Early life stress-induced miR-708-5p regulates mood disorder-associated behavioural phenotypes in mice and is a potential diagnostic biomarker for bipolar disorder

Early life stress-induced miR-708-5p regulates mood disorder-associated behavioural phenotypes in mice and is a potential diagnostic biomarker for bipolar disorder | bioRxiv /* */ /* */ <!-- <!-- /*! * yepnope1.5.4 * (c) WTFPL, GPLv2 */ (function(a,b,c){function d(a){return"[object Function]"==o.call(a)}function e(a){return"string"==typeof a}function f(){}function g(a){return!a||"loaded"==a||"complete"==a||"uninitialized"==a}function h(){var a=p.shift();q=1,a?a.t?m(function(){("c"==a.t?B.injectCss:B.injectJs)(a.s,0,a.a,a.x,a.e,1)},0):(a(),h()):q=0}function i(a,c,d,e,f,i,j){function k(b){if(!o&&g(l.readyState)&&(u.r=o=1,!q&&h(),l.onload=l.onreadystatechange=null,b)){"img"!=a&&m(function(){t.removeChild(l)},50);for(var d in y[c])y[c].hasOwnProperty(d)&&y[c][d].onload()}}var j=j||B.errorTimeout,l=b.createElement(a),o=0,r=0,u={t:d,s:c,e:f,a:i,x:j};1===y[c]&&(r=1,y[c]=[]),"object"==a?l.data=c:(l.src=c,l.type=a),l.width=l.height="0",l.onerror=l.onload=l.onreadystatechange=function(){k.call(this,r)},p.splice(e,0,u),"img"!=a&&(r||2===y[c]?(t.insertBefore(l,s?null:n),m(k,j)):y[c].push(l))}function j(a,b,c,d,f){return q=0,b=b||"j",e(a)?i("c"==b?v:u,a,b,this.i++,c,d,f):(p.splice(this.i++,0,a),1==p.length&&h()),this}function k(){var a=B;return a.loader={load:j,i:0},a}var l=b.documentElement,m=a.setTimeout,n=b.getElementsByTagName("script")[0],o={}.toString,p=[],q=0,r="MozAppearance"in l.style,s=r&&!!b.createRange().compareNode,t=s?l:n.parentNode,l=a.opera&&"[object Opera]"==o.call(a.opera),l=!!b.attachEvent&&!l,u=r?"object":l?"script":"img",v=l?"script":u,w=Array.isArray||function(a){return"[object Array]"==o.call(a)},x=[],y={},z={timeout:function(a,b){return b.length&&(a.timeout=b[0]),a}},A,B;B=function(a){function b(a){var a=a.split("!"),b=x.length,c=a.pop(),d=a.length,c={url:c,origUrl:c,prefixes:a},e,f,g;for(f=0;f<d;f++)g=a[f].split("="),(e=z[g.shift()])&&(c=e(c,g));for(f=0;f<b;f++)c=x[f](c);return c}function g(a,e,f,g,h){var i=b(a),j=i.autoCallback;i.url.split(".").pop().split("?").shift(),i.bypass||(e&&(e=d(e)?e:e[a]||e[g]||e[a.split("/").pop().split("?")[0]]),i.instead?i.instead(a,e,f,g,h):(y[i.url]?i.noexec=!0:y[i.url]=1,f.load(i.url,i.forceCSS||!i.forceJS&&"css"==i.url.split(".").pop().split("?").shift()?"c":c,i.noexec,i.attrs,i.timeout),(d(e)||d(j))&&f.load(function(){k(),e&&e(i.origUrl,h,g),j&&j(i.origUrl,h,g),y[i.url]=2})))}function h(a,b){function c(a,c){if(a){if(e(a))c||(j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}),g(a,j,b,0,h);else if(Object(a)===a)for(n in m=function(){var b=0,c;for(c in a)a.hasOwnProperty(c)&&b++;return b}(),a)a.hasOwnProperty(n)&&(!c&&!--m&&(d(j)?j=function(){var a=[].slice.call(arguments);k.apply(this,a),l()}:j[n]=function(a){return function(){var b=[].slice.call(arguments);a&&a.apply(this,b),l()}}(k[n])),g(a[n],j,b,n,h))}else!c&&l()}var h=!!a.test,i=a.load||a.both,j=a.callback||f,k=j,l=a.complete||f,m,n;c(h?a.yep:a.nope,!!i),i&&c(i)}var i,j,l=this.yepnope.loader;if(e(a))g(a,0,l,0);else if(w(a))for(i=0;i (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0];var j=d.createElement(s);var dl=l!='dataLayer'?'&l='+l:'';j.src='//www.googletagmanager.com/gtm.js?id='+i+dl;j.type='text/javascript';j.async=true;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-M677548'); Skip to main content Home About Submit ALERTS / RSS Search for this keyword Advanced Search New Results Early life stress-induced miR-708-5p regulates mood disorder-associated behavioural phenotypes in mice and is a potential diagnostic biomarker for bipolar disorder Carlotta Gilardi , Helena C. Martins , Alessandra Lo Bianco , Silvia Bicker , View ORCID Profile Pierre-Luc Germain , Fridolin Gross , Ayse Özge Sungur , Theresa M. Kisko , Frederike Stein , Susanne Meinert , Rainer K. W. Schwarting , Markus Wöhr , Udo Dannlowski , Tilo Kircher , View ORCID Profile Gerhard Schratt doi: https://doi.org/10.1101/2024.03.14.584977 Carlotta Gilardi 1 ETH Zurich , Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , 8057 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Helena C. Martins 1 ETH Zurich , Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , 8057 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Alessandra Lo Bianco 1 ETH Zurich , Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , 8057 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Silvia Bicker 1 ETH Zurich , Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , 8057 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Pierre-Luc Germain 1 ETH Zurich , Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , 8057 Zurich, Switzerland 2 Laboratory of Molecular and Behavioural Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , ETH Zürich, Switzerland 3 Lab of Statistical Bioinformatics, IMLS, University of Zürich , Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Pierre-Luc Germain Fridolin Gross 1 ETH Zurich , Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , 8057 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site Ayse Özge Sungur 4 Philipps-University of Marburg, Faculty of Psychology, Experimental and Biological Psychology, Behavioral Neuroscience , D-35032 Marburg, Germany 5 Center for Mind, Brain and Behavior , D-35032 Marburg, Germany 6 KU Leuven, Faculty of Psychology and Educational Sciences, Research Unit Brain and Cognition, Laboratory of Biological Psychology, Social and Affective Neuroscience Research Group , B-3000 Leuven, Belgium 7 KU Leuven, Leuven Brain Institute , B-3000 Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Theresa M. Kisko 4 Philipps-University of Marburg, Faculty of Psychology, Experimental and Biological Psychology, Behavioral Neuroscience , D-35032 Marburg, Germany 5 Center for Mind, Brain and Behavior , D-35032 Marburg, Germany 6 KU Leuven, Faculty of Psychology and Educational Sciences, Research Unit Brain and Cognition, Laboratory of Biological Psychology, Social and Affective Neuroscience Research Group , B-3000 Leuven, Belgium 7 KU Leuven, Leuven Brain Institute , B-3000 Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Frederike Stein 8 Department of Psychiatry and Psychotherapy, University of Marburg , Marburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Susanne Meinert 9 Institute for Translational Psychiatry, University of Münster , Münster, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Rainer K. W. Schwarting 4 Philipps-University of Marburg, Faculty of Psychology, Experimental and Biological Psychology, Behavioral Neuroscience , D-35032 Marburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Markus Wöhr 4 Philipps-University of Marburg, Faculty of Psychology, Experimental and Biological Psychology, Behavioral Neuroscience , D-35032 Marburg, Germany 5 Center for Mind, Brain and Behavior , D-35032 Marburg, Germany 6 KU Leuven, Faculty of Psychology and Educational Sciences, Research Unit Brain and Cognition, Laboratory of Biological Psychology, Social and Affective Neuroscience Research Group , B-3000 Leuven, Belgium 7 KU Leuven, Leuven Brain Institute , B-3000 Leuven, Belgium Find this author on Google Scholar Find this author on PubMed Search for this author on this site Udo Dannlowski 9 Institute for Translational Psychiatry, University of Münster , Münster, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Tilo Kircher 8 Department of Psychiatry and Psychotherapy, University of Marburg , Marburg, Germany Find this author on Google Scholar Find this author on PubMed Search for this author on this site Gerhard Schratt 1 ETH Zurich , Lab of Systems Neuroscience, Institute for Neuroscience, Department of Health Science and Technology , 8057 Zurich, Switzerland Find this author on Google Scholar Find this author on PubMed Search for this author on this site ORCID record for Gerhard Schratt For correspondence: Gerhard.schratt{at}hest.ethz.ch Abstract Full Text Info/History Metrics Supplementary material Preview PDF Abstract Mood-disorders (MDs) are caused by a complex interplay of genetic and environmental (GxE) risk factors. However, the molecular pathways engaged by GxE risk factors to trigger specific MD-associated endophenotypes are still poorly understood. Here, by using unbiased small RNA sequencing in peripheral blood mononuclear cells (PBMCs), we identified the BD-associated miR-708-5p as one of the most strongly upregulated microRNAs in peripheral blood of both healthy human subjects with a high genetic or environmental (early life stress) predisposition to develop MDs. miR-708 is also upregulated in the hippocampus of rats which underwent juvenile social isolation, a rodent model of early life stress. Furthermore, ectopic overexpression of miR-708-5p in the hippocampus of adult male mice is sufficient to elicit MD-associated behavioural endophenotypes, demonstrating a causal role for elevated miR-708-5p levels in MD development. We further show that miR-708-5p directly targets Neuronatin (Nnat), an endoplasmic reticulum (ER) resident protein involved in calcium homeostasis. Consequently, restoring Nnat expression in the hippocampus of miR-708-5p overexpressing mice rescues miR-708-5p dependent behavioural phenotypes. Finally, miR-708-5p is strongly upregulated in PBMCs derived from patients diagnosed with MD, in particular BD males. Peripheral expression of miR-708-5p, in conjunction with the previously identified miR-499-5p, allows to differentiate male BD patients from patients suffering from major depressive disorder (MDD) and healthy controls. In summary, we describe a functional role for the miR-708-5p/Nnat pathway in MD etiology and identify miR-708-5p as a potential biomarker for the differential diagnosis of MDs. Introduction MDs are a group of chronic psychiatric diseases affecting mood and cognition that include major depressive disorder (MDD) and bipolar disorder (BD). MDD is characterized by long, persistent, depressive episodes. BD has a distinct pattern of mood oscillations, from depressive episodes like MDD, and (hypo)manic phases. Furthermore, BD is subclassified in BD type 1 (mania and depression), and BD type 2 (hypomania and depression). It is known that MDs are highly heritable and share common genetic signature ( McGuffin et al , 2003 ). In particular, BD is characterized by up to 70% heritability in monozygotic twins ( Craddock & Sklar, 2013 ). However, complex polygenetic mechanisms cannot fully explain the onset of MD, and environmental factors, such as early life stress (ELS; e.g., physical and sexual abuse, emotional neglect), play an important role in the etiology and outcome of MD ( Aas et al , 2020 ; Nemeroff, 2016 ; Rodriguez et al , 2021 ). Moreover, MD have a strong sex-specific component (e.g., higher prevalence of MDD, but not BD in females) ( Noble, 2005 ) whose underlying biological mechanisms are unknown. Although recapitulating the full spectrum of MD-associated symptoms is challenging, specific endophenotypes can be reliably assessed by employing dedicated behavioural tests in rodents. These include for example behavioural despair (FST, TST), anhedonia (sucrose preference), anxiety (OFT, EPM), cognitive impairments (mazes, NOR) and compulsive/manic-like behavior (marble burying) ( Hoffman, 2013 ). Various chronic stress models have been established to mimic the impact of stress on MD symptomatology. Among them, post-weaning juvenile social isolation in rats represents a robust model to induce MD-associated endophenotypes, e.g., deficits in social communication and cognitive abilities ( Seffer et al , 2015 ; Valluy et al , 2015 ). On the other hand, haploinsufficiency for the Cacna1c cross-disorder psychiatric risk gene is frequently used to study gene x environment interactions relevant for MD ( Dedic et al , 2018 ). For example, Cacna1c (+/-) mice show decreased immobility in the tail suspension test (TST) and FST, higher preference for sucrose as well as decreased anxiety behavior, although the latter was a characteristic of female mice only ( Dao et al , 2010 ). In Cacna1c (+/-) rats, a long-term environmental impact on object recognition, spatial memory and reversal learning was observed ( Braun et al , 2019 ). microRNAs (miRNAs) are a large family of small (∼22nt), noncoding RNAs that act as posttranscriptional regulators by binding to complementary sequences in the 3’-untranslated region (UTR) of target messenger RNAs (mRNAs) (15) ( Bartel, 2018 ). Recent research has highlighted the role of miRNAs in the pathogenesis of MDs ( Martins & Schratt, 2021 ). Main pathways affected by miRNA dysregulation in the context of MD animal models include serotonergic neurotransmission (e.g., miR-16, miR-34), glucocorticoid signaling (e.g., miR-17-92 cluster, miR-15), neurotrophins (e.g., miR-182), Wnt signaling (e.g., miR-124) and synaptic plasticity (e.g., miR-134, miR-218). While these candidate studies in rodents provided important insight into biological mechanisms, the translational value for MD therapeutics and diagnostics is mostly limited by the lack of corresponding human data. In humans, miRNAs have been associated with MD etiology in expression studies of postmortem brain tissue ( Moreau et al , 2011 ) and blood samples from living patients ( Dwivedi, 2011 ). Differential expression of miRNAs in peripheral blood mononuclear cells (PBMCs) has been previously investigated for biomarker discovery in MD. For example, miR-499-5p was significantly upregulated in female and male PBMC samples of BD ( Martins et al , 2022 ). miR-124-3p was found to be significantly upregulated in PBMCs samples of MDD compared to healthy controls and it was downregulated after eight weeks of antidepressant treatment ( He et al , 2016 ). However, the overlap between these studies is usually low, and therefore these attempts so far have not yielded reliable miRNA biomarkers in MDs. We therefore decided to undertake an unbiased, back-translational approach to identify MD-associated miRNAs from a large human cohort of healthy subjects at high genetic and environmental risk to develop MD. One of the identified GxE regulated miRNAs, the BD-associated miR-708-5p ( Forstner et al , 2015 ), was further functionally characterized in rodent models and subsequently tested for its diagnostic potential in MD patient subgroups. Materials and Methods Human study Recruitment of Participants The study involved participants with BD (26 females, 37 males), MDD (18 females, 24 males), and healthy individuals (26 females, 31 males), including psychiatrically healthy subjects that had a history of childhood maltreatment (17) or a genetic predisposition to MDs (18) or no risk (18). These participants were recruited from the University of Marburg and the University of Münster in Germany, as part of the FOR2107 cohort (39) ( Kircher et al , 2019 ). Diagnoses were made using the SCID-I interview (Wittchen, 1997), and adapted to DSM-IV criteria, excluding those with substance abuse, severe neurological, or other significant medical conditions. Healthy controls were screened similarly, with inclusion in the maltreatment study if at least one of the subclasses of the Childhood Trauma Questionnaire (CTQ) reached the maltreatment threshold (Emotional Abuse ≥ 10, Physical Abuse ≥ 8, Sexual Abuse ≥ 8, Emotional Neglect ≥ 15, and Physical Neglect ≥ 8) ( Walker et al , 1999 ). All participants underwent a comprehensive neuropsychological test battery, including the d2 test of attention ( Brickenkamp, 2002 ). All necessary ethical approvals were obtained (ethics committees of the Medical Faculties of the Universities of Münster (2014-422-b-S) and Marburg (AZ: 07/14)), and participants consented to the study, which complied with the Declaration of Helsinki and the Belmont Report. Demographic and clinical data are outlined in Table 1 and in Supplementary Table 1. View this table: View inline View popup Download powerpoint Table 1. Human peripheral blood mononuclear cell (PBMC) sample processing PBMCs were obtained from 10 ml of whole blood using the LeukoLOCK technology (Thermo Scientific) at the Biomaterialbank Marburg, Germany. After sample randomization, total RNA extraction from PBMCs was carried out using the mir Vana™ kit (Thermo Fisher) or TRIzol™ Reagent (Thermo Fisher) following the manufacturer’s protocol. PBMCs processing for small RNA-sequencing For the Small RNA-sequencing, RNA was extracted with mir Vana™ kit (Thermo Fisher) and DNase treated, and it was carried out at Functional Genomic Center Zurich (FGCZ, https://fgcz.ch/ ). Small RNA-analysis Short RNA reads were processed using the ncPro 1.6.4 pipeline ( Chen et al , 2012 ) using the hg19 annotated (based on miRBase version 21). Only mature miRNA reads (accepting +2bp on either end) were considered for downstream analysis. Differential expression analysis was then performed using limma/voom 3.46.0 ( Ritchie et al , 2015 ), with a model including a covariate to correct for the two technical batches. Rat primary neuron culture Cultures of primary hippocampal and cortical neurons from rat hippocampus were established using E18 Sprague-Dawley rats obtained from Janvier Laboratories, as described previously ( Martins et al ., 2022 ). Transfection Primary hippocampal neurons were transfected with Lipofectamine™ 2000 Reagent and plasmid DNA constructs as previously described ( Martins & Schratt, 2021 ). Virus infection Primary hippocampal neurons were infected with recombinant adeno-associated virus (rAAV) containing hpCTL or hp708 constructs on days in vitro (DIV) 2 by adding 300μl of a mixture of the virus with 300µl of NBP+ per well of a 24-well plate. Cells were used for downstream analysis on DIV19-20. Luciferase Assay DIV6 hippocampal neurons were transfected with 100ng of the Nnat-3’UTR luciferase reporters and 500ng of hpCTL-p or hp708-p, spCTL-p or sp708-p. After seven days from transfection, the cells were lysed, luciferase assay was conducted using a GloMax R96 Microplate Luminometer (Promega), as previously described ( Martins et al ., 2022 ). The relative luciferase activity was determined by calculating the ratio of the Firefly signal to the Renilla signal. In Situ Hybridization Single-molecule fluorescence in situ hybridization (smFISH) for miRNA detection in hippocampal neuron cultures was conducted using the QuantiGene ViewRNA miRNA Cell Assay Kit (Thermo Fisher, QVCM0001) following the manufacturer protocol with minor adjustments. Probes for has-miR-708-5p (Alexa Fluor 546, Thermo Fisher), Nnat (Alexa Fluor 546), CamK2 (Alexa Fluor 488) and Gad2 (Alexa Fluor 488) were used for the assay. Plasmid design and rAAVs preparation For rAAV-mediated overexpression of miR-708-5p, the chimeric miR-708 hairpin was generated by polynucleotide cloning. A detailed description and characterization of this system have been published ( Christensen et al , 2010 ). To knockdown miR-708-5p, a sponge plasmid was constructed by inserting six TDMD sites predicted to bind miR-708-5p in the 3’UTR of eGFP on rAAV-hSyn-EGFP. To perform luciferase activity analysis, the wild type and mutated Nnat 3’UTR was inserted into a pmirGLO dual-luciferase expression vector (Promega). Detailed description of the design of these constructs and of the rescue constructs are listed in the supplementary material section. Viral vectors were produced by the Viral Vector Facility (VVF) of the Neuroscience Center Zurich ( https://www.vvf.uzh.ch ). Animal studies Mouse experiments Husbandry, housing, and behavioral testing All animal experiments on mice were conducted in accordance with Switzerland’s animal protection laws and received approval from local cantonal authorities (Approval ID: ZH194/21). Mice were housed collectively in cages designed for 2 to 4 individuals, with unrestricted access to food and water. The animal facility maintained an inverted light-dark cycle of 12 hours, with behavioral assessments conducted during the dark phase. Prior to experimentation, mice underwent daily handling lasting 5 minutes each over one week. Details about general behavioral procedures can be found in the supplementary materials and methods section. Stereotactic surgeries and post-operative care Stereotactic brain injections were conducted on 2-month-old C57BL/6JRj wild-type mice. The mice were anesthetized with 5% isoflurane in oxygen (1 L/min) and positioned on a stereotactic frame. Viral injections were carried out bilaterally. For dorsal hippocampus, the coordinates used were AP: -2.1mm; ML: ±1.5mm. For ventral hippocampus, the coordinates used were AP: -3.3mm; ML: ±2.7mm. Detailed description of the procedure and post-operative care can be found in the supplementary materials and methods section. Open Field Test Mice were placed in an open field box (size: L 45 x W 45 x H 40 cm, TSE System, Bad Homburg, Germany) with a 60-minute exploration window. Dim, yellow light was used in the Open field test of Fig. S2H and I, and Fig. S4C, D, E. 30 lux, white light was used for the Open Field in Fig. S2J. The experiment was recorded, and the TSE VideoMot2 analyzer software (TSE Systems, Bad Homburg, Germany) was used for the automated assessment. Elevated Plus Maze Test The mouse was placed on the center of the elevated plus maze, a cross-shaped apparatus with two open arms and two closed arms, each arm measuring L 65 x W 5.5 cm and elevated 62 cm from the ground. 20 lux white light was used. Behavior was recorded on video for five minutes. The videos were automatically analyzed using TSE VideoMot2 analyzer software (TSE Systems, Bad Homburg, Germany) Saccharin Preference Test Mice were habituated in their standard group housing to the presence of two bottles for 48 hours before the test. The test was performed by single housing the test animals, and two bottles of tap water or 0.1% Saccharin solution were given to them for 48 hours. The weight of the bottles was recorded at the beginning of the test and every 12 hours around the light change (09:00-09:15 am/pm). The position of the bottle was exchanged each time to avoid side preference. The animals were re-grouped after the test ended. Novel Object Recognition (NOR) Test The test was performed as previously described ( Daswani et al , 2022 ), with slight modifications: 1. Twenty-four hours prior to testing, the animals were habituated to the arena used for the test; 2. The break between familiarization and novelty introduction rounds was extended to five minutes or 24 hours. Marble Burying Test The test was conducted as previously described ( Levone et al , 2021 ). Passive Avoidance Test The test was conducted using the Passive Avoidance 2-Compartment light-dark arena of the TSE System. The white light was set to 500 lux. On day one, the animal was placed into the light compartment while the door to the dark one was open and it was allowed to explore; once it passed to the dark compartment, the door closed, and the animal received a 2-seconds foot shock of 0.3 mA after three seconds. On day two, the animal was placed again into the light compartment and, after 15 seconds, the door was automatically opened. The latency to enter the dark compartment was measured. Tail Suspension Test The mouse was hung by its tail for a duration of six minutes using a piece of tape affixed to the suspension metal bar, elevated approximately 50 cm from the ground. Before initiating the experiment, a climb-stopper, following a published procedure ( Can et al , 2012 ), was placed at the mouse tail base to prevent climbing. The sessions were recorded on video, and the duration of immobility was subsequently quantified. Tissue collection Animals were sacrificed by cervical dislocation and the hippocampus was collected on an ice-cold glass plate and subsequentially snap-frozen for RNA extraction (Trizol protocol) and gene expression analysis or for histological assessments after 4% paraformaldehyde fixation, cryoprotection through 30% sucrose solution, and coronal sectioning at the cryostat. Further details can be found in the supplementary materials and methods section. Rat experiments All animal experiments on rats were conducted in accordance with the National Institutes of Health Guidelines for the Care and Use of Laboratory Animals and were subject to prior authorization by the local government (MR 20/35 Nr. 19/2014 and G48/2019; Tierschutzbehörde, Regierungspräsdium Giessen, Germany). Constitutive heterozygous Cacna1c +/− animal breeding and the juvenile social isolation paradigm were conducted as described ( Martins et al ., 2022 ). RT-qPCR Total RNA extraction from mouse brain tissue, PBMC samples, and primary hippocampal cultures was performed using TRIzol™ Reagent (Thermo Fisher, 15596026), and total RNA was subsequently extracted following the manufacturer’s guidelines. Further details can be found in the supplementary materials and methods section. PolyA RNA sequencing sample preparation Total RNA extraction from mouse hippocampus was extracted TRIzol™ Reagent (Thermo Fisher, 15596026). The RNA was then treated with Turbo DNase enzyme. 600 ng of DNase-treated RNA was used for High Throughput Transcriptome sequencing. Libraries were prepared with Illumina TrueSeq mRNA protocol and the transcriptome sequencing was run on Illumina Novaseq 6000. The transcriptome sequencing was carried out by the Functional Genomic Center Zurich (FGCZ, hyperlink: https://fgcz.ch/ ). PolyA RNA sequencing analysis Reads were mapped to the GRCm39 genome with STAR 2.7.8a ( Dobin et al , 2013 ) using the GENCODE M26 annotation as reference and quantified at the gene-level using featureCounts 1.6.4 ( Liao et al , 2014 ). Genes were filtered using edgeR’s filterByExpr function before differential expression analysis with edgeR 3.32.1 ( Robinson et al , 2010 ) using likelihood ratio tests with 3 surrogate variables estimated using sva 3.38.0 ( Leek et al , 2012 ). TargetScan 7 ( Agarwal et al , 2015 ) was used to predict miRNA targets. For gene expression across different brain cell types ( Figure 4B ), the Allen 10X+smartSeq taxonomy and per-cluster expression was used, aggregating non-neuronal cell types into broader classes. Western blot Proteins from primary hippocampal neurons were isolated using ice cold RIPA lysis buffer. 20ug of protein was mixed with 4xLaemmli Sample Buffer (Biorad) and were run on a 4–20% Mini-PROTEAN® TGX™ Precast Protein Gels (Biorad). Proteins were transferred on a nitrocellulose membrane and blocked for two hours at room temperature in blocking solution and incubated in primary antibody solution for 48 hours at 4°C. Membranes were washed and incubated with secondary antibody for 1h. Membranes were washed five times in TBS-T, developed with the Clarity™ Western ECL Substrate (Bio-Rad) and visualized with the ChemiDocTM MP, Imaging System (BioRad). Further details can be found in the supplementary materials and methods section. Statistical analysis Details regarding the statistical analysis are listed in the Supplementary Materials and methods section. Results miR-708-5p is upregulated in the peripheral blood of human healthy subjects harboring an elevated genetic and environmental risk for MD We hypothesized that miRNAs whose expression correlates with environmental (ER) and genetic risk (GR) for MD in human subjects might represent strong candidates for miRNAs causally involved in MD etiology. Furthermore, by starting from a human cohort, we hoped to identify miRNA candidates with high translational potential, meaning that they could serve as targets for miRNA therapeutics and/or diagnostics in MD. To identify candidate miRNAs in an unbiased manner, we performed small-RNA sequencing with total RNA obtained from peripheral blood mononuclear cells (PBMCs) of healthy subjects (n=52) from the FOR2107 cohort (39)( Kircher et al ., 2019 ) characterized by a high genetic (at least one first degree relative diagnosed with a mood disorder; GR group; n=14) or environmental (ER group; childhood trauma based on childhood trauma questionnaire (CTQ) score; n=16) predisposition to develop MDs ( Fig. 1A ; Table 1 ). For this initial analysis, we selected females since we were able to form more homogeneous GR and ER groups with females compared to males. We then focused on miRNAs that were differentially expressed in both ER and GR compared to healthy control subjects (CTL group; no known genetic or environmental risk factors; n=18). We found that a total of six miRNAs (miR-412-5p, miR-100-5p, miR-501-3p, miR-642a-5p, miR-4999-5p, miR-708-5p) fulfilled this criterium ( Fig. 1B and C ). Among them miR-708-5p was of specific interest, since it was the most strongly upregulated miRNA. Furthermore, it was previously shown to be induced by various forms of cellular stress (26–30) ( Behrman et al , 2011 ; Lin et al , 2015 ; McIlwraith et al , 2022 ; Rodriguez-Comas et al , 2017 ; Yang et al , 2015 ) and had been associated with BD in the past (21)( Forstner et al ., 2015 ). We could confirm that miR-708-5p is significantly upregulated in the GR and ER groups using qPCR ( Fig. 1D ). Thus, we decided to focus on miR-708-5p for our further studies. Download figure Open in new tab Fig. 1 miR-708-5p is upregulated in the peripheral blood of human subjects at risk for with MDs. A. Schematic representation of the workflow to obtain PBMCs samples from the FOR2107 cohort (see also materials and methods) (39). Subjects underwent psychiatric and cognitive evaluation. After the data was collected, blood was withdrawn and PBMCs prepared followed by RNA extraction and downstream gene expression analysis. Samples were collected from psychiatrically healthy female subjects (CTL), or psychiatrically healthy female subjects with genetic predisposition for mood disorder (GR) or that suffered from childhood maltreatment (ER) (Vulnerability samples). B. and C. Volcano plot depicting miRNAs differentially expressed based on small RNA sequencing performed with RNA isolated from PBMCs of control subjects (CTL; n=18), healthy subjects who suffered childhood maltreatment (Environmental risk, ER; n=18), or healthy subjects with a genetic risk (GR; n=14). CTL vs ER (B) or GR (C): miRNAs that are significantly downregulated (logFC<0.5, p-value0.5, p-value<0.05) in blue. Each Volcano Plot is accompanied by a table showing the top 15 differentially expressed genes ordered from smallest to largest p-value. miRNAs overlapping between (B) and (C) are highlighted. C. miR-708-5p qPCR analysis of total RNA isolated from PBMCs of control subjects (CTL=18), or healthy subjects who suffered childhood Maltreatment (Environmental risk, ER= 16), or healthy subjects with a genetic risk (GR=18). One-way ANOVA with Holm-Sidak’s multiple comparisons test, HC vs ER, **, P=0.0064; HC vs GR, ***, P=0.0006; ER vs GR, ns. Data are presented as violin plots with median, quartiles and data points. miR-708-5p is expressed in rat hippocampal neurons and upregulated in the hippocampus of rat models of environmental or genetic risk for MDs To further study the functional role of miR-708-5p in MDs, we considered rodents given their extensive use in modelling psychiatric conditions. As a first step, we investigated whether miR-708-5p was expressed in different regions of the rat brain ( Fig. 2A ). We observed robust expression in regions classically implicated in MDs and cognitive function, such as the amygdala, frontal cortex, and hippocampus ( Fig. 2A ). Next, we assessed miR-708-5p expression in rat hippocampal primary neurons. Single-molecule fluorescence in situ hybridization (sm-FISH) showed that miR-708-5p is expressed in both CamK2a+ excitatory and GAD2+ inhibitory neurons in rat hippocampal cultures, illustrating its widespread neuronal distribution ( Fig. 2B and 2C ). miR-708-5p expression in rat hippocampal cultures decreased over time during in vitro development, indicating a role for miR-708-5p at early stages of neuronal development ( Fig. 2D ). Motivated by the findings of miR-708-5p elevation in peripheral blood of humans at risk to develop MDs ( Fig. 1B-D ), we asked whether miR-708-5p was similarly dysregulated in rat genetic and environmental models of MDs. As a genetic model, we chose rats heterozygous for Cacna1c ( Cacna1c +/- ), a repeatedly validated cross-disorder psychiatric risk gene (60–65) ( Bhat et al , 2012 ; Dao et al ., 2010 ; Dedic et al ., 2018 ; Harrison et al , 2022 ). Environmental risk was modelled by juvenile social isolation, a widely recognized model for early life trauma, e.g., childhood maltreatment (66) ( Seffer et al ., 2015 ) ( Fig. 2E ). miR-708-5p was significantly upregulated by social isolation in the hippocampus of juvenile wild-type rats compared to their group-housed counterparts ( Fig. 2F ), indicating parallels between the early life stress-related modulation of miR-708-5p expression in human peripheral blood and rat brain. Furthermore, Cacna1c heterozygosity led to an upregulation of miR-708-5p in the rat hippocampus independent of social housing conditions, suggesting that, like our human PBMC data ( Fig. 1C-D ), a genetic predisposition for MDs is sufficient to induce miR-708-5p expression in the rat brain ( Fig. 2F ). Our comprehensive analysis across various experimental paradigms revealed that miR-708-5p expression is linked to both environmental stressors and genetic factors associated with MDs in both humans and rodents. Therefore, manipulation of miR-708-5p in the rodent brain represents a viable strategy to study the involvement of miR-708-5p in the development of mood disorder-associated endophenotypes as well as the underlying molecular mechanisms. Download figure Open in new tab Figure 2. miR-708-5p is expressed in rat hippocampal neurons and upregulated in the hippocampus of rat models of environmental or genetic risk for MDs. A. miR-708-5p qPCR analysis using total RNA isolated from different areas of the adult female rat brain. Data are represented as bar graphs, mean ± SD (n=3 animals). B. Single-molecule fluorescence in situ hybridization (smFISH) performed in rat hippocampal neurons at 7 days in vitro (DIV7) using probes directed against miR-708-5p (red channel) and CamK2a (green channel) to identify excitatory neurons. Scale bar: 20μm. C. smFISH performed in rat hippocampal neurons at DIV7 using probes against miR-708-5p (red channel) and Gad2 (green channel) to identify inhibitory neurons. Scale bar: 20μm. D. Relative expression of miR-708-5p in primary rat hippocampal neurons at different DIV (n = 3 independent experiments) normalized to U6 snRNA. Data are represented on XY graph as mean ±SD. E. Schematic representation of the juvenile social isolation experiment, n=9 for each experimental group; PND: postnatal day. F. miR-708-5p qPCR analysis of total RNA isolated from hippocampi of Wild type (WT) or Cacna1c +/- male juvenile rats that were either group-housed or socially isolated for four weeks (n=9 in each group). WT group-housed vs WT Isolated: *, p=0.0265; WT group-housed vs Cacna1c +/- : **, p=0.0026; linear mixed model, - DeltaCq ∼ Genotype * Housing + (1| Cohort), followed by post hoc pairwise comparison with emmeans package (pairwise ∼ Genotype * Housing); ns: non-significant. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). miR-708-5p overexpression in the mouse hippocampus is sufficient to induce MD-associated endophenotypes We next investigated whether stress-induced upregulation of miR-708-5p in the rodent hippocampus is causally involved in the development of MD-associated behavioral endophenotypes. Towards this end, miR-708-5p was ectopically overexpressed in the mouse hippocampus using stereotactic injection of a recombinant rAAV expressing a miR-708-5p hairpin (hp) under the control of the human synapsin (hSYN) promoter (Fig. S1A). We chose mice four our studies since we our animal facilities are not set up for experiments with rats. The hippocampus was chosen for functional manipulation since it represents a key brain structure for cognitive and emotional processing, and our previous data indicates strong effects of juvenile social isolation on hippocampal miRNA expression ( Martins et al ., 2022 ; Valluy et al ., 2015 ) ( Fig. 2F ). To ensure the functionality of the overexpressing construct, we performed luciferase assay upon transfection of miR-708-5p overexpressing plasmid (hp708-p) together with a luciferase sensor harboring two perfect binding sites for miR-708-5p. Thereby, we detected a significant decrease in luciferase activity upon miR-708-5p overexpression compared to the control (hpCTL-p) (Fig. S1B), demonstrating efficient repression of the reporter gene by the overexpressed miR-708-5p. We went on to perform stereotactic surgeries on seven to eight-week-old mice, which then underwent a four-week recovery period before behavioral assessments were conducted ( Fig. 3A ). Our approach led to a widespread infection of both the dorsal and ventral hippocampus ( Fig. 3B ), as well as to a robust miR-708-5p overexpression compared to control infected mice ( Fig. 3C ; Fig. S1C-E). Male mice overexpressing miR-708-5p in the hippocampus exhibited a significant decrease in immobility during the tail suspension test (TST), indicative of reduced behavioral despair, while this effect was not observed in female mice ( Fig. 3D-E ). Interestingly, neither male nor female mice with miR-708-5p overexpression showed significant differences in saccharin preference compared to control groups, suggesting no alteration in anhedonia-like behavior (Fig. S1F-G). Furthermore, we observed a strong, although non-significant trend, for elevated anxiety-related compulsive behavior, as assessed by the marble burying test (MBT), in miR-708-5p overexpressing male mice (Fig. S1H). However, results from the elevated plus maze (EPM) did not reveal significant changes in general anxiety levels between the experimental groups (Fig S1I-L). We further evaluated cognitive functions since cognitive impairments are frequently observed in MD patients. Our results from the novel object recognition (NOR) ( Fig. 3G ) test revealed that miR-708-5p overexpression led to a significant impairment in object recognition memory after a 24-hour delay specifically in male mice ( Fig. 3H ; Fig. S2A, B). This impairment was also evident in short-term memory (5 min delay), as miR-708-5p overexpressing mice failed to discriminate between familiar and novel objects after a brief five-minute interval, this time in a sex-independent manner ( Fig. 3 I, J; Fig. S2C-F). Passive avoidance, however, was intact in miR-708-5p expressing male mice (Fig. S2G). Locomotor activity, assessed through the OFT remained unchanged, ruling out locomotion as a confounding factor in our behavioral assessments (Fig. S2H-J). Together, our results provide strong evidence that hippocampal overexpression of miR-708-5p leads to distinct MD-associated behaviors, including reduced behavioral despair, elevated compulsive behavior and impaired recognition memory. This behavioural “signature” is consistent with an “anti-depressant” function of miR-708-5p, for instances during the manic-like state of BD. Download figure Open in new tab Figure 3. miR-708-5p overexpression in the mouse hippocampus elicits MD-associated behavioural endophenotypes. A. Schematic timeline of the acute rAAV stereotactic injection of control (hpCTL) or miR-708-OE (hp708) virus into the dorsal and ventral hippocampus of mice at PNW 7/8, followed by behavioral testing at PNW 11/12. B. Coronal brain section of mouse brains displaying intense GFP expression upon infection with hp708 virus in the dorsal (up) and ventral (down) hippocampus. Scale bar: 2000 μm. C. miR-708-5p qPCR analysis of total RNA isolated from hippocampus of mice that underwent behavioral testing. upon miR-708-5p overexpression. hpCTL n=39, hp708 n=39. Unpaired t-Test, ****, p<0.00001. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). D. Tail Suspension Test. Time (s) male mice injected with the indicated rAAV (hpCTL or hp708, n=15 each) spent immobile. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). Unpaired t-test, ****, p<0.0001. E. Tail Suspension Test. Time (s) female mice injected with the indicated rAAV (hpCTL n= 12, or hp708, n=11) spent immobile. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). Unpaired t-test, ns. F. Schematic representation of the Novel Object Recognition (NOR) Test.mice are exposed to two familiar objects during the familiarization phase. After five minutes, they are returned to the home cage for either 24 hours or 5 minutes. They are then exposed to a novel and a familiar object during the test phase. G. Novel object recognition Test with 24 hours break in between familiarization and novelty testing. Time (s) male mice injected with the indicated rAAV (hpCTL n= 10, or hp708, n=12) explored either the familiar (F) or novel (N) object. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). Two-way RM ANOVA: Novelty x Group, ***, p=0.0004; Novelty, ****, p<0.0001; Group, ns, p=0.8830. Šídák’s post hoc test, F vs N: hpCTL, ****, p<0.0001; hp708, ns, p=0.6735. H. Novel object recognition Test with 5 minutes break in between familiarization and novelty testing. Time (s) male mice injected with the indicated rAAV (hpCTL or hp708, n=15 each) explored either the familiar (F) or novel (N) object. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). Two-way RM ANOVA: Novelty x Group, ****, p<0.0001; Novelty, ****, p<0.0001; Group, ns, p=0.8207. Šídák’s post hoc test, F vs N: hpCTL, ****, p<0.0001; hp708, ns, p=0.6975. I. Novel object recognition Test with 5 minutes break in between familiarization and novelty testing. Time (s) female mice injected with the indicated rAAV (hpCTL n= 12, or hp708, n=11) explored either the familiar (F) or novel (N) object. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). Two-way RM ANOVA: Novelty x Group, *, p<0.0130; Novelty, ***, p=0.0001; Group, ns, p=0.8143. Šídák’s post hoc test, F vs N: hpCTL, ****, p<0.0001; hp708, ns, p=0.3127. miR-708-5p directly targets Neuronatin (Nnat), an ER-resident protein involved in calcium homeostasis As a next step, we decided to explore the mechanisms underlying miR-708-5p dependent regulation of MD-associated behavior. miRNAs regulate gene expression through binding to mRNAs, predominantly leading to their degradation or inhibition of translation ( Bartel, 2018 ). Therefore, we reasoned that the analysis of the hippocampal transcriptome of miR-708-5p overexpressing mice could inform us about potential miR-708-5p downstream targets mediating behavioral effects. polyA-RNA-sequencing (seq) revealed 118 differentially expressed genes (DEG) (p-value<0.01; FDR<0.001) between the hippocampus of hp-708 and hp-control infected mice ( Fig. 4A ). Sixteen DEGs were significantly downregulated and contained predicted miR-708-5p binding sites ( Fig. 4B ), making them strong candidates for direct targets of miR-708-5p. Nnat was of particular interest, since it has been previously identified as a direct target of miR-708-5p ( Vatsa et al , 2019 ; Yang et al ., 2015 ) and its 3’UTR contains a miR-708-5p perfect binding site ( Fig. 4C ). The Nnat gene encodes for a small ER-resident protein which acts as an endogenous inhibitor of the SERCA calcium pump, thereby controlling intracellular calcium homeostasis which is disturbed especially in BD ( Harrison et al , 2019 ). Moreover, within the mouse brain, Nnat is primarily expressed in glutamatergic CA1 and CA3 neurons based on published single cell RNA-seq data ( Fig. 4B ), consistent with a functional interaction of miR-708-5p and Nnat in neurons. To further validate our RNA-seq data, we measured the levels of Nnat mRNA in the hippocampus of mice upon miR-708-5p overexpression ( Fig. 4D ) and detected a significant decrease compared to control infected mice ( Fig. 4E ). So far, our expression analysis on hippocampal tissue did not allow to distinguish between effects occurring in neuronal and non-neuronal cells. Therefore, we further extended our analysis to rat primary hippocampal neuron cultures. Like in the in vivo experiments, infection of rat neurons with hp708 resulted in a robust upregulation of miR-708-5p levels ( Fig. 4F ) and led to significant reductions in Nnat mRNA ( Fig. 4G ), and protein levels ( Fig.4H and I), demonstrating that miR-708-5p represses endogenous Nnat expression in neurons. To prove that the downregulation of Nnat is due to a direct interaction of the miR-708-5p and Nnat mRNA via the predicted miR-708-5p binding site, we performed luciferase reporter gene assay in primary rat hippocampal neurons. Luciferase reporter genes were either fused to the wild type 3’UTR of Nnat (Nnat wt) or to a 3’UTR of Nnat containing several point mutations expected to prevent miR-708-5p binding (Nnat mt). Whereas Nnat wt was efficiently downregulated by co-transfection of hp708-p, Nnat mt was insensitive to miR-708-5p overexpression ( Fig. 4J ). The results obtained from these assays therefore confirm that miR-708-5p directly suppresses Nnat expression through a 3’UTR interaction. In a complementary approach, we employed a target-directed miRNA degradation (TDMD) construct (sp708-p) to reduce endogenous miR-708-5p levels (Fig. S3A-C). Here, we detected a significant increase in luciferase activity upon transfection with sp708-p compared to control for Nnat wt, but not Nnat mt expressing neurons ( Fig. 4K ). In summary, our results from RNA-seq, qPCR and luciferase assays establish Nnat as a direct target of miR-708-5p in rodent hippocampal neurons. Download figure Open in new tab Figure 4. miR-708-5p directly targets Nnat, an ER-resident protein involved in calcium homeostasis. A. Volcano plot depicting differential gene expression in the hippocampus between hp708 and hpCTL injected mice based on polyA-RNA sequencing. B. Heatmap displaying miR-708-5p predicted targets which are significantly downregulated from (A), as well as their expression in different cell types based on single-cell RNA-seq data (Allen 10X+smartSeq taxonomy)(59). The red rectangle highlights Nnat. C. Nucleotide base pairing depicting the perfect binding of miR-708-5p with the mouse Nnat 3’-UTR. D. miR-708-5p qPCR analysis of total RNA isolated from mouse hippocampi upon miR-708-5p overexpression. hpCTL n=17, hp708 n=17. Unpaired t-Test, ****, p<0.00001. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). E. Neuronatin qPCR analysis of total RNA isolated from mouse hippocampi upon miR-708-5p overexpression. hpCTL n=13, hp708 n=15. Unpaired t-Test, ****, p=0.0269. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). F. miR-708-5p qPCR analysis of total RNA isolated from rat primary hippocampal neurons at DIV20 infected with hpCTL or hp708 for miR-708-5p overexpression. hpCTL n=4, hp708 n=5. Unpaired t-Test, *, p=0.0317. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). G. Neuronatin qPCR analysis of total RNA isolated from rat primary hippocampal neurons at DIV20 infected with hpCTL or hp708 for miR-708-5p overexpression. hpCTL n=4, hp708 n=5. Unpaired t-Test, *, p=0.0159. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). H. Representative Western blot image of Nnat (lower panel) and Tubulin (upper panel) protein expression levels in hippocampal neurons (DIV20) that were infected with hpCTL or hp708 for miR-708-5p overexpression on DIV2. Tubulin was used as a loading control. I. Quantification of the relative intensity of Nnat protein levels in hippocampal neurons (DIV20) that were infected with hpCTL or hp708 for miR-708-5p overexpression on DIV2 based on (G). Tubulin was used as normalizer. Ratio-paired t-test, **, p=0.0042. n=4 per experimental group. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). J. Relative luciferase activity of rat hippocampal neurons transfected with the indicated plasmid (Control: hpCTL-p; overexpressing miR-708-5p: hp708-p) and expressing either Nnat wild-type (wt) or miR-708-5p binding site mutant (mt) reporter genes. Data are represented as scattered dot plots with bar, mean±SD (n=4 independent experiments; Two-way ANOVA: main effect of the hairpin *p=0.0245, of the Nnat luciferase reporter **p=0.0050, and of the hairpin by Nnat luciferase reporter interaction p<0.0374. Sidak’s post hoc test: **p=0.0092). K. Relative luciferase activity of rat hippocampal neurons transfected with the indicated plasmid (Control: spCTL-p; knocking-down miR-708-5p: sp708-p) and expressing either Nnat wt or mt reporter genes. Data are represented as scattered dot plots with bar, mean±SD (n=4 independent experiments; Two-way ANOVA: main effect of the hairpin *p=0.0211, no main effect of the Nnat luciferase reporter, or of the hairpin by Nnat luciferase reporter interaction. Sidak’s post hoc test: *p=0.0287). Restoring Nnat expression in miR-708-5p overexpressing hippocampal neurons rescues BD-associated behavioral endophenotypes in mice Building upon our identification of Nnat as a potential regulatory target of miR-708-5p in the hippocampus, we hypothesized that Nnat downregulation might underlie the observed MD-associated behavioral alterations elicited by miR-708-5p overexpression. To test this hypothesis, we engineered a construct which allows to simultaneously overexpress Nnat and miR-708-5p upon viral infection. This was achieved by inserting the coding sequence of Nnat downstream of the hSYN promoter, coupled with a P2A self-cleaving peptide; this fragment was followed by the EGFP coding sequence and the overexpressing miR-708-5p hairpin. As a control, we utilized a similar construct where the mCherry coding sequence replaced Nnat followed by EGFP and hp708 or EGFP and hpCTL ( Fig 5A ). rAAV obtained from these constructs was injected into the hippocampus of seven/eight weeks old male mice by stereotaxis, followed by a four-week recovery period before behavioral assessments as described in Fig. 3A . Expression analyses confirmed the upregulation of miR-708-5p and the intended modulation of Nnat levels ( Fig. 5B , 5C; Fig. S4A, and Fig. 5D , Fig. S4B). Notably, the mCherry-P2A-hp708 construct led to a pronounced upregulation of miR-708-5p ( Fig. 5B ) and a corresponding decrease in Nnat levels, as opposed to the control and the Nnat-P2A-hp708 conditions, the latter of which restored Nnat expression to wild-type levels ( Fig. 5C and Fig. S4A, B). Behavioral analyses revealed that miR-708-5p overexpression alone impaired object discrimination ability in the NOR test as expected ( Fig. 5D , Fig. S4C). This was not observed in mice injected with the Nnat-P2A-hp708 construct, which displayed a restoration of exploratory behavior towards the novel object to a similar degree than control injected mice ( Fig. 5D , Fig. S4C). Furthermore, as expected from our previous results, the total exploration time of the animals was not affected in any of the conditions (Fig. S4D). Similarly, the TST results indicated reduced behavioral despair in miR-708-5p overexpressing mice, an effect that was normalized upon co-expression of Nnat, again mirroring control group behaviors ( Fig. 5E ). Furthermore, no significant differences between experimental groups were observed in locomotor activity in the OFT (Fig. S4E-G). Taken together, our results strongly suggest that Nnat is an important downstream target in mediating the effects on MD-associated behavioral endophenotypes caused by miR-708-5p overexpression in the mouse hippocampus. Download figure Open in new tab Figure 5. Restoring Nnat expression in miR-708-5p overexpressing hippocampal neurons rescues MD-associated behaviors in mice. A. Schematic representation of the three rAAV constructs used in the rescue experiment. B. miR-708-5p qPCR analysis of total RNA isolated from the hippocampus of male mice injected with mCherry-P2A-hpCTL (n=9), mCherry-P2A-hp708 (n12), or Nnat-P2A-hp708 (n=12) viruses. One-way ANOVA, Post hoc Tukey’s Test: mCherry-P2A-hpCTL vs mCherry-P2A-hp708: ****, p<0.0001, mCherry-P2A-hpCTL vs Nnat-P2A-hp708: ***, p<0.0001, mCherry-P2A-hp708 vs Nnat-P2A-hp708: ns, p=0.1225. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). C. Neuronatin qPCR analysis of total RNA isolated from the hippocampus of male mice injected with mCherry-P2A-hpCTL (n=9), mCherry-P2A-hp708 (n12), or Nnat-P2A-hp708 (n=12) viruses. One-way ANOVA, Post hoc Tukey’s Test: mCherry-P2A-hpCTL vs mCherry-P2A-hp708: ***, p=0.0003, mCherry-P2A-hpCTL vs Nnat-P2A-hp708: ns, p=0.7379, mCherry-P2A-hp708 vs Nnat-P2A-hp708: ****, p<0.0001. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). D. Novel object recognition Test with 5 minutes break in between familiarization and novelty testing. Time (s) male mice injected with the indicated rAAV (mCherry-P2A-hpCTL (n=9), mCherry-P2A-hp708 (n12), or Nnat-P2A-hp708 (n=12)) explored either the familiar (F) or novel (N) object. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). Two-way RM ANOVA: Novelty x Group, ****, p<0.0001; Novelty, ****, p<0.0001; Group, ns, p=0.5765. Šídák’s post hoc test, F vs N: mCherry-P2A-hpCTL, ****, p<0.0001; mCherry-P2A-hp708, ns, p=0.6534; Nnat-P2A-hp708, ****, p<0.0001. E. Tail Suspension Test. Time (s) male mice injected with the indicated rAAV (mCherry-P2A-hpCTL (n=9), mCherry-P2A-hp708 (n12), or Nnat-P2A-hp708 (n=12)) spent immobile. One-way ANOVA, Post hoc Tukey’s Test: mCherry-P2A-hpCTL vs mCherry-P2A-hp708: ****, p<0.0001, mCherry-P2A-hpCTL vs Nnat-P2A-hp708: ns, p=0.9617, mCherry-P2A-hp708 vs Nnat-P2A-hp708: ****, p<0.0001. Data are represented as box plot with whiskers and data points (+: mean, line: median; whiskers: minimum and maximum values). miR-708-5p levels negatively correlate with human cognitive processing and represent a potential biomarker for differential diagnosis in MDs Our functional analysis in mice implies an important role of miR-708-5p in the regulation of MD-associated behaviours. To explore a potential link between miR-708-5p and human behaviour, we harnessed our rich dataset of neuropsychological test results present within the FOR2107 cohort ( Kircher et al ., 2019 ). Given our previous results from mice ( Fig. 3 ), we were specifically interested in correlations between miR-708-5p expression levels (measured in PBMCs) and performances in assessments covering the neurocognitive domains (attention/concentration; executive function, verbal and visuo-spatial memory (see materials and methods). Interestingly, miR-708-5p expression in a combined human sample (n=162; both healthy and MD subjects of both sexes; Table 1 ) showed a significant negative correlation with the score from the D2 attention test ( Fig. 6A ), which suggests that high levels of miR-708-5p in human impair selective attention and cognitive processing. Significant negative correlations were also observed when considering males (n=90) and females (n=72) separately and were mostly driven by BD and to a lesser extent MDD patients (Fig. S5). These finding aligns well with our results from behavioural testing in mice, which revealed a negative role for miR-708-5p in novel object recognition memory. Download figure Open in new tab Figure 6: miR-708-5p levels negatively correlate with human cognitive processing and represent a potential biomarker for differential diagnosis in MDs A. Pearson correlation plot of miR708 peripheral levels and attention (d2 test) in all human participants (female + male, CTL + MDD + BD, n=162), **, p=.005, r=-.223. B. Same as Fig. 1A , except that samples from the FOR2107 cohort were in addition collected from male and female patients that were diagnosed with Major Depressive Disorder (MDD) or Bipolar Disorder (BD) (mood disorder samples). C. miR-708-5p qPCR analysis of total RNA isolated from PBMCs of female patients diagnosed with BD or MDD (control: CTL= 26; MDD= 18; BD= 26). Wilcoxon rank-sum test after correction for age and antidepressant treatment; linear model of the form -DeltaCq ∼ Group + Age + Antidepressant treatment. Females: CTL vs MDD, *, p<0.05; CTL vs BD, ****, p<0.0005. D. miR-708-5p qPCR analysis of total RNA isolated from PBMCs of male patients diagnosed with BD or MDD (control: CTL=31; MDD=24; BD= 37. Wilcoxon rank-sum test after correction for age and antidepressant treatment; linear model of the form -DeltaCq ∼ Group + Age + Antidepressant treatment. CTL vs MDD, p=0.0549; CTL vs BD, ****, p<0.0005). Data are presented as violin plots with median, quartiles and data points. E. miR-708-5p qPCR analysis of total RNA isolated from PBMCs of male and female patients diagnosed with Schizophrenia (male: CTL=11; SCZ=15; female: CTL=8; SCZ=8). Two-way ANOVA: Group x Sex, ns, p=0.4904; Group, ns, p=0.2383, Sex, ns, p=0.4904. Tukey’s post hoc test, ns. Data are presented as violin plots with median, quartiles and data points. F. ROC curve of combined miR-708-5p and miR-499-5p expression in female (left) and male (right) subjects to discriminate BD vs control samples. The indicated thresholds are the closest point to the optimal (i.e. top-left). G. ROC curve of combined miR-708-5p and miR-499-5p expression in female (left) and male (right) subjects to discriminate BD vs controls and MDD samples. The indicated thresholds are the closest point to the optimal (i.e. top-left). Finally, we assessed the potential of miR-708-5p as a diagnostic biomarker in MD, Therefore, we measured the levels of miR-708-5p in PBMCs obtained from MDD (n=42) and BD (n=63) patients ( Fig. 6B ; Table 1 ) by qPCR. miR-708-5p was significantly upregulated in female BD (n=26) and MDD (n=18) patients ( Fig. 6C ) compared to CTL (n=26), after correcting for age and antidepressant treatment (Fig. S6A-B), whereby the effect was more pronounced in BD compared to MDD patients. A similar picture was observed in male patients. Here, the increase was highly significant for BD subjects ( Fig. 6C , after age and antidepressant treatment correction (Fig. S6C), and only trending for patients affected by MDD ( Fig. 6D , Fig. S6D). Furthermore, miR-708-5p was significantly upregulated in both subtypes of BD in both female (Fig. S6E) and male (Fig. S6F) subjects. In female subjects, the only significant BD state for miR-708-5p upregulation was mania, although it only included two samples (Fig. S6G). There was also a significant miR-708-5p upregulation in depressive and hypomanic states in male subjects (Fig. S5H). We also investigated whether miR-708-5p correlated with the Beck’s depression inventory (BDI), in MDD male and female subjects, but no significant or trending correlation could be observed (Fig. S6I and J). miR-708-5p levels showed no correlation with the Young Mania Rating Scale (YMRS), which is used to assess manic-like symptomatology in subjects affected by BD, in female BD patients (Fig. S6K). In contrast, miR-708-5p showed a trending positive correlation with YMRS in male subjects affected by BD (Fig. S6L). Taken together, our results suggest that miR-708-5p is elevated in the peripheral blood of MD patients, with a particularly strong upregulation observed for BD males. We then asked if miR-708-5p was also differentially expressed in PBMCs obtained from patients suffering from related psychiatric disorders. For that purpose, we chose schizophrenia (SCZ), given the high genetic similarities between SCZ and BD ( Craddock et al , 2005 ). We performed qPCR on PBMCs from female (n=8) and male (n=15) SCZ patients but were unable to detect any significant change in miR-708-5p expression compared to healthy control subjects (CTL; n=19) ( Fig. 6E ). One of the biggest challenges in mood disorder diagnostics is to distinguish BD and MDD patients, especially when BD patients are in a depressive phase ( Nierenberg et al , 2023 ; Vieta et al , 2018 ). Given our results from Fig. 6C-D , we hypothesized that miR-708-5p expression in PBMCs could be used as a diagnostic tool to classify samples into BD or control group, and BD or Control and MDD group. To do this, we performed receiver operating characteristic (ROC) curve analysis ( Zweig & Campbell, 1993 ) and used miR-708-5p expression as predictor variable. We found that miR-708-5p alone performed better in male samples than in female samples when asked to discriminate BD patients from controls and MDD patients (Fig S7A and B), consistent with our previous results ( Fig. 6C-D ). We then performed the same analysis using the expression values of miR-499a-5p, which we previously found to be significantly upregulated in male BD subjects compared to healthy controls ( Martins et al ., 2022 ). Like miR-708-5p, miR-499a-5p performed better as a classifier in male subjects compared to female subjects (Fig S7C and D). Furthermore, we performed another ROC curve analysis considering both miRNAs together and found that the combined expression of miR-708-5p and miR-499a-5p performs better (AUC=0.914) in classifying male BD vs controls ( Fig. 6F ) than when the miRNAs are analyzed individually. In addition, the combination performs better (AUC=0.914) also in classifying male BD vs controls and MDD patients (AUC=0.906) ( Fig. 6G ). Taken together, our results show that the combined expression of miR-708-5p and miR-499a-5p in PBMCs shows a good predictive potential to distinguish BD patients from MDD patients and healthy controls, in particular regarding males. Discussion In this study, we found that miR-708-5p is upregulated in healthy human subjects at high risk to develop MDs, in genetic and environmental rat MD models, as well as in MD patients. Furthermore, peripheral miR-708-5p expression was negatively correlated with cognitive processing. Mimicking miR-708-5p overexpression in the mouse hippocampus was sufficient to elicit MD-associated behavioural alterations, namely memory impairments, increased compulsive anxiety and reduced depression-like behavior. Finally, peripheral levels of miR-708-5p, together with the previously characterized BD-associated miR-499-5p, efficiently discriminated male BD from MDD patients and healthy subjects, emphasizing its utility as a diagnostic biomarker in MDs. miR-708-5p regulation by genetic and environmental risk factors MDs are highly heritable psychiatric disorders. Genetic factors seem to explain about 35–45% of variance in the etiology of MDD and 65–70% of variance for BD ( Coleman et al , 2020 ; Polderman et al , 2015 ). However, genetics alone cannot explain the degree at which MD is inherited in families, suggesting that environmental factors need to be considered as well. Childhood maltreatment is the most studied environmental stressor in the context of MDs ( Aas et al ., 2020 ; Nemeroff, 2016 ). Interestingly miR-708-5p was upregulated in the juvenile social isolation rat model, an animal model for childhood maltreatment ( Seffer et al ., 2015 ). Consistent with this result, a recent study reported miR-708-5p upregulation in the PFC upon seven days of chronic social defeat in vulnerable rats compared to resilient animals ( Chen et al , 2015 ). Moreover, miR-708-5p levels are elevated in Cacna1c +/- rats, a well-established genetic model of psychiatric disorders ( Bhat et al ., 2012 ), even without environmental stressors. Taken together, these results suggest that genetic and environmental risk might impinge on common pathways triggering miR-708-5p expression. Furthermore, studies in the context of ovarian cancer revealed the presence of a glucocorticoid response element upstream of ODZ4 ( Lin et al ., 2015 ) , suggesting that the expression of the host gene and of miR-708-5p might be responsive to glucocorticoids. Moreover, studies in the context of ER stress suggest that miR-708-5p expression is induced by CHOP, a transcription factor involved in the Unfolded Protein Response (UPR) upon ER stress ( Behrman et al., 2011 ). Furthermore, metformin-induced upregulation of miR-708-5p elicits Nnat downregulation in prostate cancer, leading to the expression of ER-Stress-mediators. Finally, bisphenol A, an ubiquitous endocrine disruptive chemical, induces miR-708-5p in hypothalamic neurons in a CHOP-dependent manner, accompanied by Nnat downregulation ( Nierenberg et al., 2023 ). Thus, we speculate that increased ER stress, which has been implicated in BD, is a critical mediator of miR-708-5p upregulation in response to genetic and environmental stress in neurons. In the future, miR-708-5p manipulation in the context of a GxE rodent model should help to test this hypothesis. miR-708-5p function in MD-associated behaviors Modelling the complex symptomatology of MD in animal models is challenging and has so far only been partially achieved in mice in the context of miRNA manipulation. For example, the acute manipulation of miR-124 in the PFC of mice led to impaired social behavior and locomotor disturbances upon injection of psychostimulants ( Namkung et al , 2023 ). The hippocampal overexpression of miR-499-5p in Cacna1c +/- rats provoked memory impairments, recapitulating the cognitive deficits encountered in MD ( Martins et al ., 2022 ) which include difficulties in attention, memory and executive functions present during the different mood phases and during remission ( Huang et al , 2023 ). Accordingly, we found that in humans miR-708-5p peripheral expression is negatively correlated with D2 scoring in the attention test ( Camelo et al , 2017 ). Moreover, we found that both female and male mice overexpressing miR-708-5p in the hippocampus showed memory impairments. Furthermore, male, but not female, mice showed significantly decreased immobility in the TST. This test has been historically used to test antidepressant effects on mice but is also employed in mouse models of manic behavior. For example, BD mouse models of mania (Ankyrin-G conditional knockout, Clock19 deficient mice) show reduced immobility during TST ( Zhu et al , 2017 ) and the FST ( Roybal et al , 2007 ), suggesting that the reduced behavioral despair in our model could reflect a function of miR-708-5p in manic-like behavior. This is further supported by increased (although non-significant) marble burying, indicative of a compulsive response to anxiogenic stimuli. However, in our study, the antidepressant-like behavior was neither accompanied by increased exploratory behavior in the OFT, nor increased risk-taking during EPM testing, both of which are usually observed in mouse models of mania. We consider two possible explanations for this discrepancy. First, contrary to most of the models reported in the literature, our animal model is characterized by an acute and selective overexpression of miR-708-5p in the hippocampus. Therefore, we might expect to observe endophenotypes dependent on the hippocampus (e.g., antidepressant-like behavior, cognition), but not those related to other brain regions, such as the amygdala, prefrontal cortex, or cerebellum (e.g., risk-taking behavior, hyperactivity). Alternatively, the miR-708-5p/Nnat pathway might selectively control specific aspects of manic- and depression-like behavior. To distinguish between these possibilities, it will be important to study the role of miR-708-5p overexpression in other brain areas relevant for MD-associated behaviors in the future. Molecular and cellular mechanisms downstream of the miR-708-5p/Nnat interaction Dysregulated calcium homeostasis has been previously implicated in the pathophysiology of MD, with a special emphasis on BD ( Harrison et al ., 2019 ). Our results from this and a previous study ( Martins et al ., 2022 ) are consistent with disrupted calcium flux via L-type calcium channels, e.g., due to Cacna1c mutation, as a major underlying cause. However, ER calcium dynamics emerges as another major player. For example, store operated calcium entry (SOCE) is dysregulated in BD-induced pluripotent stem cells, leading to earlier neuronal differentiation and abnormal neurite outgrowth ( Hewitt et al , 2023 ). Furthermore, altered expression of the miR-708-5p target Nnat has been associated with defective intracellular and ER calcium levels ( Sharma et al , 2013 ; Vatsa et al ., 2019 ; Zou et al , 2023 ). Nnat is a small ER membrane protein which acts as an antagonist of the sarco-endoplasmic reticulum calcium ATPase (SERCA) pump, thereby interfering with calcium re-uptake into the ER ( Braun et al , 2021 ). Consistently, it has been reported that miR-708-5p-mediated downregulation of Nnat causes lower basal cytoplasmic calcium levels ( Vatsa et al ., 2019 ). On the other hand, excessive ER calcium re-uptake could result in ER calcium overload, which in turn leads to calcium leakage to the cytoplasm and mitochondria ( Daverkausen-Fischer & Prols, 2022 ), with negative consequences for calcium signaling and cell health, as exemplified in Alzheimer’s disease ( Bezprozvanny & Mattson, 2008 ). Clearly, further studies are warranted on the impact of aberrant miR-708-5p/Nnat signaling on intraneuronal calcium homeostasis in the context of MD. miR-708-5p as a potential biomarker in BD Our findings indicate an upregulation of miR-708-5p in PBMCs of females at risk of MDs and in patients with MDD and BD. A previous study reported a downregulated expression of miR-708-5p in leukocytes of women in depressive state compared to women in remission ( Banach et al , 2017 ). These results are in line with our detected expression difference between female euthymic patients and depressive patients. For male samples, on the other hand, our data suggest a role of miR-708-5p specifically in the manic phase of the disorder, as indicated from the YMRS correlation and the behavioral characterization. Although SCZ and BD were reported to have the highest genetic correlation among psychiatric disorders ( Craddock et al ., 2005 ), we found that miR-708-5p expression was unchanged in peripheral samples of patients diagnosed with SCZ. This suggests a rather specific expression pattern of miR-708-5p in the spectrum of MDs. Within MDs, miR-708-5p upregulation was most pronounced in BD. This is impressively illustrated by our ROC curve analysis, which shows that miR-708-5p expression, when combined with miR-499-5p, effectively distinguishes BD patients not only from healthy controls, but also from MDD patients. Although miR-708-5p is significantly upregulated in both male and female BD patient samples, the degree of increase is higher in males compared to females (8.8-fold vs. 4.23-fold change). Moreover, based on our ROC curve analysis, miR-708-5p expression discriminated male BD patients better from control and MDD compared to female patients. Together, these observations suggest a potential sex-specific role of miR-708-5p in BD, which also aligns with our results from mouse behavior. Sex differences in BD manifest across various aspects, ranging from clinical symptoms to the progression of the disorder. Males typically encounter their first manic episode at a younger age compared to females, who are more likely to experience a depressive episode at the onset of BD ( Kennedy et al , 2005 ). In contrast, females experience a higher incidence of depressive episodes and hypomania, leading to a more frequent diagnosis of BD type II ( Diflorio & Jones, 2010 ). Females are also more susceptible to mixed episodes ( Arnold et al , 2000 ), rapid cycling of mood phases ( Tondo & Baldessarini, 1998 ), and show a greater likelihood of attempting suicide ( Clements et al , 2013 ). Thus, it is tempting to speculate that miR-708-5p might play an important role in male-specific aspects of BD, e.g., the development of more common and intense manic episodes. In this regard, miRNAs have been previously implicated in the sexually dimorphic control of of circadian, cholinergic, and neurokine pathways in BD ( Lobentanzer et al , 2019 ). Taken together, we propose that miR-708-5p represents a promising candidate for the development of a biomarker that helps to stratify MD patients based on disease entity, specific phases of the disease and sex, which should greatly help with diagnosis and therapy. Author contributions Carlotta Gilardi: Investigation; writing. Helena Martins: Investigation. Pierre-Luc Germain: Formal analysis. Fridolin Gross: Formal analysis. Alessandra Lo Bianco: Investigation. Silvia Bicker: Investigation; methodology. Ayse Özge Sungur: Investigation. Theresa M. Kisko: Investigation. Rainer K.W. Schwarting: Supervision. Frederike Stein: Data curation; formal analysis. Susanne Meinert: Data curation. Udo Dannlowski: supervision. Tilo Kircher: supervision; project administration. Markus Wöhr: supervision; project administration. Gerhard Schratt: Conceptualization; supervision; writing. Conflict of Interest The authors declare that they have no conflict of interest. Data availability RNA sequencing data has been deposited to Gene Expression Omnibus (GEO, small RNA-sequencing Fig. 1B-C , accession numbers: GSE261287; RNA-sequencing Fig. 4A , accession numbers: GSE261288). Acknowledgements We greatly acknowledge the technical support in the preparation of primary hippocampal cultures by Cristina Furler, Tatjana Wüst and Dr. Roberto Fiore. We also thank Dr. Brunno Rocha Levone for support in behavioral testing and inputs on the manuscript. We also thank Darren Kelly for support in designing the TDMD construct, and David Colameo and Emanuel Sonder for support in the statistical analysis. RNA sequencing was performed at the Functional Genomics Center Zurich (FGCZ) of the University Zurich and ETH Zurich. The work in the lab of GS was supported by grants from ETH Zurich (ETH-24 18-2 Grant (NeuroSno)) and the Swiss National Science Foundation (SNSF 310030E_179651 , 32NE30_189486, 310030_205064/1). This work was further funded by the German Research Foundation (DFG grants FOR2107 KI588/14-1, and KI588/14-2, and KI588/20-1, KI588/22-1 to TK; grant FOR2107 DA1151/5-1, DA1151/5-2, DA1151/9-1, DA1151/10-1, DA1151/11-1 to UD; grant FOR2107 SCHR 1136/3-1 to GS; grant DFG WO 1732/4-1 and DFG WO 1732/4-2 to MW; grant DFG 559/14-1 and DFG 559/14-2 to RS), as well as the Fonds Wetenschappelijk Onderzoek – Vlaanderen (FWO; Research Foundation – Flanders) through a senior project to MW (G0C0522N), and the Interdisciplinary Center for Clinical Research (IZKF) of the medical faculty of Münster (grant Dan3/022/22 to UD). Biosamples and corresponding data were sampled, processed, and stored in the Marburg Biobank CBBMR. Footnotes Paper was re-written and the order of the main figures was changed (part of Fig. 1 was moved to Fig.6). An additional supplementary figure (Fig. S5) was added. 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Your Email * Your Name * Send To * Enter multiple addresses on separate lines or separate them with commas. You are going to email the following Early life stress-induced miR-708-5p regulates mood disorder-associated behavioural phenotypes in mice and is a potential diagnostic biomarker for bipolar disorder Message Subject (Your Name) has forwarded a page to you from bioRxiv Message Body (Your Name) thought you would like to see this page from the bioRxiv website. Your Personal Message CAPTCHA This question is for testing whether or not you are a human visitor and to prevent automated spam submissions. Share Early life stress-induced miR-708-5p regulates mood disorder-associated behavioural phenotypes in mice and is a potential diagnostic biomarker for bipolar disorder Carlotta Gilardi , Helena C. Martins , Alessandra Lo Bianco , Silvia Bicker , Pierre-Luc Germain , Fridolin Gross , Ayse Özge Sungur , Theresa M. Kisko , Frederike Stein , Susanne Meinert , Rainer K. W. Schwarting , Markus Wöhr , Udo Dannlowski , Tilo Kircher , Gerhard Schratt bioRxiv 2024.03.14.584977; doi: https://doi.org/10.1101/2024.03.14.584977 Share This Article: Copy Citation Tools Early life stress-induced miR-708-5p regulates mood disorder-associated behavioural phenotypes in mice and is a potential diagnostic biomarker for bipolar disorder Carlotta Gilardi , Helena C. Martins , Alessandra Lo Bianco , Silvia Bicker , Pierre-Luc Germain , Fridolin Gross , Ayse Özge Sungur , Theresa M. Kisko , Frederike Stein , Susanne Meinert , Rainer K. W. Schwarting , Markus Wöhr , Udo Dannlowski , Tilo Kircher , Gerhard Schratt bioRxiv 2024.03.14.584977; doi: https://doi.org/10.1101/2024.03.14.584977 Citation Manager Formats BibTeX Bookends EasyBib EndNote (tagged) EndNote 8 (xml) Medlars Mendeley Papers RefWorks Tagged Ref Manager RIS Zotero Tweet Widget Facebook Like Google Plus One Subject Area Neuroscience Subject Areas All Articles Animal Behavior and Cognition (7651) Biochemistry (17746) Bioengineering (13928) Bioinformatics (42066) Biophysics (21499) Cancer Biology (18650) Cell Biology (25579) Clinical Trials (138) Developmental Biology (13409) Ecology (19947) Epidemiology (2067) Evolutionary Biology (24374) Genetics (15633) Genomics (22557) Immunology (17775) Microbiology (40505) Molecular Biology (17217) Neuroscience (88796) Paleontology (667) Pathology (2845) Pharmacology and Toxicology (4836) Physiology (7664) Plant Biology (15179) Scientific Communication and Education (2047) Synthetic Biology (4304) Systems Biology (9839) Zoology (2272)