{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,5,5]],"date-time":"2026-05-05T02:53:05Z","timestamp":1777949585371,"version":"3.51.4"},"reference-count":43,"publisher":"MDPI AG","issue":"5","license":[{"start":{"date-parts":[[2026,5,2]],"date-time":"2026-05-02T00:00:00Z","timestamp":1777680000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Brain Sciences"],"abstract":"<jats:p>Background\/Objectives: High-altitude exposure represents a complex psychophysiological stressor involving hypoxia, physical effort, sleep disruption and psychological strain. The endocannabinoid system (ECS) plays a key role in stress regulation, yet its epigenetic modulation under extreme environmental conditions remains poorly characterized. This pilot and exploratory study investigated DNA methylation and descriptive microRNA (miRNA) expression patterns of CNR1 and FAAH genes, and their associations with mood and anxiety outcomes, in trekkers exposed to Himalayan high altitude. Methods: Twenty-one healthy lowlanders completed a longitudinal expedition from 2860 m to 5050 m. Psychometric measures (SVARAD, BDI, SAS, SHAPS) and saliva samples were collected at baseline (T0) and at high altitude (T1). DNA methylation of CNR1 and FAAH regulatory regions was quantified by pyrosequencing. Exosomal miRNAs targeting these genes were profiled using qRT-PCR, on pooled samples; results are presented descriptively. Results: DNA methylation analysis revealed heterogeneous, sex-specific epigenetic patterns following high-altitude exposure. A significant increase in CNR1 promoter methylation at CpG4 was observed in males at T1, whereas methylation remained largely stable in females. Descriptive miRNA expression data showed bidirectional differences between groups, consistent with context-dependent stress regulation. Convergent directional patterns between miR-23b-3p expression and CNR1 methylation in males were observed. However, given the descriptive nature of the miRNA data, this observation is purely exploratory and requires replication before any mechanistic conclusions can be drawn. Psychometrically, participants showed a mild mood decline without overt clinical symptoms. Sex-specific differences in the relationship between CNR1 methylation and psychometric outcomes were observed and warrant further investigation in adequately powered cohorts. Conclusions: These preliminary findings suggest that CNR1 epigenetic regulation warrants further investigation as a potential indicator of stress adaptation and psychological responses and underscore the need to consider sex differences when evaluating resilience and vulnerability to extreme environments.<\/jats:p>","DOI":"10.3390\/brainsci16050500","type":"journal-article","created":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T02:06:21Z","timestamp":1777860381000},"page":"500","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Sex-Specific Epigenetic Patterns in Endocannabinoid System Genes Following High-Altitude Exposure: An Exploratory Study"],"prefix":"10.3390","volume":"16","author":[{"ORCID":"https:\/\/orcid.org\/0009-0006-6953-3771","authenticated-orcid":false,"given":"Carlotta","family":"Marrangone","sequence":"first","affiliation":[{"name":"Department of Neuroscience, Imaging and Clinical Sciences, \u201cG. d\u2019Annunzio\u201d University, 66100 Chieti, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3832-1398","authenticated-orcid":false,"given":"Alessio","family":"Mosca","sequence":"additional","affiliation":[{"name":"Department of Neuroscience, Imaging and Clinical Sciences, \u201cG. d\u2019Annunzio\u201d University, 66100 Chieti, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0000-5859-5869","authenticated-orcid":false,"given":"Manuel","family":"Marzola","sequence":"additional","affiliation":[{"name":"Department of Medicine and Aging Sciences, \u201cG. d\u2019Annunzio\u201d University, 66100 Chieti, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0009-0003-9921-8690","authenticated-orcid":false,"given":"Francesca","family":"Martella","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4266-7607","authenticated-orcid":false,"given":"Martina","family":"Di Bartolomeo","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-8343-9024","authenticated-orcid":false,"given":"Vittore","family":"Verratti","sequence":"additional","affiliation":[{"name":"Department of Science, \u201cG. d\u2019Annunzio\u201d University, Italian Society of Mountain Medicine, 66100 Chieti, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-7292-2341","authenticated-orcid":false,"given":"Giovanni","family":"Martinotti","sequence":"additional","affiliation":[{"name":"Department of Neuroscience, Imaging and Clinical Sciences, \u201cG. d\u2019Annunzio\u201d University, 66100 Chieti, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-1275-098X","authenticated-orcid":false,"given":"Claudio","family":"D\u2019Addario","sequence":"additional","affiliation":[{"name":"Department of Bioscience and Technology for Food, Agriculture and Environment, University of Teramo, 64100 Teramo, Italy"}],"role":[{"role":"author","vocabulary":"crossref"}]}],"member":"1968","published-online":{"date-parts":[[2026,5,2]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"1188","DOI":"10.1111\/sms.12557","article-title":"Responses of peripheral blood mononuclear cells to moderate exercise and hypoxia","volume":"26","author":"Morabito","year":"2016","journal-title":"Scand. J. Med. Sci. Sports"},{"key":"ref_2","doi-asserted-by":"crossref","first-page":"129","DOI":"10.1007\/s00421-015-3256-z","article-title":"Gokyo Khumbu\/Ama Dablam Trek 2012: Effects of physical training and high-altitude exposure on oxidative metabolism, muscle composition, and metabolic cost of walking in women","volume":"116","author":"Tam","year":"2016","journal-title":"Eur. J. Appl. Physiol."},{"key":"ref_3","doi-asserted-by":"crossref","first-page":"57","DOI":"10.1089\/ham.2021.0029","article-title":"Neuropsychological and Neuroimaging Correlates of High-Altitude Hypoxia Trekking During the \u201cGokyo Khumbu\/Ama Dablam\u201d Expedition","volume":"23","author":"Committeri","year":"2022","journal-title":"High Alt. Med. Biol."},{"key":"ref_4","doi-asserted-by":"crossref","unstructured":"Aboouf, M.A., Thiersch, M., Soliz, J., Gassmann, M., and Gasser, E.M.S. (2023). The Brain at High Altitude: From Molecular Signaling to Cognitive Performance. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms241210179"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"637","DOI":"10.1080\/09540261.2019.1586324","article-title":"Altitude and risk of depression and anxiety: Findings from the intern health study","volume":"31","author":"Kious","year":"2019","journal-title":"Int. Rev. Psychiatry"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"388","DOI":"10.1016\/j.jad.2022.08.079","article-title":"Prevalence of depression and depressive symptoms at high altitudes: A systematic review and meta-analysis","volume":"317","year":"2022","journal-title":"J. Affect. Disord."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Boos, C.J., Bass, M., O\u2019Hara, J.P., Vincent, E., Mellor, A., Sevier, L., Abdul-Razakq, H., Cooke, M., Barlow, M., and Woods, D.R. (2018). The relationship between anxiety and acute mountain sickness. PLoS ONE, 13.","DOI":"10.1371\/journal.pone.0197147"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"336","DOI":"10.1038\/s41401-018-0051-5","article-title":"Integrating endocannabinoid signaling in the regulation of anxiety and depression","volume":"40","author":"Yin","year":"2019","journal-title":"Acta Pharmacol. Sin."},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Zhao, P., Li, S., He, Z., and Ma, X. (2024). Physiological and Genetic Basis of High-Altitude Indigenous Animals\u2019 Adaptation to Hypoxic Environments. Animals, 14.","DOI":"10.3390\/ani14203031"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"1020","DOI":"10.1016\/j.euroneuro.2016.03.003","article-title":"Genetically reduced FAAH activity may be a risk for the development of anxiety and depression in persons with repetitive childhood trauma","volume":"26","author":"Lazary","year":"2016","journal-title":"Eur. Neuropsychopharmacol."},{"key":"ref_11","doi-asserted-by":"crossref","first-page":"2426","DOI":"10.1111\/acer.13210","article-title":"FAAH Gene Variation Moderates Stress Response and Symptom Severity in Patients with Posttraumatic Stress Disorder and Comorbid Alcohol Dependence","volume":"40","author":"Spagnolo","year":"2016","journal-title":"Alcohol. Clin. Exp. Res."},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"380","DOI":"10.1016\/j.smim.2014.04.001","article-title":"Stress regulates endocannabinoid-CB1 receptor signaling","volume":"26","author":"Hillard","year":"2014","journal-title":"Semin. Immunol."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"476","DOI":"10.1016\/j.lfs.2012.06.009","article-title":"Sex differences in cannabinoid pharmacology: A reflection of differences in the endocannabinoid system?","volume":"92","author":"Craft","year":"2012","journal-title":"Life Sci."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"264","DOI":"10.1016\/j.bbr.2009.05.013","article-title":"Differential effects of chronic unpredictable stress on hippocampal CB1 receptors in male and female rats","volume":"203","author":"Reich","year":"2009","journal-title":"Behav. Brain Res."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"10506","DOI":"10.1523\/JNEUROSCI.0496-11.2011","article-title":"Recruitment of prefrontal cortical endocannabinoid signaling by glucocorticoids contributes to termination of the stress response","volume":"31","author":"Hill","year":"2011","journal-title":"J. Neurosci."},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"569","DOI":"10.1152\/japplphysiol.00645.2022","article-title":"Regulation of the endocannabinoid system by endurance and resistance exercise in hypoxia in human skeletal muscle","volume":"134","author":"Dalle","year":"2023","journal-title":"J. Appl. Physiol."},{"key":"ref_17","doi-asserted-by":"crossref","unstructured":"Rubino, T., and Parolaro, D. (2011). Sexually dimorphic effects of cannabinoid compounds on emotion and cognition. Front. Behav. Neurosci., 5.","DOI":"10.3389\/fnbeh.2011.00064"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"9","DOI":"10.1007\/s00213-020-05712-8","article-title":"Cannabidiol: Pharmacology and therapeutic targets","volume":"238","author":"Britch","year":"2021","journal-title":"Psychopharmacology"},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"431","DOI":"10.1038\/s41398-024-03151-z","article-title":"Endocannabinoid concentrations in major depression: Effects of childhood maltreatment and relation to hippocampal volume","volume":"14","author":"Mazurka","year":"2024","journal-title":"Transl. Psychiatry"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1905","DOI":"10.1111\/febs.12125","article-title":"Epigenetic mechanisms and endocannabinoid signalling","volume":"280","author":"Pucci","year":"2013","journal-title":"FEBS J."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"157","DOI":"10.1016\/j.neuroscience.2013.12.003","article-title":"Epigenetics and the regulation of stress vulnerability and resilience","volume":"264","author":"Zannas","year":"2014","journal-title":"Neuroscience"},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"23","DOI":"10.1038\/npp.2012.112","article-title":"DNA Methylation and its basic function","volume":"38","author":"Moore","year":"2013","journal-title":"Neuropsychopharmacology"},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"18","DOI":"10.1016\/j.tins.2023.09.007","article-title":"Epigenetic mechanisms underlying sex differences in the brain and behavior","volume":"47","author":"Kundakovic","year":"2024","journal-title":"Trends Neurosci."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"256","DOI":"10.1016\/j.neuropharm.2018.10.020","article-title":"miR-132\/212 is induced by stress and its dysregulation triggers anxiety-related behavior","volume":"144","author":"Aten","year":"2019","journal-title":"Neuropharmacology"},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"58","DOI":"10.1016\/j.jpsychires.2016.07.012","article-title":"The microRNA network is altered in anterior cingulate cortex of patients with unipolar and bipolar depression","volume":"82","author":"Azevedo","year":"2016","journal-title":"J. Psychiatr. Res."},{"key":"ref_26","doi-asserted-by":"crossref","unstructured":"Liu, X., Dong, L., Jiang, Z., Song, M., and Yan, P. (2024). Identifying the differentially expressed peripheral blood microRNAs in psychiatric disorders: A systematic review and meta-analysis. Front. Psychiatry, 15.","DOI":"10.3389\/fpsyt.2024.1390366"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"12","DOI":"10.1016\/j.biochi.2020.02.006","article-title":"The complex interplay between DNA methylation and miRNAs in gene expression regulation","volume":"173","author":"Fuso","year":"2020","journal-title":"Biochimie"},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"147","DOI":"10.1590\/1678-77572016-0046","article-title":"Human DNA extraction from whole saliva that was fresh or stored for 3, 6 or 12 months using five different protocols","volume":"25","author":"Garbieri","year":"2017","journal-title":"J. Appl. Oral Sci."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Di Bartolomeo, M., Aroni, S., Serra, M., Serra, V., Martella, F., Gilardini, F., Melis, M., and D\u2019addario, C. (2025). Prenatal Delta-9-Tetrahydrocannabinol Exposure Induces Transcriptional Alterations in Dopaminergic System with Associated Electrophysiological Dysregulation in the Prefrontal Cortex of Adolescent Rats. Cells, 14.","DOI":"10.3390\/cells14120904"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"107343","DOI":"10.1016\/j.phrs.2024.107343","article-title":"DNA methylation at cannabinoid type 1 and dopamine D2 receptor genes in saliva samples of psychotic subjects: Is there an effect of Cannabis use?","volume":"208","author":"Hodosy","year":"2024","journal-title":"Pharmacol. Res."},{"key":"ref_31","first-page":"40","article-title":"Dimensional psychopathology of schizophrenia: SVARAD dimensional profiles in an acute inpatient sample","volume":"53","author":"Valentini","year":"2018","journal-title":"Riv. Psichiatr."},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"75","DOI":"10.1002\/jnr.23812","article-title":"Sex-specific mechanisms for responding to stress","volume":"95","author":"Bangasser","year":"2017","journal-title":"J. Neurosci. Res."},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"705","DOI":"10.1038\/nrn4036","article-title":"The endocannabinoid system in guarding against fear, anxiety and stress","volume":"16","author":"Lutz","year":"2015","journal-title":"Nat. Rev. Neurosci."},{"key":"ref_34","doi-asserted-by":"crossref","unstructured":"Hill, M.N., and Patel, S. (2020). Endocannabinoid signaling and stress resilience. Stress Resilience: Molecular and Behavioral Aspects, Academic Press.","DOI":"10.1016\/B978-0-12-813983-7.00022-7"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"229","DOI":"10.31887\/DCNS.2020.22.3\/rmaldonado","article-title":"The endocannabinoid system in modulating fear, anxiety, and stress","volume":"22","author":"Maldonado","year":"2020","journal-title":"Dialogues Clin. Neurosci."},{"key":"ref_36","doi-asserted-by":"crossref","unstructured":"Patel, S., and Hillard, C.J. (2009). Role of endocannabinoid signaling in anxiety and depression. Behavioral Neurobiology of the Endocannabinoid System, Springer. Current Topics in Behavioral Neurosciences.","DOI":"10.1007\/978-3-540-88955-7_14"},{"key":"ref_37","doi-asserted-by":"crossref","unstructured":"Di Bartolomeo, M., Stark, T., Di Martino, S., Iannotti, F.A., Ruda-Kucerova, J., Romano, G.L., Kuchar, M., Laudani, S., Palivec, P., and Piscitelli, F. (2023). The Effects of Peripubertal THC Exposure in Neurodevelopmental Rat Models of Psychopathology. Int. J. Mol. Sci., 24.","DOI":"10.3390\/ijms24043907"},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"105357","DOI":"10.1016\/j.phrs.2020.105357","article-title":"Crosstalk between the transcriptional regulation of dopamine D2 and cannabinoid CB1 receptors in schizophrenia: Analyses in patients and in perinatal \u03949-tetrahydrocannabinol-exposed rats","volume":"164","author":"Stark","year":"2021","journal-title":"Pharmacol. Res."},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1016\/j.schres.2017.01.022","article-title":"A preliminary study of endocannabinoid system regulation in psychosis: Distinct alterations of CNR1 promoter DNA methylation in patients with schizophrenia","volume":"188","author":"Micale","year":"2017","journal-title":"Schizophr. Res."},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"7503","DOI":"10.2147\/OTT.S181706","article-title":"miR-23b-3p and miR-130a-5p affect cell growth, migration and invasion by targeting CB1R via the Wnt\/\u03b2-catenin signaling pathway in gastric carcinoma","volume":"11","author":"Xian","year":"2018","journal-title":"OncoTargets Ther."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"216","DOI":"10.1016\/j.comppsych.2016.05.006","article-title":"Correlation between the level of microRNA expression in peripheral blood mononuclear cells and symptomatology in patients with generalized anxiety disorder","volume":"69","author":"Chen","year":"2016","journal-title":"Compr. Psychiatry"},{"key":"ref_42","doi-asserted-by":"crossref","unstructured":"Rosa, J.M., Formolo, D.A., Yu, J., Lee, T.H., and Yau, S.-Y. (2022). The Role of MicroRNA and Microbiota in Depression and Anxiety. Front. Behav. Neurosci., 16.","DOI":"10.3389\/fnbeh.2022.828258"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"636","DOI":"10.1111\/pcn.13869","article-title":"Plasma circulating microRNAs and symptoms of depression: Results from a population-based study","volume":"79","author":"Kuilman","year":"2025","journal-title":"Psychiatry Clin. Neurosci."}],"container-title":["Brain Sciences"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/2076-3425\/16\/5\/500\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,5,4]],"date-time":"2026-05-04T02:28:31Z","timestamp":1777861711000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/2076-3425\/16\/5\/500"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,5,2]]},"references-count":43,"journal-issue":{"issue":"5","published-online":{"date-parts":[[2026,5]]}},"alternative-id":["brainsci16050500"],"URL":"https:\/\/doi.org\/10.3390\/brainsci16050500","relation":{},"ISSN":["2076-3425"],"issn-type":[{"value":"2076-3425","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,5,2]]}}}