Placenta-derived exosomes mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling

Placenta-derived exosomes mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (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],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Placenta-derived exosomes mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling Huiping Zhang, Miaomiao Chen, Rong Liu, Pan Zeng, Ruobing Liang, and 5 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-6587812/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Maternal depression during pregnancy adversely affects offsprings from infancy through adulthood. Placenta-derived exosomes (P exo ) play crucial roles in maternal-fetal communication. This study investigates the involvement of P exo in the intergenerational transmission of maternal depression and its underlying molecular mechanisms. P exo were isolated from placental tissue of newborns exhibiting neurobehavioral development disorders due to maternal depression during pregnancy (depression P exo , n = 90) and from healthy newborns (control P exo , n = 79). Pregnant SD rats were administrated to depression P exo (n = 6) or prenatal stress(n = 6). Behavior test, bioinformatics analyses, and molecular biology approaches were used to examine the effects of P exo . Newborns from mothers with depression during pregnancy exhibited neurobehavioral development deficits. Depression P exo inhibited hippocampal neuron proliferation in primary neuron cultures, resulting in the depressive-like behaviors and the reduced hippocampal neurogenesis in offspring. miRNA sequencing combined with RT- qPCR identified 10 differentially expressed miRNAs in depression P exo , with miRNA-485-5p emerging as a key regulator of hippocampal neurogenesis. Expression of WNT2B/β-catenin signaling was significantly decreased in offspring exposed to depression P exo or prenatal stress. WNT2B administration rescued depressive-like behavior and restored hippocampal neurogenesis impaired by depression P exo . Thus, placenta-derived exosomal miRNAs and WNT2B signaling represent promising therapeutic targets to mitigate the intergenerational effects of maternal depression. Biological sciences/Neuroscience/Molecular neuroscience Health sciences/Diseases/Psychiatric disorders/Depression maternal depression intergenerational transmission placenta exosomes miRNA-485-5p WNT2B Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction The intergenerational transmission of depression has emerged as a significant global public health concern across various countries and cultures 1 . According to the U.S. Preventive Task Force (February 2019), approximately 400,000 American mothers experience perinatal depression annually, with global prevalence rates reaching up to 13% 2 . Maternal depression adversely affects the behavioral, psychological, and physical development of offspring, influencing their health from infancy into adulthood. Clinical studies consistently identify maternal depression as a critical risk factor for transmitting depression to subsequent generations, contributing significantly to offspring depression during adolescence and early adulthood 4 . In our previous study, we found that infants born to mothers experiencing prenatal depression exhibited reduced social interactive behavior 5 . Nevertheless, the molecular mechanisms underlying the intergenerational transmission of depression remain unclear. The placenta is essential for maternal-fetal communication and significantly impacts fetal neurodevelopmental programming 6 . However, the placenta is vulnerable to various intrinsic and extrinsic stress factors that can increase the risk of fetal neurodevelopmental disorders 7 . Previous studies suggested that placental dysfunction induced by maternal depression negatively affects fetal brain development 8 . However, it is difficult to evaluate and monitor the status of the placenta functions. Placenta-derived exosomes (P exo ) have recently been recognized as indicators of placental health due to their significant roles in maternal-fetal communication 9 . Exosomes are extracellular vesicles released by cells and play a vital role in cell-to-cell communication 10 . Their ability to cross the blood-brain barrier highlights their potential relevance to mental health disorders, with emerging evidence implicating exosomes in major depressive disorder (MDD) 11 , 12 . Furthermore, Placental exosomes can traverse the placental barrier, enter fetal circulation, and alter fetal tissue functions 13 . Study has shown a reduced number of P exo in fetuses experiencing growth restriction compared to those developing normally 14 . However, the role of P exo in the intergenerational transmission of depression remains largely unexplored.. Accumulating evidence demonstrates a causal relationship between reduced hippocampal neurogenesis and depression. Clinical studies have reported reduced hippocampal volume in patients with MDD 15 , while animal models associate decreased hippocampal neurogenesis with depressive-like behaviors 16 . In rodents studies, antidepressant treatment increase hippocampal neurogenesis, and inhibition of hippocampal neurogenesis could hamper the behavioral effects of antidepressant treatment 17 . Nevertheless, the precise mechanisms underlying reduced hippocampal neurogenesis in depressive-like rodent models are not fully elucidated. Recent findings by Sharma et al. highlight that exosomes can regulate neurogenesis 18 . Therefore, we hypothesized that P exo might mediate the intergenerational transmission of depression through the modulation of hippocampal neurogenesis. In this study, we aimed to investigate whether Pexo contribute to the intergenerational transmission of depression and to explore the underlying molecular mechanisms involved. 2. Materials and Methods An extended methods section is available in the Online Data Supplement. 2.1 Subjects and sample preparation The pregnant women with term gestations (37–42 weeks) were enrolled. They were hospitalized to be ready for delivery in the department of gynecology and obstetrics, the First Affiliated Hospital of Xi’an Jiaotong University between January 2017 and August 2023. Their depressive symptoms were assessed by the Hamilton Rating Scale for Depression (HAMD). According to the scores of HAMD and willingness of the subjects, we recruited 169 mother-newborn pairs. There were normal group (n = 90) and depression group (n = 79). The placenta tissue and 15 ml umbilical cord blood was collected at delivery. We obtained and recorded demographic data and birth outcomes data from the medical records. Neonatal Behavioral Assessment Scale (NBAS) was used to assess neurobehavioral development in all the newborn infants aged 21–27 days after birth. Informed consent was obtained from all mothers. We obtained the ethical approval of the present study from Xi'an Jiaotong University (Xi’an, China). We complied with the ethical standards to perform all procedures, which was in agreement with the relevant national and institutional committees on human experimentation and the Helsinki Declaration of 1975, as revised in 2008. 2.2 Isolation and verification of exosomes 2.2.1 P exo isolation Placental tissue was cut into small pieces about 1mm 3 . Then put the small pieces into the 2ml centrifuge tubes, add type IV collagenase solution, and bathe it in water at 37℃ for 15min, shake it every 3min, until the cells forming cell clusters or single cells after shaking. Differential centrifugation were used to isolate exosomes. The samples were sequentially centrifuged at 1200 r/min 10 min and 3200 r/min 20 min at 4°C to remove cells and platelets, and then sequentially centrifuging at 15000 r/min 45 min and 30000 r/min 90 min at 4℃. The isolated P exo were resuspended in phosphate buffer saline (PBS) for further experiments. 2.2.2 Exosomes isolation from umbilical cord blood The umbilical cord samples at 4°C were centrifuged at 1600g for 20 minutes to get plasma. Ultracentrifugation was used to isolate exosomes in plasma. To remove cells and platelets, plasma samples were sequentially centrifuged at 1200 r/min 10 min and 3200 r/min 20 min at 4°C, and then sequentially centrifuging at 15000 r/min 45 min and 30000 r/min 90 min at 4℃. PBS was used to resuspend the isolated exosomes for further experiments. 2.3 miRNA Library Construction and Sequencing miRNA library preparation and sequencing were performed by a commercial service (Genergy bio-technology, China). In brief, total RNAs were extracted from isolated exosomes. Both 3’and 5’adaptors were added to each end, respectively, followed by reverse transcription and polymerase chain reaction amplification. We obtained the polymerase chain reaction products from the 18 to 30 nucleotide. Electrophoresis was used to purify RNA molecules. The illumina Hiseq3000 platform was used to sequence. 2.4 Primary hippocampal neuron cultures Sprague Dawley fetal rats aged embryonic day 18.5 were used to extract primary hippocampal neurons. We dissected the hippocampus tissue under a light microscope and aseptic conditions. A homogenized pool of hippocampus was used to obtain hippocampal neuron cells. Poly-D-lysine-coated 4-well plates (Thermo Fisher Scientific, USA) were used to plate the hippocampal neuron cells at a density of 60,000 to 80,000 cells/well. Cell cultures were maintained in a humidified incubator in an atmosphere of 5% CO 2 at 37°C by neurobasal-a medium enriched with GlutaMAX (Thermo Fisher Scientific, USA) and B27 supplement (Thermo Fisher Scientific, USA). Then 1 mM cytosine arabinoside (Sigma-Aldrich, USA) were added to each well containing a fresh growth medium. The cells were cultured for 7 days before proceeding to further experiments. 2.5 Animals We performed all animal procedures in agreement with the Institutional Animals Care and Use Committee at Xi’an Jiaotong University. Sprague-Dawley (SD) rats were from Xi’an Jiaotong University Health Science Center Measurements and were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory. The pregnant SD rats were accidentally divided to two groups, namely experimental group administrated with depression P exo and control group with control P exo from embryonic day 10–20. The offspring rats in experimental group were accidentally assigned into two groups: experimental group + LV-WNT2B and the experimental group + LV-GFP. The offspring rats in control group were accidentally divided into two groups: control group + LV-WNT2B and the control group + LV-GFP. P exo were isolated from the placental tissue of the newborn infants. Depression P exo were from the newborn infants with neurobehavioral development disorders of depressed mothers during the pregnancy period. Control P exo were from the normal newborn infants. 2.6 P exo injection P exo of newborn infants were injected to the pregnant SD rats once every two days from embryonic day 10–20 by caudal veins. 2.7 Behavioral test and molecular biology method Sucrose preference test (SPF) and forced swimming test (FST) in the offspring rats of experimental group and control group were conducted as described in Supplemental Materials and Methods. Methods of isolation of the hippocampus, RNA Isolation, RT-PCR and Western Blot Analysis were performed as previously described 19 and conducted as described in Supplemental Materials and Methods. After finishing the behavioral test, the offspring rats received consecutive i.p. injections of 50 mg/kg bromodeoxyuridine (BrdU) (Sigma Aldrich) at twice times every day for 5 consecutive days and were sacrificed 12 hours after the last injection. Methods of immunohistochemistry were described in Supplemental Materials and Methods. 2.8 Animals and Processing of Prenatal Stress (PS) The PS model was mild restraint stress and performed as previously described 20 . A transparent cylinder (6.8 cm in diameter) was used to be the device. The pregnant rats were randomly divided to four groups: control (CON) group (n = 6), PS group(n = 6), PS + miRNA-485-5p antagomir group(n = 6), and PS + miRNA control group(n = 6). The pregnant rats in the last three groups were conducted to restraint stress 3 times daily for 45 min on days 11–20 of pregnancy. The offspring rats in the PS group were administered with miRNA-485-5p antagomir or negative control antagomir. The pregnant rats in CON group were not treated at all in their home cages during the the whole period of pregnancy. 2.9 miRNA-485-5p antagomir administration For miRNA-485-5p antagomir experiments, the offspring rats in the PS group aged 21 days were intranasally administered with 5 nmol miRNA-485-5p antagomir (Shanghai GenePharma Co, China) or negative control (NC) antagomir every 3 days for four times. Then 50 mg/kg bromodeoxyuridine (BrdU) (Sigma Aldrich) were injected intraperitoneally (i.p.) at twice times every day for 5 consecutive days and were sacrificed 12 hours after the last injection to analyze newborn neurons. 2.10 Bilateral cannula implantations Intraperitoneal chloral hydrate (350 mg/kg) was used to anesthetize the offspring rats in the PS group aged 30 days. To implant the cannulae into the bilateral hippocampus, the offspring rats were positioned in a stereotaxic device (WPI 502603, USA). Their heads were fixed in a stoelting stereotaxic frame. The cannulae (AP, -3.3 mm; lateral, ± 2.1 mm; and ventral, -3.0 mm) were fastened with acrylic dental cement. The offspring rats were conducted the behavioral test and molecular biology test followed by a seven day rest period. 2.11 Lentivirus-mediated gene vector We entrusted to the commercial company (Shanghai Genechem Co. China, LTD) to construct the lentivirus vector for WNT2B overexpression (LV-WNT2B) and the negative lentivirus vector, which contained an enhanced green fluorescent protein (LV-GFP) marker. Sequencing was used to verify all constructions. The recombinant virus titer was 2.00 × 10 8 TU (transfection unit)/ml. 2.12 Microinjections 33 gauge needles were used to perform microinjections. A 2-µl syringe by PE-10 tubing was linked to the needles, which were discreetly plugged into the guide cannula. 2.5 µl suspension of Lentiviral particles expressing WNT2B or control particles expressing GFP were bilaterally microinjected into the hippocampus tissue of the offspring rats in experimental group + LV-WNT2B once a day from day 37–43. Vector stock was administrated at a rate of 0.5µl per minute for 10 days. The offspring rats were conducted the behavioral test and molecular biology test followed 24 hours after the last microinjections. The method of behavioral tests and biochemical studies were the same as before. 2.13 Statistical analysis Statistical analyses were performed by SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). All values are presented as means ± SD. The Kolmogorov-Smirnov test was used to assess normal distribution of quantitative variables. One-way ANOVA was used to compare the differences among the four groups. If the differences were significant, Fisher's LSD test was used to perform multiple comparisons. Independent sample t-test was used to perform comparisons between two groups. When the data did not comply with the normal distribution, Wilcoxon's rank sum test was used to perform comparisons between two groups. A difference was considered significant at P < 0.05 level. 3. Results 3.1 The newborn infants of maternal depression during pregnancy displayed neurobehavioral development disorders There were no significant differences in the demographic variables at baseline of pregnant women (Supplementary material Table 1) and newborn infants (Supplementary material Table 2) of between the depression group and the normal group ( P > 0.05). The newborn infants of prenatal depressed mothers had the lower scores of the social interactive items in the depression group than those of the normal group, mainly focus on difficulty in orienting to animate stimuli (face, voice, face plus voice items of the orientation cluster) ( P < 0.05) (Fig. 1B). However, there were no significant differences in the other cluster scores of the NBAS (habituation, regulation of states, range of states, and so on) in newborn infants between the depression group and the normal group ( P > 0.05) (Fig. 1). 3.2 Bioinformatics analysis and RT-qPCR verification of differentially expressed miRNAs in between control and depression P exo Electron microscopy analysis was performed to observe typical rounded particles with diameters ranging between 30 and 100 nm in isolated fractions (Fig. 2A (a1)). Exosome marker proteins CD63 and Alix were confirmed by Western blot analysis(Fig. 2A (a2-a4)). There were no significant quantitative differences between the control P exo and depression P exo . There were no significant differences in size distribution (60.54 ± 22.57versus 55.5 ± 19.26 nm) between the control P exo and depression P exo by Nanoparticle tracking analysis (NTA). NTA also showed the concentration in depression P exo was less than that in control P exo (4.28×10 10 versus 11.66×10 10 particles/ml) (Fig. 2A (a5)). 10 differentially expressed miRNAs in depression P exo were found by high-throughput sequencing(Fig. 2B). Then miRNA-485-5p stood out as the most potent molecule related to hippocampal signaling pathway and neurogenesis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway enrichment scores. Importantly, the elevation of miRNA-485-5p in both depression P exo and umbilical cord blood exosomes of newborn infants with neurobehavioral development disorders were also observed(Fig. 2C). 3.3 Depression P exo of newborn infants with neurobehavioral development disorders inhibited the proliferation activity of primary cultured hippocampal neurons Top20 function prediction of miRNA-485-5p was performed based on KEGG and GO enrichment analyses(Fig. 3A). WNT2B/β-catenin signaling was also regarded as the enrichment signaling of miRNA-485-5p by bioinformatics analysis. Primary cultured hippocampal neurons derived from fetal rats were incubated with Dil-labeled P exo (Fig. 3B). It was observed that P exo were internalized by hippocampal neurons after a 8-hour incubation as indicated by laser confocal images(Fig. 3C). We found that depression P exo derived from newborn infants with neurobehavioral development disorders inhibited the proliferation activity of primary cultured hippocampal neurons(Fig. 3D(d1). The expression level of WNT2B mRNA and protein were significantly reduced in primary cultured hippocampal neurons administrated with P exo derived from newborn infants with neurobehavioral development disorders(Fig. 3D(d2-d3). 3.4 miRNA-485-5p derived from depression P exo was identified as the key miRNA related to hippocampal neurogenesis The normal pregnant rats were randomly divided into the two groups. The pregnant rats in the experimental group were administrated with depression P exo from newborn infants with neurobehavioral development disorders once two days from embryonic day 10–20. And the pregnant rats in the control group were administrated with P exo from normal newborn infants. The offspring rats in the experimental group displayed depression-like behavior, and they showed the longer immobile time in FST and the decrease of sucrose consumption in SPF compared with the control group (Fig. 4A). The expression levels of WNT2B/β-catenin mRNA and protein were significantly reduced in the offspring rats of the experimental group than that the control group (Fig. 4B-C). We found that there were less numbers of BrdU + positive cells and BrdU + /NeuN + positive cells in the dentate gyrus (DG) of the hippocampus in the experimental group than that in the control group (Fig. 4D-E). We next used the prenatal stress (PS) animal model to further assess the functional involvement of exosomal miRNA-485-5p in intergenerational transmission of depression. We observed that the offspring rats in the PS group displayed depression-like behavior, and they showed the longer immobile time in FST and the decrease of sucrose consumption in SPF compared with the control group (Fig. 4F). The expression level of exosomal miRNA-485-5p in the placenta of the pregnant rats during 20 days of pregnancy in the PS group was higher than that in the control group, which is consistent with our clinical findings (Fig. 4G). To assess the functional involvement of exosomal miRNA-485-5p upregulation in intergenerational transmission of depression, we intranasally administered 5nmol miRNA-485-5p antagomir in the pregnant rats of the PS group to inhibit exosomal miRNA-485-5p expression. Depressive-like behaviors in the offspring rats of the PS group were rescued by intranasal miRNA-485-5p antagomir administration as assessed by FST and SPF(Fig. 4F). The expression level of WNT2B mRNA and protein were significantly increased in the offspring rats of the PS + miRNA-485-5p antagomir administration group than that the control group(Fig. 4H). We found that there were more BrdU + /NeuN + positive cells in the dentate gyrus (DG) of the hippocampus in the offspring rats of the PS + miRNA-485-5p antagomir administration group than that in the control group(Fig. 4I-J). 3.5 P exo mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling The normal pregnant rats were randomly divided into the two groups. The pregnant rats in the experimental group were administrated with depression P exo from newborn infants with neurobehavioral development disorders once two days from embryonic day 10–20, and the pregnant rats in the control group with P exo from normal newborn infants. The offspring rats in both the experimental group and control group were injected with LV-WNT2B or LV-GFP once a day from day 37–43. We found that depressive-like behaviors in the offspring rats of the experimental group were rescued by LV-WNT2B administration as assessed by FST and SPF(Fig. 5A). The decrease of β-catenin mRNA and protein in the offspring rats of the experimental group were prevented by LV-WNT2B administration (Fig. 5B). There were more number of BrdU + /NeuN + positive cells in the dentate gyrus (DG) of the offsprings hippocampus in the experimental group + LV-WNT2B group than that in the experimental group + LV-GFP group (Fig. 5C-D). It indicated that administration of WNT2B could prevent the depression-like behavior of offspring rats induced by depression P exo of newborn infants with neurobehavioral development disorders. Therefore, we proposed that P exo may mediate the intergenerational transmission of depression by regulating WNT2B/β-catenin signaling. 4. Discussion In the present study, we firstly isolated placenta-derived exosomes (P exo ) from newborn infants with neurobehavioral development disorders of depressed mothers (depression P exo ) and provided a comprehensive evaluation of P exo in intergenerational transmission of depression. The findings reveal a novel mechanism that maternal depression during pregnancy could be transmitted to the offsprings by P exo . Maternal prenatal depression could have the negative effects on offspring behavioral and physical development 21 . These effects can appear as early as childhood or even in infancy. For example, the infants of depressed mothers developed negative affective states that bias their interactions with others 22 . In this study, we found that the newborn infants of maternal depression during pregnancy displayed reduced social interactive behavior. However, the underlying molecular mechanisms for maternal depression inducing the behavioral and mental changes of offsprings remain unclear. The placenta is an important micro-environmental factor in fetal neurodevelopment by maternal-fetal communication. P exo was associated with intergenerational transmission of diseases, since the composition of P exo reflect the alterations of maternal status during pregnancy 23 . In the present study, the pregnant rats were administrated with depression P exo from newborn infants, then the offspring rats displayed depressive-like behaviors. We firstly proposed that P exo could play a crucial role in the intergenerational transmission of depression. Exosomes contain proteins, miRNAs and so on. Growing attention has been focused on exosomal miRNAs. Increasing evidence has shown that exosomal miRNAs was involved in mental disorders 24 . For example, the increased levels of miRNA-139-5p in blood exosomes were observed in the patients with major depressive disorder 12 . In the present study, miRNA-485-5p was identified in depression P exo as a key molecule that was associated with the intergenerational transmission of depression. Increased expression levels of miRNA-485-5p were observed in both depression P exo and umbilical cord blood exosomes of newborn infants with neurobehavioral development disorders, and also observed in P exo from the placenta of the pregnant rats of the PS group. These results demonstrated that maternal depression during pregnancy induced the secretion of P exo enriched with miRNA-485-5p. We found that depressive-like behaviors in the offspring rats of the PS group were rescued by miRNA-485-5p antagomir administration by preventing the decrease in hippocampal neurogenesis. Jonathan E found that miRNA-485-5p was developmentally regulated in the hippocampus and could affect synaptic plasticity and neurite outgrowth, and miRNA-485-5p overexpression dramatically reduced the extent of axonal outgrowth in hippocampal neurons 25 . It is possible that maternal depression during pregnancy led to placental function disorders, then induced the secretion of P exo enriched with miRNA-485-5p, which could cross the placental barrier and enter into the fetal circulation. Nextly, P exo enriched with miRNA-485-5p in fetal circulation could cross the blood-brain barrier, then resulting in structural and functional changes of hippocampal neurons, such as decreased hippocampal neurogenesis. Therefore, we suggested that P exo was involved in the intergenerational transmission of depression by regulating hippocampal neurogenesis via miRNA-485-5p. Bioinformatic and quantification analysis showed that WNT2B/β-catenin signaling were targets of miRNA-485-5p related to neurogenesis and hippocampal signaling pathway. We found that both depression P exo administration enriched with miRNA-485-5p and PS reduced WNT2B expression level. miRNA-485-5p antagomir administration could rescue the decrease of WNT2B expression level induced by PS. Moreover, administration of WNT2B could prevent the depression-like behavior of offspring rats induced by depression P exo and rescue the decrease of hippocampal neurogenesis. It is well established that WNT2B is the key element of the canonical WNT signaling pathway playing a pivotal role in the generation of newborn neurons in the hippocampal dentate gyrus 26 . Inhibition of WNT signaling within the dentate gyrus almost completely abolished the formation of newborn neurons 27 . Importantly, WNT2B gene plays important roles in developing human hippocampus 28 . It is possible that the decrease of WNT2B gene in early life could impair structural and functional of hippocampus. Furthermore, it was reported that WNT2B gene was associated with molecular mechanisms for depression by regulating neuroplasticity 29 . We suggest that P exo enriched with miRNA-485-5p in fetal circulation may result in the decrease of WNT2B expression level in hippocampus of offspring of depressed mothers in early life, which then inducing structural and functional changes of hippocampus. Therefore, depression P exo caused depressive-like behaviors in offspring resulting from decreased hippocampal neurogenesis regulated by WNT2B/β-catenin signaling. The present study has some limitations. First, we should measure and analyze the expression of P exo in neonatal peripheral blood and cerebrospinal fluid of newborn infants with neurobehavioral development disorders of depressed mothers. We should further observe the expression of depression P exo in peripheral blood, cerebrospinal fluid and hippocampus of the offspring rats of the mother rats administrated with depression P exo from newborn infants. Second, there is no vivo-specific pharmacological inhibitors or model animals to inhibit exosomes production. It is difficult to perform in vivo loss-of-function studies on depression P exo from newborn infants. In summary, maternal depression during pregnancy induced the secretion of depression P exo . The offspring rats of pregnant rats administrated with depression P exo displayed depression-like behavior accompanied by reduced hippocampal neurogenesis, indicating a crosstalk between P exo and reduced hippocampal neurogenesis in the offsprings. We identified miRNA-485-5p in depression P exo as a key molecule which reduced hippocampal neurogenesis. Administration of WNT2B could rescue the depression-like behavior of offspring rats induced by depression P exo . These findings present a novel mechanism underlying intergenerational transmission of depression with miRNA-485-5p in P exo as a potential target and highlight the therapeutic potential of WNT2B regulated by miRNA-485-5p in the treatment of intergenerational transmission of depression. Declarations Supplementary Material Refer to Web version on PubMed Central for supplementary material. Funding and Disclosure This work was supported by National Natural Science Foundation of China (No. 82371547). All the authors report no biomedical financial interests or potential conflicts of interest. Acknowledgements We are very grateful to all the mother-infant pairs that took part in this study and the graduate students for their help in recruiting the subjects and follow up data. Our team includes obstetricians, pediatrician, nurses, interviewers, laboratory technicians, basic researchers and volunteers. The named authors will serve as guarantors for the contents of this paper. Author Contributions Huiping Zhang drafted the manuscript and made substantial contributions to the conception or design of the work. Miaomiao Chen, Rong Liu, Pan Zeng, Ruobing Liang, Jiao Liu made substantial contributions to interpretation of data for the work. Qiaomian Zhu，Minjie Fu, Siliang Chen made substantial contributions to revising the work critically for important intellectual content. Huiping Zhang and Zhuoyue Shi made substantial contributions to the conception or design of the work and revised the manuscript. References Weissman MM. Intergenerational study of depression: a convergence of findings and opportunities. Psychological medicine 50,170–172(2020). O'connor E, Senger CA, Henninger ML, Coppola E, Gaynes BN. Interventions to Prevent Perinatal Depression: Evidence Report and Systematic Review for the US Preventive Services Task Force. Jama 321,588–601(2019). Goodman SH, Garber J. Evidence-Based Interventions for Depressed Mothers and Their Young Children. 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Journal of neuroscience research 70,200–208(2002). Breitfeld J, Scholl C, Steffens M, Laje G, Stingl JC. Gene expression and proliferation biomarkers for antidepressant treatment resistance. Translational psychiatry 7,e1061(2017). Additional Declarations The authors have declared there is NO conflict of interest to disclose Supplementary Files supplementarymaterials.docx Placenta-derived exosomes mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. 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Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-6587812","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":462869995,"identity":"a068a268-28fc-4813-a272-87bf7d565ae4","order_by":0,"name":"Huiping Zhang","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA5ElEQVRIiWNgGAWjYDACCSBmbGDgAXM+GNjYkaaFcUZBWjLRWsCAmefDIRgbN5Cf3fzs4dcdh2XMJdKffbYxOMDMwH746AZ8WhjnHDM3lj1zmMdyRkLy7ByDO3wMPGlpN/BpYZZIMJOWbDvMY3A74TBzjsEzZgYJHjO8Wtgk0r9BtSQ2M1sYHGZsIKSFRyLHTPIjWEsyMzMDMVokJHLKpBnb0nks5z9jZuwxSEtmI+QX+Rnp2yR/tlnbm/Mcf8zw44+NHT/74WN4tYAAMygeDeC+I6QcBBh/IGsZBaNgFIyCUYAOACHjRYi7bGhJAAAAAElFTkSuQmCC","orcid":"https://orcid.org/0000-0002-9003-0996","institution":"the Affiliated Children's Hospital of Xi'an Jiaotong University, Xi'an, Shaanxi, China","correspondingAuthor":true,"prefix":"","firstName":"Huiping","middleName":"","lastName":"Zhang","suffix":""},{"id":462869996,"identity":"3a1e7dde-c8c1-42c8-8f1f-552ffa7aa455","order_by":1,"name":"Miaomiao Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Miaomiao","middleName":"","lastName":"Chen","suffix":""},{"id":462869997,"identity":"21c69fb2-e4cc-4789-b797-e6f9797be931","order_by":2,"name":"Rong Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Rong","middleName":"","lastName":"Liu","suffix":""},{"id":462869998,"identity":"0d383b48-4b09-46a0-be66-976cd9d0573d","order_by":3,"name":"Pan Zeng","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Pan","middleName":"","lastName":"Zeng","suffix":""},{"id":462869999,"identity":"015fed31-5afa-47a2-b734-118f52a7ee84","order_by":4,"name":"Ruobing Liang","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Ruobing","middleName":"","lastName":"Liang","suffix":""},{"id":462870000,"identity":"98112e32-9e01-41cf-8c56-7d66f28ff1ec","order_by":5,"name":"Jiao Liu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Jiao","middleName":"","lastName":"Liu","suffix":""},{"id":462870001,"identity":"2df6c0dc-34e6-4466-9cf9-a8ff18600a07","order_by":6,"name":"Qiaomian Zhu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Qiaomian","middleName":"","lastName":"Zhu","suffix":""},{"id":462870002,"identity":"6d1d6741-e9b8-427f-9b4f-f0c7cc2a5b2c","order_by":7,"name":"Minjie Fu","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Minjie","middleName":"","lastName":"Fu","suffix":""},{"id":462870003,"identity":"710d884b-d0e5-4c06-88bf-13905f578651","order_by":8,"name":"Siliang Chen","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Siliang","middleName":"","lastName":"Chen","suffix":""},{"id":462870004,"identity":"1a606f65-ae38-40e4-9254-853e6971a14c","order_by":9,"name":"Zhuyue Shi","email":"","orcid":"","institution":"","correspondingAuthor":false,"prefix":"","firstName":"Zhuyue","middleName":"","lastName":"Shi","suffix":""}],"badges":[],"createdAt":"2025-05-04 09:55:08","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-6587812/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-6587812/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":83687272,"identity":"e4c6ee46-4334-40f5-918c-e2da1bd9f95c","added_by":"auto","created_at":"2025-05-30 18:51:31","extension":"jpg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":173926,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eThe score of NBAS in the newborns between the normal group and the depression group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Habituation; B. Social interactive organization; C. Motor system; D. Range of states; E. Regulation of states; F. Autonomic stability. * \u003cem\u003eP\u003c/em\u003e\u0026lt;0.05.\u003c/p\u003e","description":"","filename":"1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-6587812/v1/193274397dfe5096895f581e.jpg"},{"id":83687274,"identity":"66104d1e-32ac-4280-aaa0-7de613d9b77f","added_by":"auto","created_at":"2025-05-30 18:51:31","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":272801,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDifferentially expressed miRNAs of P\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexo\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e in the newborns between the normal group\u003c/strong\u003e \u003cstrong\u003eand the depression group\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA. Characterization and functional validation of P\u003csub\u003eexo\u003c/sub\u003e in the newborns between the normal group (n=6) and the depression group (n=6). (a1) Representative electron micrograph of isolated P\u003csub\u003eexo\u003c/sub\u003e. Scale bar: 200 nm. (a2-a4) Representative blots of exosomal marker proteins CD63 and Alix in P\u003csub\u003eexo\u003c/sub\u003e purified from the normal group (control P\u003csub\u003eexo \u003c/sub\u003e(n=6)) and the depression group (depression P\u003csub\u003eexo \u003c/sub\u003e(n=6)); quantified data obtained from quantitative densitometry of protein blots. (a5) Representative results of nanoparticle tracking analysis demonstrating size distribution of P\u003csub\u003eexo\u003c/sub\u003e.\u003c/p\u003e\n\u003cp\u003eB. Differentially expressed miRNAs of P\u003csub\u003eexo\u003c/sub\u003e in the newborns between the normal group and the depression group. (b1) Boxplot showing abundance level of miRNAs in P\u003csub\u003eexo\u003c/sub\u003e. Group 1: normal group (red color) (n=3), Group 2: depression group (blue color) (n=3). (b2) Principal Component Analysis (PCA) diagram of the sample. (b3) Heat map-clustering analysis on top-10 differentially expressed miRNAs in P\u003csub\u003eexo\u003c/sub\u003e; Quantitative reverse transcription polymerase chain reaction (qRT-PCR) analysis of the 10 differentially expressed miRNAs in P\u003csub\u003eexo\u003c/sub\u003e. (b4) Differential miRNAs volcanic map.\u003c/p\u003e\n\u003cp\u003eC. miRNA-485-5p expression level in P\u003csub\u003eexo\u003c/sub\u003e and exosomes of umbilical cord blood in newborns. (c1) miRNA-485-5p expression level in control P\u003csub\u003eexo\u003c/sub\u003e (n=6) and depression P\u003csub\u003eexo\u003c/sub\u003e (n=6). (c2) miRNA-485-5p expression level in exosomes of umbilical cord blood in newborns between the normal group (n=6) and the depression group (n=6).\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-6587812/v1/4b6ff98c9b3c50071ed77fbc.png"},{"id":83687722,"identity":"4babfdc0-7422-40a3-a93a-dd59f777c1f8","added_by":"auto","created_at":"2025-05-30 19:07:31","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":841363,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eDepression P\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexo\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e of newborns with neurobehavioral development disorder inhibited the proliferation activity of primary cultured hippocampal neurons\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp; Top20 prediction of miRNA-485-5p function. (a1) Top 20 functional enrichment in GO database. (a2) Top 20 functional enrichment in KEGG database.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp; Primary cultured hippocampal neurons. (b1) Optical microscope, Scale bars:100 μm.(b) laser scanning confocal microscopy (Phalloidin staining).\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp; Depression P\u003csub\u003eexo\u003c/sub\u003e were internalized by primary cultured hippocampal neurons after a 8-hour incubation as indicated by laser confocal images.\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp; Administration of depression P\u003csub\u003eexo\u003c/sub\u003e reduced proliferation activity of primary cultured hippocampal neurons and WNT2B expression level in hippocampal neurons. (d1) Proliferation activity of primary cultured hippocampal neurons (n=6 in each group). (d2) The expression level of WNT2B gene (n=6 in each group). (d3) The expression level of WNT2B protein (n=6 in each group).\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-6587812/v1/0b4a818dfa6c89f949bc9227.png"},{"id":83687634,"identity":"ff93429b-d25c-4390-93e4-9d6ff7eed3d7","added_by":"auto","created_at":"2025-05-30 18:59:31","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":660591,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003emiR-342-5p in depression P\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexo\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e was essential in the intergenerational transmission of depression\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp; Administration of depression P\u003csub\u003eexo \u003c/sub\u003eresulted in depression-like behavior of the offspring rats (n=6 in each group). (a1) Sucrose preference test. (a2) Immobile time in the forced swimming test.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp; Administration of depression P\u003csub\u003eexo \u003c/sub\u003ereduced the expression level of WNT2B gene and protein in hippocampus of the offspring rats(n=6 in each group).\u003c/p\u003e\n\u003cp\u003eC.\u0026nbsp;\u0026nbsp;\u0026nbsp; Administration of depression P\u003csub\u003eexo \u003c/sub\u003ereduced the expression level of β-catenin gene and protein in hippocampus of the offspring rats(n=6 in each group).\u003c/p\u003e\n\u003cp\u003eD.\u0026nbsp;\u0026nbsp;\u0026nbsp; Administration of depression P\u003csub\u003eexo\u003c/sub\u003e reduced the numbers of BrdU\u003csup\u003e+\u003c/sup\u003e positive cells in the dentate gyrus of the hippocampus(n=6 in each group).\u003c/p\u003e\n\u003cp\u003eE.\u0026nbsp;\u0026nbsp;\u0026nbsp; Administration of depression P\u003csub\u003eexo\u003c/sub\u003e reduced the numbers of BrdU\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e positive cells in the dentate gyrus of the hippocampus(n=6 in each group).\u003c/p\u003e\n\u003cp\u003eF.\u0026nbsp;\u0026nbsp;\u0026nbsp;\u0026nbsp; The effect of prenatal stress or administration of miRNA-485-5p antagomir on the depressive-like behaviors in the offspring rats(n=6 in each group). (f1) Sucrose preference test. (f2) Immobile time in the forced swimming test.\u003c/p\u003e\n\u003cp\u003eG.\u0026nbsp;\u0026nbsp;\u0026nbsp; The effect of prenatal stress or administration of miRNA-485-5p antagomir on the expression level of exosomal miRNA-485-5p from the placenta of the pregnant rats during 20 days of pregnancy(n=6 in each group).\u003c/p\u003e\n\u003cp\u003eH.\u0026nbsp;\u0026nbsp;\u0026nbsp; The effect of prenatal stress or administration of miRNA-485-5p antagomir on the expression level of WNT2B in hippocampus of the offspring rats. (h1) The expression level of WNT2B gene (n=6 in each group). (h2) The expression level of WNT2B protein (n=6 in each group).\u003c/p\u003e\n\u003cp\u003eI-J. The effect of prenatal stress or administration of miRNA-485-5p antagomir on the numbers of BrdU\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e positive cells in the dentate gyrus of the hippocampus (n=6 in each group).\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-6587812/v1/6d03599381d97b0fad74c06d.png"},{"id":83687288,"identity":"b2f3a1fc-49f9-4081-bc30-87d01bc6fff1","added_by":"auto","created_at":"2025-05-30 18:51:31","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":1295348,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eP\u003c/strong\u003e\u003csub\u003e\u003cstrong\u003eexo\u003c/strong\u003e\u003c/sub\u003e\u003cstrong\u003e mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA.\u0026nbsp;\u0026nbsp;\u0026nbsp; The effect of administration of depression P\u003csub\u003eexo \u003c/sub\u003eor LV-WNT2B on the depression-like behavior of the offspring rats (n=6 in each group). (a1) Sucrose preference test. (a2) Immobile time in the forced swimming test.\u003c/p\u003e\n\u003cp\u003eB.\u0026nbsp;\u0026nbsp;\u0026nbsp; The effect of administration of depression P\u003csub\u003eexo \u003c/sub\u003eor LV-WNT2B on the expression level of β-catenin in hippocampus of the offspring rats. (b1) The expression level of β-catenin gene (n=6 in each group). (b2) The expression level of β-catenin protein (n=6 in each group).\u003c/p\u003e\n\u003cp\u003eC-D. The effect of administration of depression P\u003csub\u003eexo \u003c/sub\u003eor LV-WNT2B on the numbers of BrdU\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e positive cells in the dentate gyrus of the hippocampus (n=6 in each group).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-6587812/v1/42d005ad7ad8ad2b20fbcc77.png"},{"id":105752166,"identity":"0d3f945f-4664-499a-b8db-af6726cba123","added_by":"auto","created_at":"2026-03-30 15:55:20","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":4078563,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-6587812/v1/6cefe3a9-b6d5-4c05-a555-ecd716f4665a.pdf"},{"id":83687632,"identity":"d0d812fe-18fa-4eed-a2e9-f97879689fa1","added_by":"auto","created_at":"2025-05-30 18:59:31","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":27505,"visible":true,"origin":"","legend":"Placenta-derived exosomes mediate the intergenerational transmission of depression by regulating WNT2B /\u0026#x03B2;-catenin signaling","description":"","filename":"supplementarymaterials.docx","url":"https://assets-eu.researchsquare.com/files/rs-6587812/v1/97da6b07ea3db8dd4e0618f0.docx"}],"financialInterests":"The authors have declared there is \u003cb\u003eNO\u003c/b\u003e conflict of interest to disclose","formattedTitle":"Placenta-derived exosomes mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eThe intergenerational transmission of depression has emerged as a significant global public health concern across various countries and cultures \u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. According to the U.S. Preventive Task Force (February 2019), approximately 400,000 American mothers experience perinatal depression annually, with global prevalence rates reaching up to 13% \u003csup\u003e2\u003c/sup\u003e. Maternal depression adversely affects the behavioral, psychological, and physical development of offspring, influencing their health from infancy into adulthood. Clinical studies consistently identify maternal depression as a critical risk factor for transmitting depression to subsequent generations, contributing significantly to offspring depression during adolescence and early adulthood \u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e. In our previous study, we found that infants born to mothers experiencing prenatal depression exhibited reduced social interactive behavior \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the molecular mechanisms underlying the intergenerational transmission of depression remain unclear.\u003c/p\u003e \u003cp\u003eThe placenta is essential for maternal-fetal communication and significantly impacts fetal neurodevelopmental programming \u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e\u003c/sup\u003e. However, the placenta is vulnerable to various intrinsic and extrinsic stress factors that can increase the risk of fetal neurodevelopmental disorders\u003csup\u003e\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. Previous studies suggested that placental dysfunction induced by maternal depression negatively affects fetal brain development \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. However, it is difficult to evaluate and monitor the status of the placenta functions.\u003c/p\u003e \u003cp\u003ePlacenta-derived exosomes (P\u003csub\u003eexo\u003c/sub\u003e) have recently been recognized as indicators of placental health due to their significant roles in maternal-fetal communication \u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Exosomes are extracellular vesicles released by cells and play a vital role in cell-to-cell communication \u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e. Their ability to cross the blood-brain barrier highlights their potential relevance to mental health disorders, with emerging evidence implicating exosomes in major depressive disorder (MDD) \u003csup\u003e\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Furthermore, Placental exosomes can traverse the placental barrier, enter fetal circulation, and alter fetal tissue functions \u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e. Study has shown a reduced number of P\u003csub\u003eexo\u003c/sub\u003e in fetuses experiencing growth restriction compared to those developing normally \u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e. However, the role of P\u003csub\u003eexo\u003c/sub\u003e in the intergenerational transmission of depression remains largely unexplored..\u003c/p\u003e \u003cp\u003eAccumulating evidence demonstrates a causal relationship between reduced hippocampal neurogenesis and depression. Clinical studies have reported reduced hippocampal volume in patients with MDD \u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e, while animal models associate decreased hippocampal neurogenesis with depressive-like behaviors \u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. In rodents studies, antidepressant treatment increase hippocampal neurogenesis, and inhibition of hippocampal neurogenesis could hamper the behavioral effects of antidepressant treatment\u003csup\u003e\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u003c/sup\u003e. Nevertheless, the precise mechanisms underlying reduced hippocampal neurogenesis in depressive-like rodent models are not fully elucidated. Recent findings by Sharma et al. highlight that exosomes can regulate neurogenesis \u003csup\u003e\u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u003c/sup\u003e. Therefore, we hypothesized that P\u003csub\u003eexo\u003c/sub\u003e might mediate the intergenerational transmission of depression through the modulation of hippocampal neurogenesis.\u003c/p\u003e \u003cp\u003eIn this study, we aimed to investigate whether Pexo contribute to the intergenerational transmission of depression and to explore the underlying molecular mechanisms involved.\u003c/p\u003e"},{"header":"2. Materials and Methods","content":" \u003cp\u003eAn extended methods section is available in the Online Data Supplement.\u003c/p\u003e \u003cdiv id=\"Sec2\" class=\"Section2\"\u003e \u003ch2\u003e2.1 Subjects and sample preparation\u003c/h2\u003e \u003cp\u003eThe pregnant women with term gestations (37\u0026ndash;42 weeks) were enrolled. They were hospitalized to be ready for delivery in the department of gynecology and obstetrics, the First Affiliated Hospital of Xi\u0026rsquo;an Jiaotong University between January 2017 and August 2023. Their depressive symptoms were assessed by the Hamilton Rating Scale for Depression (HAMD). According to the scores of HAMD and willingness of the subjects, we recruited 169 mother-newborn pairs. There were normal group (n\u0026thinsp;=\u0026thinsp;90) and depression group (n\u0026thinsp;=\u0026thinsp;79).\u003c/p\u003e \u003cp\u003eThe placenta tissue and 15 ml umbilical cord blood was collected at delivery. We obtained and recorded demographic data and birth outcomes data from the medical records. Neonatal Behavioral Assessment Scale (NBAS) was used to assess neurobehavioral development in all the newborn infants aged 21\u0026ndash;27 days after birth. Informed consent was obtained from all mothers. We obtained the ethical approval of the present study from Xi'an Jiaotong University (Xi\u0026rsquo;an, China). We complied with the ethical standards to perform all procedures, which was in agreement with the relevant national and institutional committees on human experimentation and the Helsinki Declaration of 1975, as revised in 2008.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.2 Isolation and verification of exosomes\u003c/h2\u003e \u003cdiv id=\"Sec4\" class=\"Section3\"\u003e \u003ch2\u003e2.2.1 P\u003csub\u003eexo\u003c/sub\u003e isolation\u003c/h2\u003e \u003cp\u003ePlacental tissue was cut into small pieces about 1mm\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e. Then put the small pieces into the 2ml centrifuge tubes, add type IV collagenase solution, and bathe it in water at 37℃ for 15min, shake it every 3min, until the cells forming cell clusters or single cells after shaking. Differential centrifugation were used to isolate exosomes. The samples were sequentially centrifuged at 1200 r/min 10 min and 3200 r/min 20 min at 4\u0026deg;C to remove cells and platelets, and then sequentially centrifuging at 15000 r/min 45 min and 30000 r/min 90 min at 4℃. The isolated P\u003csub\u003eexo\u003c/sub\u003e were resuspended in phosphate buffer saline (PBS) for further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section3\"\u003e \u003ch2\u003e2.2.2 Exosomes isolation from umbilical cord blood\u003c/h2\u003e \u003cp\u003eThe umbilical cord samples at 4\u0026deg;C were centrifuged at 1600g for 20 minutes to get plasma. Ultracentrifugation was used to isolate exosomes in plasma. To remove cells and platelets, plasma samples were sequentially centrifuged at 1200 r/min 10 min and 3200 r/min 20 min at 4\u0026deg;C, and then sequentially centrifuging at 15000 r/min 45 min and 30000 r/min 90 min at 4℃. PBS was used to resuspend the isolated exosomes for further experiments.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.3 miRNA Library Construction and Sequencing\u003c/h2\u003e \u003cp\u003emiRNA library preparation and sequencing were performed by a commercial service (Genergy bio-technology, China). In brief, total RNAs were extracted from isolated exosomes. Both 3\u0026rsquo;and 5\u0026rsquo;adaptors were added to each end, respectively, followed by reverse transcription and polymerase chain reaction amplification. We obtained the polymerase chain reaction products from the 18 to 30 nucleotide. Electrophoresis was used to purify RNA molecules. The illumina Hiseq3000 platform was used to sequence.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.4 Primary hippocampal neuron cultures\u003c/h2\u003e \u003cp\u003eSprague Dawley fetal rats aged embryonic day 18.5 were used to extract primary hippocampal neurons. We dissected the hippocampus tissue under a light microscope and aseptic conditions. A homogenized pool of hippocampus was used to obtain hippocampal neuron cells. Poly-D-lysine-coated 4-well plates (Thermo Fisher Scientific, USA) were used to plate the hippocampal neuron cells at a density of 60,000 to 80,000 cells/well. Cell cultures were maintained in a humidified incubator in an atmosphere of 5% CO\u003csub\u003e2\u003c/sub\u003e at 37\u0026deg;C by neurobasal-a medium enriched with GlutaMAX (Thermo Fisher Scientific, USA) and B27 supplement (Thermo Fisher Scientific, USA).\u003c/p\u003e \u003cp\u003eThen 1 mM cytosine arabinoside (Sigma-Aldrich, USA) were added to each well containing a fresh growth medium. The cells were cultured for 7 days before proceeding to further experiments.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.5 Animals\u003c/h2\u003e \u003cp\u003e We performed all animal procedures in agreement with the Institutional Animals Care and Use Committee at Xi\u0026rsquo;an Jiaotong University. Sprague-Dawley (SD) rats were from Xi\u0026rsquo;an Jiaotong University Health Science Center Measurements and were performed in accordance with the National Institutes of Health (NIH) Guide for the Care and Use of Laboratory. The pregnant SD rats were accidentally divided to two groups, namely experimental group administrated with depression P\u003csub\u003eexo\u003c/sub\u003e and control group with control P\u003csub\u003eexo\u003c/sub\u003e from embryonic day 10\u0026ndash;20. The offspring rats in experimental group were accidentally assigned into two groups: experimental group\u0026thinsp;+\u0026thinsp;LV-WNT2B and the experimental group\u0026thinsp;+\u0026thinsp;LV-GFP. The offspring rats in control group were accidentally divided into two groups: control group\u0026thinsp;+\u0026thinsp;LV-WNT2B and the control group\u0026thinsp;+\u0026thinsp;LV-GFP.\u003c/p\u003e \u003cp\u003eP\u003csub\u003eexo\u003c/sub\u003e were isolated from the placental tissue of the newborn infants. Depression P\u003csub\u003eexo\u003c/sub\u003e were from the newborn infants with neurobehavioral development disorders of depressed mothers during the pregnancy period. Control P\u003csub\u003eexo\u003c/sub\u003e were from the normal newborn infants.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.6 P\u003csub\u003eexo\u003c/sub\u003e injection\u003c/h2\u003e \u003cp\u003eP\u003csub\u003eexo\u003c/sub\u003e of newborn infants were injected to the pregnant SD rats once every two days from embryonic day 10\u0026ndash;20 by caudal veins.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.7 Behavioral test and molecular biology method\u003c/h2\u003e \u003cp\u003eSucrose preference test (SPF) and forced swimming test (FST) in the offspring rats of experimental group and control group were conducted as described in Supplemental Materials and Methods. Methods of isolation of the hippocampus, RNA Isolation, RT-PCR and Western Blot Analysis were performed as previously described \u003csup\u003e\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e\u003c/sup\u003e and conducted as described in Supplemental Materials and Methods. After finishing the behavioral test, the offspring rats received consecutive i.p. injections of 50 mg/kg bromodeoxyuridine (BrdU) (Sigma Aldrich) at twice times every day for 5 consecutive days and were sacrificed 12 hours after the last injection. Methods of immunohistochemistry were described in Supplemental Materials and Methods.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.8 Animals and Processing of Prenatal Stress (PS)\u003c/h2\u003e \u003cp\u003eThe PS model was mild restraint stress and performed as previously described \u003csup\u003e\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e\u003c/sup\u003e. A transparent cylinder (6.8 cm in diameter) was used to be the device. The pregnant rats were randomly divided to four groups: control (CON) group (n\u0026thinsp;=\u0026thinsp;6), PS group(n\u0026thinsp;=\u0026thinsp;6), PS\u0026thinsp;+\u0026thinsp;miRNA-485-5p antagomir group(n\u0026thinsp;=\u0026thinsp;6), and PS\u0026thinsp;+\u0026thinsp;miRNA control group(n\u0026thinsp;=\u0026thinsp;6). The pregnant rats in the last three groups were conducted to restraint stress 3 times daily for 45 min on days 11\u0026ndash;20 of pregnancy. The offspring rats in the PS group were administered with miRNA-485-5p antagomir or negative control antagomir. The pregnant rats in CON group were not treated at all in their home cages during the the whole period of pregnancy.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.9 miRNA-485-5p antagomir administration\u003c/h2\u003e \u003cp\u003eFor miRNA-485-5p antagomir experiments, the offspring rats in the PS group aged 21 days were intranasally administered with 5 nmol miRNA-485-5p antagomir (Shanghai GenePharma Co, China) or negative control (NC) antagomir every 3 days for four times. Then 50 mg/kg bromodeoxyuridine (BrdU) (Sigma Aldrich) were injected intraperitoneally (i.p.) at twice times every day for 5 consecutive days and were sacrificed 12 hours after the last injection to analyze newborn neurons.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003e2.10 Bilateral cannula implantations\u003c/h2\u003e \u003cp\u003eIntraperitoneal chloral hydrate (350 mg/kg) was used to anesthetize the offspring rats in the PS group aged 30 days. To implant the cannulae into the bilateral hippocampus, the offspring rats were positioned in a stereotaxic device (WPI 502603, USA). Their heads were fixed in a stoelting stereotaxic frame. The cannulae (AP, -3.3 mm; lateral, \u0026plusmn;\u0026thinsp;2.1 mm; and ventral, -3.0 mm) were fastened with acrylic dental cement. The offspring rats were conducted the behavioral test and molecular biology test followed by a seven day rest period.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e\u003cb\u003e2.11 Lentivirus-mediated gene vector\u003c/b\u003e\u003c/h2\u003e \u003cp\u003eWe entrusted to the commercial company (Shanghai Genechem Co. China, LTD) to construct the lentivirus vector for WNT2B overexpression (LV-WNT2B) and the negative lentivirus vector, which contained an enhanced green fluorescent protein (LV-GFP) marker. Sequencing was used to verify all constructions. The recombinant virus titer was 2.00 \u0026times; 10\u003csup\u003e8\u003c/sup\u003e TU (transfection unit)/ml.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e2.12 Microinjections\u003c/h2\u003e \u003cp\u003e33 gauge needles were used to perform microinjections. A 2-\u0026micro;l syringe by PE-10 tubing was linked to the needles, which were discreetly plugged into the guide cannula. 2.5 \u0026micro;l suspension of Lentiviral particles expressing WNT2B or control particles expressing GFP were bilaterally microinjected into the hippocampus tissue of the offspring rats in experimental group\u0026thinsp;+\u0026thinsp;LV-WNT2B once a day from day 37\u0026ndash;43. Vector stock was administrated at a rate of 0.5\u0026micro;l per minute for 10 days. The offspring rats were conducted the behavioral test and molecular biology test followed 24 hours after the last microinjections. The method of behavioral tests and biochemical studies were the same as before.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e2.13 Statistical analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed by SPSS software (version 20.0, SPSS Inc., Chicago, IL, USA). All values are presented as means\u0026thinsp;\u0026plusmn;\u0026thinsp;SD. The Kolmogorov-Smirnov test was used to assess normal distribution of quantitative variables. One-way ANOVA was used to compare the differences among the four groups. If the differences were significant, Fisher's LSD test was used to perform multiple comparisons. Independent sample t-test was used to perform comparisons between two groups. When the data did not comply with the normal distribution, Wilcoxon's rank sum test was used to perform comparisons between two groups. A difference was considered significant at \u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05 level.\u003c/p\u003e \u003c/div\u003e"},{"header":"3. Results","content":"\u003cdiv id=\"Sec18\" class=\"Section2\"\u003e \u003ch2\u003e3.1 The newborn infants of maternal depression during pregnancy displayed neurobehavioral development disorders\u003c/h2\u003e \u003cp\u003eThere were no significant differences in the demographic variables at baseline of pregnant women (Supplementary material Table\u0026nbsp;1) and newborn infants (Supplementary material Table\u0026nbsp;2) of between the depression group and the normal group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05). The newborn infants of prenatal depressed mothers had the lower scores of the social interactive items in the depression group than those of the normal group, mainly focus on difficulty in orienting to animate stimuli (face, voice, face plus voice items of the orientation cluster) (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026lt;\u0026thinsp;0.05) (Fig.\u0026nbsp;1B). However, there were no significant differences in the other cluster scores of the NBAS (habituation, regulation of states, range of states, and so on) in newborn infants between the depression group and the normal group (\u003cem\u003eP\u003c/em\u003e\u0026thinsp;\u0026gt;\u0026thinsp;0.05) (Fig.\u0026nbsp;1).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e3.2 Bioinformatics analysis and RT-qPCR verification of differentially expressed miRNAs in between control and depression P\u003csub\u003eexo\u003c/sub\u003e\u003c/h2\u003e \u003cp\u003eElectron microscopy analysis was performed to observe typical rounded particles with diameters ranging between 30 and 100 nm in isolated fractions (Fig.\u0026nbsp;2A (a1)). Exosome marker proteins CD63 and Alix were confirmed by Western blot analysis(Fig.\u0026nbsp;2A (a2-a4)). There were no significant quantitative differences between the control P\u003csub\u003eexo\u003c/sub\u003e and depression P\u003csub\u003eexo\u003c/sub\u003e. There were no significant differences in size distribution (60.54\u0026thinsp;\u0026plusmn;\u0026thinsp;22.57versus 55.5\u0026thinsp;\u0026plusmn;\u0026thinsp;19.26 nm) between the control P\u003csub\u003eexo\u003c/sub\u003e and depression P\u003csub\u003eexo\u003c/sub\u003e by Nanoparticle tracking analysis (NTA). NTA also showed the concentration in depression P\u003csub\u003eexo\u003c/sub\u003e was less than that in control P\u003csub\u003eexo\u003c/sub\u003e (4.28\u0026times;10\u003csup\u003e10\u003c/sup\u003e versus 11.66\u0026times;10\u003csup\u003e10\u003c/sup\u003e particles/ml) (Fig.\u0026nbsp;2A (a5)). 10 differentially expressed miRNAs in depression P\u003csub\u003eexo\u003c/sub\u003e were found by high-throughput sequencing(Fig.\u0026nbsp;2B). Then miRNA-485-5p stood out as the most potent molecule related to hippocampal signaling pathway and neurogenesis based on Kyoto Encyclopedia of Genes and Genomes (KEGG) and gene ontology (GO) pathway enrichment scores. Importantly, the elevation of miRNA-485-5p in both depression P\u003csub\u003eexo\u003c/sub\u003e and umbilical cord blood exosomes of newborn infants with neurobehavioral development disorders were also observed(Fig.\u0026nbsp;2C).\u003c/p\u003e \u003cp\u003e \u003cb\u003e3.3 Depression P\u003c/b\u003e \u003csub\u003e \u003cb\u003eexo\u003c/b\u003e \u003c/sub\u003e \u003cb\u003eof newborn infants with neurobehavioral development disorders inhibited the proliferation activity of primary cultured hippocampal neurons\u003c/b\u003e\u003c/p\u003e \u003cp\u003eTop20 function prediction of miRNA-485-5p was performed based on KEGG and GO enrichment analyses(Fig.\u0026nbsp;3A). WNT2B/β-catenin signaling was also regarded as the enrichment signaling of miRNA-485-5p by bioinformatics analysis. Primary cultured hippocampal neurons derived from fetal rats were incubated with Dil-labeled P\u003csub\u003eexo\u003c/sub\u003e(Fig.\u0026nbsp;3B). It was observed that P\u003csub\u003eexo\u003c/sub\u003e were internalized by hippocampal neurons after a 8-hour incubation as indicated by laser confocal images(Fig.\u0026nbsp;3C). We found that depression P\u003csub\u003eexo\u003c/sub\u003e derived from newborn infants with neurobehavioral development disorders inhibited the proliferation activity of primary cultured hippocampal neurons(Fig.\u0026nbsp;3D(d1). The expression level of WNT2B mRNA and protein were significantly reduced in primary cultured hippocampal neurons administrated with P\u003csub\u003eexo\u003c/sub\u003e derived from newborn infants with neurobehavioral development disorders(Fig.\u0026nbsp;3D(d2-d3).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e3.4 miRNA-485-5p derived from depression P\u003csub\u003eexo\u003c/sub\u003e was identified as the key miRNA related to hippocampal neurogenesis\u003c/h2\u003e \u003cp\u003eThe normal pregnant rats were randomly divided into the two groups. The pregnant rats in the experimental group were administrated with depression P\u003csub\u003eexo\u003c/sub\u003e from newborn infants with neurobehavioral development disorders once two days from embryonic day 10\u0026ndash;20. And the pregnant rats in the control group were administrated with P\u003csub\u003eexo\u003c/sub\u003e from normal newborn infants. The offspring rats in the experimental group displayed depression-like behavior, and they showed the longer immobile time in FST and the decrease of sucrose consumption in SPF compared with the control group (Fig.\u0026nbsp;4A). The expression levels of WNT2B/β-catenin mRNA and protein were significantly reduced in the offspring rats of the experimental group than that the control group (Fig.\u0026nbsp;4B-C). We found that there were less numbers of BrdU\u003csup\u003e+\u003c/sup\u003e positive cells and BrdU\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e positive cells in the dentate gyrus (DG) of the hippocampus in the experimental group than that in the control group (Fig.\u0026nbsp;4D-E).\u003c/p\u003e \u003cp\u003eWe next used the prenatal stress (PS) animal model to further assess the functional involvement of exosomal miRNA-485-5p in intergenerational transmission of depression. We observed that the offspring rats in the PS group displayed depression-like behavior, and they showed the longer immobile time in FST and the decrease of sucrose consumption in SPF compared with the control group (Fig.\u0026nbsp;4F). The expression level of exosomal miRNA-485-5p in the placenta of the pregnant rats during 20 days of pregnancy in the PS group was higher than that in the control group, which is consistent with our clinical findings (Fig.\u0026nbsp;4G).\u003c/p\u003e \u003cp\u003eTo assess the functional involvement of exosomal miRNA-485-5p upregulation in intergenerational transmission of depression, we intranasally administered 5nmol miRNA-485-5p antagomir in the pregnant rats of the PS group to inhibit exosomal miRNA-485-5p expression. Depressive-like behaviors in the offspring rats of the PS group were rescued by intranasal miRNA-485-5p antagomir administration as assessed by FST and SPF(Fig.\u0026nbsp;4F).\u003c/p\u003e \u003cp\u003eThe expression level of WNT2B mRNA and protein were significantly increased in the offspring rats of the PS\u0026thinsp;+\u0026thinsp;miRNA-485-5p antagomir administration group than that the control group(Fig.\u0026nbsp;4H). We found that there were more BrdU\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e positive cells in the dentate gyrus (DG) of the hippocampus in the offspring rats of the PS\u0026thinsp;+\u0026thinsp;miRNA-485-5p antagomir administration group than that in the control group(Fig.\u0026nbsp;4I-J).\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e3.5 P\u003csub\u003eexo\u003c/sub\u003e mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling\u003c/h2\u003e \u003cp\u003eThe normal pregnant rats were randomly divided into the two groups. The pregnant rats in the experimental group were administrated with depression P\u003csub\u003eexo\u003c/sub\u003e from newborn infants with neurobehavioral development disorders once two days from embryonic day 10\u0026ndash;20, and the pregnant rats in the control group with P\u003csub\u003eexo\u003c/sub\u003e from normal newborn infants. The offspring rats in both the experimental group and control group were injected with LV-WNT2B or LV-GFP once a day from day 37\u0026ndash;43. We found that depressive-like behaviors in the offspring rats of the experimental group were rescued by LV-WNT2B administration as assessed by FST and SPF(Fig.\u0026nbsp;5A). The decrease of β-catenin mRNA and protein in the offspring rats of the experimental group were prevented by LV-WNT2B administration (Fig.\u0026nbsp;5B). There were more number of BrdU\u003csup\u003e+\u003c/sup\u003e/NeuN\u003csup\u003e+\u003c/sup\u003e positive cells in the dentate gyrus (DG) of the offsprings hippocampus in the experimental group\u0026thinsp;+\u0026thinsp;LV-WNT2B group than that in the experimental group\u0026thinsp;+\u0026thinsp;LV-GFP group (Fig.\u0026nbsp;5C-D). It indicated that administration of WNT2B could prevent the depression-like behavior of offspring rats induced by depression P\u003csub\u003eexo\u003c/sub\u003e of newborn infants with neurobehavioral development disorders. Therefore, we proposed that P\u003csub\u003eexo\u003c/sub\u003e may mediate the intergenerational transmission of depression by regulating WNT2B/β-catenin signaling.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Discussion","content":"\u003cp\u003eIn the present study, we firstly isolated placenta-derived exosomes (P\u003csub\u003eexo\u003c/sub\u003e) from newborn infants with neurobehavioral development disorders of depressed mothers (depression P\u003csub\u003eexo\u003c/sub\u003e) and provided a comprehensive evaluation of P\u003csub\u003eexo\u003c/sub\u003e in intergenerational transmission of depression. The findings reveal a novel mechanism that maternal depression during pregnancy could be transmitted to the offsprings by P\u003csub\u003eexo\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eMaternal prenatal depression could have the negative effects on offspring behavioral and physical development \u003csup\u003e\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e. These effects can appear as early as childhood or even in infancy. For example, the infants of depressed mothers developed negative affective states that bias their interactions with others \u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In this study, we found that the newborn infants of maternal depression during pregnancy displayed reduced social interactive behavior. However, the underlying molecular mechanisms for maternal depression inducing the behavioral and mental changes of offsprings remain unclear. The placenta is an important micro-environmental factor in fetal neurodevelopment by maternal-fetal communication. P\u003csub\u003eexo\u003c/sub\u003e was associated with intergenerational transmission of diseases, since the composition of P\u003csub\u003eexo\u003c/sub\u003e reflect the alterations of maternal status during pregnancy \u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e. In the present study, the pregnant rats were administrated with depression P\u003csub\u003eexo\u003c/sub\u003e from newborn infants, then the offspring rats displayed depressive-like behaviors. We firstly proposed that P\u003csub\u003eexo\u003c/sub\u003e could play a crucial role in the intergenerational transmission of depression.\u003c/p\u003e \u003cp\u003eExosomes contain proteins, miRNAs and so on. Growing attention has been focused on exosomal miRNAs. Increasing evidence has shown that exosomal miRNAs was involved in mental disorders \u003csup\u003e\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e. For example, the increased levels of miRNA-139-5p in blood exosomes were observed in the patients with major depressive disorder \u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. In the present study, miRNA-485-5p was identified in depression P\u003csub\u003eexo\u003c/sub\u003e as a key molecule that was associated with the intergenerational transmission of depression. Increased expression levels of miRNA-485-5p were observed in both depression P\u003csub\u003eexo\u003c/sub\u003e and umbilical cord blood exosomes of newborn infants with neurobehavioral development disorders, and also observed in P\u003csub\u003eexo\u003c/sub\u003e from the placenta of the pregnant rats of the PS group. These results demonstrated that maternal depression during pregnancy induced the secretion of P\u003csub\u003eexo\u003c/sub\u003e enriched with miRNA-485-5p. We found that depressive-like behaviors in the offspring rats of the PS group were rescued by miRNA-485-5p antagomir administration by preventing the decrease in hippocampal neurogenesis. Jonathan E found that miRNA-485-5p was developmentally regulated in the hippocampus and could affect synaptic plasticity and neurite outgrowth, and miRNA-485-5p overexpression dramatically reduced the extent of axonal outgrowth in hippocampal neurons \u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. It is possible that maternal depression during pregnancy led to placental function disorders, then induced the secretion of P\u003csub\u003eexo\u003c/sub\u003e enriched with miRNA-485-5p, which could cross the placental barrier and enter into the fetal circulation. Nextly, P\u003csub\u003eexo\u003c/sub\u003e enriched with miRNA-485-5p in fetal circulation could cross the blood-brain barrier, then resulting in structural and functional changes of hippocampal neurons, such as decreased hippocampal neurogenesis. Therefore, we suggested that P\u003csub\u003eexo\u003c/sub\u003e was involved in the intergenerational transmission of depression by regulating hippocampal neurogenesis via miRNA-485-5p.\u003c/p\u003e \u003cp\u003eBioinformatic and quantification analysis showed that WNT2B/β-catenin signaling were targets of miRNA-485-5p related to neurogenesis and hippocampal signaling pathway. We found that both depression P\u003csub\u003eexo\u003c/sub\u003e administration enriched with miRNA-485-5p and PS reduced WNT2B expression level. miRNA-485-5p antagomir administration could rescue the decrease of WNT2B expression level induced by PS. Moreover, administration of WNT2B could prevent the depression-like behavior of offspring rats induced by depression P\u003csub\u003eexo\u003c/sub\u003e and rescue the decrease of hippocampal neurogenesis. It is well established that WNT2B is the key element of the canonical WNT signaling pathway playing a pivotal role in the generation of newborn neurons in the hippocampal dentate gyrus \u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e. Inhibition of WNT signaling within the dentate gyrus almost completely abolished the formation of newborn neurons \u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Importantly, WNT2B gene plays important roles in developing human hippocampus \u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. It is possible that the decrease of WNT2B gene in early life could impair structural and functional of hippocampus. Furthermore, it was reported that WNT2B gene was associated with molecular mechanisms for depression by regulating neuroplasticity \u003csup\u003e\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e\u003c/sup\u003e. We suggest that P\u003csub\u003eexo\u003c/sub\u003e enriched with miRNA-485-5p in fetal circulation may result in the decrease of WNT2B expression level in hippocampus of offspring of depressed mothers in early life, which then inducing structural and functional changes of hippocampus. Therefore, depression P\u003csub\u003eexo\u003c/sub\u003e caused depressive-like behaviors in offspring resulting from decreased hippocampal neurogenesis regulated by WNT2B/β-catenin signaling.\u003c/p\u003e \u003cp\u003eThe present study has some limitations. First, we should measure and analyze the expression of P\u003csub\u003eexo\u003c/sub\u003e in neonatal peripheral blood and cerebrospinal fluid of newborn infants with neurobehavioral development disorders of depressed mothers. We should further observe the expression of depression P\u003csub\u003eexo\u003c/sub\u003e in peripheral blood, cerebrospinal fluid and hippocampus of the offspring rats of the mother rats administrated with depression P\u003csub\u003eexo\u003c/sub\u003e from newborn infants. Second, there is no vivo-specific pharmacological inhibitors or model animals to inhibit exosomes production. It is difficult to perform in vivo loss-of-function studies on depression P\u003csub\u003eexo\u003c/sub\u003e from newborn infants.\u003c/p\u003e \u003cp\u003eIn summary, maternal depression during pregnancy induced the secretion of depression P\u003csub\u003eexo\u003c/sub\u003e. The offspring rats of pregnant rats administrated with depression P\u003csub\u003eexo\u003c/sub\u003e displayed depression-like behavior accompanied by reduced hippocampal neurogenesis, indicating a crosstalk between P\u003csub\u003eexo\u003c/sub\u003e and reduced hippocampal neurogenesis in the offsprings. We identified miRNA-485-5p in depression P\u003csub\u003eexo\u003c/sub\u003e as a key molecule which reduced hippocampal neurogenesis. Administration of WNT2B could rescue the depression-like behavior of offspring rats induced by depression P\u003csub\u003eexo\u003c/sub\u003e. These findings present a novel mechanism underlying intergenerational transmission of depression with miRNA-485-5p in P\u003csub\u003eexo\u003c/sub\u003e as a potential target and highlight the therapeutic potential of WNT2B regulated by miRNA-485-5p in the treatment of intergenerational transmission of depression.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eSupplementary Material\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eRefer to Web version on PubMed Central for supplementary material.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding and Disclosure\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by National Natural Science Foundation of China (No. 82371547). All the authors report no biomedical financial interests or potential conflicts of interest.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe are very grateful to all the mother-infant pairs that took part in this study and the graduate students for their help in recruiting the subjects and follow up data. Our team includes obstetricians, pediatrician, nurses, interviewers, laboratory technicians, basic researchers and volunteers. The named authors will serve as guarantors for the contents of this paper.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eHuiping Zhang drafted the manuscript and made substantial contributions to the conception or design of the work. Miaomiao Chen, Rong Liu, Pan Zeng, Ruobing Liang, Jiao Liu made substantial contributions to interpretation of data for the work. Qiaomian Zhu，Minjie Fu, Siliang Chen made substantial contributions to revising the work critically for important intellectual content. Huiping Zhang and Zhuoyue Shi made substantial contributions to the conception or design of the work and revised the manuscript.\u0026nbsp;\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eWeissman MM. Intergenerational study of depression: a convergence of findings and opportunities. Psychological medicine 50,170\u0026ndash;172(2020).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eO'connor E, Senger CA, Henninger ML, Coppola E, Gaynes BN. Interventions to Prevent Perinatal Depression: Evidence Report and Systematic Review for the US Preventive Services Task Force. Jama 321,588\u0026ndash;601(2019).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGoodman SH, Garber J. Evidence-Based Interventions for Depressed Mothers and Their Young Children. 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Journal of neuroscience research 70,200\u0026ndash;208(2002).\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBreitfeld J, Scholl C, Steffens M, Laje G, Stingl JC. Gene expression and proliferation biomarkers for antidepressant treatment resistance. Translational psychiatry 7,e1061(2017).\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"maternal depression, intergenerational transmission, placenta, exosomes, miRNA-485-5p, WNT2B","lastPublishedDoi":"10.21203/rs.3.rs-6587812/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-6587812/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eMaternal depression during pregnancy adversely affects offsprings from infancy through adulthood. Placenta-derived exosomes (P\u003csub\u003eexo\u003c/sub\u003e) play crucial roles in maternal-fetal communication. This study investigates the involvement of P\u003csub\u003eexo\u003c/sub\u003e in the intergenerational transmission of maternal depression and its underlying molecular mechanisms. P\u003csub\u003eexo\u003c/sub\u003e were isolated from placental tissue of newborns exhibiting neurobehavioral development disorders due to maternal depression during pregnancy (depression P\u003csub\u003eexo\u003c/sub\u003e, n\u0026thinsp;=\u0026thinsp;90) and from healthy newborns (control P\u003csub\u003eexo\u003c/sub\u003e, n\u0026thinsp;=\u0026thinsp;79). Pregnant SD rats were administrated to depression P\u003csub\u003eexo\u003c/sub\u003e (n\u0026thinsp;=\u0026thinsp;6) or prenatal stress(n\u0026thinsp;=\u0026thinsp;6). Behavior test, bioinformatics analyses, and molecular biology approaches were used to examine the effects of P\u003csub\u003eexo\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eNewborns from mothers with depression during pregnancy exhibited neurobehavioral development deficits. Depression P\u003csub\u003eexo\u003c/sub\u003e inhibited hippocampal neuron proliferation in primary neuron cultures, resulting in the depressive-like behaviors and the reduced hippocampal neurogenesis in offspring. miRNA sequencing combined with RT- qPCR identified 10 differentially expressed miRNAs in depression P\u003csub\u003eexo\u003c/sub\u003e, with miRNA-485-5p emerging as a key regulator of hippocampal neurogenesis. Expression of WNT2B/β-catenin signaling was significantly decreased in offspring exposed to depression P\u003csub\u003eexo\u003c/sub\u003e or prenatal stress. WNT2B administration rescued depressive-like behavior and restored hippocampal neurogenesis impaired by depression P\u003csub\u003eexo\u003c/sub\u003e.\u003c/p\u003e \u003cp\u003eThus, placenta-derived exosomal miRNAs and WNT2B signaling represent promising therapeutic targets to mitigate the intergenerational effects of maternal depression.\u003c/p\u003e","manuscriptTitle":"Placenta-derived exosomes mediate the intergenerational transmission of depression by regulating WNT2B /β-catenin signaling","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-05-30 18:51:26","doi":"10.21203/rs.3.rs-6587812/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"c7b68312-f050-430f-b2c2-ad990b8b2c26","owner":[],"postedDate":"May 30th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":49147894,"name":"Biological sciences/Neuroscience/Molecular neuroscience"},{"id":49147895,"name":"Health sciences/Diseases/Psychiatric disorders/Depression"}],"tags":[],"updatedAt":"2026-03-30T15:17:21+00:00","versionOfRecord":[],"versionCreatedAt":"2025-05-30 18:51:26","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-6587812","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-6587812","identity":"rs-6587812","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}