This is the first description of the effects of exercise on cerebrospinal fluid miRNA expression in healthy subjects. Exercise diminished miR-328 and miR-608 in all subjects suggesting a general effect on the brain (Fig. 8, Supplementary Table S5). Exercise caused distinct patterns of miRNA changes in CFS and the START and STOPP phenotypes of GWI indicating significant pathophysiological differences between conditions. 
 
 Unlike our starting hypothesis, there were no differences in miRNA levels between the nonexercise groups of control, CFS and GWI subjects. Therefore, baseline levels of cerebrospinal fluid miRNAs may not be useful for diagnosis of CFS or GWI. 
 
 The only significant differences between groups after exercise were diminished miR-22-3p in START compared to SC and STOPP, and elevated miR-9-3p in START compared to STOPP (Fig. 6). These differences between START and STOPP support our 2 phenotypes of GWI25. 
 
 The most striking findings were the changes between post-exercise groups and their appropriate nonexercise comparison groups. SC had 5 elevated miRNAs after exercise, compared to 3 for START, 1 for STOPP, and none in CFS (Fig. 7, Supplementary Table S4). 
 
 The reduction of miR-608 after exercise has implications for the cholinergic hypothesis of GWI pathophysiology because it targets acetylcholinesterase and interleukin-6 (IL6) mRNAs. miR-608 binds weakly to the single-nucleotide polymorphism rs17228616 allele in the 3′-untranslated region of acetylcholinesterase mRNA44. Homozygotes for rs17228616 have reduced affinity for miR-608. This promotes mRNA stability and increases acetylcholinesterase protein translation. As a consequence, more miR-608 is available to bind to IL6 mRNA and reduces its translation. This allele also contributes to reduced cortisol and elevated blood pressure. Because rs17228616 promotes higher acetylcholinestase activity, it may be relatively protective against nerve agent and pyridostimine bromide exposure. 
 
 miR-let-7i-5p, miR-93-3p and miR-200a-5p were significantly diminished after exercise in START, STOPP and CFS, but not SC (Fig. 9). This was consistent with a cardinal finding in CFS and GWI: function may appear normal when rested, but will deteriorate after a physiological stressor1,2,3,4,7,25,45,46. miR-let-7i was reduced in plasma after exercise in athletes47. miR-let-7i has decreased expression in the prefrontal cortex of FSL rats in a model of depression48. IL6 is a target of miR-let-7i, and, as predicted, this cytokine was significantly elevated in the brains of these rats. When FSL rats were given access to running wheels, their miR-let-7i expression was increased and IL6 reduced. Modulation of miR-let-7i and IL-6 may contribute to exercise-induced benefits in “inflammatory” depression48. miR-let-7i also contributes to the regulation of acetylcholine’s muscarinic and α4β2 nicotinic receptors and epigenetic regulation of acetylcholinesterase. These animal models may not be appropriate for CFS or GWI because human subjects develop exertional exhaustion after exercise, and are unlikely to significantly increase spontaneous exercise levels when provided with a treadmill45. 
 
 The CFS group had 12 miRNAs reduced after exercise. miR-186-3p was decreased in aging mice49 where it targets β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) mRNA. Diminished miR-186-3p allows increased BACE1 mRNA translation and cleavage of amyloid peptides that increase the risk for brain disease. miR-19b-3p was reduced in serum from Alzheimer’s patients, and targeted signal transduction and activator of transcription 3 (STAT3) mRNA in a murine model50. miR-92a-3p was increased in glioblastoma and targeted BCL2L11 to reduce tumor apoptosis51. Its reduction after exercise may promote apoptosis in CFS. miR-126-5p was highly expressed in endothelial cells where it targets vascular (VCAM), intercellular (ICAM) and activated leukocyte (ALCAM) cell adhesion molecule mRNAs and so reduces transendothelial migration52,53. This is relevant for immune cell influx into the brain, and hypotheses of neuroinflammation in CFS pathogenesis. 
 
 Neurons may be the sources of miR-124-3p, miR-127-3p, miR-433, and miR-323b-5p (Figs 4 and 10)54. There was little overlap with the miRNAs synthesized in astrocytes, oligodendrocytes and microglia. 
 
 The choroid plexus epithelium may be a primary source of miRNAs in cerebrospinal fluid55,56,57. Epithelial cells form a monolayer linked by tight junctions that creates the “shrink wrapped” cellular barrier around fenestrated capillaries58. Interferon-gamma and other mediators generated by exercise, inflammation, and other stressors act directly on choroid plexus to modulate barrier permeability, plasma protein transport, protein synthesis and secretion of nutrients into cerebrospinal fluid59,60,61. miR-328, which was present in all subjects and reduced after exercise (Fig. 8), binds to the 3′-untranslated regions of CD44 and collagen type 1α1 mRNAs to modulate extracellular barrier functions62. Choroid plexus miRNAs55 are packaged into extracellular vesicles and released into cerebrospinal fluid56,57,63,64. Downstream targets include subventricular neural stem cells, mature neurons, astrocytes, oligodendrocytes, microglia, meningeal and central immune cells56,57,63,65,66,67. Blockade of extracellular vesicle secretion from choroid plexus cells decreased brain inflammation in a mouse model of lipopolysaccharide-induced inflammation56. Choroid plexus miRNAs may be novel drug targets to modulate acute illness behaviours, fever, and chronic pain in systemic illnesses. 
 
 Choroid plexus is dysfunctional in Alzheimer’s disease68. This provides the rationale to consider the role of the blood – cerebrospinal fluid barrier in the cognitive dysfunction of CFS and GWI. There are numerous reports of elevated and diminished miRNAs in cerebrospinal fluid in Alzheimer’s disease36,69,70,71,72,73,74, but none matched the patterns of our groups. miR-let-7i-5p36 was elevated in Alzheimer’s, but levels were equivalent in nonexercise groups (Fig. 8). 
 
 Depression is in the differential diagnosis because of the shared ancillary diagnostic criteria (Fig. 1)75,76,77,78. Major depressive disorder is defined by affective dysfunction with sadness, flat affect and anhedonia as essential features, followed by secondary criteria including fatigue, cognitive, sleep, and somatic dysfunction78. However, screening questionnaires for depression emphasize somatic symptoms79,80. Complaints of fatigue, sleep, and cognitive dysfunction will inflate total questionnaire scores, and may lead to false positive inference of major depressive disorder even if anhedonia or affective complaints are absent81,82,83. This is particularly problematic in CFS and GWI where these features are diagnostic criteria (Fig. 1). As a result, Center for Epidemiology – Depression (CESD)33 scores were significantly elevated in GWI (78.3%), CFS (64.0%) and control (25.0%) groups (Table 1). 
 
 Quantitative PCR of miRNAs offers a more objective solution84. miR-16 in cerebrospinal fluid was significantly lower in major depressive disorder patients than control subjects85. However, this was not confirmed in an independent group who had a different pattern of 11 significantly elevated and 5 reduced miRNAs86. Our data did not confirm either of these findings because only 3 of the miRNAs were detected with Ct ≤ 35 in more than two thirds of our nonexercise group. miR-425-3p was significantly reduced in depression patients84,85,86, and was detected in about half of all nonexercise subjects. It was increased after exercise in SC, START and STOPP but not CFS (Fig. 7). This lack of reproducibility highlights the need to independently verify miRNA findings, and supports our rationale for strict statistical criteria to define potential miRNA biomarkers. 
 
 The pain and tenderness of GWI subjects (Table 1) indicated systemic hyperalgesia and suggested parallels with fibromyalgia34. Nine miRNAs were virtually undetectable in 10 fibromyalgia women compared to 8 healthy control women87. miR-99b-5p and miR-29a-3p were absent in fibromyalgia, but were detected in more than two thirds of our participants. miR-99b-5p was significantly increased after exercise in SC, START and STOPP (Fig. 7). The other 7 miRNAs were detected in less than half of our specimens. This suggested that GWI and CFS were distinct from fibromyalgia. 
 
 Limitations to the diagnostic use of quantitative miRNA analysis in cerebrospinal fluid include the remarkable lack of consensus about miRNA levels in control subjects. This can be remedied by standardization of reagents and protocols72, open source sharing of study outcomes, and meta-analysis of the raw data. The yield of extracted miRNA88 and detectability were improved with 0.5 ml instead of 0.2 ml of cerebrospinal fluid36,87. QPCR with Ct cut-offs ≤ 35 cycles reduced amplification artifacts73,87. The wide range of miR-22-3p Ct values (Fig. 6) may be due to commercial changes in reagents designed to improve miRNA detection. Highly abundant miRNAs that were detected with Ct < 35 in all subjects were used as normalizers (Fig. 4) rather than the global average level, miR-423-5p, miR-124-3p or U636,71,73,86,89. Constraints included (i) significant ANOVA and Tukey HSD between groups, (ii) significant FDR to correct for multiple comparisons, and (iii) focusing on miRNAs that were detected in more than two thirds of subjects per group that may be viable biomarker candidates for use in the general population36,71,86. Ages were comparable between groups74 (Table 1) and there were no differences in expression between males and females43,87. Next generation sequencing is an excellent discovery tool but needs careful internal standardization to be as sensitive as QPCR for quantification90,91,92. Adequate sample sizes were essential because our initial findings with about a dozen subjects per group showed differences between START and STOPP after exercise93, but these differences eventually regressed to the mean as more subjects were analyzed. This is especially pertinent to smaller studies examining the differential diagnosis of CFS and GWI85,86,87,94,95. 
 
 Limitations of the testing paradigm include the intensive nature of the exercise and magnetic resonance imaging characterization of GWI subjects to determine their phenotypes. Lumbar puncture was required to obtain the cerebrospinal fluid miRNA biomarkers, but this procedure is not a contraindication to making an objective diagnosis of GWI. On the contrary, magnetic resonance imaging with cerebrospinal fluid QPCR miRNA profiling may be complementary tools for diagnosis of CFS, GWI and their subtypes. 

 WASHINGTON -- Researchers at Georgetown University Medical Center have found distinct molecular signatures in two brain disorders long thought to be psychological in origin -- chronic fatigue syndrome (CFS) and Gulf War Illness (GWI). 
 
 In addition, the work supports a previous observation by GUMC investigators of two variants of GWI. The disorders share commonalities, such as pain, fatigue, cognitive dysfunction and exhaustion after exercise. 
 
 Their study, published in Scientific Reports, lays groundwork needed to understand these disorders in order to diagnosis and treat them effectively, says senior investigator, James N. Baraniuk, MD, professor of medicine at Georgetown University School of Medicine. Narayan Shivapurkar, PhD, assistant professor of oncology at the medical school worked with Baraniuk on the research. 
 
 The changes in brain chemistry -- observed in levels of miRNAs that turn protein production on or off -- were seen 24 hours after riding a stationary bike for 25 minutes. 
 
 "We clearly see three different patterns in the brain's production of these molecules in the CFS group and the two GWI phenotypes," says Baraniuk. "This news will be well received by patients who suffer from these disorders who are misdiagnosed and instead may be treated for depression or other mental disorders." 
 
 Chronic fatigue syndrome affects between 836,000 and 2.5 million Americans, according to a National Academy of Medicine report. The disorder was thought to be psychosomatic until a 2015 review of 9,000 articles over 64 years of research pointed to unspecified biological causes. Still, no definitive diagnosis or treatment is available. 
 
 Gulf War Illness has developed in more than one-fourth of the 697,000 veterans deployed to the 1990-1991 Persian Gulf War, Baraniuk and his colleagues have reported in earlier work. 
 
 Gulf War veterans were exposed to combinations of nerve agents, pesticides and other toxic chemicals that may have triggered the chronic pain, cognitive, gastrointestinal and other problems, Baraniuk says. Although the mechanisms remain unknown, the study provides significant insights into brain chemistry that can now be investigated. 
 
 This study focused on spinal fluid of CFS, GWI and control subjects who agreed to have a lumbar puncture. Spinal taps before exercise showed miRNA levels were the same in all participants. In contrast, miRNA levels in spinal fluid were significantly different after exercise. The CFS, control and two subtypes of GWI groups had distinct patterns of change. For example, CFS subjects who exercised had reduced levels of 12 different mRNAs, compared to those who did not exercise. 
 
 The miRNA changes in the two GWI subtypes add to other differences caused by exercise. One subgroup developed jumps in heart rate of over 30 beats when standing up that lasted for two to three days after exercise. Magnetic resonance imaging showed they had smaller brainstems in regions that control heart rate, and did not activate their brains when doing a cognitive task. In contrast, the other subgroup did not have any heart rate or brainstem changes, but did recruit additional brain regions to complete a memory test. The two groups were as different from each other as they were from the control group. 
 
 Finding two distinct pathophysiological miRNA brain patterns in patients reporting Gulf War disease "adds another layer of evidence to support neuropathology in the two different manifestations of Gulf War disease," he says. 
 
 Baraniuk adds that miRNA levels in these disorders were different from the ones that are altered in depression, fibromyalgia, and Alzheimer's disease, further suggesting CFS and GWI are distinct diseases. 
 
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 The study was supported by funding from The Sergeant Sullivan Center, Dr. Barbara Cottone, Dean Clarke Bridge Prize, Department of Defense Congressionally Directed Medical Research Program (CDMRP) W81XWH-15-1-0679, and National Institute of Neurological Diseases and Stroke R21NS088138 and RO1NS085131. 
 
 Baraniuk and Shivapurkar are named as inventors on a patent application that has been filed by Georgetown University related to the technology described. 
 
 About Georgetown University Medical Center 
 
 Georgetown University Medical Center (GUMC) is an internationally recognized academic medical center with a three-part mission of research, teaching and patient care (through MedStar Health). GUMC's mission is carried out with a strong emphasis on public service and a dedication to the Catholic, Jesuit principle of cura personalis -- or "care of the whole person." The Medical Center includes the School of Medicine and the School of Nursing & Health Studies, both nationally ranked; Georgetown Lombardi Comprehensive Cancer Center, designated as a comprehensive cancer center by the National Cancer Institute; and the Biomedical Graduate Research Organization, which accounts for the majority of externally funded research at GUMC including a Clinical and Translational Science Award from the National Institutes of Health. Connect with GUMC on Facebook (Facebook.com/GUMCUpdate), Twitter (@gumedcenter) and Instagram (@gumedcenter).
– More than a quarter of the nearly 700,000 US veterans who served in the Persian Gulf War in the early 1990s have been diagnosed with Gulf War Illness. And up to 2.5 million Americans are believed to be affected by chronic fatigue syndrome. Despite symptoms ranging from pain to cognitive dysfunction to exhaustion after exercise, both disorders were long believed to be psychological in nature, according to a press release. But in a study published Friday in Scientific Reports, researchers say they've found unique molecular patterns in the brain that hint at a physical cause for the brain disorders. And that's a first step toward figuring out a way to both diagnose and treat Gulf War Illness and chronic fatigue syndrome. Researchers observed subjects 24 hours after riding a stationary bike for 25 minutes. They found different changes in the levels of miRNAs in the brain between chronic fatigue syndrome patients, two subsets of Gulf War Illness sufferers, and a control group. MiRNAs are responsible for turning protein production on and off. But that wasn't the only difference between the various groups. Researchers also found one subgroup of Gulf War Illness patients saw their heart rate increase over 30 beats while standing for up to three days after exercising; the other subgroup required additional brain regions to complete a memory test following exercise. There is no settled-on diagnosis or treatment for chronic fatigue syndrome, which was considered psychosomatic until 2015. And the exact cause of Gulf War Illness remains unknown.