 patterns because of increased ADAR p150 editing activity and/or interference between different 222 ADARs. 223 224 Appropriate ADAR editing is required for proper neuronal development 225 Severe CNS phenotypes are observed in ADAR knockouts and ADAR deficiency or loss of 226 function studies (e.g., [57]), including embryonic lethality [58], epilepsy, motor neuron death [59], 227 microcephaly and neural tube defects [60], as well as shortened life-span in worms [61]. Likewise, 228 decreased ADARB1 editing activity plays a role in the pathogenesis of a common motor neuron 229 disease, sporadic amyotrophic lateral sclerosis (ALS) [62], and various brain and neurological 230 cancers [52, 63]. 231 232 Mutations and polymorphisms in ADAR loci, such as the ADARB2 locus polymorphisms, have 233 been implicated in complex neurological phenotypes such as migraines [64], autism [65], and 234 genetic epilepsy [66]. Likewise, mutations in the ADAR gene that affect efficacy of RNA binding 235 and/or of A-to-I editing [67] have been associated with a rare human Mendelian disease, Aicardi-

9

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 236 Goutieres syndrome (AGS6) [68]. In its typical form, the damage caused by AGS resembles 237 neurodevelopmental pathology due to in utero infection [69], and is associated with an increased 238 level of interferon and upregulation of interferon-stimulated genes [70]. 239 240 ADAR editing is enriched in the nervous system [71]. One prominent example of ADAR editing 241 in the brain is the Q/R site of the glutamate receptor subunit GRIA2 (also known as GluA2), 242 where virtually complete (~100%) editing [72] of genomic glutamine codon 607 (Q607) to 243 arginine (R) results in multiple changes of the receptor properties, including cellular permeability 244 to calcium (Ca2+) [73]. Underediting of Q607R GRIA2 site (e.g., in the case of ADARB1 245 deficiency) results in increased influx of calcium, and neuronal death [74]. GRIA2 has an 246 additional ADAR editing site, R764G, which is edited mostly by ADAR, and results in different 247 electrophysiological properties. Editing at R764G gradually increases during neural 248 development, with the edited isoform fraction in adult brains ranging from 50% to 85% [75]. 249 250 Another well-documented ADAR editing event (via ADARB1) results in I-to-M, Q-to-R, and Y- 251 to-C amino acid changes in the mammalian calcium voltage-gated channel subunit alpha1 D

252 channel CACNA1D (also known as CaV1.3) within the IQ domain, a calmodulin-binding site 253 responsible for regulating sensitivity to inhibitory Ca2+-feedback (CDI) [76]. Edited CACNA1D 254 proteins are expressed in both brain tissue and the surface membranes of primary neurons; 255 neurons with edited CACNA1D proteins show decreased CDI, as part of likely selective fine- 256 tuning of Ca2+ feedback for low-voltage activated Ca2+ influx [76]. 257 258 GRIA2 and CACNA1D are not the only ADAR editing targets critical for neural function. 259 Functionally relevant editing targets include other glutamate receptors, such as glutamate 260 receptor subunit GRIA3 (also known as GluA3) [77], receptors for other neurotransmitters, 261 including excitatory and inhibitory neurotransmitters, such as serotonin 2C receptor 5HT2CR [78] 262 and the alpha3 subunit of gamma-aminobutyric acid (GABA) receptor (Gabra3) [79], respectively, 263 as well as ion channels (e.g., voltage-dependent potassium channel KCNA1 [80]), and 264 microRNAs [81]. Disruption of editing of these targets is known to lead to changes in excitability, 265 kinetics and other crucial functions of these receptors and ion channels, ultimately resulting in 266 neurodevelopmental defects, decreased proliferation and neuronal death [45]. In other words,

10

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 267 seemingly minor amino acid changes in these proteins have the potential to significantly change 268 electrophysiology, calcium permeability and metabolism, among other properties, in neural (and 269 likely glial) cells, thus initiating a broad array of downstream neurophysiological changes such 270 as those observed in CZS and GBS. Indeed, because of glutamate’s fundamental role as a major 271 excitatory neurotransmitter in as many as 80 to 90% of brain neurons, most neurons and many 272 glial cells have glutamate receptors in their membranes [82]. Glutamatergic signaling also plays a 273 critical role in regulation of neural development, including neurotoxic effects of increased 274 calcium permeability due to, for example, inappropriate editing of GRIA2 [83]. 275 276 These examples offer a potential common explanation to the breadth of the observed clinical 277 symptoms and defects in both fetal and adult ZIKV infections, where variation in specific 278 manifestations across developmental stage at the time of infection can be attributed to the 279 nuanced spatio-temporal regulation of ADAR editing – and/or its infection-mediated 280 dysregulation, particularly in neural cells [84]. 281 282 ADAR is part of the antiviral response 283 Expression of ADARs, specifically, the ADAR1 p150 isoform, is regulated by interferon as part 284 of cell-autonomous immunity [85], with ADAR editing playing a role in an anti-viral activity, as 285 part of the innate immune response [86]. Recent experimental ZIKV studies have shown that the 286 interferon (IFN) type I response plays a role in ZIKV infection, that is that in human ZIKV 287 infections, expression of IFN cytokines increases, followed by increased expression of IFN- 288 regulated genes, including ADARs [54]. Because the interactions between ZIKV and IFN pathway 289 activation are complex (e.g., may include mechanisms that suppress IFN signaling, such as 290 through action of viral NS5 protein [87], or by targeting STAT1 [88] and STAT2 [89]), and may 291 vary between ZIKV strains and/or specific variants of human genes or combination of the two 292 phenomena, that may help explain why not all ZIKV-positive pregnancies – or even fetuses from 293 the same pregnancy – show neurodevelopmental defects [90]. 294 295 Our recent work showed that ADAR editing plays a role in the molecular evolution of RNA 296 viruses [91], including ZIKV [92]. These results indicate that ADAR editing is active during ZIKV 297 infection. While it remains to be determined whether observed ADAR editing footprints were

11

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 298 due to ADAR action in the mosquito or mammalian host (or both) [92], studies of the mammalian 299 immune response support the hypothesis that ADAR (specifically, the IFN-induced ADARp150 300 isoform) is activated in ZIKV infections. 301 Dysregulation of ADAR editing could potentially contribute to ZIKV pathogenesis via decreased 302 host editing (i.e., if ADAR editing activity is re-directed from the host editome to ZIKV) or via 303 increased host editing due to elevated expression of ADAR p150. ADAR p150 might directly 304 alter editing patterns, or do so indirectly if a stoichiometric change between ADAR p150 and 305 other ADARs modifies editing patterns. As noted previously, in mice both ADAR and ADARB1 306 knockouts can be fatal, e.g., due to underediting of key transcripts such as GRIA2 [58, 74, 93]. There 307 is mounting evidence that off-target editing by ADAR is also deleterious in humans [94]. 308 309 Thus, one of the potential explanations for mechanisms of congenital Zika syndrome may be 310 attributed to the disruption of editing and regulatory function of ADAR genes due to ZIKV- 311 mediated IFN pathway activation (Figure 1). Specifically, physiological consequences of ADAR 312 editing dysregulation may be particularly critical for key synaptic signaling proteins, which in 313 turn result in shifts in calcium metabolism and homeostasis in the developing brain. For example, 314 changes in ADAR editing patterns can be linked to changes in (i) calcium permeability, and thus, 315 channel conductivity of GRIA2 and GRIA3 [95], and (ii) changes in how multiple isoforms of 5- [96] 316 HT2C – that have different binding affinities and functional potencies of agonists – are 317 distributed within the brain. Appropriate ADAR editing thus could influence synaptic 318 transmission, as well as regulation of cell proliferation and neuronal differentiation [97], in [98] 319 proliferative zones of the developing brain . The changes of 5-HT2C editing have been linked 320 to mental disorders and suicide [99], although the full extent of editing consequences remains to 321 be elucidated [100]. Dysregulation of ADAR activity can also be potentially connected to the 322 neurodegeneration observed in GBS, where IFN-triggered elevated expression of ADAR results 323 in abnormal editing of proteins involved in synaptic signaling, thereby changing the ion 324 homeostasis of neurons, and hence, triggering the subsequent cascade of effects, similar to what 325 is observed in CZS. 326 327 328

12

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 329 Conclusions and prospects 330 We posit that the experimental evidence from both the CZS and the GBS cases available so far is 331 consistent with the expectations of the hypothesis presented here. However, a causal link 332 between ADAR editing and subsequent neurodevelopmental or neurodegenerative sequelae in 333 CZS or GBS remains to be demonstrated. A critical first step would be to document ADAR 334 expression and ADAR editing levels in ZIKV infection in pregnancy and GBS, including self- 335 regulation aspects of ADAR loci themselves [101], as well as expression and activity of calcium 336 homeostasis and metabolism proteins. Simultaneously, we need to elucidate the patterns and 337 regulatory processes involved in nuanced spatio-temporal changes in ADAR editing in brain 338 development during normal as well as ZIKV-positive pregnancies, and how these changes are 339 linked to observed clinical symptoms, because we still have only a limited understanding of the 340 complex regulation of ADAR editing across cells, tissues, developmental stages and individuals. 341 The observation of differential editing activity and its ramp-up during brain development [102] 342 must be supplemented by deeper understanding of how editing is regulated during different 343 stages of brain development and/or different locations in the CNS. This can be greatly aided by 344 the recently developed single-cell transcriptomics approaches [103]. 345 Understanding how ADAR editing influences brain development and function may lead to 346 development of treatment strategies that can be applied to newly infected ZIKV-positive 347 pregnant women, as well as to infants born or later diagnosed with CZS. It is likely that in utero 348 treatment strategies would vary depending on the stage of pregnancy (stemming from the 349 changing role of ADAR-edited proteins) at different stages of prenatal development. Treatment 350 strategies might range from (tailored) applications of existing immunotherapies to regulate 351 expression of interferon and/or other players of the cell-autonomous immunity pathway that are 352 upstream regulators of ADAR expression [85], to development of biomarkers of editing as well as 353 compounds targeting the editing process itself [104]. Similarly, we need to better understand the 354 role of ADAR editing and the functional implications of it (including quantitative aspects of how 355 much gets edited during any given time) on its respective targets during neurodegeneration of 356 GBS. 357 358 There might be an opportunity to intervene in the regulation of ADAR editing during/shortly 359 after ZIKV infection with the aim of preventing/diminishing the spectrum of ZIKV

13

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 360 pathogenic/neurodevelopmental sequelae. For example, during pregnancy, within two-three 361 weeks of diagnosed ZIKV infection (i.e., within two-three weeks after the initial rash appears 362 [105]) a short course of drugs leading to the suppression of the type I interferon pathway can be 363 expected to result in down-regulation (or prevention of activation) of fetal ADAR activity. This 364 can be achieved by administering treatments currently used, e.g., to treat lupus [106]. Decisions to 365 use such treatments will be complicated by the fact that not all pregnant women exhibit clinical 366 signs of ZIKV infection [19]. However, the timing of asymptomatic infections can be inferred if 367 there is a finite window of exposure, by definition problematic in ZIKV-endemic areas. A similar 368 course of treatment can be applied if the early GBS symptoms were to appear a few weeks post- 369 ZIKV infection (similar to that in HCV infections, [107]). Furthermore, the role of the recently 370 discovered member of the type III interferon pathway in the immune response needs to be 371 considered too [108]. Additionally, development of treatments specifically aimed at the reversal 372 of deleterious editing effects can also be pursued, e.g., via a combination of short-term 373 stimulation of expression of ADAR editing targets, administration of antagonists to the edited 374 protein versions and/or suppression of ADAR editing. 375 376 The same molecular mechanism postulated here to explain ZIKV pathogenesis may underlie

377 other conditions. For example, the increased editing of the calcium channel CaV1.3 has been 378 observed in dopamine-containing neurons affected in Parkinson’s disease, a second most 379 frequent neurodegenerative disease in the world [109], in which degeneration and death of these 380 (‘dopaminergic’) neurons is associated with motor symptoms of the disease [110]. Currently we 381 have neither a cure for it, nor a firm understanding of its underlying mechanisms [110, 111], 382 although it appears that (some form of) calcium dysregulation likely plays a prominent role [112]. 383 This dysregulation would result in oxidative and proteolytic stress and neuroinflammation that 384 may lead to mitochondrial dysfunction or vice versa [112, 113]. Evidence suggests that even in early 385 stages of Parkinson’s the calcium homeostasis is disturbed, in part attributable to what appears to [114] 386 be a shift in cells reliance on (editable) CaV1.3 in lieu of CaV1.2 . It is unclear whether the

387 changes in channel kinetics are due to CaV1.3 editing, and/or differential expression of [115] 388 alternatively spliced isoforms CaV1.3 , as the study lacked the needed antibody resolution 389 [114]. In either case, this scenario offers an explanation of yet another neurodegenerative condition 390 (in addition to GBS) with unclear aetiology that could potentially be linked to possible changes

14

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 391 in ADAR editing patterns induced by prior infections (e.g., [116]), likely of minor viral variety, 392 which have mild to subclinical symptoms that nonetheless have the potential to (over)activate 393 ADARs as part of cell-autonomous immunity. Could changes in edited calcium channels also 394 explain the brain calcification observed following prenatal CMV (a herpesvirus) infection [117]? 395 Interestingly, brain calcification is also a common finding in CZS infants, born with or without 396 microcephaly [32], again suggesting a role for ADAR editing in ZIKV pathogenesis. 397 Ultimately, we would like to call for systematic, concerted and broad efforts to understand the 398 nuances of spatio-temporal distribution of ADARs and associated editing activity throughout the 399 brain and primary neurons, both in pregnancy and postnatally, as well the influence of viral 400 infections (including ZIKV) on such activity and subsequent clinical consequences. 401 402 Funding 403 This work was partially supported Kent State University Research Council Seed Award, and 404 Brain Health Institute Pilot Award to HP. MLW was supported by the National Institutes of 405 Health grant number GM083192. 406 407

15

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 Figure 1. A

B

408

16

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 409 410 Figure 1. Simplified (A) and detailed (B) summaries of our new hypothesis linking ZIKV 411 infection to congenital Zika syndrome and Guillain-Barre syndrome. (B) Overview of ZIKV 412 infection and the proposed mechanistic model of ZIKV-mediated pathogenesis. Upon infection, 413 ZIKV activates Toll-like-Receptor 3 (TLR3), which in turn activates the interferon (IFN) 414 pathway [118, 119]. Interferon-sensitive response elements (ISREs) upregulate interferon-stimulated 415 genes (ISGs), including the ADAR p150 isoform [85]. Thus, in addition to ADAR editing of viral 416 RNA as part of cell-autonomous immunity, ZIKV-driven activation of ADARp150 may also 417 lead to dysregulation of ADAR editing within host cells. These changes in levels of ADAR 418 editing may be due to increased abundance of ADAR p150 enzyme copies and/or due to 419 interference between ADAR p150 and other ADARs, e.g., through substrate competition with 420 ADARB1 [52, 101]. In turn, ZIKV-induced changes in editing patterns of host transcripts – such as 421 neurotransmitter receptors and transporters - can result in physiological changes in host proteins, 422 such as changes in ion permeability and excitability as illustrated by GRIA2 and GRIA3 [73, 77], in 423 turn leading to excitotoxicity and neuronal demise [45, 46]. Artistic drawing is by Peggy Muddles, 424 @vexedmuddler.

425

17

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 426 References used 427 428 [1] S. A. Rasmussen, D. J. Jamieson, M. A. Honein, L. R. Petersen, N Engl J Med 2016, 374, 429 1981. 430 [2] GBD 2016 DALYs and HALE Collaborators, Lancet 2017, 390, 1211. 431 [3] GBD 2016 DALYs and HALE Collaborators, Lancet 2017, 390, 1260. 432 [4] E. Oehler, L. Watrin, P. Larre, I. Leparc-Goffart, S. Lastere, F. Valour, L. Baudouin, H. 433 Mallet, D. Musso, F. Ghawche, Euro Surveill 2014, 19, pii: 20720. 434 [5] V. M. Cao-Lormeau, A. Blake, S. Mons, S. Lastere, C. Roche, J. Vanhomwegen, T. Dub, 435 L. Baudouin, A. Teissier, P. Larre, A. L. Vial, C. Decam, V. Choumet, S. K. Halstead, H. J. 436 Willison, L. Musset, J. C. Manuguerra, P. Despres, E. Fournier, H. P. Mallet, D. Musso, A. 437 Fontanet, J. Neil, F. Ghawche, Lancet 2016, 387, 1531. 438 [6] B. van den Berg, C. Walgaard, J. Drenthen, C. Fokke, B. C. Jacobs, P. A. van Doorn, Nat 439 Rev Neurol 2014, 10, 469. 440 [7] M. R. Duffy, T. H. Chen, W. T. Hancock, A. M. Powers, J. L. Kool, R. S. Lanciotti, M. 441 Pretrick, M. Marfel, S. Holzbauer, C. Dubray, L. Guillaumot, A. Griggs, M. Bel, A. J. Lambert, 442 J. Laven, O. Kosoy, A. Panella, B. J. Biggerstaff, M. Fischer, E. B. Hayes, N Engl J Med 2009, 443 360, 2536. 444 [8] A. Delaney, C. Mai, A. Smoots, J. Cragan, S. Ellington, P. Langlois, R. Breidenbach, J. 445 Fornoff, J. Dunn, M. Yazdy, N. Scotto-Rosato, J. Sweatlock, D. Fox, J. Palacios, N. Forestieri, 446 V. Leedom, M. Smiley, A. Nance, H. Lake-Burger, P. Romitti, C. Fall, M. V. Prado, J. Barton, J. 447 M. Bryan, W. Arias, S. V. Brown, J. Kimura, S. Mann, B. Martin, L. Orantes, A. Taylor, J. 448 Nahabedian, A. Akosa, Z. Song, S. Martin, R. Ramlal, C. Shapiro-Mendoza, J. Isenburg, C. A. 449 Moore, S. Gilboa, M. A. Honein, MMWR Morb Mortal Wkly Rep 2018, 67, 91. 450 [9] C. A. Moore, J. E. Staples, W. B. Dobyns, A. Pessoa, C. V. Ventura, E. B. Fonseca, E. 451 M. Ribeiro, L. O. Ventura, N. N. Neto, J. F. Arena, S. A. Rasmussen, JAMA Pediatr 2017, 171, 452 288. 453 [10] A. S. Melo, R. S. Aguiar, M. M. Amorim, M. B. Arruda, F. O. Melo, S. T. Ribeiro, A. G. 454 Batista, T. Ferreira, M. P. Dos Santos, V. V. Sampaio, S. R. Moura, L. P. Rabello, C. E. 455 Gonzaga, G. Malinger, R. Ximenes, P. S. de Oliveira-Szejnfeld, F. Tovar-Moll, L. Chimelli, P.

18

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 456 P. Silveira, R. Delvechio, L. Higa, L. Campanati, R. M. Nogueira, A. M. Filippis, J. Szejnfeld, C. 457 M. Voloch, O. C. Ferreira, Jr., R. M. Brindeiro, A. Tanuri, JAMA Neurol 2016, 73, 1407. 458 [11] J. R. de Oliveira Dias, C. V. Ventura, B. de Paula Freitas, J. Prazeres, L. O. Ventura, V. 459 Bravo-Filho, T. Aleman, A. I. Ko, A. Zin, R. Belfort, M. Maia, Progress in Retinal and Eye 460 Research 2018, pii: S1350. 461 [12] M. C. Leal, L. F. Muniz, T. S. Ferreira, C. M. Santos, L. C. Almeida, V. Van Der Linden, 462 R. C. Ramos, L. C. Rodrigues, S. S. Neto, MMWR Morb Mortal Wkly Rep 2016, 65, 917. 463 [13] M. R. Reynolds, A. M. Jones, E. E. Petersen, E. H. Lee, M. E. Rice, A. Bingham, S. R. 464 Ellington, N. Evert, S. Reagan-Steiner, T. Oduyebo, C. M. Brown, S. Martin, N. Ahmad, J. 465 Bhatnagar, J. Macdonald, C. Gould, A. D. Fine, K. D. Polen, H. Lake-Burger, C. L. Hillard, N. 466 Hall, M. M. Yazdy, K. Slaughter, J. N. Sommer, A. Adamski, M. Raycraft, S. Fleck-Derderian, 467 J. Gupta, K. Newsome, M. Baez-Santiago, S. Slavinski, J. L. White, C. A. Moore, C. K. Shapiro- 468 Mendoza, L. Petersen, C. Boyle, D. J. Jamieson, D. Meaney-Delman, M. A. Honein, MMWR 469 Morb Mortal Wkly Rep 2017, 66, 366. 470 [14] C. G. Victora, M. C. Castro, G. V. Franca, L. Schuler-Faccini, F. C. Barros, Lancet 2017, 471 389, 152. 472 [15] G. V. Franca, L. Schuler-Faccini, W. K. Oliveira, C. M. Henriques, E. H. Carmo, V. D. 473 Pedi, M. L. Nunes, M. C. Castro, S. Serruya, M. F. Silveira, F. C. Barros, C. G. Victora, Lancet 474 2016, 388, 891. 475 [16] K. M. Adams Waldorf, B. R. Nelson, J. E. Stencel-Baerenwald, C. Studholme, R. P. 476 Kapur, B. Armistead, C. L. Walker, S. Merillat, J. Vornhagen, J. Tisoncik-Go, A. Baldessari, M. 477 Coleman, M. K. Dighe, D. W. W. Shaw, J. A. Roby, V. Santana-Ufret, E. Boldenow, J. Li, X. 478 Gao, M. A. Davis, J. A. Swanstrom, K. Jensen, D. G. Widman, R. S. Baric, J. T. Medwid, K. A. 479 Hanley, J. Ogle, G. M. Gough, W. Lee, C. English, W. M. Durning, J. Thiel, C. Gatenby, E. C. 480 Dewey, M. R. Fairgrieve, R. D. Hodge, R. F. Grant, L. Kuller, W. B. Dobyns, R. F. Hevner, M. 481 Gale, Jr., L. Rajagopal, Nat Med 2018, 24, 368. 482 [17] F. Grischott, M. Puhan, C. Hatz, P. Schlagenhauf, Travel Med Infect Dis 2016, 14, 313. 483 [18] K. A. Caillouet, S. R. Michaels, X. Xiong, I. Foppa, D. M. Wesson, Emerg Infect Dis 484 2008, 14, 804. 485 [19] M. A. Honein, A. L. Dawson, E. E. Petersen, A. M. Jones, E. H. Lee, M. M. Yazdy, N. 486 Ahmad, J. Macdonald, N. Evert, A. Bingham, JAMA 2017, 317, 59.

19

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 487 [20] E. Dirlikov, C. G. Major, N. A. Medina, R. Lugo-Robles, D. Matos, J. L. Munoz-Jordan, 488 C. Colon-Sanchez, M. Garcia, M. Olivero-Segarra, G. Malave, G. M. Rodriguez-Vega, D. L. 489 Thomas, S. H. Waterman, J. J. Sejvar, C. A. Luciano, T. M. Sharp, B. Rivera-Garcia, JAMA 490 Neurol 2018, 75, 1089. 491 [21] M. D. Landry, S. R. Raman, K. Kennedy, J. P. Bettger, D. Magnusson, Phys Ther 2017, 492 97, 275. 493 [22] J. Li, C. Y. Chong, N. W. Tan, C. F. Yung, N. W. Tee, K. C. Thoon, Clin Infect Dis 494 2017, 64, 1445. 495 [23] S. N. Slavov, A. K. Matsuno, F. U. de Melo, K. K. Otaguiri, V. d. M. P. Balbão, D. T. 496 Covas, S. Kashima, Journal of Clinical Virology 2016, 82, S9. 497 [24] A. Landais, A. Césaire, M. Fernandez, S. Breurec, C. Herrmann, F. Delion, P. Desprez, 498 Journal of the Neurological Sciences 2017, 383, 211. 499 [25] A. Satterfield-Nash, K. Kotzky, J. Allen, J. Bertolli, C. A. Moore, I. O. Pereira, A. 500 Pessoa, F. Melo, A. Santelli, C. A. Boyle, G. Peacock, MMWR Morb Mortal Wkly Rep 2017, 66, 501 1347. 502 [26] P. J. Hotez, JAMA Pediatr 2016, 170, 787; A. Soares de Souza, C. Moraes Dias, F. D. 503 Braga, A. C. Terzian, C. F. Estofolete, A. H. Oliani, G. H. Oliveira, C. C. Brandao de Mattos, L. 504 C. de Mattos, M. L. Nogueira, D. C. Vaz-Oliani, Clin Infect Dis 2016, 63, 1622; A. S. Oliveira 505 Melo, G. Malinger, R. Ximenes, P. O. Szejnfeld, S. Alves Sampaio, A. M. Bispo de Filippis, 506 Ultrasound Obstet Gynecol 2016, 47, 6. 507 [27] M. C. Leal, V. van der Linden, T. P. Bezerra, L. de Valois, A. C. G. Borges, M. M. C. 508 Antunes, K. G. Brandt, C. X. Moura, L. C. Rodrigues, C. R. Ximenes, Emerg Infect Dis 2017, 509 23, 1253. 510 [28] J. Oliveira-Filho, R. Felzemburgh, F. Costa, N. Nery, A. Mattos, D. F. Henriques, A. I. 511 Ko, T. For The Salvador Zika Response, Am J Trop Med Hyg 2018, 98, 1860. 512 [29] M. C. Leal, L. F. Muniz, S. D. Caldas Neto, V. van der Linden, R. C. Ramos, Braz J 513 Otorhinolaryngol 2016, S1808-8694, 30127. 514 [30] M. Kumar, L. Ching, J. Astern, E. Lim, A. J. Stokes, M. Melish, V. R. Nerurkar, PLoS 515 Negl Trop Dis 2016, 10, e0005262. 516 [31] D. J. Platt, J. J. Miner, Curr Opin Virol 2017, 27, 1.

20

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 517 [32] V. van der Linden, A. Pessoa, W. Dobyns, A. J. Barkovich, H. V. Junior, E. L. Filho, E. 518 M. Ribeiro, M. C. Leal, P. P. Coimbra, M. F. Aragao, I. Vercosa, C. Ventura, R. C. Ramos, D. 519 D. Cruz, M. T. Cordeiro, V. M. Mota, M. Dott, C. Hillard, C. A. Moore, MMWR Morb Mortal 520 Wkly Rep 2016, 65, 1343. 521 [33] S. B. Mulkey, G. Vezina, D. I. Bulas, Z. Khademian, A. Blask, Y. Kousa, C. Cristante, L. 522 Pesacreta, A. J. du Plessis, R. L. DeBiasi, Pediatr Neurol 2018, 78, 75. 523 [34] P. Brasil, P. C. Sequeira, A. D. Freitas, H. E. Zogbi, G. A. Calvet, R. V. de Souza, A. M. 524 Siqueira, M. C. de Mendonca, R. M. Nogueira, A. M. de Filippis, T. Solomon, Lancet 2016, 387, 525 1482. 526 [35] H. J. Willison, B. C. Jacobs, P. A. van Doorn, Lancet 2016, 388, 717. 527 [36] R. A. Hughes, A. V. Swan, J. C. Raphael, D. Annane, R. van Koningsveld, P. A. van 528 Doorn, Brain 2007, 130, 2245. 529 [37] J. M. Leger, R. Guimaraes-Costa, C. Muntean, Neurotherapeutics 2016, 13, 96. 530 [38] N. van Leeuwen, H. F. Lingsma, A. M. Vanrolleghem, M. C. Sturkenboom, P. A. van 531 Doorn, E. W. Steyerberg, B. C. Jacobs, PLoS One 2016, 11, e0143837. 532 [39] L. Ruts, J. Drenthen, J. L. Jongen, W. C. Hop, G. H. Visser, B. C. Jacobs, P. A. van 533 Doorn, Neurology 2010, 75, 1439. 534 [40] S. B. Mulkey, C. M. Glasier, B. El-Nabbout, W. D. Walters, C. Ionita, M. H. McCarthy, 535 G. B. Sharp, R. M. Shbarou, Pediatr Neurol 2010, 43, 263. 536 [41] M. E. Rice, R. R. Galang, N. M. Roth, S. R. Ellington, C. A. Moore, M. Valencia-Prado, 537 E. M. Ellis, A. J. Tufa, L. A. Taulung, J. M. Alfred, J. Perez-Padilla, C. A. Delgado-Lopez, S. R. 538 Zaki, S. Reagan-Steiner, J. Bhatnagar, J. F. Nahabedian, 3rd, M. R. Reynolds, M. Yeargin- 539 Allsopp, L. J. Viens, S. M. Olson, A. M. Jones, M. A. Baez-Santiago, P. Oppong-Twene, K. 540 VanMaldeghem, E. L. Simon, J. T. Moore, K. D. Polen, B. Hillman, R. Ropeti, L. Nieves-Ferrer, 541 M. Marcano-Huertas, C. A. Masao, E. J. Anzures, R. L. Hansen, Jr., S. I. Perez-Gonzalez, C. P. 542 Espinet-Crespo, M. Luciano-Roman, C. K. Shapiro-Mendoza, S. M. Gilboa, M. A. Honein, 543 MMWR Morb Mortal Wkly Rep 2018, 67, 858. 544 [42] N. Yuki, H. P. Hartung, N Engl J Med 2012, 366, 2294. 545 [43] M. T. Medina, M. Medina-Montoya, J Neurol Sci 2017, 383, 214. 546 [44] R. Brusa, F. Zimmermann, D. S. Koh, D. Feldmeyer, P. Gass, P. H. Seeburg, R. 547 Sprengel, Science 1995, 270, 1677.

21

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 548 [45] I. Gaisler-Salomon, E. Kravitz, Y. Feiler, M. Safran, A. Biegon, N. Amariglio, G. 549 Rechavi, Neurobiol Aging 2014, 35, 1785. 550 [46] J. J. Rosenthal, P. H. Seeburg, Neuron 2012, 74, 432. 551 [47] B. L. Bass, Annu Rev Biochem 2002, 71, 817. 552 [48] S. N. Deffit, H. A. Hundley, Wiley Interdiscip Rev RNA 2016, 7, 113. 553 [49] J. B. Li, G. M. Church, Nat Neurosci 2013, 16, 1518. 554 [50] C. R. Walkley, B. Liddicoat, J. C. Hartner, in Adenosine Deaminases Acting on RNA 555 (ADARs) and A-to-I Editing, Springer, 2011, 197. 556 [51] Y. A. Savva, L. E. Rieder, R. A. Reenan, Genome Biol 2012, 13, 252. 557 [52] C. Cenci, R. Barzotti, F. Galeano, S. Corbelli, R. Rota, L. Massimi, C. Di Rocco, M. A. 558 O'Connell, A. Gallo, J Biol Chem 2008, 283, 7251. 559 [53] A. Gallo, D. Vukic, D. Michalik, M. A. O'Connell, L. P. Keegan, Hum Genet 2017, 136, 560 1265. 561 [54] E. L. McGrath, S. L. Rossi, J. Gao, S. G. Widen, A. C. Grant, T. J. Dunn, S. R. Azar, C. 562 M. Roundy, Y. Xiong, D. J. Prusak, B. D. Loucas, T. G. Wood, Y. Yu, I. Fernandez-Salas, S. C. 563 Weaver, N. Vasilakis, P. Wu, Stem Cell Reports 2017, 8, 715. 564 [55] W. Garncarz, A. Tariq, C. Handl, O. Pusch, M. F. Jantsch, RNA Biol 2013, 10, 192. 565 [56] M. M. Jacobs, R. L. Fogg, R. B. Emeson, G. D. Stanwood, Dev Neurosci 2009, 31, 223. 566 [57] E. M. Riedmann, S. Schopoff, J. C. Hartner, M. F. Jantsch, RNA 2008, 14, 1110. 567 [58] Q. Wang, J. Khillan, P. Gadue, K. Nishikura, Science 2000, 290, 1765. 568 [59] T. Yamashita, C. Tadami, Y. Nishimoto, T. Hideyama, D. Kimura, T. Suzuki, S. Kwak, 569 Neurosci Res 2012, 73, 42. 570 [60] G. Lanzi, E. Fazzi, S. D'Arrigo, S. Orcesi, I. Maraucci, C. Uggetti, E. Bertini, P. Lebon, 571 Neurology 2005, 64, 1621. 572 [61] H. Q. Zhao, P. Zhang, H. Gao, X. He, Y. Dou, A. Y. Huang, X. M. Liu, A. Y. Ye, M. Q. 573 Dong, L. Wei, Genome Res 2015, 25, 66. 574 [62] S. Kwak, T. Hideyama, T. Yamashita, H. Aizawa, Neuropathology 2010, 30, 182. 575 [63] F. Galeano, A. Leroy, C. Rossetti, I. Gromova, P. Gautier, L. P. Keegan, L. Massimi, C. 576 Di Rocco, M. A. O'Connell, A. Gallo, Int J Cancer 2010, 127, 127. 577 [64] H. C. Cox, R. A. Lea, C. Bellis, M. Carless, T. D. Dyer, J. Curran, J. Charlesworth, S. 578 Macgregor, D. Nyholt, D. Chasman, Neurogenetics 2012, 13, 261.

22

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 579 [65] E. Ben-David, E. Granot-Hershkovitz, G. Monderer-Rothkoff, E. Lerer, S. Levi, M. 580 Yaari, R. P. Ebstein, N. Yirmiya, S. Shifman, Hum Mol Genet 2011, 20, 3632. 581 [66] L. Mittaz, S. E. Antonarakis, M. Higuchi, H. S. Scott, Hum Genet 1997, 100, 398. 582 [67] A. J. Fisher, P. A. Beal, RNA Biol 2017, 14, 164. 583 [68] Y. J. Crow, N. Manel, Nat Rev Immunol 2015, 15, 429; F. Goutieres, J. Aicardi, P. G. 584 Barth, P. Lebon, Ann Neurol 1998, 44, 900. 585 [69] G. I. Rice, P. R. Kasher, G. M. Forte, N. M. Mannion, S. M. Greenwood, M. 586 Szynkiewicz, J. E. Dickerson, S. S. Bhaskar, M. Zampini, T. A. Briggs, E. M. Jenkinson, C. A. 587 Bacino, R. Battini, E. Bertini, P. A. Brogan, L. A. Brueton, M. Carpanelli, C. De Laet, P. de 588 Lonlay, M. del Toro, I. Desguerre, E. Fazzi, A. Garcia-Cazorla, A. Heiberg, M. Kawaguchi, R. 589 Kumar, J. P. Lin, C. M. Lourenco, A. M. Male, W. Marques, Jr., C. Mignot, I. Olivieri, S. 590 Orcesi, P. Prabhakar, M. Rasmussen, R. A. Robinson, F. Rozenberg, J. L. Schmidt, K. Steindl, T. 591 Y. Tan, W. G. van der Merwe, A. Vanderver, G. Vassallo, E. L. Wakeling, E. Wassmer, E. 592 Whittaker, J. H. Livingston, P. Lebon, T. Suzuki, P. J. McLaughlin, L. P. Keegan, M. A. 593 O'Connell, S. C. Lovell, Y. J. Crow, Nat Genet 2012, 44, 1243. 594 [70] J. H. Livingston, Y. J. Crow, Neuropediatrics 2016, 47, 355. 595 [71] E. Y. Levanon, M. Hallegger, Y. Kinar, R. Shemesh, K. Djinovic-Carugo, G. Rechavi, 596 M. F. Jantsch, E. Eisenberg, Nucleic Acids Res 2005, 33, 1162. 597 [72] Y. Kawahara, K. Ito, H. Sun, H. Aizawa, I. Kanazawa, S. Kwak, Nature 2004, 427, 801. 598 [73] T. Hideyama, T. Yamashita, H. Aizawa, S. Tsuji, A. Kakita, H. Takahashi, S. Kwak, 599 Neurobiol Dis 2012, 45, 1121. 600 [74] A. Tariq, M. F. Jantsch, Front Neurosci 2012, 6, 99. 601 [75] S. Pachernegg, Y. Munster, E. Muth-Kohne, G. Fuhrmann, M. Hollmann, Front Cell 602 Neurosci 2015, 9, 69. 603 [76] H. Huang, B. Z. Tan, Y. Shen, J. Tao, F. Jiang, Y. Y. Sung, C. K. Ng, M. Raida, G. Kohr, 604 M. Higuchi, H. Fatemi-Shariatpanahi, B. Harden, D. T. Yue, T. W. Soong, Neuron 2012, 73, 605 304. 606 [77] D. Bonini, A. Filippini, L. La Via, C. Fiorentini, F. Fumagalli, M. Colombi, A. Barbon, 607 RNA Biol 2015, 12, 43. 608 [78] T. D. Werry, R. Loiacono, P. M. Sexton, A. Christopoulos, Pharmacol Ther 2008, 119, 7.

23

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 609 [79] E. Y. Rula, A. H. Lagrange, M. M. Jacobs, N. Hu, R. L. Macdonald, R. B. Emeson, J 610 Neurosci 2008, 28, 6196. 611 [80] M. Holmgren, J. J. Rosenthal, Curr Issues Mol Biol 2015, 17, 23. 612 [81] K. Nishikura, Nat Rev Mol Cell Biol 2016, 17, 83. 613 [82] N. C. Danbolt, D. N. Furness, Y. Zhou, Neurochem Int 2016, 98, 29. 614 [83] P. H. Seeburg, M. Higuchi, R. Sprengel, Brain Res Brain Res Rev 1998, 26, 217. 615 [84] M. T. Veno, J. B. Bramsen, C. Bendixen, F. Panitz, I. E. Holm, M. Ohman, J. Kjems, 616 RNA Biol 2012, 9, 1054; M. A. Huntley, M. Lou, L. D. Goldstein, M. Lawrence, G. J. Dijkgraaf, 617 J. S. Kaminker, R. Gentleman, BMC Genomics 2016, 17, 61. 618 [85] J. D. MacMicking, Nat Rev Immunol 2012, 12, 367. 619 [86] C. E. Samuel, Virology 2011, 411, 180; G. C. Jayan, J. L. Casey, J Virol 2002, 76, 3819. 620 [87] A. Grant, S. S. Ponia, S. Tripathi, V. Balasubramaniam, L. Miorin, M. Sourisseau, M. C. 621 Schwarz, M. P. Sanchez-Seco, M. J. Evans, S. M. Best, A. Garcia-Sastre, Cell Host Microbe 622 2016, 19, 882. 623 [88] J. R. Bowen, K. M. Quicke, M. S. Maddur, J. T. O'Neal, C. E. McDonald, N. B. 624 Fedorova, V. Puri, R. S. Shabman, B. Pulendran, M. S. Suthar, PLoS Pathog 2017, 13, 625 e1006164. 626 [89] S. Tripathi, V. R. Balasubramaniam, J. A. Brown, I. Mena, A. Grant, S. V. Bardina, K. 627 Maringer, M. C. Schwarz, A. M. Maestre, M. Sourisseau, R. A. Albrecht, F. Krammer, M. J. 628 Evans, A. Fernandez-Sesma, J. K. Lim, A. Garcia-Sastre, PLoS Pathog 2017, 13, e1006258. 629 [90] N. R. Matevosyan, Critical Care Obstetrics and Gynecology 2016; V. V. Linden, H. V. J. 630 Linden, M. C. Leal, E. L. F. Rolim, A. V. Linden, M. Aragao, A. M. Brainer-Lima, D. Cruz, L. 631 O. Ventura, T. L. T. Florencio, M. T. Cordeiro, S. D. S. N. Caudas, R. C. Ramos, Arq 632 Neuropsiquiatr 2017, 75, 381. 633 [91] H. Piontkivska, L. F. Matos, S. Paul, B. Scharfenberg, W. G. Farmerie, M. M. Miyamoto, 634 M. L. Wayne, Genome Biol Evol 2016, 8, 2952. 635 [92] H. Piontkivska, M. Frederick, M. M. Miyamoto, M. L. Wayne, Ecol Evol 2017, 7, 4475. 636 [93] S. Kwak, Y. Nishimoto, T. Yamashita, RNA Biol 2008, 5, 193. 637 [94] G. X. Xu, J. Z. Zhang, P Natl Acad Sci USA 2014, 111, 3769.

24

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 638 [95] A. Sailer, G. T. Swanson, I. Perez-Otano, L. O'Leary, S. A. Malkmus, R. H. Dyck, H. 639 Dickinson-Anson, H. H. Schiffer, C. Maron, T. L. Yaksh, F. H. Gage, S. O'Gorman, S. F. 640 Heinemann, J Neurosci 1999, 19, 8757. 641 [96] J. G. Hensler, in Basic Neurochemistry. Molecular, Cellular and Medical Aspects., (Ed: 642 G. S. Scott Brady, R. Wayne Albers, Donald Price), Elsevier, 2012, 300. 643 [97] M. Tohda, M. Nomura, Y. Nomura, J Pharmacol Sci 2006, 100, 427. 644 [98] S. De Lucchini, M. Ori, M. Nardini, S. Marracci, I. Nardi, Brain Res Mol Brain Res 645 2003, 115, 196. 646 [99] R. Lyddon, A. J. Dwork, M. Keddache, L. J. Siever, S. Dracheva, World J Biol 647 Psychiatry 2013, 14, 590. 648 [100] A. Gatsiou, N. Vlachogiannis, F. F. Lunella, M. Sachse, K. Stellos, Antioxid Redox 649 Signal 2018, 29, 846. 650 [101] E. Oakes, A. Anderson, A. Cohen-Gadol, H. A. Hundley, J Biol Chem 2017, 292, 4326. 651 [102] H. Wahlstedt, C. Daniel, M. Enstero, M. Ohman, Genome Res 2009, 19, 978. 652 [103] E. Picardi, D. S. Horner, G. Pesole, RNA 2017, 23, 860. 653 [104] S. van der Laan, N. Salvetat, D. Weissmann, F. Molina, Drug Discov Today 2017, 22, 654 1056. 655 [105] M. S. Majumder, R. Hess, R. Ross, H. Piontkivska, F1000Res 2018, 7, 159. 656 [106] R. Furie, M. Khamashta, J. T. Merrill, V. P. Werth, K. Kalunian, P. Brohawn, G. G. Illei, 657 J. Drappa, L. Wang, S. Yoo, Arthritis Rheumatol 2017, 69, 376. 658 [107] E. Doncel-Perez, L. Mateos-Hernandez, E. Pareja, A. Garcia-Forcada, M. Villar, R. 659 Tobes, F. Romero Ganuza, V. Vila Del Sol, R. Ramos, I. G. Fernandez de Mera, J. de la Fuente, 660 J Immunol 2016, 196, 1102. 661 [108] T. R. O'Brien, L. Prokunina-Olsson, R. P. Donnelly, J Interferon Cytokine Res 2014, 34, 662 829. 663 [109] L. M. de Lau, M. M. Breteler, Lancet Neurol 2006, 5, 525. 664 [110] C. S. Chan, J. N. Guzman, E. Ilijic, J. N. Mercer, C. Rick, T. Tkatch, G. E. Meredith, D. 665 J. Surmeier, Nature 2007, 447, 1081. 666 [111] S. Fahn, Parkinsonism Relat Disord 2018, 46 Suppl 1, S1. 667 [112] R. Knorle, Neurotox Res 2018, 33, 515. 668 [113] J. M. Taylor, B. S. Main, P. J. Crack, Neurochem Int 2013, 62, 803.

25

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018 669 [114] M. J. Hurley, B. Brandon, S. M. Gentleman, D. T. Dexter, Brain 2013, 136, 2077. 670 [115] G. Bock, M. Gebhart, A. Scharinger, W. Jangsangthong, P. Busquet, C. Poggiani, S. 671 Sartori, M. E. Mangoni, M. J. Sinnegger-Brauns, S. Herzig, J. Striessnig, A. Koschak, J Biol 672 Chem 2011, 286, 42736. 673 [116] A. Cucca, H. A. Migdadi, A. Di Rocco, Parkinsonism Relat Disord 2018, 46 Suppl 1, 674 S83. 675 [117] K. R. Fink, M. M. Thapa, G. E. Ishak, S. Pruthi, Radiographics 2010, 30, 1779; L. 676 Gabrielli, M. P. Bonasoni, T. Lazzarotto, S. Lega, D. Santini, M. P. Foschini, B. Guerra, F. 677 Baccolini, G. Piccirilli, A. Chiereghin, E. Petrisli, G. Gardini, M. Lanari, M. P. Landini, J Clin 678 Virol 2009, 46 Suppl 4, S16. 679 [118] J. Dang, S. K. Tiwari, G. Lichinchi, Y. Qin, V. S. Patil, A. M. Eroshkin, T. M. Rana, Cell 680 Stem Cell 2016, 19, 258; 681 [119] R. Hamel, P. Ferraris, S. Wichit, F. Diop, L. Talignani, J. Pompon, D. Garcia, F. 682 Liegeois, A. A. Sall, H. Yssel, D. Misse, Infect Genet Evol 2017, 49, 134. 683

684

26

PeerJ Preprints | https://doi.org/10.7287/peerj.preprints.27401v1 | CC BY 4.0 Open Access | rec: 3 Dec 2018, publ: 3 Dec 2018