Traumatic brain injury increases levels of miR-21 in extracellular vesicles: implications for neuroinflammation

Traumatic brain injury (TBI) is an important health concern and effective treatment strategies remain elusive. Understanding the complex multicellular response to TBI may provide new avenues for intervention. In the context of TBI, cell–cell communication is critical. One relatively unexplored form of cell–cell communication in TBI is extracellular vesicles (EVs). These membrane‐bound vesicles can carry many different types of cargo between cells. Recently, miRNA in EVs have been shown to mediate neuroinflammation and neuronal injury. To explore the role of EV‐associated miRNA in TBI, we isolated EVs from the brain of injured mice and controls, purified RNA from brain EVs, and performed miRNA sequencing. We found that the expression of miR‐212 decreased, while miR‐21, miR‐146, miR‐7a, and miR‐7b were significantly increased with injury, with miR‐21 showing the largest change between conditions. The expression of miR‐21 in the brain was primarily localized to neurons near the lesion site. Interestingly, adjacent to these miR‐21‐expressing neurons were activated microglia. The concurrent increase in miR‐21 in EVs with the elevation of miR‐21 in neurons, suggests that miR‐21 is secreted from neurons as potential EV cargo. Thus, this study reveals a new potential mechanism of cell–cell communication not previously described in TBI

Correction: MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease.

[This corrects the article DOI: 10.1371/journal.ppat.1005032.]

Traumatic brain injury increases levels of miR‐21 in extracellular vesicles: implications for neuroinflammation

Traumatic brain injury (TBI) is an important health concern and effective treatment strategies remain elusive. Understanding the complex multicellular response to TBI may provide new avenues for intervention. In the context of TBI, cell–cell communication is critical. One relatively unexplored form of cell–cell communication in TBI is extracellular vesicles (EVs). These membrane‐bound vesicles can carry many different types of cargo between cells. Recently, miRNA in EVs have been shown to mediate neuroinflammation and neuronal injury. To explore the role of EV‐associated miRNA in TBI, we isolated EVs from the brain of injured mice and controls, purified RNA from brain EVs, and performed miRNA sequencing. We found that the expression of miR‐212 decreased, while miR‐21, miR‐146, miR‐7a, and miR‐7b were significantly increased with injury, with miR‐21 showing the largest change between conditions. The expression of miR‐21 in the brain was primarily localized to neurons near the lesion site. Interestingly, adjacent to these miR‐21‐expressing neurons were activated microglia. The concurrent increase in miR‐21 in EVs with the elevation of miR‐21 in neurons, suggests that miR‐21 is secreted from neurons as potential EV cargo. Thus, this study reveals a new potential mechanism of cell–cell communication not previously described in TBI

Downregulation of an Evolutionary Young miR-1290 in an iPSC-Derived Neural Stem Cell Model of Autism Spectrum Disorder

The identification of several evolutionary young miRNAs, which arose in primates, raised several possibilities for the role of such miRNAs in human-specific disease processes. We previously have identified an evolutionary young miRNA, miR-1290, to be essential in neural stem cell proliferation and neuronal differentiation. Here, we show that miR-1290 is significantly downregulated during neuronal differentiation in reprogrammed induced pluripotent stem cell- (iPSC-) derived neurons obtained from idiopathic autism spectrum disorder (ASD) patients. Further, we identified that miR-1290 is actively released into extracellular vesicles. Supplementing ASD patient-derived neural stem cells (NSCs) with conditioned media from differentiated control-NSCs spiked with “artificial EVs” containing synthetic miR-1290 oligonucleotides significantly rescued differentiation deficits in ASD cell lines. Based on our earlier published study and the observations from the data presented here, we conclude that miR-1290 regulation could play a critical role during neuronal differentiation in early brain development

MiR-21 in Extracellular Vesicles Leads to Neurotoxicity via TLR7 Signaling in SIV Neurological Disease

<div><p>Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes. In the present study, we isolated and characterized EVs from the brains of rhesus macaques, both with and without simian immunodeficiency virus (SIV) induced central nervous system (CNS) disease. Small RNA sequencing revealed increased miR-21 levels in EVs from SIV encephalitic (SIVE) brains. In situ hybridization revealed increased miR-21 expression in neurons and macrophage/microglial cells/nodules during SIV induced CNS disease. In vitro culture of macrophages revealed that miR-21 is released into EVs and is neurotoxic when compared to EVs derived from miR-21^-/- knockout animals. A mutation of the sequence within miR-21, predicted to bind TLR7, eliminates this neurotoxicity. Indeed miR-21 in EV activates TLR7 in a reporter cell line, and the neurotoxicity is dependent upon TLR7, as neurons isolated from TLR7^-/- knockout mice are protected from neurotoxicity. Further, we show that EVs isolated from the brains of monkeys with SIV induced CNS disease activates TLR7 and were neurotoxic when compared to EVs from control animals. Finally, we show that EV-miR-21 induced neurotoxicity was unaffected by apoptosis inhibition but could be prevented by a necroptosis inhibitor, necrostatin-1, highlighting the actions of this pathway in a growing number of CNS disorders.</p></div

In vitro neurotoxicity assays with exosomes from bone marrow derived macrophage (BMDM) cultures.

<p><b>(A)</b> Quantitative reat-time PCR (qRT-PCR) for miR-21 was performed on EVs isolated from WT and miR-21^-/- BMDMs. Raw CT values confirm the absence of miR-21 in the EVs isolated from miR-21^-/- BMDMs. <b>(B)</b> WT mouse hippocampal neurons were incubated with 1 μg of EVs isolated from WT (WT-Exo) and miR-21 ^-/- (miR-21KO-Exo) littermate BMDMs for 24 hr. LDH assay was performed to assess the neuronal viability Results indicate a significantly higher in cell death with WT EVs than with miR-21^-/- EVs. Statistical analyses were performed on data from six independent experiments. Error bars = SEM; ^**<i>P</i> < 0.01; unpaired t-test. <b>(C)</b> Cultured hippocampal neurons (DIV 7) from WT and TLR7-/- mice were treated with CL075 (6μM) and vehicle for 6h and harvested for real time PCR using GAPDH as an internal control to quantify the levels of IL6 and TNFα. Error bars = SEM; ^*<i>P</i> < 0.05; ****P < 0.0001; Two-way ANOVA with Bonferroni post-hoc test. <b>(D)</b> LDH assay was performed on WT and TLR7^-/- mice hippocampal cultures with WT and miR-21^-/- littermate BMDM derived EVs. A significant increase in neuronal cell death is seen with WT-EVs when compared to miR-21^-/- EVs. No miR-21-EV induced toxicity was found when hippocampal neurons from TLR7^-/- mice were used. Error bars = SEM; ^**<i>P</i> < 0.01; Two-way ANOVA with Bonferroni post-hoc test.</p

Isolation and characterization of brain derived EVs.

<p><b>(A)</b> Left, Electron microscopic (TEM) morphological analysis of EVs derived from uninfected (control) macaque brain. EVs show a size range from 100–150 nm. Scale bar = 100 nm. Right, Western blots for flotillin, CD9, CD63, CD81, HSP70, TSG101 markers for EVs. Non-EV fractions from sucrose gradients were used as negative controls for the EV proteins, brain lysates were used as positive controls for the synaptic proteins. <b>(B)</b> Small RNA sequencing performed on RNA isolated from uninfected, SIV and SIVE brains. Analysis revealed significantly increased expression of miR-21-5p, miR-100-5p and miR-146-5p, and decreased expression miR-126-5p, in SIVE. Error bars = SEM; * <i>P</i> <0.05, ** <i>P</i> <0.01*** <i>P</i> <0.001, **** <i>P</i> < 0.0001; ANOVA with Tukey’s post-hoc test. <b>(C)</b> qRT-PCR validation of miR-21 expression in EVs. Relative quantification was performed based on a standard curve. Statistical analyses were performed on log-transformed values. One-way ANOVA showed p = 0.0024 with Tukey’s <0.01 for uninfected vs. SIVE, and SIV vs. SIVE.</p

Combined FISH and IF for miR-21, CD163 and GFAP.

<p>In SIVE brain sections containing macrophage/microglial nodules, miR-21 (magenta) partially colocalized with the macrophage/microglia marker CD163 (green). No colocalization was observed with GFAP (red), an astrocyte marker. DAPI (blue) was used to label nuclei; a scrambled miRNA probe (Scrm) was used as negative control for hybridization; and U6 probe (a non-coding snRNA) was used as a positive control. Scale bars = 20 μm for all panels except 5 μm for SIVE-Mag panels.</p