TNFα and IL-1β modify the miRNA cargo of astrocyte shed extracellular vesicles to regulate neurotrophic signaling in neurons - PubMed (original) (raw)

TNFα and IL-1β modify the miRNA cargo of astrocyte shed extracellular vesicles to regulate neurotrophic signaling in neurons

Amrita Datta Chaudhuri et al. Cell Death Dis. 2018.

Abstract

Astrocytes are known to be critical regulators of neuronal function. However, relatively few mediators of astrocyte to neuron communication have been identified. Recent advancements in the biology of extracellular vesicles have begun to implicate astrocyte derived extracellular vesicles (ADEV) as mediators of astrocyte to neuron communication, suggesting that alterations in the release and/or composition of ADEVs could influence gliotransmission. TNFα and IL-1β are key mediators of glial activation and neuronal damage, but the effects of these cytokines on the release or molecular composition of ADEVs is unknown. We found that ADEVs released in response to IL-1β (ADEV-IL-1β) and TNFα (ADEV-TNFα) were enriched with miRNAs that target proteins involved in neurotrophin signaling. We confirmed that miR-125a-5p and miR-16-5p (both enriched in ADEV-IL-1β and ADEV-TNFα) targeted NTKR3 and its downstream effector Bcl2. Downregulation of these targets in neurons was associated with reductions in dendritic growth, dendritic complexity, reduced spike rates, and burst activity. Molecular interference of miR-125a-5p and miR-16-5p prevented ADEV-IL-1β from reducing dendritic complexity, spike, and burst rates. These findings suggest that astrocytes respond to inflammatory challenge by modifying the miRNA cargo of ADEVs to diminish the activity of target neurons by regulating the translational expression of proteins controlling programs essential for synaptic stability and neuronal excitability.

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Conflict of interest statement

The authors declare that they have no conflict of interest.

Figures

Fig. 1

Fig. 1. ADEV-ATP enhances dendritic arborization.

a Representative fluorescent images of MAP2 immunopositive hippocampal neurons (top panels) and Neurolucida dendrite tracings (bottom panels) following a 48 h exposure to the indicated concentrations of ADEV-ATP. Quantitative data show b dendritic complexity, c surface area, d dendritic length, e number of ends, f nodes, and g total dendrite number for the indicated concentrations of ADEV-ATP. Data are mean ± SEM of 15–20 neurons from three independent experiments. One-way ANOVA with Tukey’s post hoc comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 compared to control

Fig. 2

Fig. 2. ADEV-IL-1β and ADEV-TNFα dose-dependently reduce dendritic complexity.

a Representative fluorescent images of MAP2 immunopositive hippocampal neurons (top panels) and Neurolucida dendrite tracings (bottom panels) following a 48 h exposure to the indicated concentrations of ADEV-IL-1β. Quantitative data show (b) dendritic complexity, c surface area, d dendritic length, e number of ends, f nodes, and g total dendrite number for the indicated concentrations of ADEV-IL-1β. h Representative fluorescent images of MAP2 immunopositive hippocampal neurons (top panels) and neurite tracings (bottom panels) following a 48 h exposure to the indicated concentrations of ADEV-IL-1β. Quantitative data show i dendritic complexity, j surface area, k dendritic length, l number of ends, m nodes, and n total dendrite number for the indicated concentrations of ADEV-IL-1β. Data are mean ± SEM of 15–20 neurons from three independent experiments. One-way ANOVA followed by Tukey’s post hoc comparisons. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to control

Fig. 3

Fig. 3. Stimulus-depended enrichment of miRNA cargo in ADEVs.

a Heatmap and hierarchical clustering showing relative levels for microRNAs enriched >1.5-fold in ADEVs shed in response to the indicated treatment conditions compared with ADEV-CR. b qRT-PCR validation of the miRNAs most enriched in ADEV-IL-1β. c qRT-PCR validation of the miRNAs most enriched in ADEV-TNFα. d qRT-PCR of the microRNAs most enriched in astrocyte ADEV-ATP. e qRT-PCR showing levels of the indicated miRNAs in cultured astrocytes following treatment with ATP (10 μM), IL-1β or TNFα (200 ng/ml) for 2 h. Data are mean ± SEM of n = 3 independent experiments. One-way ANOVA with Tukey’s post hoc comparisons. *p < 0.05, **p < 0.01, ***p < 0.001 compared with CR

Fig. 4

Fig. 4. Bioinformatic interrogation of miRNAs enriched in ADEV-IL-1β and ADEV-TNFα identified several neuronal-specific pathways.

a Pathway analysis of predicted miRNA targets enriched in ADEV-IL-1β using DIANA miRPATH. b Pathway analysis of predicted miRNA targets enriched in ADEV-TNFα using DIANA miRPATH. Heat maps depict levels of miRNA enrichment for the indicated signaling pathways. Darker colors (orange to red) indicate more enrichment of predicted microRNA targets belonging to the corresponding pathway. Several predicted pathways were associated with neurite outgrowth, dopamine and glutamatergic synapses, long-term potentiation, long-term depression, and neurotrophin signaling (shown in red)

Fig. 5

Fig. 5. MiR-125a-5p and miR-16-5p target NTRK3 and Bcl2.

a Schematic diagram of the neurotrophin-signaling pathway. The mRNAs in red text and yellow highlight are predicted targets of at least two miRNAs (miR-125a-5p and miR-16-5p) enriched in ADEV-TNFα, or ADEV-IL-1β. The mRNAs in magenta text are predicted targets of 1 miRNA enriched in ADEV-TNFα, or ADEV-IL-1β. b Representative western blot for NTRK3 and Bcl2 in primary neurons treated with ADEV-CR or ADEV-IL-1β at the indicated particle dose. Densitometry quantitation of (c) NTRK3, and (d) Bcl2 levels normalized to β-actin. Schematic diagrams show (e) miR-125a-5p binding site on NTRK3 3′-UTR, f miR-125a-5p binding site on BCL2 3′-UTR, g miR-16-5p binding site on NTRK3 3’UTR, and h miR-16-5p binding site on BCL2 3′-UTR. i 3′-UTR luciferase reporter assay showing functional binding of miR-125a-5p to the 3′-UTR of NTRK3 and Bcl2. j 3′-UTR luciferase reporter assay showing functional binding of miR-16-5p to the 3′-UTR of NTRK3 and Bcl2. k 3′-UTR luciferase reporter assay in cells transfected with NTRK3 3′-UTR followed by treatment with ADEV-CR, ADEV-ATP or ADEV-IL-1β at the indicated ADEV dose. Data are mean ± SEM of a minimum n = 3 independent experiments per condition. One-way ANOVA with Tukey’s post hoc comparisons. #p < 0.05, ##p < 0.01, ###p < 0.001 compared to control

Fig. 6

Fig. 6. Molecular interference of miR-125a-5p and miR-16-5p prevents ADEV-IL-1β from reducing NTRK3 and BCl2 expression.

a Representative western blot showing NTKR3 and Bcl2 protein expression in neurons 24 h following treatment with ADEV-CR or ADEV-IL-1β alone or in combination with antisense oligonucleotide inhibitors for miR-125 and miR-16 (20 pmole each; Combined In). Scrambled oligonucleotide inhibitors were used as a control (Scr In). Quantitative densitometry analysis of (b) NTRK3, and (c) Bcl2 for the indicated treatment conditions. d Representative western blot showing NTKR3 and Bcl2 protein expression in neurons 24 h following treatment with ADEV-CR or ADEV-IL-1β alone or in combination with antisense oligonucleotide inhibitors either miR-1 25 or miR-16 alone or in combination (20 pmole each; Combined In). Scrambled oligonucleotide inhibitors were used as a control (Scr In). Quantitative densitometry analysis of (e) NTRK3, and (f) Bcl2 for the indicated treatment conditions. Data are mean ± SEM of n = 3 independent experiments. One-way ANOVA with Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 increased compared to no treatment (NT), #p < 0.05, ###p < 0.001 decreased compared with NT

Fig. 7

Fig. 7. Molecular interference of miR-125a-5p and miR-16-5p prevents ADEV-IL-1β from reducing dendritic arborization.

a Representative fluorescent images of MAP2 immunopositive hippocampal neurons (top panels) and Neurolucida dendrite tracings (bottom panels) following a 48 h exposure to the indicated treatment conditions. Quantitative data show (b) dendritic complexity, c surface area, d dendritic length, e number of ends, f nodes, and g total dendrite number for the indicated treatment conditions. ADEV-IL-1β particle dose was 50 ADEVs/cell, and antisense oligonucleotide inhibitors for miR-125 and miR-16 were used at 20 pmole. Data are mean ± SEM for the indicated treatment conditions. One-way ANOVA followed by Tukey’s post hoc test. *p < 0.05, **p < 0.01, ***p < 0.001 increased compared to NT. #p < 0.05, ##p < 0.01, ###p < 0.001 decreased compared to NT

Fig. 8

Fig. 8. ADEV-IL-1β reduces neuronal activity but not connectivity.

a Representative recording of population spikes (each purple vertical line), and bursts (purple clusters with >4 spikes/s) from an individual electrode of a multichannel electrode array. Representative tracings show averages of spikes/second/electrode, and associated scatter plots show quantitation of spike and burst rates for (b–e) Control, (f–i) ADEV-IL-1β (particle dose of 50 ADEVs/cell), (j–m) ADEV-IL-1β+ Scrambled oligonucleotide (Scr In, 20 pmole), and (n–q) ADEV-IL-1β+ oligonucleotide inhibitors for miR-125 and miR-16 (Combined In, 20 pmole each). Data are mean ± SEM. Paired _t_-tests were performed to compare spike and burst rate of each electrode before and after treatment. ***p < 0.001 increased compared to baseline and ###p < 0.001 decreased compared to baseline

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