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Inflammatory gene expression signatures in idiopathic intracranial hypertension: possible implications in microgravity-induced ICP elevation

Inflammatory gene expression signatures in idiopathic intracranial hypertension: possible... www.nature.com/npjmgrav ARTICLE OPEN Inflammatory gene expression signatures in idiopathic intracranial hypertension: possible implications in microgravity-induced ICP elevation 1 2 2 2 3 3 3 Susana B. Zanello , Vasisht Tadigotla , James Hurley , Johan Skog , Brian Stevens , Eusebia Calvillo and Eric Bershad The visual impairment and intracranial pressure (VIIP) syndrome is a neuro–ophthalmologic condition described in astronauts returning from long duration space missions. Idiopathic intracranial hypertension (IIH), also known as pseudotumor cerebri, is characterized by a chronic elevation of intracranial pressure (ICP) in the absence of an intracranial mass lesion. Because VIIP and IIH share some neurologic and ophthalmologic manifestations, the latter might be used as a model to study some of the processes underlying VIIP. This work constitutes a preliminary investigation of the molecular pathways associated with the elevation of ICP in IIH. Gene expression signatures were obtained from exosomes collected from CSF and plasma in patients with possible signs of IIH. The gene expression targets focused on inflammatory genes and miRNAs. The results suggest that inflammatory cytokine-driven processes and immune cell migration are activated when ICP is elevated in IIH patients, either as a cause or effect of the ICP increase. Several miRNAs appear to be involved in this response, among which miR-9 and miR-16 are upregulated in CSF and plasma of higher ICP subjects. This study provides evidence in support of neurophysiological alterations and neuro- immunomodulation in this condition. If similar changes are seen in astronauts manifesting with the VIIP syndrome, an underlying pathophysiological basis may be discovered. npj Microgravity (2018) 4:1 ; doi:10.1038/s41526-017-0036-6 INTRODUCTION implications in understanding neurophysiologic changes during spaceflight. Human space exploration involves multi-system health risks. Neuro-ophthalmologic symptoms, including elevated intracranial pressure (ICP) upon return to Earth, have been observed in RESULTS astronauts participating in long-duration missions, a condition Subject information named visual impairment and intracranial pressure syndrome Patient information is presented in Table 1 and the Supplementary (VIIP). On Earth, these neuroanatomical findings concur with those 2 Table e-1. The average age was 33.8 ± 9.7 years (range 18–51), 2 in idiopathic intracranial hypertension (IIH). The etiology of IIH is a males and 20 females. No subject within the lower ICP group was topic of debate. The moderately elevated ICP over many years has diagnosed with IIH. The subjects in the higher ICP group were been linked to cognition losses relieved by lumbar tapping to diagnosed with IIH and exhibited typical ocular symptoms. No 3,4 reduce ICP. While most cases are seen in overweight women of correlation was found between ICP and RNFL thickness (Pearson child-bearing age, there is no specific condition on Earth that correlation coefficient between ICP and the average RNFL of both shares more similarities to VIIP, constituting a reasonable analog eyes was less than 0.4). to study the effects of chronically elevated ICP at the molecular level. 5–7 miRNA and mRNA expression in higher and lower ICP subjects A number of performance and neurologic signs (“space In plasma, the average detection rate reached almost 45% of the fog”) have also been linked to spaceflight and might also result targets in the TaqMan Open Array miRNA panel and 75% for the from elevation of ICP. In this paper, we test the hypothesis that mRNA Inflammation Open Array. In CSF, the average detection these symptoms are caused by disturbances in the neurophysiol- rate for miRNAs was approximately 15% of the targets and ogy of the brain and correlated with molecular markers in the approximately 25% for mRNA targets. Very few samples (two or cerebrospinal fluid (CSF) by studying gene expression profiles less) showed a lower detection rate and those were excluded from from CSF and plasma in individuals with suspected IIH. The gene analysis. Table 2 lists the differentially expressed miRNAs and expression signatures found suggest a systemic inflammatory mRNAs between subjects in the higher and lower ICP groups, after status corresponded by local brain inflammatory processes in a Mann–Whitney test was applied with p < 0.05 (FDR not applied). subjects with elevated ICP. Putative biomarker candidates are The sensitivity and reproducibility of the mRNA assay was very discussed, although further studies are necessary to validate their high as evaluated by multiple extractions and qRT-PCRs (R > 0.95 possible research and clinical use. These findings may have and 0.9 in plasma and CSF samples, respectively). 1 2 3 KBRwyle, NASA Johnson Space Center, Houston, TX, USA; Exosome Diagnostics, Cambridge, MA, USA and Baylor College of Medicine, Houston, TX, USA Correspondence: Susana B. Zanello (susana.b.zanello@nasa.gov) Received: 25 April 2017 Revised: 6 November 2017 Accepted: 13 December 2017 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Gene expression signatures in intracranial hypertension S B Zanello et al. Table 1. Subject distribution summarizing the mean ICP and standard Table 2. Differentially expressed genes between subjects with deviation, ICP range and retinal nerve fiber layer (RNFL) measured by normal-to-mildly elevated ICP and with ICP higher than 18 mmHg optical coherence tomography (OCT) and averaged between the two Plasma eyes on each individual miRNA U p Ratio (higher ICP/lower Fold change (log2 Lower ICP group Higher ICP group value value ICP) ratio) dme-miR-7 80 0.007 29.53 4.88 N 714 hsa-miR-938 78.5 0.009 48.11 5.59 Mean ICP 14.6 mmHg ± 2.5 24.0 mmHg ± 4.1 hsa-miR-143 76.5 0.013 579.88 9.18 ICP range 11.0–17.9 mmHg 18.8–31.4 mmHg hsa-let-7i 77 0.013 16.73 4.06 Mean RNFL 147.6 µm ± 119.9 195.2 µm ± 116.3 hsa-miR-618 14.5 0.014 0.009 −6.80 hsa-miR-374 76 0.015 78.96 6.30 Of 22 subjects recruited, ICP was not able to be determined in one of them hsa-miR-657 15 0.017 0.07 −3.84 due to technically difficult LP. That subject was therefore not included in hsa-miR-190b 15 0.017 0.003 −8.38 the study and data collection was completed in 21 subjects. The cut off between normal and elevated ICP was established at 18 mm Hg hsa-miR-10b 16 0.021 0.18 −2.47 hsa-miR-589 74 0.025 16.38 4.03 hsa-miR-16-1 74 0.026 20.45 4.35 Pathway analysis hsa-miR-298 18.5 0.027 0.0001 −13.29 miRNA Target Filter analysis in IPA was performed for the hsa-miR-551b 73 0.029 594.24 9.21 complete gene expression data from the miRNA and Inflammation Hsa-miR-545 73 0.031 17.08 4.09 mRNA panels in CSF and plasma. The process allows the Hsa-miR-375 19 0.038 0.003 −8.38 combination of experimental gene expression data with the Hsa-miR-10b 19 0.039 0.29 −1.79 existing knowledge base of predicted and validated miRNA-target Hsa-miR-145 71.5 0.042 30.74 4.94 relationships. Figure 1 summarizes the result of this process, Hsa-miR-483- 19.5 0.043 0.4 −1.32 depicting miRNAs, their targets and the most represented 5p canonical pathways related to inflammation, Hsa-let-7a 70.5 0.044 42.02 5.39 eicosanoid–phospholipase signaling and immune cell migration. Mmu-miR- 71 0.045 31.91 5.00 A comparison was run between the CSF and plasma expression data sets. Of 205 genes with expression data in plasma and 162 Hsa-miR-154 20.5 0.045 0.008 −6.97 genes in CSF, only 31 were represented in both, and only two, Gene target miRNA-16 and CD80 (CD86), were differentially expressed in both CXCR3 10 0.0008 0.05 −4.32 CSF and plasma (Table 2). miR-9 and miR-16 were upregulated by TLR3 13 0.016 0.19 −2.39 approximately 4.7 fold in CSF and plasma of higher ICP subjects, LEFTY2 70 0.02 2.64 1.4 and CD80/86 was downregulated by −5.0 fold and −2.5 fold in CSF TNFSF4 67.5 0.035 6.01 2.59 and plasma, respectively. CD70 17 0.038 0.12 −3.06 EREG 67 0.038 51.93 5.70 CD80 17 0.038 0.17 −2.56 DISCUSSION This paper reports a gene expression survey of exosomal RNA in CSF CSF and plasma from patients with elevated ICP (>18 mmHg) compared to patients with normal to moderately elevated ICP miRNA U p Ratio (higher ICP/ Fold change (log2 (11–18 mmHg). All subjects recruited for the study reported to the value value lower ICP) ratio) neurology clinic, therefore none represented a completely hsa-miR-9 75.5 0.018 24.89 4.64 “healthy” volunteer and results need to take into account the hsa-miR- 71 0.045 25.89 4.69 existence of underlying conditions. Although this study only examined a limited panel of mRNAs Gene target and miRNAs, it is expected that a similar proportion of overlapping genes would have been found with a wider expression screening, STAT5B 70.5 0.006 106.98 6.74 like whole genome microarray or RNAseq. In spite of the small TGM2 67.5 0.011 58.04 5.86 overlap in the set of differentially expressed genes from CSF and CD86 12.5 0.018 0.03 −5.06 plasma, the identified pathways associated with increased ICP (CD80) were similar. Pro-inflammatory pathways such as acute phase S100A9 62.5 0.036 91.53 6.52 response and interleukin signaling were highly represented. Both RIPK2 16 0.048 0.11 3.18 gene sets suggest that inflammatory processes are acting both locally in the brain as well as systemically, perhaps contributing to The differentially expressed miRNAs and mRNAs with p < 0.05 as IIH pathophysiology. determined by the Mann–Whitney test are shown for both plasma and Pathway analysis at the CSF level also suggested processes of CSF samples. Ratio values that are <1 indicate downregulation. The list can brain cell death, possibly targeted by miR-9. This hypothesis is also be further filtered for U values lesser than the U critical value (20 for plasma and 22 for CSF), providing more stringency supported by evidence that involves miRNA-9 in degenerative diseases, as well as promoting glial activation via the NFkB pathway. Interestingly, miR-9 is itself targeted by miR-16, which was also observed to be upregulated in the high ICP group. which has been implicated in progressive inflammatory Circulating miRNAs are increasingly gaining attention as screening myelopathy. biomarkers in central nervous system (CNS) diseases. In this For a long time, the CNS has been considered a site of immune work, identified candidates warrant monitoring in larger cohort privilege, suffering from costly physiologic consequences if studies to investigate their biomarker value, i.e., miR-16, also subject to inflammation due to trauma or infection. Currently, investigated as a biomarker for glioblastoma, and CD86 (CD80), this paradigm is being reexamined by work highlighting the npj Microgravity (2018) 1 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; Gene expression signatures in intracranial hypertension S B Zanello et al. miRNA, Molecular Targets and Pathways in High ICP VEGFA* Amyotrophic Lateral Sclerosis Signaling TNFSF13B* Agranulocyte Adhesion and Diapedesis CMTM4* VEGF Signaling GDF11* Hypoxia Signaling in the Cardiovascular System Axonal Guidance Signaling SPRED1* Altered T and B Cell Signaling in Rheumatoid Arthritis CACNB2* CXCR4* Role of Macrophages in Rheumatoid Arthritis IL-8 Signaling ABCF1* Endothelin-1 Signaling IL-10 Signaling miR-16-5p PRKCA* Acute Phase Response Signaling Huntington’s Disease Signaling miR-9-5p Aldosterone Signaling in Epithelial Cells HMOX1* NRF2-mediated Oxidative Stress Response Androgen Signaling JUN* TBP* Renin-Angiotensin Signaling 14-3-3-mediated Signaling 4-1BB Signaling in T Lymphocytes DNAJC8* NFKB1* Adipogenesis Pathway HDAC5* IL-17 Signaling GSK3B* B Cell Receptor Signaling BCL6* NR3C1* Amyloid Processing VEGFA* IL-6 Signaling KITLG* OSM* ErbB Signaling PLA2G5* CMTM4* Neuregulin Signaling EREG* CD80* Oncostatin M Signaling NRG1* IRAK2* IL6ST* Acute Phase Response Signaling ADORA2A* AOC3* Role of Macrophages in Rheumatoid Arthritis miR-16-5p Phospholipase C Signaling miR-1291 Axonal Guidance Signaling PLCD1 miR-543-3p Phospholipases miR-92a-3p Endothelin-1 Signaling miR-483-5p 14-3-3-meditated Signaling PLCB4* miR-643 Aldosterone Signaling in Epithelial Cells miR-190a-5p miR-182-5p Cardiac Hypertrophy Signaling Fig. 1 Main miRNA and canonical pathways represented in patients with elevated ICP. miRNA Target Filter Analysis (Ingenuity Pathways Analysis, IPA , QIAGEN Redwood City, www.qiagen.com/ingenuity) was applied to the differentially expressed gene data set from CSF (a) and plasma (b). miRNA and target relationships are shown by arrows, as well as the main possibly affected canonical pathways Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 1 Plasma Plasma Cytoplasm Nucleus Cytoplasm membrane membrane Gene expression signatures in intracranial hypertension S B Zanello et al. protective action that a controlled inflammatory response may Additionally, subjects had 10 ml blood drawn for plasma separation in K EDTA BD Vacutainer blood collection tubes with gel barrier. All plasma have on the CNS. For example, it has been shown that mice 2 and CSF samples were pre-filtered through a 0.8-µm syringe filter prior to deficient in functional T cells underperform normal mice in microvesicular processing, aliquoted and stored at −80 °C in a freezer at cognitive tasks and this pro-cognitive effect may be mediated by the Center for Space Medicine at BCM and then shipped to the Exosome 14,15 IL-4. Our work supports the existence of an underlying Diagnostics facilities in Cambridge, MA. subclinical inflammatory status in IIH. Further studies should be directed towards assessing the extent of these processes and their Exosome and RNA isolation correlation with neurocognitive parameters. CSF at volume of 5 ml and plasma at volume of 4 ml (pre-filtered) were This study has some limitations. First, it constitutes an used for exosome isolation by the ExosomeDx spin column-based method exploratory study with small sample size. Second, the lower ICP (expRNAeasy SerumPlasma Maxi kit, Qiagen, Valencia, CA). The method average, 14.6 mmHg, lies on the higher limit of what is considered allows the reproducible isolation of high-quality exosome-specific RNA, a normal ICP (5–15 mmHg), and therefore the comparison with which includes both mRNA and miRNA fractions. Briefly, the pre-filtered high ICP may not yield as many differentially expressed genes as sample was mixed 1:1 with 2× binding buffer (XBP) and added to the would be expected in comparison with a normal ICP group. Third, exoEasy membrane affinity column to bind the exosomes to the membrane. After centrifugation, the flow-through was discarded and IIH patients are arguably a distinct population from the normal wash buffer (XWP) was added to the column to wash off non-specifically healthy astronaut. Since IIH is more prevalent in women, this study retained material. After another centrifugation and discarding of the flow- contains a majority of females, also deviating from the sample through, the vesicles were lysed by adding QIAzol solution and following population of astronauts so far screened for VIIP. the addition of chloroform, thorough mixing and centrifugation to Our findings support new concepts regarding the communica- separate organic and aqueous phases, the aqueous phase was recovered tion between the immune and nervous systems. The IIH patients and mixed with ethanol. The sample–ethanol mixture was added to an evidenced signs of generalized inflammation that may be RNAeasy MinElute spin column and centrifuged. The column was washed once with buffer RWT, and then twice with buffer RPE followed by elution affecting neural physiology at various levels, from neural signaling of RNA in a final volume of 14 μL water. to cognition. These patients were predominantly obese (average BMI = 34.7 ± 7.4 kg/m ), with likely associated metabolic syn- drome, chronic low-grade inflammation, and insulin resistance MicroRNA analysis and other obesity-associated complications, for which pathways Reverse transcription (RT) of miRNA was performed using the Megaplex™ and processes were identified in the analysis. RT Primers (ThermoFisher Scientific, Waltham, MA) to prepare cDNA for real-time PCR analysis on a TaqMan MicroRNA Array, including a pre- The value of this study predominantly lies in being the first amplification step. Collectively, the Human Pool Set v3.0 containing two evaluation of the molecular players in IIH and the first comparison Megaplex™ RT Primer Pools, Pools A v2.1 and B v3.0 cover 754 unique between expression profiles from exosomes in CSF and plasma for microRNAs. The RT reaction for each primer pool was set as follows: 1× this condition. Because elevation of ICP is a hallmark of the Megaplex RT Primers (Pool A or B), 2 mM dNTPs (with dTTP), 10 U/μL disease, findings from this study may be relevant to the VIIP MultiScribe reverse transcriptase, 1.5 mM MgCl2, 1× RT buffer, 0.25 U/ μL syndrome. The results from the CSF survey suggest RNAse inhibitor, in a final volume of 15 μL per reaction. The reaction was neuro–immunomodulatory processes, and further work including run through the temperature sequence per the manufacturer’s instructions. astronauts exposed to microgravity and the concomitant fluid Preamplification was performed in a 50 μL reaction with a 10 μL aliquot shifts with ICP increase, is proposed to test this hypothesis on a of the RT reaction as a template, 1× TaqMan PreAmp Master Mix and 1× more relevant sample population. Megaplex™ PreAmp Primers (Pool A or Pool B), in a temperature cycling (16 cycles) according to the manufacturer. Pre-amplified samples were ® ® diluted 1:10 in 0.1× TE pH 8.0. The TaqMan OpenArray Human MicroRNA METHODS Panel was used for final PCR detection. Sample collection In this final step, the DNA polymerase of the TaqMan Universal PCR Methods were performed in accordance with relevant regulations and Master Mix amplifies the specific cDNA targets using sequence-specific primers and probe on the TaqMan microRNA Array. 25 µL TaqMan guidelines. The study was reviewed and approved by the institutional OpenArray Real-Time PCR Master Mix and 13 µL 0.1× buffer TE pH 8.0 review boards at Johnson Space Center and Baylor College of Medicine were loaded into each of two adjacent wells per sample on a clean 96-well (BCM), Houston, TX. Subject recruitment was done at BCM and written plate. For each sample, 12 µL of diluted Pool A PreAmp was pipetted into informed consent was obtained from all subjects. one well of each pair and 12 µL of diluted Pool B PreAmp was pipetted into The study population was a pool of neurological patients with suspected the other well for a total volume of 50 µL in each well. For each sample and IIH, requiring diagnostic or therapeutic LP. Subjects were recruited on the primer pool set (Pool A or Pool B), 5 µL of each reaction mixture was basis of the modified Dandy criteria for IIH and the IIH treatment trial, pipetted into each of eight wells on an OpenArray 384-well sample plate. consisting in the presence of signs and symptoms of raised ICP (>20 cm Samples were loaded onto OpenArray plates using the standard Accufill™ H O = 14.7 mmHg), absence of localized findings except those from protocol. Amplification was performed according to the protocol increased ICP, or abnormal neuroimaging except for empty sella turcica ® ® established for the TaqMan OpenArray Human MicroRNA panel. and distended optic nerve sheath. The subject was awake and alert, and no other cause of increased ICP was present. For the LP, the subjects lay in the lateral decubitus position with legs slightly extended. Once the needle was mRNA analysis in the thecal sac at the L3-L4 or L4-L5 region, the manometer was The RT reaction was carried out in a final volume of 20 μL containing: 14 μL observed for the presence of pulse and respiratory waves to indicate RNA sample, 2 μL Superscript reverse transcriptase enzyme mix, and 4 μL patent communication between needle and subarachnoid space. Next, the VILO Master Mix (which includesRNaseOUT™ Recombinant Ribonuclease opening pressure was monitored over a period of 5 min, ICP was measured Inhibitor, MgCl2, dNTPs and random primers). The reaction proceeded for every minute, and the average calculated. CSF was then drained as per the 10 min at 95 °C, 15 s at 95 °C, 4 min at 60 °C, followed by inactivation at 95 ° normal clinical procedure. A volume of 5 ml was collected specifically for C for 10 min. the purpose of this study. Pre-amplification was done using Human Inflammation PreAmp Primers Of 22 subjects recruited, ICP was not able to be determined in one of (Pool A or B) 5 μL, TaqMan PreAmp Master Mix 10 μL, and 5 μLofRT them due to technically difficult LP. That subject was therefore not product (cDNA) in a final volume of 50 μL. The temperature sequence was included in the study and data collection was completed in 21 subjects. the following: 10 min at 95 °C, 15 s at 95 °C and 4 min at 60 °C (for 14 The cut off between normal and elevated ICP was established at 18 mm cycles), followed by inactivation at 95 °C for 10 min. The pre-amplified Hg. Patients were grouped into “normal to moderately high ICP” subjects samples were stored at 4 °C until the PCR was performed. ® ® (less than 18 mmHg) and high ICP (higher than 18 mmHg). Seven “normal The TaqMan OpenArray Human Inflammation Panel (Applied Biosys- to mild ICP” and 14 high ICP subjects were compared in the study. tems, ThermoFisher Scientific) was used for final PCR detection. These npj Microgravity (2018) 1 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Gene expression signatures in intracranial hypertension S B Zanello et al. contain TaqMan gene expression assays dried down in 384-well TaqMan Competing interests: Dr Zanello is the recipient of the NASA award funding this Array microfluidic cards targeting pathway relevant gene targets. The study, as well as other research support from the agency. Dr Tadigotla is employed by panel covers 586 genes that have been studied as targets for a range of Exosome Diagnostics, Inc. and holds stock options from Exosome Diagnostics. Dr inflammatory diseases, plus 21 endogenous control genes. James Hurley is employed by Exosome Diagnostics, Inc. and holds stock options from PreAmp products for each sample were mixed and diluted 1:10 with 0.1× Exosome Diagnostics. Dr Skog is Chief ScientificOfficer at Exosome Diagnostics, Inc. ® ® TE pH = 8.0. For each sample, 35 μL of 2× TaqMan OpenArray Real-Time and holds stock options from Exosome Diagnostics. Mr Brian Stevens and Ms Eusebia PCR Master Mix was mixed with 35 μL mixed, diluted PreAmp. 5 μL of each Calvillo report no disclosures. Dr Bershad performs neurology clinical practice, and reaction mixture was pipetted into each of 12 wells on an OpenArray 384- conducts several research projects funded by NASA and the NSBRI (National Space well sample plate. Samples were loaded onto OpenArray plates using the Biomedical Research Institute) standard Accufill™ protocol. Amplification was performed according to the ® ® protocol established for the TaqMan OpenArray Human Inflammation Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims Panel with the QuantStudio™ 12 K Flex Real-time PCR system. in published maps and institutional affiliations. Data analysis and bioinformatics REFERENCES The mRNA data comprises cycle threshold (Ct) values from the OpenArray 1. Mader, T. H. et al. Optic disc edema, globe flattening, choroidal folds, and QuantStudio™ 12 K Flex Real-time PCR system, which is converted to gene hyperopic shifts observed in astronauts after long-duration space flight. Oph- expression levels for a panel of 607 inflammation-related genes and thalmology 118, 2058–2069 (2011). endogenous control genes. The miRNA data set consists of 754 different 2. Peng, K. P., Fuh, J. L. & Wang, S. J. 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The NORDIC Idiopathic Intracranial Hypertension Study Group Writing Commit- of Houston College of Optometry for performing the ophthalmological evaluations, tee. Effect of Acetazolamide on visual function in patients with idiopathic intra- and John Blackwell, USRA-LPI, for artwork. This study was funded by NASA (Grant cranial hypertension and mild visual lossthe idiopathic intracranial hypertension NNX15AW48G). Other Contributors (non-authors): Rosa Tang, MD, MPH (University treatment trial. JAMA 311, 1641–1651 (2014). Eye Institute, University of Houston; ophthalmic examinations); B Gutierrez Flores, MD (University Eye Institute, University of Houston; ophthalmic examinations) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, AUTHOR CONTRIBUTIONS adaptation, distribution and reproduction in any medium or format, as long as you give S.Z., study concept and design, study supervision, analysis and interpretation of data, appropriate credit to the original author(s) and the source, provide a link to the Creative manuscript writing, funded investigator. V.T., gene expression data and statistical Commons license, and indicate if changes were made. The images or other third party analysis. J.H., sample processing and analysis. J.S., study supervision, concept and material in this article are included in the article’s Creative Commons license, unless design, data analysis. E.C., research coordination. B.S., sampling handling, subject data indicated otherwise in a credit line to the material. If material is not included in the analysis. E.B., sample collection, study concept and design, critical revision of article’s Creative Commons license and your intended use is not permitted by statutory manuscript. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ADDITIONAL INFORMATION Supplementary information accompanies the paper on the npj Microgravity website © The Author(s) 2018 (https://doi.org/10.1038/s41526-017-0036-6). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 1 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png npj Microgravity Springer Journals

Inflammatory gene expression signatures in idiopathic intracranial hypertension: possible implications in microgravity-induced ICP elevation

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www.nature.com/npjmgrav ARTICLE OPEN Inflammatory gene expression signatures in idiopathic intracranial hypertension: possible implications in microgravity-induced ICP elevation 1 2 2 2 3 3 3 Susana B. Zanello , Vasisht Tadigotla , James Hurley , Johan Skog , Brian Stevens , Eusebia Calvillo and Eric Bershad The visual impairment and intracranial pressure (VIIP) syndrome is a neuro–ophthalmologic condition described in astronauts returning from long duration space missions. Idiopathic intracranial hypertension (IIH), also known as pseudotumor cerebri, is characterized by a chronic elevation of intracranial pressure (ICP) in the absence of an intracranial mass lesion. Because VIIP and IIH share some neurologic and ophthalmologic manifestations, the latter might be used as a model to study some of the processes underlying VIIP. This work constitutes a preliminary investigation of the molecular pathways associated with the elevation of ICP in IIH. Gene expression signatures were obtained from exosomes collected from CSF and plasma in patients with possible signs of IIH. The gene expression targets focused on inflammatory genes and miRNAs. The results suggest that inflammatory cytokine-driven processes and immune cell migration are activated when ICP is elevated in IIH patients, either as a cause or effect of the ICP increase. Several miRNAs appear to be involved in this response, among which miR-9 and miR-16 are upregulated in CSF and plasma of higher ICP subjects. This study provides evidence in support of neurophysiological alterations and neuro- immunomodulation in this condition. If similar changes are seen in astronauts manifesting with the VIIP syndrome, an underlying pathophysiological basis may be discovered. npj Microgravity (2018) 4:1 ; doi:10.1038/s41526-017-0036-6 INTRODUCTION implications in understanding neurophysiologic changes during spaceflight. Human space exploration involves multi-system health risks. Neuro-ophthalmologic symptoms, including elevated intracranial pressure (ICP) upon return to Earth, have been observed in RESULTS astronauts participating in long-duration missions, a condition Subject information named visual impairment and intracranial pressure syndrome Patient information is presented in Table 1 and the Supplementary (VIIP). On Earth, these neuroanatomical findings concur with those 2 Table e-1. The average age was 33.8 ± 9.7 years (range 18–51), 2 in idiopathic intracranial hypertension (IIH). The etiology of IIH is a males and 20 females. No subject within the lower ICP group was topic of debate. The moderately elevated ICP over many years has diagnosed with IIH. The subjects in the higher ICP group were been linked to cognition losses relieved by lumbar tapping to diagnosed with IIH and exhibited typical ocular symptoms. No 3,4 reduce ICP. While most cases are seen in overweight women of correlation was found between ICP and RNFL thickness (Pearson child-bearing age, there is no specific condition on Earth that correlation coefficient between ICP and the average RNFL of both shares more similarities to VIIP, constituting a reasonable analog eyes was less than 0.4). to study the effects of chronically elevated ICP at the molecular level. 5–7 miRNA and mRNA expression in higher and lower ICP subjects A number of performance and neurologic signs (“space In plasma, the average detection rate reached almost 45% of the fog”) have also been linked to spaceflight and might also result targets in the TaqMan Open Array miRNA panel and 75% for the from elevation of ICP. In this paper, we test the hypothesis that mRNA Inflammation Open Array. In CSF, the average detection these symptoms are caused by disturbances in the neurophysiol- rate for miRNAs was approximately 15% of the targets and ogy of the brain and correlated with molecular markers in the approximately 25% for mRNA targets. Very few samples (two or cerebrospinal fluid (CSF) by studying gene expression profiles less) showed a lower detection rate and those were excluded from from CSF and plasma in individuals with suspected IIH. The gene analysis. Table 2 lists the differentially expressed miRNAs and expression signatures found suggest a systemic inflammatory mRNAs between subjects in the higher and lower ICP groups, after status corresponded by local brain inflammatory processes in a Mann–Whitney test was applied with p < 0.05 (FDR not applied). subjects with elevated ICP. Putative biomarker candidates are The sensitivity and reproducibility of the mRNA assay was very discussed, although further studies are necessary to validate their high as evaluated by multiple extractions and qRT-PCRs (R > 0.95 possible research and clinical use. These findings may have and 0.9 in plasma and CSF samples, respectively). 1 2 3 KBRwyle, NASA Johnson Space Center, Houston, TX, USA; Exosome Diagnostics, Cambridge, MA, USA and Baylor College of Medicine, Houston, TX, USA Correspondence: Susana B. Zanello (susana.b.zanello@nasa.gov) Received: 25 April 2017 Revised: 6 November 2017 Accepted: 13 December 2017 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Gene expression signatures in intracranial hypertension S B Zanello et al. Table 1. Subject distribution summarizing the mean ICP and standard Table 2. Differentially expressed genes between subjects with deviation, ICP range and retinal nerve fiber layer (RNFL) measured by normal-to-mildly elevated ICP and with ICP higher than 18 mmHg optical coherence tomography (OCT) and averaged between the two Plasma eyes on each individual miRNA U p Ratio (higher ICP/lower Fold change (log2 Lower ICP group Higher ICP group value value ICP) ratio) dme-miR-7 80 0.007 29.53 4.88 N 714 hsa-miR-938 78.5 0.009 48.11 5.59 Mean ICP 14.6 mmHg ± 2.5 24.0 mmHg ± 4.1 hsa-miR-143 76.5 0.013 579.88 9.18 ICP range 11.0–17.9 mmHg 18.8–31.4 mmHg hsa-let-7i 77 0.013 16.73 4.06 Mean RNFL 147.6 µm ± 119.9 195.2 µm ± 116.3 hsa-miR-618 14.5 0.014 0.009 −6.80 hsa-miR-374 76 0.015 78.96 6.30 Of 22 subjects recruited, ICP was not able to be determined in one of them hsa-miR-657 15 0.017 0.07 −3.84 due to technically difficult LP. That subject was therefore not included in hsa-miR-190b 15 0.017 0.003 −8.38 the study and data collection was completed in 21 subjects. The cut off between normal and elevated ICP was established at 18 mm Hg hsa-miR-10b 16 0.021 0.18 −2.47 hsa-miR-589 74 0.025 16.38 4.03 hsa-miR-16-1 74 0.026 20.45 4.35 Pathway analysis hsa-miR-298 18.5 0.027 0.0001 −13.29 miRNA Target Filter analysis in IPA was performed for the hsa-miR-551b 73 0.029 594.24 9.21 complete gene expression data from the miRNA and Inflammation Hsa-miR-545 73 0.031 17.08 4.09 mRNA panels in CSF and plasma. The process allows the Hsa-miR-375 19 0.038 0.003 −8.38 combination of experimental gene expression data with the Hsa-miR-10b 19 0.039 0.29 −1.79 existing knowledge base of predicted and validated miRNA-target Hsa-miR-145 71.5 0.042 30.74 4.94 relationships. Figure 1 summarizes the result of this process, Hsa-miR-483- 19.5 0.043 0.4 −1.32 depicting miRNAs, their targets and the most represented 5p canonical pathways related to inflammation, Hsa-let-7a 70.5 0.044 42.02 5.39 eicosanoid–phospholipase signaling and immune cell migration. Mmu-miR- 71 0.045 31.91 5.00 A comparison was run between the CSF and plasma expression data sets. Of 205 genes with expression data in plasma and 162 Hsa-miR-154 20.5 0.045 0.008 −6.97 genes in CSF, only 31 were represented in both, and only two, Gene target miRNA-16 and CD80 (CD86), were differentially expressed in both CXCR3 10 0.0008 0.05 −4.32 CSF and plasma (Table 2). miR-9 and miR-16 were upregulated by TLR3 13 0.016 0.19 −2.39 approximately 4.7 fold in CSF and plasma of higher ICP subjects, LEFTY2 70 0.02 2.64 1.4 and CD80/86 was downregulated by −5.0 fold and −2.5 fold in CSF TNFSF4 67.5 0.035 6.01 2.59 and plasma, respectively. CD70 17 0.038 0.12 −3.06 EREG 67 0.038 51.93 5.70 CD80 17 0.038 0.17 −2.56 DISCUSSION This paper reports a gene expression survey of exosomal RNA in CSF CSF and plasma from patients with elevated ICP (>18 mmHg) compared to patients with normal to moderately elevated ICP miRNA U p Ratio (higher ICP/ Fold change (log2 (11–18 mmHg). All subjects recruited for the study reported to the value value lower ICP) ratio) neurology clinic, therefore none represented a completely hsa-miR-9 75.5 0.018 24.89 4.64 “healthy” volunteer and results need to take into account the hsa-miR- 71 0.045 25.89 4.69 existence of underlying conditions. Although this study only examined a limited panel of mRNAs Gene target and miRNAs, it is expected that a similar proportion of overlapping genes would have been found with a wider expression screening, STAT5B 70.5 0.006 106.98 6.74 like whole genome microarray or RNAseq. In spite of the small TGM2 67.5 0.011 58.04 5.86 overlap in the set of differentially expressed genes from CSF and CD86 12.5 0.018 0.03 −5.06 plasma, the identified pathways associated with increased ICP (CD80) were similar. Pro-inflammatory pathways such as acute phase S100A9 62.5 0.036 91.53 6.52 response and interleukin signaling were highly represented. Both RIPK2 16 0.048 0.11 3.18 gene sets suggest that inflammatory processes are acting both locally in the brain as well as systemically, perhaps contributing to The differentially expressed miRNAs and mRNAs with p < 0.05 as IIH pathophysiology. determined by the Mann–Whitney test are shown for both plasma and Pathway analysis at the CSF level also suggested processes of CSF samples. Ratio values that are <1 indicate downregulation. The list can brain cell death, possibly targeted by miR-9. This hypothesis is also be further filtered for U values lesser than the U critical value (20 for plasma and 22 for CSF), providing more stringency supported by evidence that involves miRNA-9 in degenerative diseases, as well as promoting glial activation via the NFkB pathway. Interestingly, miR-9 is itself targeted by miR-16, which was also observed to be upregulated in the high ICP group. which has been implicated in progressive inflammatory Circulating miRNAs are increasingly gaining attention as screening myelopathy. biomarkers in central nervous system (CNS) diseases. In this For a long time, the CNS has been considered a site of immune work, identified candidates warrant monitoring in larger cohort privilege, suffering from costly physiologic consequences if studies to investigate their biomarker value, i.e., miR-16, also subject to inflammation due to trauma or infection. Currently, investigated as a biomarker for glioblastoma, and CD86 (CD80), this paradigm is being reexamined by work highlighting the npj Microgravity (2018) 1 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA 1234567890():,; Gene expression signatures in intracranial hypertension S B Zanello et al. miRNA, Molecular Targets and Pathways in High ICP VEGFA* Amyotrophic Lateral Sclerosis Signaling TNFSF13B* Agranulocyte Adhesion and Diapedesis CMTM4* VEGF Signaling GDF11* Hypoxia Signaling in the Cardiovascular System Axonal Guidance Signaling SPRED1* Altered T and B Cell Signaling in Rheumatoid Arthritis CACNB2* CXCR4* Role of Macrophages in Rheumatoid Arthritis IL-8 Signaling ABCF1* Endothelin-1 Signaling IL-10 Signaling miR-16-5p PRKCA* Acute Phase Response Signaling Huntington’s Disease Signaling miR-9-5p Aldosterone Signaling in Epithelial Cells HMOX1* NRF2-mediated Oxidative Stress Response Androgen Signaling JUN* TBP* Renin-Angiotensin Signaling 14-3-3-mediated Signaling 4-1BB Signaling in T Lymphocytes DNAJC8* NFKB1* Adipogenesis Pathway HDAC5* IL-17 Signaling GSK3B* B Cell Receptor Signaling BCL6* NR3C1* Amyloid Processing VEGFA* IL-6 Signaling KITLG* OSM* ErbB Signaling PLA2G5* CMTM4* Neuregulin Signaling EREG* CD80* Oncostatin M Signaling NRG1* IRAK2* IL6ST* Acute Phase Response Signaling ADORA2A* AOC3* Role of Macrophages in Rheumatoid Arthritis miR-16-5p Phospholipase C Signaling miR-1291 Axonal Guidance Signaling PLCD1 miR-543-3p Phospholipases miR-92a-3p Endothelin-1 Signaling miR-483-5p 14-3-3-meditated Signaling PLCB4* miR-643 Aldosterone Signaling in Epithelial Cells miR-190a-5p miR-182-5p Cardiac Hypertrophy Signaling Fig. 1 Main miRNA and canonical pathways represented in patients with elevated ICP. miRNA Target Filter Analysis (Ingenuity Pathways Analysis, IPA , QIAGEN Redwood City, www.qiagen.com/ingenuity) was applied to the differentially expressed gene data set from CSF (a) and plasma (b). miRNA and target relationships are shown by arrows, as well as the main possibly affected canonical pathways Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 1 Plasma Plasma Cytoplasm Nucleus Cytoplasm membrane membrane Gene expression signatures in intracranial hypertension S B Zanello et al. protective action that a controlled inflammatory response may Additionally, subjects had 10 ml blood drawn for plasma separation in K EDTA BD Vacutainer blood collection tubes with gel barrier. All plasma have on the CNS. For example, it has been shown that mice 2 and CSF samples were pre-filtered through a 0.8-µm syringe filter prior to deficient in functional T cells underperform normal mice in microvesicular processing, aliquoted and stored at −80 °C in a freezer at cognitive tasks and this pro-cognitive effect may be mediated by the Center for Space Medicine at BCM and then shipped to the Exosome 14,15 IL-4. Our work supports the existence of an underlying Diagnostics facilities in Cambridge, MA. subclinical inflammatory status in IIH. Further studies should be directed towards assessing the extent of these processes and their Exosome and RNA isolation correlation with neurocognitive parameters. CSF at volume of 5 ml and plasma at volume of 4 ml (pre-filtered) were This study has some limitations. First, it constitutes an used for exosome isolation by the ExosomeDx spin column-based method exploratory study with small sample size. Second, the lower ICP (expRNAeasy SerumPlasma Maxi kit, Qiagen, Valencia, CA). The method average, 14.6 mmHg, lies on the higher limit of what is considered allows the reproducible isolation of high-quality exosome-specific RNA, a normal ICP (5–15 mmHg), and therefore the comparison with which includes both mRNA and miRNA fractions. Briefly, the pre-filtered high ICP may not yield as many differentially expressed genes as sample was mixed 1:1 with 2× binding buffer (XBP) and added to the would be expected in comparison with a normal ICP group. Third, exoEasy membrane affinity column to bind the exosomes to the membrane. After centrifugation, the flow-through was discarded and IIH patients are arguably a distinct population from the normal wash buffer (XWP) was added to the column to wash off non-specifically healthy astronaut. Since IIH is more prevalent in women, this study retained material. After another centrifugation and discarding of the flow- contains a majority of females, also deviating from the sample through, the vesicles were lysed by adding QIAzol solution and following population of astronauts so far screened for VIIP. the addition of chloroform, thorough mixing and centrifugation to Our findings support new concepts regarding the communica- separate organic and aqueous phases, the aqueous phase was recovered tion between the immune and nervous systems. The IIH patients and mixed with ethanol. The sample–ethanol mixture was added to an evidenced signs of generalized inflammation that may be RNAeasy MinElute spin column and centrifuged. The column was washed once with buffer RWT, and then twice with buffer RPE followed by elution affecting neural physiology at various levels, from neural signaling of RNA in a final volume of 14 μL water. to cognition. These patients were predominantly obese (average BMI = 34.7 ± 7.4 kg/m ), with likely associated metabolic syn- drome, chronic low-grade inflammation, and insulin resistance MicroRNA analysis and other obesity-associated complications, for which pathways Reverse transcription (RT) of miRNA was performed using the Megaplex™ and processes were identified in the analysis. RT Primers (ThermoFisher Scientific, Waltham, MA) to prepare cDNA for real-time PCR analysis on a TaqMan MicroRNA Array, including a pre- The value of this study predominantly lies in being the first amplification step. Collectively, the Human Pool Set v3.0 containing two evaluation of the molecular players in IIH and the first comparison Megaplex™ RT Primer Pools, Pools A v2.1 and B v3.0 cover 754 unique between expression profiles from exosomes in CSF and plasma for microRNAs. The RT reaction for each primer pool was set as follows: 1× this condition. Because elevation of ICP is a hallmark of the Megaplex RT Primers (Pool A or B), 2 mM dNTPs (with dTTP), 10 U/μL disease, findings from this study may be relevant to the VIIP MultiScribe reverse transcriptase, 1.5 mM MgCl2, 1× RT buffer, 0.25 U/ μL syndrome. The results from the CSF survey suggest RNAse inhibitor, in a final volume of 15 μL per reaction. The reaction was neuro–immunomodulatory processes, and further work including run through the temperature sequence per the manufacturer’s instructions. astronauts exposed to microgravity and the concomitant fluid Preamplification was performed in a 50 μL reaction with a 10 μL aliquot shifts with ICP increase, is proposed to test this hypothesis on a of the RT reaction as a template, 1× TaqMan PreAmp Master Mix and 1× more relevant sample population. Megaplex™ PreAmp Primers (Pool A or Pool B), in a temperature cycling (16 cycles) according to the manufacturer. Pre-amplified samples were ® ® diluted 1:10 in 0.1× TE pH 8.0. The TaqMan OpenArray Human MicroRNA METHODS Panel was used for final PCR detection. Sample collection In this final step, the DNA polymerase of the TaqMan Universal PCR Methods were performed in accordance with relevant regulations and Master Mix amplifies the specific cDNA targets using sequence-specific primers and probe on the TaqMan microRNA Array. 25 µL TaqMan guidelines. The study was reviewed and approved by the institutional OpenArray Real-Time PCR Master Mix and 13 µL 0.1× buffer TE pH 8.0 review boards at Johnson Space Center and Baylor College of Medicine were loaded into each of two adjacent wells per sample on a clean 96-well (BCM), Houston, TX. Subject recruitment was done at BCM and written plate. For each sample, 12 µL of diluted Pool A PreAmp was pipetted into informed consent was obtained from all subjects. one well of each pair and 12 µL of diluted Pool B PreAmp was pipetted into The study population was a pool of neurological patients with suspected the other well for a total volume of 50 µL in each well. For each sample and IIH, requiring diagnostic or therapeutic LP. Subjects were recruited on the primer pool set (Pool A or Pool B), 5 µL of each reaction mixture was basis of the modified Dandy criteria for IIH and the IIH treatment trial, pipetted into each of eight wells on an OpenArray 384-well sample plate. consisting in the presence of signs and symptoms of raised ICP (>20 cm Samples were loaded onto OpenArray plates using the standard Accufill™ H O = 14.7 mmHg), absence of localized findings except those from protocol. Amplification was performed according to the protocol increased ICP, or abnormal neuroimaging except for empty sella turcica ® ® established for the TaqMan OpenArray Human MicroRNA panel. and distended optic nerve sheath. The subject was awake and alert, and no other cause of increased ICP was present. For the LP, the subjects lay in the lateral decubitus position with legs slightly extended. Once the needle was mRNA analysis in the thecal sac at the L3-L4 or L4-L5 region, the manometer was The RT reaction was carried out in a final volume of 20 μL containing: 14 μL observed for the presence of pulse and respiratory waves to indicate RNA sample, 2 μL Superscript reverse transcriptase enzyme mix, and 4 μL patent communication between needle and subarachnoid space. Next, the VILO Master Mix (which includesRNaseOUT™ Recombinant Ribonuclease opening pressure was monitored over a period of 5 min, ICP was measured Inhibitor, MgCl2, dNTPs and random primers). The reaction proceeded for every minute, and the average calculated. CSF was then drained as per the 10 min at 95 °C, 15 s at 95 °C, 4 min at 60 °C, followed by inactivation at 95 ° normal clinical procedure. A volume of 5 ml was collected specifically for C for 10 min. the purpose of this study. Pre-amplification was done using Human Inflammation PreAmp Primers Of 22 subjects recruited, ICP was not able to be determined in one of (Pool A or B) 5 μL, TaqMan PreAmp Master Mix 10 μL, and 5 μLofRT them due to technically difficult LP. That subject was therefore not product (cDNA) in a final volume of 50 μL. The temperature sequence was included in the study and data collection was completed in 21 subjects. the following: 10 min at 95 °C, 15 s at 95 °C and 4 min at 60 °C (for 14 The cut off between normal and elevated ICP was established at 18 mm cycles), followed by inactivation at 95 °C for 10 min. The pre-amplified Hg. Patients were grouped into “normal to moderately high ICP” subjects samples were stored at 4 °C until the PCR was performed. ® ® (less than 18 mmHg) and high ICP (higher than 18 mmHg). Seven “normal The TaqMan OpenArray Human Inflammation Panel (Applied Biosys- to mild ICP” and 14 high ICP subjects were compared in the study. tems, ThermoFisher Scientific) was used for final PCR detection. These npj Microgravity (2018) 1 Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA Gene expression signatures in intracranial hypertension S B Zanello et al. contain TaqMan gene expression assays dried down in 384-well TaqMan Competing interests: Dr Zanello is the recipient of the NASA award funding this Array microfluidic cards targeting pathway relevant gene targets. The study, as well as other research support from the agency. Dr Tadigotla is employed by panel covers 586 genes that have been studied as targets for a range of Exosome Diagnostics, Inc. and holds stock options from Exosome Diagnostics. Dr inflammatory diseases, plus 21 endogenous control genes. James Hurley is employed by Exosome Diagnostics, Inc. and holds stock options from PreAmp products for each sample were mixed and diluted 1:10 with 0.1× Exosome Diagnostics. Dr Skog is Chief ScientificOfficer at Exosome Diagnostics, Inc. ® ® TE pH = 8.0. For each sample, 35 μL of 2× TaqMan OpenArray Real-Time and holds stock options from Exosome Diagnostics. Mr Brian Stevens and Ms Eusebia PCR Master Mix was mixed with 35 μL mixed, diluted PreAmp. 5 μL of each Calvillo report no disclosures. Dr Bershad performs neurology clinical practice, and reaction mixture was pipetted into each of 12 wells on an OpenArray 384- conducts several research projects funded by NASA and the NSBRI (National Space well sample plate. Samples were loaded onto OpenArray plates using the Biomedical Research Institute) standard Accufill™ protocol. Amplification was performed according to the ® ® protocol established for the TaqMan OpenArray Human Inflammation Publisher's note: Springer Nature remains neutral with regard to jurisdictional claims Panel with the QuantStudio™ 12 K Flex Real-time PCR system. in published maps and institutional affiliations. Data analysis and bioinformatics REFERENCES The mRNA data comprises cycle threshold (Ct) values from the OpenArray 1. Mader, T. H. et al. Optic disc edema, globe flattening, choroidal folds, and QuantStudio™ 12 K Flex Real-time PCR system, which is converted to gene hyperopic shifts observed in astronauts after long-duration space flight. Oph- expression levels for a panel of 607 inflammation-related genes and thalmology 118, 2058–2069 (2011). endogenous control genes. The miRNA data set consists of 754 different 2. Peng, K. P., Fuh, J. L. & Wang, S. J. 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The NORDIC Idiopathic Intracranial Hypertension Study Group Writing Commit- of Houston College of Optometry for performing the ophthalmological evaluations, tee. Effect of Acetazolamide on visual function in patients with idiopathic intra- and John Blackwell, USRA-LPI, for artwork. This study was funded by NASA (Grant cranial hypertension and mild visual lossthe idiopathic intracranial hypertension NNX15AW48G). Other Contributors (non-authors): Rosa Tang, MD, MPH (University treatment trial. JAMA 311, 1641–1651 (2014). Eye Institute, University of Houston; ophthalmic examinations); B Gutierrez Flores, MD (University Eye Institute, University of Houston; ophthalmic examinations) Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, AUTHOR CONTRIBUTIONS adaptation, distribution and reproduction in any medium or format, as long as you give S.Z., study concept and design, study supervision, analysis and interpretation of data, appropriate credit to the original author(s) and the source, provide a link to the Creative manuscript writing, funded investigator. V.T., gene expression data and statistical Commons license, and indicate if changes were made. The images or other third party analysis. J.H., sample processing and analysis. J.S., study supervision, concept and material in this article are included in the article’s Creative Commons license, unless design, data analysis. E.C., research coordination. B.S., sampling handling, subject data indicated otherwise in a credit line to the material. If material is not included in the analysis. E.B., sample collection, study concept and design, critical revision of article’s Creative Commons license and your intended use is not permitted by statutory manuscript. regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons. org/licenses/by/4.0/. ADDITIONAL INFORMATION Supplementary information accompanies the paper on the npj Microgravity website © The Author(s) 2018 (https://doi.org/10.1038/s41526-017-0036-6). Published in cooperation with the Biodesign Institute at Arizona State University, with the support of NASA npj Microgravity (2018) 1

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