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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 artificial EVs.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of synthetic miRNAs; miR-21 (miR21-WT), miR-21 containing a mutation in TLR7 binding site (miR21-Mut) and a known TLR7 activator, Let-7b, and DOTAP artificial EVs. Neurons were incubated for 24 hr and then stained with NeuN, a cell body marker for neurons (Left). The number of NeuN positive neurons was counted and the relative neuronal viability to untreated cultures was calculated (Middle). The result indicates no significant cell death by naked synthetic miRNAs. LDH assay was performed to assess the neuronal viability (Right). Results indicate no difference in cell death. (B) Synthetic miRNAs were mixed with DOTAP liposomal formulations creating “artificial EVs” and WT hippocampal neurons were incubated with 1 μg of synthetic miRNAs within artificial EVs for 24 hr. NeuN staining was performed, and the results indicated increased neuronal loss as seen in fewer numbers of green NeuN positive neurons in miR21-WT and Let-7b treated cultures when compared to miR21-mut and DOTAP treated hippocampal cultures (Left). Quantification (middle bar graph) shows a significant cell death in cultures treated with miR-21-WT and Let-7b when compared to DOTAP control. LDH assay was performed to assess the neuronal viability. Results indicate a significantly higher cell death with miR-21-WT and Let-7b than with miR-21-Mut and DOTAP control. Statistical analyses were performed on data from six independent experiments for NeuN counting and three independent experiments for LDH assay. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. (C) Immunostaining was performed for the neuronal (neurite) marker, MAP2 and staining reveals loss of neurites in cultures treated with miR-21-WT and Let-7b artificial EVs compared to DOTAP only or in the miR-21-Mut treated cultures. Statistical analyses were performed on data from three independent experiments. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. Scale bar = 20 μm.
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ppat.1005032.g003: In vitro neurotoxicity assays with artificial EVs.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of synthetic miRNAs; miR-21 (miR21-WT), miR-21 containing a mutation in TLR7 binding site (miR21-Mut) and a known TLR7 activator, Let-7b, and DOTAP artificial EVs. Neurons were incubated for 24 hr and then stained with NeuN, a cell body marker for neurons (Left). The number of NeuN positive neurons was counted and the relative neuronal viability to untreated cultures was calculated (Middle). The result indicates no significant cell death by naked synthetic miRNAs. LDH assay was performed to assess the neuronal viability (Right). Results indicate no difference in cell death. (B) Synthetic miRNAs were mixed with DOTAP liposomal formulations creating “artificial EVs” and WT hippocampal neurons were incubated with 1 μg of synthetic miRNAs within artificial EVs for 24 hr. NeuN staining was performed, and the results indicated increased neuronal loss as seen in fewer numbers of green NeuN positive neurons in miR21-WT and Let-7b treated cultures when compared to miR21-mut and DOTAP treated hippocampal cultures (Left). Quantification (middle bar graph) shows a significant cell death in cultures treated with miR-21-WT and Let-7b when compared to DOTAP control. LDH assay was performed to assess the neuronal viability. Results indicate a significantly higher cell death with miR-21-WT and Let-7b than with miR-21-Mut and DOTAP control. Statistical analyses were performed on data from six independent experiments for NeuN counting and three independent experiments for LDH assay. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. (C) Immunostaining was performed for the neuronal (neurite) marker, MAP2 and staining reveals loss of neurites in cultures treated with miR-21-WT and Let-7b artificial EVs compared to DOTAP only or in the miR-21-Mut treated cultures. Statistical analyses were performed on data from three independent experiments. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. Scale bar = 20 μm.

Mentions: Recent studies have found that certain microRNAs containing a GU-rich sequence could activate TLR7. Neurotoxicity and neuronal and non-neuronal cell activation has been found with such free microRNAs and with synthetic EVs of lipid-encapsulated microRNAs [25–27]. First, we asked if the presence of extracellular miR-21 could render neurotoxicity. To do so, we used miRNA oligonucleotides (oligos) of wildtype miR-21 (miR-21-WT), a mutant miR-21 (miR-21-Mut) containing a point mutation in one of the uridine residues in a small G/U sequence in the TLR binding motif (U to G, since uridines are more crucial ligands to TLRs [29]). Another characterized microRNA, the TLR7 ligand let-7b, was used as a positive control. First, we added the free “naked” oligos directly to the hippocampal neuronal cultures. Results indicated no significant cell death observed either in miR-21-WT, miR-21-Mut, or let-7b, assessed with NeuN counting or LDH assay (Fig 3A, middle and right). Next, we tested whether these microRNAs, when encased in EV-like vesicles, could have an effect on neurons. Interestingly, when the neuronal cultures were treated with these synthetic EVs, significant neuronal cell death was observed with miR-21-WT and let-7b but not with miR-21-Mut, again demonstrated by both NeuN cell counting assay and LDH assay (Fig 3B). Staining with the neuronal marker MAP2 also revealed a loss in neurites (Fig 3C). In clear distinction to what we saw with free miR-21, the delivery of miR-21 in EV-like vesicles is essential to elicit neurotoxicity.


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 artificial EVs.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of synthetic miRNAs; miR-21 (miR21-WT), miR-21 containing a mutation in TLR7 binding site (miR21-Mut) and a known TLR7 activator, Let-7b, and DOTAP artificial EVs. Neurons were incubated for 24 hr and then stained with NeuN, a cell body marker for neurons (Left). The number of NeuN positive neurons was counted and the relative neuronal viability to untreated cultures was calculated (Middle). The result indicates no significant cell death by naked synthetic miRNAs. LDH assay was performed to assess the neuronal viability (Right). Results indicate no difference in cell death. (B) Synthetic miRNAs were mixed with DOTAP liposomal formulations creating “artificial EVs” and WT hippocampal neurons were incubated with 1 μg of synthetic miRNAs within artificial EVs for 24 hr. NeuN staining was performed, and the results indicated increased neuronal loss as seen in fewer numbers of green NeuN positive neurons in miR21-WT and Let-7b treated cultures when compared to miR21-mut and DOTAP treated hippocampal cultures (Left). Quantification (middle bar graph) shows a significant cell death in cultures treated with miR-21-WT and Let-7b when compared to DOTAP control. LDH assay was performed to assess the neuronal viability. Results indicate a significantly higher cell death with miR-21-WT and Let-7b than with miR-21-Mut and DOTAP control. Statistical analyses were performed on data from six independent experiments for NeuN counting and three independent experiments for LDH assay. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. (C) Immunostaining was performed for the neuronal (neurite) marker, MAP2 and staining reveals loss of neurites in cultures treated with miR-21-WT and Let-7b artificial EVs compared to DOTAP only or in the miR-21-Mut treated cultures. Statistical analyses were performed on data from three independent experiments. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. Scale bar = 20 μm.
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ppat.1005032.g003: In vitro neurotoxicity assays with artificial EVs.(A) Wildtype (WT) mouse hippocampal neurons were incubated with 1 μg of synthetic miRNAs; miR-21 (miR21-WT), miR-21 containing a mutation in TLR7 binding site (miR21-Mut) and a known TLR7 activator, Let-7b, and DOTAP artificial EVs. Neurons were incubated for 24 hr and then stained with NeuN, a cell body marker for neurons (Left). The number of NeuN positive neurons was counted and the relative neuronal viability to untreated cultures was calculated (Middle). The result indicates no significant cell death by naked synthetic miRNAs. LDH assay was performed to assess the neuronal viability (Right). Results indicate no difference in cell death. (B) Synthetic miRNAs were mixed with DOTAP liposomal formulations creating “artificial EVs” and WT hippocampal neurons were incubated with 1 μg of synthetic miRNAs within artificial EVs for 24 hr. NeuN staining was performed, and the results indicated increased neuronal loss as seen in fewer numbers of green NeuN positive neurons in miR21-WT and Let-7b treated cultures when compared to miR21-mut and DOTAP treated hippocampal cultures (Left). Quantification (middle bar graph) shows a significant cell death in cultures treated with miR-21-WT and Let-7b when compared to DOTAP control. LDH assay was performed to assess the neuronal viability. Results indicate a significantly higher cell death with miR-21-WT and Let-7b than with miR-21-Mut and DOTAP control. Statistical analyses were performed on data from six independent experiments for NeuN counting and three independent experiments for LDH assay. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. (C) Immunostaining was performed for the neuronal (neurite) marker, MAP2 and staining reveals loss of neurites in cultures treated with miR-21-WT and Let-7b artificial EVs compared to DOTAP only or in the miR-21-Mut treated cultures. Statistical analyses were performed on data from three independent experiments. Error bar = SEM; ***P<0.001; One-way ANOVA with Dunnett’s post-hoc test. Scale bar = 20 μm.
Mentions: Recent studies have found that certain microRNAs containing a GU-rich sequence could activate TLR7. Neurotoxicity and neuronal and non-neuronal cell activation has been found with such free microRNAs and with synthetic EVs of lipid-encapsulated microRNAs [25–27]. First, we asked if the presence of extracellular miR-21 could render neurotoxicity. To do so, we used miRNA oligonucleotides (oligos) of wildtype miR-21 (miR-21-WT), a mutant miR-21 (miR-21-Mut) containing a point mutation in one of the uridine residues in a small G/U sequence in the TLR binding motif (U to G, since uridines are more crucial ligands to TLRs [29]). Another characterized microRNA, the TLR7 ligand let-7b, was used as a positive control. First, we added the free “naked” oligos directly to the hippocampal neuronal cultures. Results indicated no significant cell death observed either in miR-21-WT, miR-21-Mut, or let-7b, assessed with NeuN counting or LDH assay (Fig 3A, middle and right). Next, we tested whether these microRNAs, when encased in EV-like vesicles, could have an effect on neurons. Interestingly, when the neuronal cultures were treated with these synthetic EVs, significant neuronal cell death was observed with miR-21-WT and let-7b but not with miR-21-Mut, again demonstrated by both NeuN cell counting assay and LDH assay (Fig 3B). Staining with the neuronal marker MAP2 also revealed a loss in neurites (Fig 3C). In clear distinction to what we saw with free miR-21, the delivery of miR-21 in EV-like vesicles is essential to elicit neurotoxicity.

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