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Cited by 354Extracellular vesicles in diseaseBarrier Structure and Function StudiesMicroRNA in disease regulation

Neurons secrete miR-132-containing exosomes to regulate brain vascular integrity

Open access
Xu, Bing, Zhang, Yu, Du, Xu-Fei, Li, Jia, Zi, Hua-Xing, Bu, Ji-Wen, Yan, Yong, Han, Hua +1 more·2017·Cell Research
DOI 10.1038/cr.2017.62PMC PMC5518987
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Abstract

Vascular integrity helps maintain brain microenvironment homeostasis, which is critical for the normal development and function of the central nervous system. It is known that neural cells can regulate brain vascular integrity. However, due to the high complexity of neurovascular interactions involved, understanding of the neural regulation of brain vascular integrity is still rudimentary. Using intact zebrafish larvae and cultured rodent brain cells, we find that neurons transfer miR-132, a highly conserved and neuron-enriched microRNA, via secreting exosomes to endothelial cells (ECs) to maintain brain vascular integrity. Following translocation to ECs through exosome internalization, miR-132 regulates the expression of vascular endothelial cadherin (VE-cadherin), an important adherens junction protein, by directly targeting eukaryotic elongation factor 2 kinase (eef2k). Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity. Our study indicates that miR-132 acts as an intercellular signal mediating neural regulation of the brain vascular integrity and suggests that the neuronal exosome is a novel avenue for neurovascular communication.

AI snapshot

Written from the paper’s full text.

TL;DR

Neurons secrete exosomes containing miR-132 that are internalized by brain endothelial cells, where miR-132 targets eef2k to maintain Cdh5/VE-cadherin and brain vascular integrity. Disrupting neuronal miR-132, exosome secretion, or vascular eef2k causes intracranial hemorrhage and vascular leakage in zebrafish.

Key findings

  • miR-132 knockdown or mutation in zebrafish caused intracranial hemorrhage (Ctrl MO: 3.3% ± 0.7%; miR-132 MO: 38.0% ± 2.8%; miR-132 loop MO: 37.1% ± 7.9%; P < 0.001) and vascular leakage, with reduced Cdh5 and β-catenin but not Claudin-5, Occludin, ZO-1, N-cadherin, or α-catenin.
  • Neuron-specific miR-132 sponge caused hemorrhage, DAPI leakage, and reduced Cdh5; neuronal miR-132 manipulation coordinately changed endothelial miR-132, and EC-specific miR-132 sponge also caused hemorrhage and reduced Cdh5.
  • Neuronal exosomes (average diameter 112 ± 60 nm) contained miR-132, were internalized by b.End3 endothelial cells, increased endothelial miR-132 and Cdh5, and nSMase2 inhibition/knockdown caused hemorrhage with decreased endothelial miR-132.
  • eef2k was a direct miR-132 target; miR-132 repressed eef2k 3′UTR luciferase activity, eEF2K inhibitor NH125 alleviated hemorrhage in miR-132 morphants, and EC-specific Eef2k overexpression caused hemorrhage, DAPI leakage, and reduced Cdh5. Microarray analysis identified 1,253 upregulated genes in miR-132 morphants, 848 predicted targets, and 21 overlapping candidates (12 annotated).

Methods

  • Zebrafish larvae and cultured rodent brain cells were used, including rat primary cortical neurons and mouse brain microvascular endothelial b.End3 cells; manipulations included morpholino knockdown, CRISPR/Cas9 mutation, transient plasmid expression, and drug treatments.
  • Exosomes were purified and characterized by TEM, nanoparticle tracking, and Western blotting; uptake, flow cytometry, real-time PCR, luciferase reporter assays, in vivo confocal imaging, TEM, DAPI/dextran leakage, and fluorescent bead leakage were used.
  • Statistics used unpaired two-tailed Student’s t-test, one-way ANOVA with Bonferroni’s or Tukey’s multiple comparison test, and Page’s trend test, with P < 0.05 considered significant.

Limitations

  • Stated: HuC promoter-driven transient expression labeled less than 5% of neurons, so effects of neuronal manipulations on endothelial miR-132 are underestimated; the paper also notes no direct evidence for astrocytes in zebrafish larvae and that roles of miR-132 in pericytes/glial cells and other exosomal factors remain to be examined.
  • Evident: the study relies heavily on zebrafish and cultured rodent cells, with no mammalian in vivo validation; only about 40% of miR-132 morphants showed severe intracranial hemorrhage, although non-hemorrhagic morphants also had DAPI leakage, and miR-132 levels were comparable between hemorrhagic and non-hemorrhagic morphants.

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

Integrity signals

Registry

Crossref · 10.1038/cr.2017.62

Cites

Statistics

Nothing to recompute.

Claims the paper makes

0 of 62 cited sources checked · 0 supported · 0 flagged

  • Findingown data

    Exosomes purified from neurons expressing CD63-GFP appeared in b.End3 cells.

    “This lead to the appearance of GFP-expressing exosomes in b.End3 cells (Figure 4F).”
    Quote in the paperSubstantial claim
  • Findingown data

    miR-132 level in isolated neuronal exosomes positively correlated with that in neurons.

    “By overexpressing or downregulating miR-132 in cultured cortical neurons, we found that the miR-132 level in isolated neuronal exosomes positively correlated with that in neurons (Figure 4J and 4K; P < 0.01).”
    Quote in the paperSubstantial claim
  • Findingown data

    Purified pellets from conditioned medium of cultured primary rat cortical neurons contained small membrane vesicles with an average diameter of 112 ± 60 nm.

    “TEM and nanoparticle tracing analysis of the purified pellets identified small membrane vesicles with an average diameter of 112 ± 60 nm (mean ± SD; Figure 4A and 4B), suggesting the identity of exosomes.”
    Quote in the paperSubstantial claim
  • Findingown data

    Red fluorescent beads leaked from brain vessels of miR-132 morphants.

    “red fluorescent beads were readily observed to leak out from the brain vessels of miR-132 morphants (Supplementary information, Figure S1I and S1J).”
    Quote in the paperSubstantial claim
  • Findingcitation

    miR-132-S efficiently inhibited miR-132 function by chelating miR-132.

    “The sponge contained ten repeats of the miR-132 antisense sequence and efficiently inhibited miR-132 function by chelating miR-132 (Supplementary information, Figure S6; P < 0.001)31.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [31] Ebert MS, Neilson JR, Sharp PA . MicroRNA sponges: competitive inhibitors of small RNAs in mammalian cells. Nat Methods 2007; 4:721–726. source
  • May combine two claims. 30
  • Classified as probably not a substantial claim. 27

  • May combine two claims.

    Findingown data

    Cdh5 morphants displayed increased intracranial hemorrhage and DAPI leakage.

    “Cdh5 morphants also displayed increased intracranial hemorrhage (see also ref.27) and DAPI leakage (Figure 2D, 2E and Supplementary information, Figure S5; P < 0.001).”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites ref.27

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    Neuronal exosomes repressed luciferase activity of a miR-132 sensor in ECs, indicating transferred neuronal exosomal miR-132 is functional in ECs.

    “Using a luciferase assay, we found that neuronal exosomes repressed the luciferase activity of a miR-132 sensor in ECs, indicating that the transferred neuronal exosomal miR-132 is functional in ECs (Supplementary information, Figure S8B; P < 0.05).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-132-S expression in ECs resulted in severe intracranial hemorrhage and reduction of Cdh5 expression.

    “miR-132-S expression in ECs resulted in severe intracranial hemorrhage (Figure 3H; P < 0.01) and reduction of Cdh5 expression (Figure 3I; P < 0.01).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    EC-EC contacts in control larvae showed numerous and extensive junctions whereas junctions between brain ECs in hemorrhagic miR-132 morphants were discontinuous, junction density was reduced, and the cleft between ECs was increased.

    “EC-EC contacts in control larvae showed numerous and extensive junctions (Figure 1G and 1H) whereas junctions between brain ECs in hemorrhagic miR-132 morphants was discontinuous, junction density was reduced, and the cleft between ECs was increased (Figure 1G and 1I).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    miR-132 was detected in isolated neuronal exosomes and its level increased with increased amount of exosomes.

    “miR-132 was detected in the isolated neuronal exosome and its level was increased with the increased amount of exosomes (Figure 4D; P < 0.001).”
    Quote in the paperSubstantial claim

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

    Findingcitation

    MiR-132 is evolutionarily conserved among animal species and highly expressed in the brain of zebrafish larvae.

    “MiR-132 is evolutionarily conserved among animal species and highly expressed in the brain of zebrafish larvae (Supplementary information, Figure S1A and S1B; see also ref.22).”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites ref.22

      Couldn’t be matched to a paper in Crossref, so it can’t be checked.

  • May combine two claims.

    Findingown data

    miR-132 morphants showed a significant increase of DAPI-positive parenchymal cell nuclei and dextran leakage.

    “A significant increase of DAPI-positive parenchymal cell nuclei as well as dextran leakage was observed in miR-132 morphants (Figure 1D, 1E and Supplementary information, Figure S1H; P < 0.001).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    The increase in miR-132 in b.End3 cells was not affected by co-treatment with DRB, suggesting neuronal exosomes can transfer mature miR-132 to ECs.

    “This increase was not affected by co-treatment with the RNA polymerase II inhibitor DRB (5, 6-dichloro-1-b-D-ribofuranosylbenzimidazole; Figure 4H; P = 0.9), suggesting neuronal exosomes can transfer mature miR-132 to ECs.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of β-catenin was reduced in miR-132 morphants, whereas no significant change for α-catenin was observed.

    “The expression of β-catenin was reduced in miR-132 morphants (P < 0.05), whereas we observed no significant change for α-catenin (Figure 2A and 2B).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Vesicle preparations showed intensive expression of exosomal markers Alix and CD63 but not the endoplasmic reticulum marker Calnexin.

    “vesicle preparations showed intensive expression of the exosomal markers Alix and CD63 but not the endoplasmic reticulum marker Calnexin (Figure 4C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity.

    “Disruption of neuronal miR-132 expression or exosome secretion, or overexpression of vascular eef2k impairs VE-cadherin expression and brain vascular integrity.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Larvae with neurons expressing miR-132-S exhibited severe intracranial hemorrhage, increased DAPI leakage, and reduced Cdh5 expression.

    “larvae with neurons expressing miR-132-S exhibited severe intracranial hemorrhage (Figure 3B and 3C; P < 0.01) and increased DAPI leakage (Figure 3D and 3E; P < 0.05) and reduced Cdh5 expression (Figure 3F; P < 0.05).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Microarray analysis identified 1,253 upregulated genes in miR-132 morphants and Microcosm database predicted 848 target genes, with 21 candidate genes (12 annotated) overlapping.

    “we identified 1 253 upregulated genes in miR-132 morphants by performing microarray analysis (accession number for the microarray data: GSE85291 in NCBI) and predicted 848 target genes by using the Microcosm database. Among these predicted targets, 21 candidate genes (12 annotated) overlapped (Supplementary Information, Figure S9A).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    EC-specific overexpression of Eef2k caused intracranial hemorrhage, DAPI leakage, and reduction of Cdh5 expression.

    “EC-specific overexpression of Eef2k (“Flk1:tdT-P2A-Eef2k”) also caused intracranial hemorrhage (Figure 6K; P < 0.01), DAPI leakage (Figure 6L and Supplementary information, Figure S10D; P < 0.001), and reduction of Cdh5 expression (Figure 6M; P < 0.01).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Treatment with nSMase2 inhibitor spiroepoxide or nSMase2 knockdown with MO led to severe intracranial hemorrhage accompanied with decreased expression of miR-132 in ECs of zebrafish larvae.

    “Treatment with the nSMase2 inhibitor spiroepoxide (10 μM) or nSMase2 knockdown with MO led to severe intracranial hemorrhage accompanied with decreased expression of miR-132 in ECs of zebrafish larvae (Figure 5B, 5C and Supplementary information, Figure S8D-S8F; P < 0.05).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: The nSMase2 inhibitor spiroepoxide specifically inhibits exosome release in zebrafish larvae without off-target effects that could cause intracranial hemorrhage.

  • May combine two claims.

    Findingown data

    Treatment with eEF2K non-specific activator rapamycin led to intracranial hemorrhage in wildtype larvae and further aggravated the hemorrhagic defect of miR-132 morphants.

    “treatment with the eEF2K non-specific activator rapamycin (2 μM) led to intracranial hemorrhage in wildtype larvae (Figure 6F; P < 0.05) and further aggravated the hemorrhagic defect of miR-132 morphants (Figure 6F; P < 0.001).”
    Quote in the paperSubstantial claim
    • Rests on an assumption: Rapamycin's effects on intracranial hemorrhage in zebrafish larvae are due to activation of eEF2K rather than other targets.

  • May combine two claims.

    Findingown data

    Knockdown of eef2k alleviated intracranial hemorrhage, DAPI leakage, and reduction of Cdh5 expression in miR-132 morphants.

    “knockdown of eef2k alleviated the intracranial hemorrhage (Figure 6G; P < 0.01), DAPI leakage (Figure 6H and Supplementary information, Figure S10C; P < 0.05) and reduction of Cdh5 expression (Figure 6I; P < 0.05) in miR-132 morphants.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Both in vitro and in vivo reporter assays showed that eef2k 3′UTR was sufficient to confer miR-132 regulation, and the second binding site was indispensable.

    “Both in vitro and in vivo reporter assays showed that eef2k 3′ UTR was sufficient to confer miR-132 regulation (Figure 6B and Supplementary information, Figure S10A and S10B; P < 0.001), and the second binding site was indispensable (Figure 6A and 6C).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Findingown data

    Expression of eEF2K in zebrafish larvae was decreased or increased by overexpression or knockdown of miR-132, respectively.

    “the expression of eEF2K in zebrafish larvae was decreased or increased by the overexpression or knockdown of miR-132, respectively (Figure 6D and 6E; P < 0.05).”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    miR-132 regulates brain vascular integrity through affecting adherens junction proteins rather than transcytosis or pericytes.

    “Collectively, these results suggest that miR-132 regulates the brain vascular integrity through affecting adherens junction proteins rather than transcytosis or pericytes.”
    Quote in the paperSubstantial claim

  • May combine two claims.

    Interpretationown data

    miR-132 in ECs can regulate expression of Cdh5 and the integrity of the brain vasculature.

    “This indicates that miR-132 in ECs can regulate expression of Cdh5 and the integrity of the brain vasculature.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Both pericytes and astrocytes can release Angiopoietin-1 that binds to endothelial Tie-2 receptors to improve brain vascular integrity.

    “both pericytes and astrocytes can release Angiopoietin-1 that binds to endothelial Tie-2 receptors to improve brain vascular integrity45,46.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [45] Prat A, Biernacki K, Wosik K, Antel JP . Glial cell influence on the human blood-brain barrier. Glia 2001; 36:145–155. source
    • cites [46] Saharinen P, Eklund L, Miettinen J, et al. Angiopoietins assemble distinct Tie2 signalling complexes in endothelial cell-cell and cell-matrix contacts. Nat Cell Biol 2008; 10:527–537. source

  • May combine two claims.

    Interpretationown data

    Knockdown of miR-132 did not affect pericyte coverage, suggesting no direct involvement of pericytes in the regulation of brain vascular integrity by miR-132.

    “Here we found that knockdown of miR-132 did not affect the pericyte coverage, suggesting no direct involvement of pericytes in the regulation of brain vascular integrity by miR-132.”
    Quote in the paperSubstantial claim

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

    Premisecitation

    Exosomes released by Schwann cells and oligodendrocytes can be taken up by neuronal axons to increase neurite growth, enhance axonal regeneration and improve neuronal survival.

    “Exosomes released by Schwann cells and oligodendrocytes can be taken up by neuronal axons to increase neurite growth, enhance axonal regeneration and improve neuronal survival49,50.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [49] Fruhbeis C, Frohlich D, Kuo WP, et al. Neurotransmitter-triggered transfer of exosomes mediates oligodendrocyte-neuron communication. PLoS Biol 2013; 11:e1001604. source
    • cites [50] Lopez-Verrilli MA, Picou F, Court FA . Schwann cell-derived exosomes enhance axonal regeneration in the peripheral nervous system. Glia 2013; 61:1795–1806. source

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

    Premisecitation

    Wnt and Sonic Hedgehog pathways were found to be implicated in neural regulation of brain vasculature.

    “several signaling pathways, in particular the Wnt and Sonic Hedgehog (Shh) pathways, were found to be implicated in this neural regulation4,5.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [4] Obermeier B, Daneman R, Ransohoff RM . Development, maintenance and disruption of the blood-brain barrier. Nat Med 2013; 19:1584–1596. source

      Not checked in the free run.

    • cites [5] Zhao Z, Nelson AR, Betsholtz C, Zlokovic BV . Establishment and dysfunction of the blood-brain barrier. Cell 2015; 163:1064–1078. source

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

    Premisecitation

    During embryogenesis, neural progenitors secrete Wnt ligands, which activate canonical Wnt signaling in ECs and contribute to maturation of brain vasculature.

    “During embryogenesis, neural progenitors secrete Wnt ligands, which activate canonical Wnt signaling in ECs and contribute to the maturation of brain vasculature6,7,8.”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [6] Stenman JM, Rajagopal J, Carroll TJ, Ishibashi M, McMahon J, McMahon AP . Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science 2008; 322:1247–1250. source
    • cites [7] Daneman R, Agalliu D, Zhou L, Kuhnert F, Kuo C, Barres BA . Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci USA 2009; 106:641–646. source
    • cites [8] Liebner S, Corada M, Bangsow T, et al. Wnt/beta-catenin signaling controls development of the blood-brain barrier. J Cell Biol 2008; 183:409–417. source

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

    Premisecitation

    During postnatal development, astrocytes can release Shh to promote brain vascular integrity by acting on EC Patched-1 receptors.

    “During postnatal development, astrocytes can release Shh to promote the brain vascular integrity by acting on EC Patched-1 receptors9.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Alvarez Jl, Dodelet-Devillers A, Kebir H, et al. The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 2011; 334:1727–1731. source

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

    Premisecitation

    Previous reports showed miR-132 was also expressed in pericytes and glial cells.

    “However, previous reports showed that miR-132 was also expressed in pericytes and glial cells43,44.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [43] Katare R, Riu F, Mitchell K, et al. Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132. Circ Res 2011; 109:894–906. source
    • cites [44] Kong H, Yin F, He F, et al. The Effect of miR-132, miR-146a, and miR-155 on MRP8/TLR4-induced astrocyte-related inflammation. J Mol Neurosci 2015; 57:28–37. source

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

    Premisecitation

    Motor neuron terminals in Drosophila larvae release exosomes to promote maturation of neuromuscular junctions.

    “motor neuron terminals in Drosophila larvae release exosomes to promote the maturation of neuromuscular junctions11,18.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] Korkut C, Li Y, Koles K, et al. Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron 2013; 77:1039–1046. source
    • cites [18] Korkut C, Ataman B, Ramachandran P, et al. Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell 2009; 139:393–404. source

  • Probably not a substantial claim: may not pass S4 (based).

    Premisenone given

    The development of brain vasculature, including its integrity, is tightly regulated by surrounding neural tissues.

    “It is generally believed that the development of brain vasculature, including its integrity, is tightly regulated by surrounding neural tissues.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    eef2k encodes a calcium/calmodulin-dependent protein kinase, which phosphorylates eukaryotic elongation factor 2 (eEF2) to inhibit translational elongation.

    “The eef2k encodes a calcium/calmodulin-dependent protein kinase, which phosphorylates eukaryotic elongation factor 2 (eEF2) to inhibit translational elongation34,35.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [34] Pigott CR, Mikolajek H, Moore CE, et al. Insights into the regulation of eukaryotic elongation factor 2 kinase and the interplay between its domains. Biochem J 2012; 442:105–118. source
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  • Probably not a substantial claim: may not pass S3 (load-bearing).

    Premisecitation

    Neurons can transfer miR-124a in exosomes to astrocytes to regulate expression of astroglial glutamate transporters.

    “neurons can transfer miR-124a in exosomes to astrocytes to regulate the expression of astroglial glutamate transporters, which is important for the maintenance of extracellular glutamate levels47.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [47] Morel L, Regan M, Higashimori H, et al. Neuronal exosomal miRNA-dependent translational regulation of astroglial glutamate transporter GLT1. J Biol Chem 2013; 288:7105–7116. source

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

    Premisecitation

    The exosome is a suitable vehicle for drug delivery because of its high biocompatibility and low immunogenicity.

    “The exosome is a suitable vehicle for drug delivery because of its high biocompatibility and low immunogenicity55.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [55] EL Andaloussi S, Mager I, Breakefield XO, Wood MJ . Extracellular vesicles: biology and emerging therapeutic opportunities. Nat Rev Drug Discov 2013; 12:347–357. source

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

    Premisecitation

    Exosome-mediated intercellular communication plays important roles in both physiological and pathological processes in neural systems.

    “Increasing evidence indicates that exosome-mediated intercellular communication plays important roles in both physiological and pathological processes in neural systems16,17.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [16] Rajendran L, Bali J, Barr MM, et al. Emerging roles of extracellular vesicles in the nervous system. J Neurosci 2014; 34:15482–15489. source
    • cites [17] Budnik V, Ruiz-Canada C, Wendler F . Extracellular vesicles round off communication in the nervous system. Nat Rev Neurosci 2016; 17:160–172. source

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

    Premisecitation

    At postnatal stages, astrocyte-secreted Shh promotes vascular integrity of the brain by activating Hedgehog signaling in ECs through the receptor Patched-1.

    “At postnatal stages, astrocyte-secreted Shh further promotes vascular integrity of the brain by activating Hedgehog signaling in ECs through the receptor Patched-19.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [9] Alvarez Jl, Dodelet-Devillers A, Kebir H, et al. The Hedgehog pathway promotes blood-brain barrier integrity and CNS immune quiescence. Science 2011; 334:1727–1731. source

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

    Premisecitation

    Exosomes are small membrane vesicles that originate from inward budding of endosomal membranes.

    “Exosomes are small membrane vesicles that originate from the inward budding of endosomal membranes10.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [10] Colombo M, Raposo G, Thery C . Biogenesis, secretion, and intercellular interactions of exosomes and other extracellular vesicles. Annu Rev Cell Dev Biol 2014; 30:255–289. source

  • May combine two claims.

    Premisecitation

    Impairment of brain vascular integrity will lead to ingress of plasma components into the brain and compromise synaptic and neuronal functions.

    “As the impairment of brain vascular integrity will lead to the ingress of plasma components into the brain and compromise synaptic and neuronal functions1,2”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [1] Abbott NJ, Ronnback L, Hansson E . Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci 2006; 7:41–53. source
    • cites [2] Zlokovic BV . The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 2008; 57:178–201. source

      Queued — the background check reaches it, usually within a day.

  • Classified as probably not a substantial claim.

    Premisecitation

    Exosome-mediated material transfer has been recognized as an important route for intercellular communication.

    “Exosome-mediated material transfer has been recognized as an important route for intercellular communication15.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [15] Kourembanas S . Exosomes: vehicles of intercellular signaling, biomarkers, and vectors of cell therapy. Annu Rev Physiol 2015; 77:13–27. source

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

    Premisecitation

    The blood-brain barrier restricts material exchange between the brain and blood vessels to maintain homeostasis of the brain microenvironment.

    “The BBB restricts material exchange between the brain and blood vessels to maintain the homeostasis of the brain microenvironment, which is crucial for the normal development and function of the central nervous system (CNS)1,2,3,4.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [1] Abbott NJ, Ronnback L, Hansson E . Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci 2006; 7:41–53. source
    • cites [2] Zlokovic BV . The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 2008; 57:178–201. source

      Queued — the background check reaches it, usually within a day.

    • cites [3] Tam SJ, Watts RJ . Connecting vascular and nervous system development: angiogenesis and the blood-brain barrier. Annu Rev Neurosci 2010; 33:379–408. source
    • cites [4] Obermeier B, Daneman R, Ransohoff RM . Development, maintenance and disruption of the blood-brain barrier. Nat Med 2013; 19:1584–1596. source

      Queued — the background check reaches it, usually within a day.

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

    Premisecitation

    Brain vascular endothelial cells connect with each other through junction proteins and are enveloped by surrounding pericytes and astrocytes to form the blood-brain barrier.

    “Brain vascular endothelial cells (ECs) connect with each other through junction proteins and are enveloped by surrounding pericytes and astrocytes to form the blood-brain barrier (BBB)1,2,3,4.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [1] Abbott NJ, Ronnback L, Hansson E . Astrocyte-endothelial interactions at the blood-brain barrier. Nat Rev Neurosci 2006; 7:41–53. source
    • cites [2] Zlokovic BV . The blood-brain barrier in health and chronic neurodegenerative disorders. Neuron 2008; 57:178–201. source

      Queued — the background check reaches it, usually within a day.

    • cites [3] Tam SJ, Watts RJ . Connecting vascular and nervous system development: angiogenesis and the blood-brain barrier. Annu Rev Neurosci 2010; 33:379–408. source
    • cites [4] Obermeier B, Daneman R, Ransohoff RM . Development, maintenance and disruption of the blood-brain barrier. Nat Med 2013; 19:1584–1596. source

      Queued — the background check reaches it, usually within a day.

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

    Study design and conductnone given

    All clones were verified by sequencing analysis.

    “All clones were verified by sequencing analysis.”
    Quote in the paperNot a substantial claim

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

    Premisecitation

    During embryogenesis, neural progenitor cells can secrete Wnt ligands to regulate development of brain vasculature and BBB through activation of canonical Wnt signaling in ECs.

    “During embryogenesis, neural progenitor cells can secrete Wnt ligands to regulate the development of brain vasculature and BBB through activation of canonical Wnt signaling in ECs6,7.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [6] Stenman JM, Rajagopal J, Carroll TJ, Ishibashi M, McMahon J, McMahon AP . Canonical Wnt signaling regulates organ-specific assembly and differentiation of CNS vasculature. Science 2008; 322:1247–1250. source
    • cites [7] Daneman R, Agalliu D, Zhou L, Kuhnert F, Kuo C, Barres BA . Wnt/beta-catenin signaling is required for CNS, but not non-CNS, angiogenesis. Proc Natl Acad Sci USA 2009; 106:641–646. source

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

    Premisecitation

    In vivo studies in Drosophila larvae showed motor neurons can secrete Wingless-associated and Synaptotagmin 4-containing exosomes to promote synaptic development and plasticity.

    “In vivo studies in Drosophila larvae showed that motor neurons can secrete Wingless-associated and Synaptotagmin 4-containing exosomes to promote the synaptic development and plasticity11,18.”
    Quote in the paperNot a substantial claim0 of 2 sources checked
    • cites [11] Korkut C, Li Y, Koles K, et al. Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron 2013; 77:1039–1046. source
    • cites [18] Korkut C, Ataman B, Ramachandran P, et al. Trans-synaptic transmission of vesicular Wnt signals through Evi/Wntless. Cell 2009; 139:393–404. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Neurons can secrete exosomes to regulate neural development.

    “Recent studies show that neurons can secrete exosomes to regulate neural development16,17.”
    Quote in the paperSubstantial claim0 of 2 sources checked
    • cites [16] Rajendran L, Bali J, Barr MM, et al. Emerging roles of extracellular vesicles in the nervous system. J Neurosci 2014; 34:15482–15489. source
    • cites [17] Budnik V, Ruiz-Canada C, Wendler F . Extracellular vesicles round off communication in the nervous system. Nat Rev Neurosci 2016; 17:160–172. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Cultured embryonic rat cortical neurons can release exosomes.

    “During neural development, cultured embryonic rat cortical neurons can release exosomes54.”
    Quote in the paperSubstantial claim0 of 1 sources checked
    • cites [54] Faure J, Lachenal G, Court M, et al. Exosomes are released by cultured cortical neurones. Mol Cell Neurosci 2006; 31:642–648. source

  • May combine two claims.

    Premisecitation

    Formulated nanoparticle-mediated miR-132 delivery into ECs can improve EC survival in vitro and EC transplantation efficiency in mice.

    “formulated nanoparticle-mediated miR-132 delivery into ECs can improve EC survival in vitro and EC transplantation efficiency in mice56.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [56] Devalliere J, Chang WG, Andrejecsk JW, et al. Sustained delivery of proangiogenic microRNA-132 by nanoparticle transfection improves endothelial cell transplantation. FASEB J 2014; 28:908–922. source

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

    Premisecitation

    miR-132 expression is positively regulated by sensory experience and neural activity.

    “As the expression of miR-132 is positively regulated by sensory experience and neural activity38,39,40”
    Quote in the paperNot a substantial claim0 of 3 sources checked
    • cites [38] Wayman GA, Davare M, Ando H, et al. An activity-regulated microRNA controls dendritic plasticity by down-regulating p250GAP. Proc Natl Acad Sci USA 2008; 105:9093–9098. source

      Not checked in the free run.

    • cites [39] Mellios N, Sugihara H, Castro J, et al. miR-132, an experience-dependent microRNA, is essential for visual cortex plasticity. Nat Neurosci 2011; 14:1240–1242. source
    • cites [40] Tognini P, Putignano E, Coatti A, Pizzorusso T . Experience-dependent expression of miR-132 regulates ocular dominance plasticity. Nat Neurosci 2011; 14:1237–1239. source

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

    Premisecitation

    Under pathological conditions, exosomes increase propagation of neurotoxic proteins in neurodegenerative diseases and aggravate brain tumor metastasis and progression.

    “Under pathological conditions, exosomes have been found to increase the propagation of neurotoxic proteins in neurodegenerative diseases51,52,53, and aggravate brain tumor metastasis and progression14.”
    Quote in the paperNot a substantial claim0 of 4 sources checked
    • cites [51] Aguzzi A, Rajendran L . The transcellular spread of cytosolic amyloids, prions, and prionoids. Neuron 2009; 64:783–790. source
    • cites [52] Emmanouilidou E, Melachroinou K, Roumeliotis T, et al. Cell-produced alpha-synuclein is secreted in a calcium-dependent manner by exosomes and impacts neuronal survival. J Neurosci 2010; 30:6838–6851. source

      Not checked in the free run.

    • cites [53] Bellingham SA, Guo BB, Coleman BM, Hill AF . Exosomes: vehicles for the transfer of toxic proteins associated with neurodegenerative diseases? Front Physiol 2012; 3:124. source
    • cites [14] Zhang L, Zhang S, Yao J, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 2015; 527:100–104. source

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

    Premisecitation

    Synapsin 1-containing exosomes secreted by astrocytes can be taken up by neurons and promote neurite outgrowth and neuronal survival.

    “Synapsin 1-containing exosomes secreted by astrocytes can be taken up by neurons and promote neurite outgrowth and neuronal survival48.”
    Quote in the paperNot a substantial claim0 of 1 sources checked
    • cites [48] Wang S, Cesca F, Loers G, et al. Synapsin I is an oligomannose-carrying glycoprotein, acts as an oligomannose-binding lectin, and promotes neurite outgrowth and neuronal survival when released via glia-derived exosomes. J Neurosci 2011; 31:7275–7290. source

  • Classified as probably not a substantial claim.

    Premisecitation

    Diverse types of cells including neurons can secrete exosomes, which transfer functional proteins, mRNAs and/or microRNAs to recipient cells.

    “Diverse types of cells including neurons can secrete exosomes, which transfer functional proteins11,12, mRNAs and/or microRNAs (miRNAs)13,14 to recipient cells.”
    Quote in the paperSubstantial claim0 of 4 sources checked
    • cites [11] Korkut C, Li Y, Koles K, et al. Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron 2013; 77:1039–1046. source
    • cites [12] Kucharzewska P, Christianson HC, Welch JE, et al. Exosomes reflect the hypoxic status of glioma cells and mediate hypoxia-dependent activation of vascular cells during tumor development. Proc Natl Acad Sci USA 2013; 110:7312–7317. source
    • cites [13] Valadi H, Ekstrom K, Bossios A, Sjostrand M, Lee JJ, Lotvall JO . Exosome-mediated transfer of mRNAs and microRNAs is a novel mechanism of genetic exchange between cells. Nat Cell Biol 2007; 9:654–659. source
    • cites [14] Zhang L, Zhang S, Yao J, et al. Microenvironment-induced PTEN loss by exosomal microRNA primes brain metastasis outgrowth. Nature 2015; 527:100–104. source
50 of 50

  • The paper relies on this without support.

    The nSMase2 inhibitor spiroepoxide specifically inhibits exosome release in zebrafish larvae without off-target effects that could cause intracranial hemorrhage.

    “Treatment with the nSMase2 inhibitor spiroepoxide (10 μM) or nSMase2 knockdown with MO led to severe intracranial hemorrhage accompanied with decreased expression of miR-132 in ECs of zebrafish larvae (Figure 5B, 5C and Supplementary information, Figure S8D-S8F; P < 0.05).”

  • The paper relies on this without support.

    The eEF2K inhibitor NH125 specifically inhibits eEF2K in zebrafish larvae without off-target effects that could alleviate hemorrhage.

    “Application of the eEF2K inhibitor NH125 (2.5 μM) significantly alleviated the hemorrhagic defect in miR-132 morphants (Figure 6F; P < 0.001).”

  • The paper relies on this without support.

    Rapamycin's effects on intracranial hemorrhage in zebrafish larvae are due to activation of eEF2K rather than other targets.

    “treatment with the eEF2K non-specific activator rapamycin (2 μM) led to intracranial hemorrhage in wildtype larvae (Figure 6F; P < 0.05) and further aggravated the hemorrhagic defect of miR-132 morphants (Figure 6F; P < 0.001).”

  • The paper relies on this without support.

    The mouse brain microvascular EC line b.End3 is a valid model of brain endothelial cells for exosome uptake and miR-132 function.

    “we first labeled purified exosomes with the green fluorescent lipid dye PKH67 and incubated them with the mouse brain microvascular EC line (b.End3).”

  • The paper relies on this without support.

    Cultured primary rat cortical neurons are a valid model for neuronal exosome secretion and miR-132 transfer relevant to zebrafish brain vascular integrity.

    “we isolated exosomes from the conditioned medium of cultured primary rat cortical neurons (see Materials and Methods).”
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?

  • Recent findings of a long-range crosstalk between periphery and CNS via miRNA-containing exosomes position the BBB in the center of this communication under physiological and pathophysiological conditions.

    “Recent findings of a long-range crosstalk between periphery and CNS via miRNA-containing exosomes position the BBB in the center of this communication (685) under physiological (635, 660) and pathophysiological (378, 493, 498, 587) conditions.”

    From Blood-Brain Barrier: From Physiology to Disease and Back (2019) · cites this paper as [660]

    The Results section (“Exosomes transfer neuronal miR-132 from neurons to ECs”; page unavailable) shows neuronal miR-132-containing exosomes entering brain endothelial cells and affecting brain vascular integrity. That supports exosome-mediated communication involving the BBB, but the study concerns local neuron-to-endothelial signaling, not long-range communication between the periphery and CNS. The physiological and pathophysiological conditions are attached to other citations in the citing sentence. (LLM, conf=0.86)

    Experimental — accuracy not yet measured

  • Embryonic neurons in zebrafish larvae produce miR-132 in exosomes, which regulates BBB integrity by controlling expression of endothelial VE-cadherin, an adherens junction (AJ) protein at the BBB.

    “For instance, embryonic neurons in zebrafish larvae produce miR-132 in exosomes, which regulates BBB integrity by controlling expression of endothelial VE-cadherin, an adherens junction (AJ) protein at the BBB (660).”

    From Blood-Brain Barrier: From Physiology to Disease and Back (2019) · cites this paper as [660]

  • Exosomes can transport across the blood-brain barrier and play a key role in cellular communication, neurogenesis, and synaptic plasticity in the central nervous system.

    “The capacity for transport of exosomes across the blood-brain barrier enables them to play a key role in cellular communication, neurogenesis, and synaptic plasticity in the central nervous system.13,14”

    From Microglia secrete miR-146a-5p-containing exosomes to regulate neurogenesis in depression (2022) · cites this paper as [14]

The citation lists are queued

They are fetched later and appear here then.

References 63 · Cited by 354

Authors
Xu, B., Zhang, Y., Du, X.-F., Li, J., Zi, H.-X., Bu, J.-W., Yan, Y., Han, H., & Du, J.-L.
Affiliations
Chinese Academy of Sciences (CN)
ORCID
Venue
Cell Research
Publisher
Springer Science and Business Media LLC
Published
2017-04-21
Volume
27
Issue
7
Pages
882-897
ISSN
  • 1001-0602 (ISSN-L)
  • 1748-7838
DOI
10.1038/cr.2017.62
PMC
PMC5518987
OpenAlex
W2606039495
Cited by
354
References
63
Citations by year
2026: 22 · 2025: 37 · 2024: 38 · 2023: 41 · 2022: 58 · 2021: 49 · 2020: 41 · 2019: 36 · 2018: 24 · 2017: 7
FWCI
12.07
Type
journal article
Language
en
Open access
Licence
Locations
  • Cell Research (publishedVersion, cc-by)
  • Europe PMC (PubMed Central) (publishedVersion, cc-by)
  • PubMed Central (publishedVersion, cc-by)
  • PubMed (publishedVersion)
Funders
  • National Natural Science Foundation of China (31325011)
  • Science and Technology Commission of Shanghai Municipality (14YF1406600)
  • Postdoctoral Research Foundation of China
Topics
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Extracellular vesicles in disease
  • Life Sciences › Neuroscience › Neurology › Barrier Structure and Function Studies
  • Life Sciences › Biochemistry, Genetics and Molecular Biology › Cancer Research › MicroRNA in disease regulation
Keywords
miR-132, exosomes, endothelial cells, VE-cadherin, eEF2K, microRNA transfer, adherens junctions, zebrafish larvae, intercellular communication
MeSH terms
Cadherin 5, Analysis of Variance, Animals, Brain, Humans, Larva, Neurons, Zebrafish, Antigens, CD, Cadherins, Rats, Sprague-Dawley, Intracranial Hemorrhages, MicroRNAs, Mice, Rats, Elongation Factor 2 Kinase, Exosomes, Primary Cell Culture, Human Umbilical Vein Endothelial Cells

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

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