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

Yelamanchili SV, Lamberty BG, Rennard DA, Morsey BM, Hochfelder CG, Meays BM, Levy E, Fox HS - PLoS Pathog. (2015)

Bottom Line: Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, United States of America. syelamanchili@unmc.edu

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

In vitro neurotoxicity assays with exosomes from bone marrow derived macrophage (BMDM) cultures.(A) 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) 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; **P < 0.01; unpaired t-test. (C) 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; *P < 0.05; ****P < 0.0001; Two-way ANOVA with Bonferroni post-hoc test. (D) 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; **P < 0.01; Two-way ANOVA with Bonferroni post-hoc test.
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ppat.1005032.g004: In vitro neurotoxicity assays with exosomes from bone marrow derived macrophage (BMDM) cultures.(A) 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) 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; **P < 0.01; unpaired t-test. (C) 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; *P < 0.05; ****P < 0.0001; Two-way ANOVA with Bonferroni post-hoc test. (D) 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; **P < 0.01; Two-way ANOVA with Bonferroni post-hoc test.

Mentions: To further examine whether EV-miR-21 activates the TLR7 pathway, we isolated EVs from bone marrow derived macrophage cultures prepared from wildtype (WT) and miR-21-/- mice and used these, differing in the presence of miR-21, to examine potential neurotoxicity (Fig 4A). Indeed, there is a significant increase in neuronal cell death when cultures were treated with EVs derived from WT than from miR-21-/- macrophage cultures (Fig 4B). In order to examine if this neurotoxicity is dependent on TLR7, we performed the neurotoxicity studies on neurons derived from TLR7-/- animals. To confirm that TLR7 -/- neurons do not respond to ligands, we treated the hippocampal neurons isolated from WT and TLR7 -/- mice with TLR7 agonist CL075. Quantitative RT-PCR on confirms the expression of pro-inflammatory cytokine genes such as IL6 and TNFα only in WT neurons confirming that TLR7 -/- neurons did not respond to TLR7 ligand stimulation (Fig 4C). Treating the TLR7 -/- neurons with WT-EVs and miR-21-/- EVs demonstrated that toxicity depended not only on the presence of miR-21 in the EVs but also upon the presence of TLR7 in the neurons (Fig 4D). These results clearly indicate that both miR-21 and TLR7 are required for the activation of neurotoxic pathways.


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

Yelamanchili SV, Lamberty BG, Rennard DA, Morsey BM, Hochfelder CG, Meays BM, Levy E, Fox HS - PLoS Pathog. (2015)

In vitro neurotoxicity assays with exosomes from bone marrow derived macrophage (BMDM) cultures.(A) 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) 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; **P < 0.01; unpaired t-test. (C) 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; *P < 0.05; ****P < 0.0001; Two-way ANOVA with Bonferroni post-hoc test. (D) 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; **P < 0.01; Two-way ANOVA with Bonferroni post-hoc test.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4496044&req=5

ppat.1005032.g004: In vitro neurotoxicity assays with exosomes from bone marrow derived macrophage (BMDM) cultures.(A) 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) 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; **P < 0.01; unpaired t-test. (C) 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; *P < 0.05; ****P < 0.0001; Two-way ANOVA with Bonferroni post-hoc test. (D) 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; **P < 0.01; Two-way ANOVA with Bonferroni post-hoc test.
Mentions: To further examine whether EV-miR-21 activates the TLR7 pathway, we isolated EVs from bone marrow derived macrophage cultures prepared from wildtype (WT) and miR-21-/- mice and used these, differing in the presence of miR-21, to examine potential neurotoxicity (Fig 4A). Indeed, there is a significant increase in neuronal cell death when cultures were treated with EVs derived from WT than from miR-21-/- macrophage cultures (Fig 4B). In order to examine if this neurotoxicity is dependent on TLR7, we performed the neurotoxicity studies on neurons derived from TLR7-/- animals. To confirm that TLR7 -/- neurons do not respond to ligands, we treated the hippocampal neurons isolated from WT and TLR7 -/- mice with TLR7 agonist CL075. Quantitative RT-PCR on confirms the expression of pro-inflammatory cytokine genes such as IL6 and TNFα only in WT neurons confirming that TLR7 -/- neurons did not respond to TLR7 ligand stimulation (Fig 4C). Treating the TLR7 -/- neurons with WT-EVs and miR-21-/- EVs demonstrated that toxicity depended not only on the presence of miR-21 in the EVs but also upon the presence of TLR7 in the neurons (Fig 4D). These results clearly indicate that both miR-21 and TLR7 are required for the activation of neurotoxic pathways.

Bottom Line: Recent studies have found that extracellular vesicles (EVs) play an important role in normal and disease processes.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Pharmacology and Experimental Neuroscience, University of Nebraska Medical Center, Omaha, Nebraska, United States of America. syelamanchili@unmc.edu

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus