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Cited by 169Extracellular vesicles in diseaseNeuroinflammation and Neurodegeneration MechanismsTryptophan and brain disorders

Neuron secrete exosomes containing miR-9-5p to promote polarization of M1 microglia in depression

Open access
Xian, Xian, Cai, Li-Li, Li, Yang, Wang, Ran-Chao, Xu, Yu-Hao, Chen, Ya-Jie, Xie, Yu-Hang, Zhu, Xiao-Lan +1 more·2022·Journal of Nanobiotechnology
DOI 10.1186/s12951-022-01332-wPMC PMC8905830
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Abstract

Background: Neuroinflammation is an important component mechanism in the development of depression. Exosomal transfer of MDD-associated microRNAs (miRNAs) from neurons to microglia might exacerbate neuronal cell inflammatory injury. Results: By sequence identification, we found significantly higher miR-9-5p expression levels in serum exosomes from MDD patients than healthy control (HC) subjects. Then, in cultured cell model, we observed that BV2 microglial cells internalized PC12 neuron cell-derived exosomes while successfully transferring miR-9-5p. MiR-9-5p promoted M1 polarization in microglia and led to over releasing of proinflammatory cytokines, such as interleukin-1β (IL-1β), interleukin-6 (IL-6) and tumor necrosis factor-alpha (TNF-α), which exacerbated neurological damage. Furthermore, we identified suppressor of cytokine signaling 2 (SOCS2) as a direct target of miR-9-5p. Overexpression of miR-9-5p suppressed SOCS2 expression and reactivated SOCS2-repressed Janus kinase (JAK)/signal transducer and activator of transcription 3 (STAT3) pathways. Consistently, we confirmed that adeno-associated virus (AAV)-mediated overexpression of miR-9-5p polarized microglia toward the M1 phenotype and exacerbated depressive symptoms in chronic unpredictable mild stress (CUMS) mouse mode. Conclusion: MiR-9-5p was transferred from neurons to microglia in an exosomal way, leading to M1 polarization of microglia and further neuronal injury. The expression and secretion of miR-9-5p might be novel therapeutic targets for MDD. Graphical Abstract:

AI snapshot

Written from the paper’s full text.

TL;DR

Serum exosomes from MDD patients and depressed neuronal cells are enriched in miR-9-5p, which is transferred to microglia and promotes M1 polarization. Mechanistically, miR-9-5p suppresses SOCS2 and reactivates JAK/STAT3 signaling, exacerbating neuroinflammation and depressive-like behavior in mice.

Key findings

  • Serum exosomes from MDD patients promoted BV2 microglial M1 polarization, with increased iNOS+ cells and upregulated IL-1β, IL-6, and TNF-α; GW4869 treatment inhibited this effect.
  • miRNA profiling of serum exosomes from 3 HC subjects and 6 MDD patients identified 251 differentially expressed miRNAs: 167 increased and 84 decreased; miR-9-5p was the only top upregulated miRNA validated by RT-PCR.
  • PC12 neuron-derived exosomes transferred miR-9-5p to BV2 microglia, increasing M1 microglia and inflammatory cytokines; miR-9-5p overexpression in BV2 cells increased iNOS+ cells and IL-1β, IL-6, and TNF-α.
  • miR-9-5p directly targeted SOCS2; overexpression suppressed SOCS2 and activated JAK/STAT3, while JAK/STAT3 inhibition by cucurbitacin I rescued p-STAT3 activation and M1 polarization. AAV-mediated miR-9-5p overexpression in CUMS mice increased depressive-like behavior, M1 microglia, and hippocampal [18F]DPA-714 uptake.

Methods

  • Human serum exosomes were isolated from 3 healthy controls and 6 MDD patients; exosomes were characterized by TEM, western blotting for CD9/CD63, and NTA.
  • In vitro models used PC12 neuronal cells, BV2 microglia, primary hippocampal neurons, and primary microglia; CORT (200 μM) was used to model depression in PC12 cells, with Transwell co-culture, miRNA mimics/inhibitors, qRT-PCR, western blot, ELISA, and immunofluorescence.
  • In vivo studies used male C57BL/6 J mice in a CUMS model, stereotactic hippocampal injection of AAV miR-9-5p or inhibitor, behavioral tests (OFT, FST), immunofluorescence, NIRFI, and [18F]DPA-714 PET/CT imaging.

Limitations

  • The paper states that the main limitation of the MDD inflammation hypothesis is the lack of measurements of neurological inflammation in MDD patient brain, and that further work is needed on the dynamic M1/M2 balance and possible malignant neuroinflammatory feedback loops.
  • Evident from the text: the human exosome analysis used a very small sample (3 HC, 6 MDD), limiting generalizability.
  • Evident from the text: most mechanistic and behavioral evidence comes from PC12/BV2 cell lines and CUMS mice, not human brain tissue or clinical intervention studies.

Model-written summary. Check it against the paper before relying on it.

Integrity signals

Registry

Crossref · 10.1186/s12951-022-01332-w

Cites

Statistics

Nothing to recompute.

Claims the paper makes

9 of 39 cited sources checked · 4 supported · 1 flagged · accuracy not yet measured

  • Findingown data

    Exosome treatment increases mean [18F]DPA-714 uptake in the hippocampus, indicating increased neuroinflammatory signals.

    “We also observed increased mean [18F]DPA-714 uptake in the hippocampus (Fig. 1l), indicating an increased expression of neuroinflammatory signals in our experimental setting [32].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [32] Zinnhardt B, Belloy M, Fricke IB, Orije J, Guglielmetti C, Hermann S, Wagner S, Schafers M, Van der Linden A, Jacobs AH. Molecular imaging of immune cell dynamics during de- and remyelination in the cuprizone model of multiple sclerosis by [(18)F]DPA-714 PET and MRI. Theranostics. 2019;9(6):1523–37. source
  • Premisecitation

    Microglia are highly activated in the brains of patients with depression.

    “It was reported that immune cell microglia were highly activated in the brains of patients with depression, indicating that neuroinflammation played a key role in pathogenic mechanisms of the depressed brain [5].”
    Quote in the paperSubstantial claim1 of 1 sources checked
    • cites [5] Brites D, Fernandes A. Neuroinflammation and depression: microglia activation, extracellular microvesicles and microRNA dysregulation. Front Cell Neurosci. 2015;9:476. source
  • Findingown data

    Primary neurons secrete miR-9-5p-containing exosomes.

    “We then confirmed that primary neurons secreted miR-9-5p-containing exosomes as well (Additional file 1: Fig. S3c).”
    Quote in the paperSubstantial claim
  • Findingown data

    Exosome treatment increases the proportion of iNOS+ cells in the mouse hippocampus.

    “The proportion of iNOS+ cells was also increased in the treated group (Fig. 1k).”
    Quote in the paperSubstantial claim
  • Findingown data

    AAV-mediated overexpression of miR-9-5p in MDD mice increases depression-like behavior in the open field test and forced swimming test.

    “Comparing to control group, MDD mice injected with AAV carrying miR-9-1 showed increased depression-like behavior in the OFT and FST (Fig. 4d–f, Additional file 1: Fig. S4e).”
    Quote in the paperSubstantial claim
  • Classified as probably not a substantial claim. 24
  • May combine two claims. 22

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    In physiological status, microglia are commonly in the resting M2 (CD206-positive) state in the central nervous system.

    “In physiological status, microglia were commonly in the resting M2 (CD206-positive) state in the central nervous system (CNS) [14].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [14] Dey A, Allen J, Hankey-Giblin PA. Ontogeny and polarization of macrophages in inflammation: blood monocytes versus tissue macrophages. Front Immunol. 2014;5:683. source

      The “Microglia: Resident CNS Macrophages” section describes healthy-brain microglia as quiescent or down-regulated, but says resident microglia have an expression profile distinct from M1- or M2-polarized microglia. It describes M2 as a subdivision of an activated state, not the usual resting state. The cited passages do not identify resting microglia as CD206-positive. (LLM, conf=0.90)

      Checked by a reasoning model — may be wrong.

      Experimental — accuracy not yet measured

  • May combine two claims.

    Premisecitation

    SOCS2 is involved in STAT3 pathway activation and can mediate microglial polarization regulation.

    “We identified miR-9-5p binding sites in the 3'-UTR of SOCS2 mRNA (Fig. 5a), which was involved in STAT3 pathway activation and able to mediate microglial polarization regulation [35–37].”
    Quote in the paperSubstantial claim3 of 3 sources checked
    • cites [35] Kwon SH, Han JK, Choi M, Kwon YJ, Kim SJ, Yi EH, Shin JC, Cho IH, Kim BH, Jeong Kim S, et al. Dysfunction of microglial STAT3 alleviates depressive behavior via neuron-microglia interactions. Neuropsychopharmacology. 2017;42(10):2072–86. source

      The Introduction and Results discuss microglial STAT3 signaling and the effects of STAT3 depletion on neuron–microglia interactions. Our copy does not address SOCS2 or show that it activates STAT3 or mediates microglial polarization (searched: SOCS2, suppressor of cytokine signaling 2, microglial polarization). (LLM, conf=0.72)

      Experimental — accuracy not yet measured
    • cites [36] Xiaoying G, Guo M, Jie L, Yanmei Z, Ying C, Shengjie S, Haiyan G, Feixiang S, Sihua Q, Jiahang S. CircHivep2 contributes to microglia activation and inflammation via miR-181a-5p/SOCS2 signalling in mice with kainic acid-induced epileptic seizures. J Cell Mol Med. 2020;24(22):12980–93. source

      The Results section on microglial activation (page not provided) links the circHivep2/miR-181a-5p/SOCS2 pathway to activation in KA-treated BV-2 cells. It does not assess microglial polarization. The Discussion mentions STAT3 in connection with other pathways and SOCS1/SOCS3, but does not establish that SOCS2 activates STAT3. (LLM, conf=0.86)

      Experimental — accuracy not yet measured
    • cites [37] Basrai HS, Christie KJ, Turbic A, Bye N, Turnley AM. Suppressor of cytokine signaling-2 (SOCS2) regulates the microglial response and improves functional outcome after traumatic brain injury in mice. PLoS ONE. 2016;11(4):e0153418. source

      The Discussion reports more M2-like macrophages/microglia in SOCS2-overexpressing mice after traumatic brain injury, supporting a potential role for SOCS2 in regulating the microglial response. It says the mechanism is unclear and does not establish STAT3 pathway activation; the claim’s STAT3 component is therefore unsupported by this paper. (LLM, conf=0.88)

      Experimental — accuracy not yet measured

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Depressive symptoms are positively associated with chronic inflammatory disorders.

    “The correlation between psychiatric disorders and neuroinflammation was reported decades ago: increased levels of inflammatory markers often occurred in patients with depression, while the presence of depressive symptoms was positively associated with patients suffering from chronic inflammatory disorders [4, 5].”
    Quote in the paperNot a substantial claim2 of 2 sources checked
    • cites [4] Haapakoski R, Ebmeier KP, Alenius H, Kivimaki M. Innate and adaptive immunity in the development of depression: an update on current knowledge and technological advances. Prog Neuropsychopharmacol Biol Psychiatry. 2016;66:63–72. source

      The section on inflammatory and neuroendocrine mechanisms (page unavailable) describes a high prevalence of immune-related chronic disorders, such as diabetes, among depressed patients. The Evidence on immune–brain relations section also links depression with inflammatory markers. Neither passage establishes the claimed positive association between depressive symptoms and chronic inflammatory disorders as a group. (LLM, conf=0.78)

      Experimental — accuracy not yet measured
    • cites [5] Brites D, Fernandes A. Neuroinflammation and depression: microglia activation, extracellular microvesicles and microRNA dysregulation. Front Cell Neurosci. 2015;9:476. source

  • May combine two claims.

    Premisecitation

    [18F]DPA-714 has the advantages of a high binding affinity and target-to-background ratio.

    “Moreover, [18F]DPA-714 has the advantages of a high binding affinity and target-to-background ratio [57].”
    Quote in the paperNot a substantial claim1 of 1 sources checked
    • cites [57] Wang YL, Han QQ, Gong WQ, Pan DH, Wang LZ, Hu W, Yang M, Li B, Yu J, Liu Q. Microglial activation mediates chronic mild stress-induced depressive- and anxiety-like behavior in adult rats. J Neuroinflammation. 2018;15(1):21. source

  • May combine two claims.

    Findingown data

    251 miRNAs are significantly differentially expressed in serum exosomes of MDD patients versus HC subjects, including 167 increased and 84 decreased.

    “Volcano plot showed 251 significantly differentially expressed miRNAs, including 167 miRNAs with increased expression and 84 miRNAs with decreased expression in MDD patients (Fig. 2b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    AAV-mediated overexpression of miR-9-5p increases M1 microglial number, [18F]DPA-714 signals in the hippocampus, and M1 polarization in primary microglia.

    “Moreover, both the M1 microglial number and [18F]DPA-714 signals were increased in the hippocampus (Fig. 4g, h, Additional file 1: Fig. S4f), and M1 polarization in primary microglia was also enhanced. (Additional file 1: Fig. S4g).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    M1 microglia pretreated with LPS, serum exosomes from MDD patients, or exosomes from MDD cellular models directly shorten neuronal synapses and inhibit cell proliferation when co-cultured with PC12 cells.

    “M1 microglia, pretreated with LPS, serum exosomes from MDD patients or exosomes from MDD cellular models, directly shortened neuronal synapses and inhibited cell proliferation when co-cultured with PC12 cells.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Isolated exosome samples express high levels of CD9 and CD63, while the remaining serum supernatant does not.

    “We observed high expression of the exosomal markers CD9 and CD63 in the isolated samples, but undetectable in the remaining serum supernatant (Fig. 1b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MiR-9-5p mimic attenuates SOCS2 mRNA and protein levels, while miR-9-5p inhibitor elevates them.

    “As shown in Fig. 5b, c and Additional file 1: Fig. S5a–c, the SOCS2 mRNA and protein levels were attenuated in the mimic group and elevated in the inhibitor group.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    BV2 cells internalize PC12-derived exosomes, leading to increased miR-9 levels.

    “As shown, BV2 cells internalized PC12-derived exosomes, leading to increase of miR-9 levels (Fig. 3b).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MiR-9-5p overexpression in BV2 cells significantly increases the proportion of iNOS+ cells and upregulates IL-1β, IL-6, and TNF-α expression.

    “As expected, in miR-9-5p-overexpressed BV2 cells, the proportion of iNOS+ cells was significantly increased (Fig. 4b, Additional file 1: Fig. S4a), and the expression of IL-1β, IL-6, and TNF-α was upregulated (Fig. 4c).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Exosomes from a MDD cellular model injected into the mouse hippocampus induce depressive-like behavior, as evidenced by avoidance of the central part of the arena in the open field test and increased immobility in the forced swimming test.

    “Comparing to control group, exosomes treated mice showed depressive-like behavior, as evidenced by significant avoidance of the central part of the arena in the open field test (OFT) and increased immobility in the forced swimming test (FST) (Fig. 1i, j, Additional file 1: Fig. S1h).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    MDD patient serum and serum exosomes have similar function on promoting M1 polarization in microglia, but the effect of serum exosomes is more pronounced.

    “In summary, MDD patients-derived serum and serum exosomes had similar function on promoting M1 polarization in microglia, but the effect of serum exosomes was more pronounced.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Enriched miR-9-5p in these exosomes suppresses SOCS2 expression and activates JAK/STAT3 pathways in BV2 microglia.

    “BV2 microglia phagocytosed these exosomes, in which enriched miR-9-5p, suppressed SOCS2 expression and activated JAK/STAT3 pathways, leading to polarization into M1 type.”
    Quote in the paperSubstantial claim
    • Rests on an assumption: PC12 cells are a valid model of depressed neurons.
    • Rests on an assumption: Exosomes derived from PC12 cells are representative of exosomes from depressed neurons.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Secreted extracellular vehicles (EVs), including exosomes and ectosomes ranging in size from 50 to 200 nm, are important carriers in mediating intercellular signaling.

    “Growing evidence showed that secreted extracellular vehicles (EVs), including exosomes and ectosomes ranging in size from 50 to 200 nm, were important carriers in mediating intercellular signaling.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [16] Pegtel DM, Peferoen L, Amor S. Extracellular vesicles as modulators of cell-to-cell communication in the healthy and diseased brain. Philos Trans R Soc Lond B Biol Sci. 2014. https://doi.org/10.1098/rstb.2013.0516. source

      Not checked in the free run.

  • May combine two claims.

    Premisecitation

    Circular RNA DYM (circDYM) levels are significantly decreased both in the peripheral blood of MDD patients and in the CUMS mouse models.

    “Circular RNA DYM (circDYM) levels were significantly decreased both in the peripheral blood of MDD patients and in the CUMS mouse models.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [49] Zhang Y, Du L, Bai Y, Han B, He C, Gong L, Huang R, Shen L, Chao J, Liu P, et al. CircDYM ameliorates depressive-like behavior by targeting miR-9 to regulate microglial activation via HSP90 ubiquitination. Mol Psychiatry. 2020;25(6):1175–90. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Increased levels of inflammatory markers often occur in patients with depression.

    “The correlation between psychiatric disorders and neuroinflammation was reported decades ago: increased levels of inflammatory markers often occurred in patients with depression, while the presence of depressive symptoms was positively associated with patients suffering from chronic inflammatory disorders [4, 5].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Haapakoski R, Ebmeier KP, Alenius H, Kivimaki M. Innate and adaptive immunity in the development of depression: an update on current knowledge and technological advances. Prog Neuropsychopharmacol Biol Psychiatry. 2016;66:63–72. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [5] Brites D, Fernandes A. Neuroinflammation and depression: microglia activation, extracellular microvesicles and microRNA dysregulation. Front Cell Neurosci. 2015;9:476. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing). May combine two claims.

    Premisecitation

    MiR-9 is expressed in the developing embryonic CNS, is highly conserved, and facilitates neurogenesis and differentiation.

    “MiR-9, which is expressed in the developing embryonic CNS, is highly conserved and facilitates neurogenesis and differentiation [45–48].”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [45] Delaloy C, Liu L, Lee JA, Su H, Shen F, Yang GY, Young WL, Ivey KN, Gao FB. MicroRNA-9 coordinates proliferation and migration of human embryonic stem cell-derived neural progenitors. Cell Stem Cell. 2010;6(4):323–35. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [46] Zhao C, Sun G, Li S, Shi Y. A feedback regulatory loop involving microRNA-9 and nuclear receptor TLX in neural stem cell fate determination. Nat Struct Mol Biol. 2009;16(4):365–71. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [47] Leucht C, Stigloher C, Wizenmann A, Klafke R, Folchert A, Bally-Cuif L. MicroRNA-9 directs late organizer activity of the midbrain-hindbrain boundary. Nat Neurosci. 2008;11(6):641–8. source
    • cites [48] Shibata M, Nakao H, Kiyonari H, Abe T, Aizawa S. MicroRNA-9 regulates neurogenesis in mouse telencephalon by targeting multiple transcription factors. J Neurosci. 2011;31(9):3407–22. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    LPS injection induces miR-9 expression in Iba-1-labeled mouse microglia.

    “LPS-injection also induced miR-9 expression in Iba-1-labeled mouse microglia [51].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [51] Akerblom M, Sachdeva R, Quintino L, Wettergren EE, Chapman KZ, Manfre G, Lindvall O, Lundberg C, Jakobsson J. Visualization and genetic modification of resident brain microglia using lentiviral vectors regulated by microRNA-9. Nat Commun. 2013;4:1770. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Anti-inflammatory drugs have therapeutic efficacy in reducing depressive symptoms.

    “On the other hand, the “inflammation hypothesis” of MDD was also supported by clinical findings that anti-inflammatory drugs have therapeutic efficacy in reducing depressive symptoms [7].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [7] Lehrer S, Rheinstein P. Nonsteroidal anti-inflammatory drugs (NSAIDs) reduce suicidal ideation and depression. Discov Med. 2019;28(154):205–12.

  • Classified as probably not a substantial claim.

    Premisecitation

    Microglia participate in the initiation and progression of the brain-derived inflammatory microenvironment.

    “Microglia participated in the initiation and progression of the brain-derived inflammatory microenvironment [8].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [8] Santos LE, Beckman D, Ferreira ST. Microglial dysfunction connects depression and Alzheimer’s disease. Brain Behav Immun. 2016;55:151–65. source

  • May combine two claims.

    Interpretationown data

    Depressed neuronal exosomes activate microglia and release large amounts of inflammatory factors, causing cumulative inflammatory damage and perturbing the neuronal cell cycle.

    “In our work, depressed neuronal exosomes activated microglia and released large amounts of inflammatory factors, causing cumulative inflammatory damage and perturbing the neuronal cell cycle.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Secretion of IL-1β, IL-6 and TNF-α with macrophage-associated proinflammatory cytokines is synchronized with M1 activation in MDD patients.

    “Secretion of IL-1β, IL-6 and TNF-α with macrophage-associated proinflammatory cytokines was synchronized with M1 activation in MDD patients [15].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [15] Felger JC, Lotrich FE. Inflammatory cytokines in depression: neurobiological mechanisms and therapeutic implications. Neuroscience. 2013;246:199–229. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    CircDYM acts as an endogenous miR-9 sponge to restrain its activity.

    “The function of CircDYM was acting as an endogenous miR-9 sponge to restrain its activity [49].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [49] Zhang Y, Du L, Bai Y, Han B, He C, Gong L, Huang R, Shen L, Chao J, Liu P, et al. CircDYM ameliorates depressive-like behavior by targeting miR-9 to regulate microglial activation via HSP90 ubiquitination. Mol Psychiatry. 2020;25(6):1175–90. source

      Not checked in the free run.

  • May combine two claims.

    Interpretationown data

    MiR-9-5p induces neuroinflammation and exacerbates depression.

    “These results indicated that miR-9-5p induced neuroinflammation and exacerbated depression.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Brain miR-9 levels in mood disorder patients are positively correlated with its levels in peripheral blood.

    “Brain miR-9 levels in mood disorder patients was positively correlated to its levels in peripheral blood [50], which is consistent with our observation in serum exosomes from MDD.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [50] He C, Bai Y, Wang Z, Fan D, Wang Q, Liu X, Zhang H, Zhang H, Zhang Z, Yao H, et al. Identification of microRNA-9 linking the effects of childhood maltreatment on depression using amygdala connectivity. Neuroimage. 2021;224:117428. source

      Checked before — its earlier verdict is held back until a new check runs.

  • May combine two claims.

    Interpretationown data

    MiR-9-5p activates JAK/STAT3 by suppressing SOCS2, thereby inducing M1 polarization.

    “In this study, we confirmed that miR-9-5p activated JAK/STAT3 by suppressing SOCS2, thereby inducing M1 polarization.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationreasoning

    Activated microglia-induced inflammatory factors are delivered to the CNS, stimulating neuronal cells and developing depressive symptoms.

    “Our findings suggested that activated microglia-induced inflammatory factors were delivered to the CNS, stimulating neuronal cells and developing depressive symptoms.”
    Quote in the paperSubstantial claim

  • Classified as probably not a substantial claim. May combine two claims.

    Premisecitation

    Microglia are classified into M1 (classically activated) cells that secrete proinflammatory cytokines and M2 (alternatively activated) cells that promote tissue regeneration and repair.

    “Microglia are classified into two subtypes: classically activated (M1) cells, which secrete proinflammatory cytokines, and alternatively activated (M2) cells, which promote tissue regeneration and repair [9–12].”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [9] Lan X, Han X, Li Q, Yang QW, Wang J. Modulators of microglial activation and polarization after intracerebral haemorrhage. Nat Rev Neurol. 2017;13(7):420–33. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [10] Zeng F, Wu Y, Li X, Ge X, Guo Q, Lou X, Cao Z, Hu B, Long NJ, Mao Y, et al. Custom-made ceria nanoparticles show a neuroprotective effect by modulating phenotypic polarization of the microglia. Angew Chem Int Ed Engl. 2018;57(20):5808–12. source
    • cites [11] Song WM, Colonna M. The identity and function of microglia in neurodegeneration. Nat Immunol. 2018;19(10):1048–58. source
    • cites [12] Italiani P, Boraschi D. From monocytes to M1/M2 macrophages: phenotypical vs. functional differentiation. Front Immunol. 2014;5:514. source

      Not checked in the free run.

  • Classified as probably not a substantial claim.

    Premisecitation

    Activation and polarization of microglia are regulated by the brain-derived microenvironment.

    “Based on the fact that the activation and polarization of microglia are regulated by the brain-derived microenvironment [19], we first isolated exosomes from MDD patients, and examined their function on microglia polarization.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [19] Dey A, Hankey Giblin PA. Insights into macrophage heterogeneity and cytokine-induced neuroinflammation in major depressive disorder. Pharmaceuticals. 2018. https://doi.org/10.3390/ph11030064. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Premisecitation

    Neuroinflammation and inflammation are tightly associated with depression deterioration.

    “A series of longitudinal experiments in animals and humans have elaborated that neuroinflammation and inflammation, which are relevant biological factors interacting with neurophysiological mechanisms and external stimuli, are tightly associated to depression deteriorate [6].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [6] Ignacio ZM, da Silva RS, Plissari ME, Quevedo J, Reus GZ. Physical exercise and neuroinflammation in major depressive disorder. Mol Neurobiol. 2019;56(12):8323–35. source

  • May combine two claims.

    Interpretationown data

    Raised miR-9-5p contributes to microglial M1 polarization and neuroinflammation in vivo and in vitro.

    “To sum up, raised miR-9-5p contributed to microglial M1 polarization and neuroinflammation in vivo and in vitro.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    Exosomes from a MDD cellular model promote M1 polarization and exacerbate depressive-like behaviors and neuroinflammation.

    “These results confirmed that exosomes from a MDD cellular model promoted M1 polarization and exacerbated depressive-like behaviors and neuroinflammation.”
    Quote in the paperSubstantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    MDD severity was assessed using the Hamilton Depression Rating Scale (HAMD) and Montgomery Asperger's Depression Rating Scale (MADRS).

    “The Hamilton Depression Rating Scale (HAMD) and Montgomery Asperger's Depression Rating Scale (MADRS) were used to assess the severity of MDD.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    All animal experiment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Medical School of Jiangsu University.

    “All animal experiment protocols were approved by the Institutional Animal Care and Use Committee (IACUC) of the Medical School of Jiangsu University, according to the laboratory guidelines for the ethical review of animal welfare.”
    Quote in the paperNot a substantial claim

  • May combine two claims.

    Premisecitation

    Anti-miR-9 microinjection inactivates microglia in the CNS upon LPS stimulation, resulting in alleviated neuroinflammation in mice.

    “In addition, anti-miR-9 microinjection inactivated microglia in the CNS upon LPS stimulation, resulting in alleviated neuroinflammation in mice [52].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [52] Yao H, Ma R, Yang L, Hu G, Chen X, Duan M, Kook Y, Niu F, Liao K, Fu M, et al. MiR-9 promotes microglial activation by targeting MCPIP1. Nat Commun. 2014;5:4386. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Premisecitation

    The pathophysiology of MDD, as an inflammatory CNS disease, can be better understood by PET/CT imaging.

    “The pathophysiology of MDD, as an inflammatory CNS disease, can be better understood by PET/CT imaging [57].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [57] Wang YL, Han QQ, Gong WQ, Pan DH, Wang LZ, Hu W, Yang M, Li B, Yu J, Liu Q. Microglial activation mediates chronic mild stress-induced depressive- and anxiety-like behavior in adult rats. J Neuroinflammation. 2018;15(1):21. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    STAT3 plays a significant role in controlling the inflammatory response, innate immunity and adaptive immunity.

    “The JAK-STAT pathway is a common signaling cascade in which STAT3 plays a significant role in controlling the inflammatory response, innate immunity and adaptive immunity [55, 56].”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [55] Morris R, Kershaw NJ, Babon JJ. The molecular details of cytokine signaling via the JAK/STAT pathway. Protein Sci. 2018;27(12):1984–2009. source

      Checked before — its earlier verdict is held back until a new check runs.

    • cites [56] Sha C, Jia G, Jingjing Z, Yapeng H, Zhi L, Guanghui X. miR-486 is involved in the pathogenesis of acute myeloid leukemia by regulating JAK-STAT signaling. Naunyn Schmiedebergs Arch Pharmacol. 2021;394(1):177–87. source

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    The study was approved by the Ethics Committee of the Fifth People's Hospital of Zhenjiang, China.

    “The study was approved by the Ethics Committee of the Fifth People's Hospital of Zhenjiang, China, and written informed consent was obtained from all participants or their legally authorized representatives.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing), S4 (based).

    Interpretationreasoning

    Neuronal damage may have also induced microglial inflammation.

    “Second, we identified that polarized microglia caused neuronal damage and that neuronal damage may have also induced microglial inflammation.”
    Quote in the paperNot a substantial claim
    • Rests on an assumption: The CUMS mouse model is a valid model of MDD.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    SOCS negatively regulates JAK-STAT signaling.

    “SOCS is characterized by possessing a central Src homology 2 (SH2) structural domain and a carbon-terminal SOCS box that negatively regulates JAK-STAT signaling [54].”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [54] Kazi JU, Kabir NN, Flores-Morales A, Ronnstrand L. SOCS proteins in regulation of receptor tyrosine kinase signaling. Cell Mol Life Sci. 2014;71(17):3297–310. source

      Checked before — its earlier verdict is held back until a new check runs.

  • Classified as probably not a substantial claim.

    Premisecitation

    [18F]DPA-714 is a biomarker of neuroinflammation in various human CNS disorders.

    “[18F]DPA-714 was extensively studied in preclinical and clinical models [58–61], as a biomarker of neuroinflammation in various human CNS disorders [58].”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [58] Hamelin L, Lagarde J, Dorothee G, Leroy C, Labit M, Comley RA, de Souza LC, Corne H, Dauphinot L, Bertoux M, et al. Early and protective microglial activation in Alzheimer’s disease: a prospective study using 18F-DPA-714 PET imaging. Brain. 2016;139(Pt 4):1252–64. source

      Not checked in the free run.

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    At least three biological replicates were performed for each experiment.

    “In our study, at least three biological replicates were performed for each experiment, with data expressed as the mean ± SD.”
    Quote in the paperNot a substantial claim

  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Study design and conductnone given

    Written informed consent was obtained from all participants or their legally authorized representatives.

    “The study was approved by the Ethics Committee of the Fifth People's Hospital of Zhenjiang, China, and written informed consent was obtained from all participants or their legally authorized representatives.”
    Quote in the paperNot a substantial claim
44 of 44

  • The paper relies on this without support.

    The role of microglia-neuron interactions in MDD is unclear.

    “Recently, more studies made rapid progress in understanding the neurobiology of astrocytes and microglia, but the role of microglia-neuron interactions in MDD is unclear.”

  • The paper relies on this without support.

    To date, there was less study using PET/CT imaging to measure neuroinflammatory changes in depression mouse models.

    “However, to date, there was less study using PET/CT imaging to measure neuroinflammatory changes in depression mouse models.”

  • The paper relies on this without support.

    PC12 cells are a valid model of depressed neurons.

    “The PC12 cell line, a commonly used rat neuronal cell line, was acquired from the Central Laboratory of Jiangsu University, Zhenjiang.”

  • The paper relies on this without support.

    The CUMS mouse model is a valid model of MDD.

    “An animal model of CUMS was chosen in our study.”

  • The paper relies on this without support.

    Exosomes derived from PC12 cells are representative of exosomes from depressed neurons.

    “In this study, we demonstrated that enhancing miRNA-9-5p levels in PC12-derived exosomes can exacerbate cognitive and behavioral impairments in MDD mice, along with neuronal cell damage and neuroinflammation.”
5 of 5

Claims other papers make about this one

Each is checked against this paper’s own text: does it say what the citing paper says it does?

  • Expression of miR-124, miR-532-5p, and miR-9-5p alters in tissues from depression patients or animal models, activating STAT3 and mediating disruption of neuronal differentiation and proliferation in depression.

    “the expression of miR-124, miR-532-5p, and miR-9-5p alters in tissues from depression patients/animal models, activating signal transducer and activator of transcription 3 (STAT3) and mediating the disruption of neuronal differentiation and proliferation in depression.259–262”

    From Epigenetic regulation in major depression and other stress-related disorders: molecular mechanisms, clinical relevance and therapeutic potential (2023) · cites this paper as [262]

    The Discussion reports higher exosomal miR-9-5p in a depression cell model, and the Results show that miR-9-5p activates STAT3 through SOCS2 suppression. The reported outcome is microglial M1 polarization, not disruption of neuronal differentiation or proliferation. This paper does not establish the sentence’s claims about miR-124 or miR-532-5p, which may be covered by the other citations in the cluster. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

Self-check

Percentages, sums and reported differences re-computed from the paper’s own numbers.

Each contradiction between two passages listed here was confirmed by an independent check.

  • Figure 3d cytokines described as intracellular and extracellular

    Minor

    The results describe the cytokine levels in Fig. 3d as intracellular, while the figure caption describes the same measurements as extracellular.

1 of 1

The citation lists are queued

They are fetched later and appear here then.

References 62 · Cited by 169

Authors
Xian, X., Cai, L.-L., Li, Y., Wang, R.-C., Xu, Y.-H., Chen, Y.-J., Xie, Y.-H., Zhu, X.-L., & Li, Y.-F.
Affiliations
Affiliated Hospital of Jiangsu University (CN)
ORCID
Li, Yang
Venue
Journal of Nanobiotechnology
Publisher
Springer Science and Business Media LLC
Published
2022-03-09
Volume
20
Issue
1
Pages
122-122
ISSN
1477-3155 (ISSN-L)
DOI
10.1186/s12951-022-01332-w
PMC
PMC8905830
OpenAlex
W4221075588
Cited by
169
References
62
Citations by year
2026: 36 · 2025: 62 · 2024: 40 · 2023: 28 · 2022: 2
FWCI
10.77
Type
journal article
Language
en
Open access
Licence
Locations
  • Journal of Nanobiotechnology (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
  • DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion, cc-by-sa)
Funders
  • National Natural Science Foundation of China (82172838, 81871343)
  • Natural Science Foundation of Jiangsu Province (BK20181226, BK20201227)
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Neuroscience › Neurology › Neuroinflammation and Neurodegeneration Mechanisms
  • Life Sciences › Neuroscience › Biological Psychiatry › Tryptophan and brain disorders
Keywords
miR-9-5p, exosomes, microglial M1 polarization, major depressive disorder, neuroinflammation, neuron-microglia communication, SOCS2, JAK/STAT3 signaling, inflammatory cytokines, chronic unpredictable mild stress, neuronal injury, therapeutic target
MeSH terms
Animals, Depression, Humans, Neurons, Microglia, MicroRNAs, Mice, Exosomes

About these checks

Registry, retraction, Cites and statistics results are computed, not judged, with one exception: a printed reference that no index lists is read once by a model before it counts as a citation error. Claims are extracted by a model and kept only when their quote appears verbatim in the text; each cited source is then checked, with a model’s help, for whether it says what the claim relies on. Those verdicts can be wrong — read the claim’s quote and the cited source before relying on one.

Claims read by deepseek-v4.1-flash under definition version 3 (extract-v3) on 2026-11-06.

Classified by jev-1.13-20260917 (gold-guideline-v0.2) on 2026-11-06.

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