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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

EVs from SIVE brains can elicit neurotoxicity and can activate TLR7 pathway.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. LDH assay was performed to assess the neuronal viability; results indicate a significant increase in cell death with SIVE than with uninfected EVs. Statistical analyses were performed on data from three independent sets. Error bars = SEM; *P < 0.05; unpaired t-test. (B) HEK-Blue Null (HEK-control) or TLR7 overexpressing (HEK-TLR7) cells were incubated with increasing concentrations of EVs from uninfected and SIVE brains and with CL264. Dose response curves show a clear increase in the SEAP activity with SIVE EVs in TLR7 (SIVE-EV (TLR7)), comparable to the TLR7 ligand, CL264. No response was seen with EVs from control monkey brains (Control-EV (TLR7)) cells nor in any of the conditions using the cells without TLR7 expression (Null). (C) Similar to (B), cells were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. Increased activity was found in HEK-TLR7 cells treated with SIVE EVs when compared to uninfected EVs. No change in absorbance was observed in HEK-Null (Control) cells. Statistical analyses were performed on data from three independent experiments. Error bars = SEM; *P < 0.05, Two-way ANOVA with Sidak’s multiple comparison was performed.
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ppat.1005032.g005: EVs from SIVE brains can elicit neurotoxicity and can activate TLR7 pathway.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. LDH assay was performed to assess the neuronal viability; results indicate a significant increase in cell death with SIVE than with uninfected EVs. Statistical analyses were performed on data from three independent sets. Error bars = SEM; *P < 0.05; unpaired t-test. (B) HEK-Blue Null (HEK-control) or TLR7 overexpressing (HEK-TLR7) cells were incubated with increasing concentrations of EVs from uninfected and SIVE brains and with CL264. Dose response curves show a clear increase in the SEAP activity with SIVE EVs in TLR7 (SIVE-EV (TLR7)), comparable to the TLR7 ligand, CL264. No response was seen with EVs from control monkey brains (Control-EV (TLR7)) cells nor in any of the conditions using the cells without TLR7 expression (Null). (C) Similar to (B), cells were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. Increased activity was found in HEK-TLR7 cells treated with SIVE EVs when compared to uninfected EVs. No change in absorbance was observed in HEK-Null (Control) cells. Statistical analyses were performed on data from three independent experiments. Error bars = SEM; *P < 0.05, Two-way ANOVA with Sidak’s multiple comparison was performed.

Mentions: Since miR-21 is increased in EVs from the brains of monkeys with SIVE, and EV associated miR-21 (EV-miR-21) is associated with neurotoxicity, we then assessed whether EVs isolated from the SIVE (SIVE-EV) and uninfected (control-EV) brains would show differences in neurotoxicity. Indeed, treatment of neuronal cultures with SIVE-EV significantly increased neuronal death as compared to control-EV (Fig 5A).


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)

EVs from SIVE brains can elicit neurotoxicity and can activate TLR7 pathway.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. LDH assay was performed to assess the neuronal viability; results indicate a significant increase in cell death with SIVE than with uninfected EVs. Statistical analyses were performed on data from three independent sets. Error bars = SEM; *P < 0.05; unpaired t-test. (B) HEK-Blue Null (HEK-control) or TLR7 overexpressing (HEK-TLR7) cells were incubated with increasing concentrations of EVs from uninfected and SIVE brains and with CL264. Dose response curves show a clear increase in the SEAP activity with SIVE EVs in TLR7 (SIVE-EV (TLR7)), comparable to the TLR7 ligand, CL264. No response was seen with EVs from control monkey brains (Control-EV (TLR7)) cells nor in any of the conditions using the cells without TLR7 expression (Null). (C) Similar to (B), cells were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. Increased activity was found in HEK-TLR7 cells treated with SIVE EVs when compared to uninfected EVs. No change in absorbance was observed in HEK-Null (Control) cells. Statistical analyses were performed on data from three independent experiments. Error bars = SEM; *P < 0.05, Two-way ANOVA with Sidak’s multiple comparison was performed.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4496044&req=5

ppat.1005032.g005: EVs from SIVE brains can elicit neurotoxicity and can activate TLR7 pathway.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. LDH assay was performed to assess the neuronal viability; results indicate a significant increase in cell death with SIVE than with uninfected EVs. Statistical analyses were performed on data from three independent sets. Error bars = SEM; *P < 0.05; unpaired t-test. (B) HEK-Blue Null (HEK-control) or TLR7 overexpressing (HEK-TLR7) cells were incubated with increasing concentrations of EVs from uninfected and SIVE brains and with CL264. Dose response curves show a clear increase in the SEAP activity with SIVE EVs in TLR7 (SIVE-EV (TLR7)), comparable to the TLR7 ligand, CL264. No response was seen with EVs from control monkey brains (Control-EV (TLR7)) cells nor in any of the conditions using the cells without TLR7 expression (Null). (C) Similar to (B), cells were incubated with 1 μg of EVs isolated from uninfected and SIVE brains for 24 hr. Increased activity was found in HEK-TLR7 cells treated with SIVE EVs when compared to uninfected EVs. No change in absorbance was observed in HEK-Null (Control) cells. Statistical analyses were performed on data from three independent experiments. Error bars = SEM; *P < 0.05, Two-way ANOVA with Sidak’s multiple comparison was performed.
Mentions: Since miR-21 is increased in EVs from the brains of monkeys with SIVE, and EV associated miR-21 (EV-miR-21) is associated with neurotoxicity, we then assessed whether EVs isolated from the SIVE (SIVE-EV) and uninfected (control-EV) brains would show differences in neurotoxicity. Indeed, treatment of neuronal cultures with SIVE-EV significantly increased neuronal death as compared to control-EV (Fig 5A).

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