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. 2017 Jun 14;36(12):1770-1787.
doi: 10.15252/embj.201695748. Epub 2017 May 9.

A microRNA-129-5p/Rbfox Crosstalk Coordinates Homeostatic Downscaling of Excitatory Synapses

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Free PMC article

A microRNA-129-5p/Rbfox Crosstalk Coordinates Homeostatic Downscaling of Excitatory Synapses

Marek Rajman et al. EMBO J. .
Free PMC article

Abstract

Synaptic downscaling is a homeostatic mechanism that allows neurons to reduce firing rates during chronically elevated network activity. Although synaptic downscaling is important in neural circuit development and epilepsy, the underlying mechanisms are poorly described. We performed small RNA profiling in picrotoxin (PTX)-treated hippocampal neurons, a model of synaptic downscaling. Thereby, we identified eight microRNAs (miRNAs) that were increased in response to PTX, including miR-129-5p, whose inhibition blocked synaptic downscaling in vitro and reduced epileptic seizure severity in vivo Using transcriptome, proteome, and bioinformatic analysis, we identified the calcium pump Atp2b4 and doublecortin (Dcx) as miR-129-5p targets. Restoring Atp2b4 and Dcx expression was sufficient to prevent synaptic downscaling in PTX-treated neurons. Furthermore, we characterized a functional crosstalk between miR-129-5p and the RNA-binding protein (RBP) Rbfox1. In the absence of PTX, Rbfox1 promoted the expression of Atp2b4 and Dcx. Upon PTX treatment, Rbfox1 expression was downregulated by miR-129-5p, thereby allowing the repression of Atp2b4 and Dcx. We therefore identified a novel activity-dependent miRNA/RBP crosstalk during synaptic scaling, with potential implications for neural network homeostasis and epileptogenesis.

Keywords: RNA‐binding protein; epilepsy; homeostatic plasticity; microRNA; synaptic scaling.

Figures

Figure 1
Figure 1. A specific set of miRNAs is upregulated during synaptic downscaling in vitro

Western blot analysis of surface GluA1 expression after cross‐linking in lysates from vehicle (Veh)‐ or picrotoxin (PTX, 48 h)‐treated hippocampal neurons. Left: Representative Western blot. Tubulin was used as a loading control. Right: Quantification of Western blot data from three independent experiments. Dot‐blot presentation with mean optical band density (OD) normalized to tubulin ± s.e.m. (independent two‐sample t‐test, two‐tailed, homoscedastic variance; *P = 0.026).

Average changes in miRNA levels (PTX/Veh; log2) as determined by small RNA‐seq (n = 3,442 miRNAs; correction for multiple comparisons by FDR, P < 0.05). miRNAs significantly upregulated by PTX are highlighted.

TaqMan qPCR analysis of miR‐129‐5p and miR‐132‐3p in PTX‐ and vehicle‐treated hippocampal neurons. Dot‐plot presentation with mean relative miRNA levels (PTX/Veh) normalized to miR‐99b ± s.e.m. (n = 6; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; *P = 0.022, **P = 0.010).

Source data are available online for this figure.
Figure 2
Figure 2. miR‐129‐5p is required for synaptic downscaling in vitro

Upper panel: Representative confocal microscopy images of dendritic segments from PTX‐ or vehicle (Veh)‐treated hippocampal neurons transfected with indicated anti‐miRs (scale bar = 5 μm). Bottom panel: Quantification of dendritic spine size from three independent experiments. Dot‐plot presentation with mean relative spine size (Ptx/Veh) ± s.e.m. (n = 3; 8 neurons, ˜200 spines/neuron per experimental condition; one‐way ANOVA; F (2,6) = 29.31, P = 0.001; groups were compared by Bonferroni post hoc test; **P = 0.0030, ***P = 0.001, ns: not significant).

Upper panel: Representative traces of mEPSC recordings from bicuculline (Bic)‐ or vehicle‐treated neurons transfected with indicated anti‐miRs. Bottom panel: Quantification of mEPSC amplitudes from multiple neurons, data are presented as box plots (anti‐miR Control Veh/Bic n = 13/12; anti‐miR‐129 Veh/Bic n = 14/13; GLM model; activity P = 0.015; estimated means of specific experimental conditions were compared by pairwise comparison in SPSS; anti‐miR Control Veh vs. Bic *P = 0.023; anti‐miR‐129‐5p Veh vs. Bic P = 0.242; Veh anti‐miR Control vs. anti‐miR‐129‐5p P = 0.827; Bic anti‐miR Control vs. anti‐miR‐129‐5p P = 0.307; ns: not significant). The boundary of the box closest to zero indicates the 25th percentile, a line within the box marks the median, and the boundary of the box farthest from zero indicates the 75th percentile. Error bars above and below the box indicate the 90th and 10th percentiles.

Figure 3
Figure 3. miR‐129‐5p is involved in the regulation of epileptic seizures in vivo

Small RNA‐seq analysis of Ago2‐associated miR‐129‐5p from the dentate gyrus of rats at indicated times after perforant path stimulation (PPS) or after the occurrence of the first seizure (post‐spontaneous seizure). Data are presented as fold (log2) induction over control‐treated rats in a dot plot with mean ± s.e.m. (one‐way ANOVA, correction for multiple comparisons by Benjamini–Hochberg FDR, P = 0.005; n = 3 per experimental condition, *P < 0.05, **P < 0.01). Average levels of Ago2‐associated miR‐129‐5p in control group were set to 0 (log2).

Representative heatmap showing frequency (Hz) and amplitude (μV) of EEG recordings over time (in minutes) for a control (PBS)‐ and anti‐miR‐129‐5p‐injected mice, followed by the respective EEG traces immediately below.

Graphs show EEG total power (C), amplitude (D), depicting % of increase from each animal's own baseline data, and time spent in seizures (E) during 40 min of observation after KA‐induced status epilepticus in mice pretreated (24 h) with PBS or anti‐miR‐129‐5p. Dot‐plot presentation with mean ± s.e.m. (n = 4–5 mice per group; independent two‐sample t‐test, two‐tailed, homoscedastic variance; total power *P = 0.0421, amplitude *P = 0.041, time in seizures **P = 0.0012).

Representative photomicrographs from Fluoro‐Jade B (FJB) stainings of the mouse dorsal hippocampus 24 h after status epilepticus. Mice were injected with PBS or anti‐miR‐129‐5p 24 h before KA administration. Scale bar = 100 μm.

Quantification of FJB‐positive cells from multiple slices performed in (F). Dot‐plot presentation with mean cell count ± s.e.m. (n = 4–5 mice per group; independent two‐sample t‐test, two‐tailed, homoscedastic variance; **P = 0.009).

TaqMan qPCR analysis of miR‐129‐5p levels in the hippocampus of human temporal lobe epilepsy (TLE) patients displaying hippocampal sclerosis (HS) (n = 6) or healthy controls (n = 10). Dot‐plot presentation with mean relative miR‐129‐5p levels (log2) ± s.e.m. (independent two‐sample t‐test, two‐tailed, homoscedastic variance; **P = 0.004).

Figure 4
Figure 4. Systematic miRNA target identification using a combination of RNA‐seq and quantitative proteomics

Average changes in mRNA levels (PTX/Veh; log2) as determined by polyA RNA‐seq (n = 3; q‐values represent FDR‐corrected P‐values; q < 0.05); 495 genes were significantly upregulated and 462 genes were significantly downregulated upon PTX treatment.

GO term analysis for 957 genes with differentially regulated mRNA levels upon 48‐h PTX treatment (Fig 4A). The color of the central column elements represents GO term enrichment in the analyzed dataset. Numbers in the central column represent total number of genes associated with the specific GO term. Percentage in the downregulated or upregulated group stands for identified number of genes associated with specific GO term in the experimental dataset.

qPCR analysis of indicated candidate genes using polyA RNA from PTX (48 h)‐ or vehicle‐treated hippocampal neurons. Dot‐plot presentation with mean relative RNA levels (PTX/Veh (log2)) ± s.e.m. (n = 4 independent experiments; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Atp2b4 *P = 0.033, Camk2a *P = 0.023, Dcx P = 0.062, Camk2b P = 0.450; ns: not significant).

Left panel: Timeline of pSILAC experiment in primary hippocampal neurons cultured for indicated number of days in vitro (DIV). Right panel: Average changes in new protein synthesis (PTX/Veh; log2) plotted vs. P‐value (log2) (n = 3; 1,225 proteins; independent one‐sample t‐test, two‐tailed, heteroscedastic variance). Proteins with P < 0.05 were considered as differentially synthesized (total of 182 proteins, 97↓, 85↑).

Western blot analysis of Atp2b4, Camk2a, Syn1, Dcx, GluA1, Ago2, and 4E‐BP1 expression in lysates from vehicle (Veh)‐ and PTX (48 h)‐treated hippocampal neurons. Left: Representative Western blot. Tubulin was used as a loading control. Right: Quantification of four independent experiments except Ago2 (n = 3) and Syn1 (n = 5). Dot‐plot presentation with mean relative protein levels (PTX/Veh) ± s.e.m. (independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Atp2b4 *P = 0.012, Camk2a **P = 0.005, Dcx *P = 0.026, 4E‐BP1 P = 0.09, Syn1 **P = 0.005, Ago2 **P = 0.005, GluA1 *P = 0.03; ns: not significant).

Overlap of genes displaying differential mRNA levels (panel A) and new protein synthesis (panel D). mRNA fold changes (PTX/Veh; log2) are plotted vs. fold changes in new protein synthesis (PTX/Veh; log2). Pearson's correlation was used to compare changes at the mRNA and protein synthesis level.

Box plot representation of the 3′UTR length of genes that are significantly regulated by PTX based on pSILAC (left; panel D) or RNA‐seq (right; panel A) (Mann–Whitney U‐test; ***P < 0.001).

Abundance of binding sites (8mer‐1a and 7mer‐m8 seeds) for differentially regulated miRNAs (Fig 1B) in 3′UTRs of genes that are significantly regulated by PTX based on pSILAC (panel D) or RNA‐seq (panel A) according to TargetScan‐Human version 6.2. D: decreased; I: increased. Seeds were classified as poorly conserved (≤ 3 species), conserved (4–9 species), or highly conserved (≥ 10 species). Bar graphs represent total number of miRNA binding sites per group. Numbers above bars represent the ratio of total miRNA binding sites between decreased and increased genes.

Source data are available online for this figure.
Figure 5
Figure 5. Atp2b4 and Dcx are miR‐129‐5p target genes

List of predicted miR‐129‐5p targets (TargetScan‐Human version 6.2) that were significantly downregulated by PTX in both pSILAC (Fig 4D) and RNA‐seq (Fig 4A) experiments.

Localization of mir‐129‐5p binding sites (as predicted in Fig 4H) in the 3′UTRs of Atp2b4 and Dcx. Color code indicates different degree of conservation.

Western blot analysis of Atp2b4, Dcx, and actin protein expression in hippocampal neurons transfected with miR‐129‐5p or control duplex RNA. Left: Representative Western blot. Tubulin was used as a loading control. Right: Quantification of multiple experiments. Dot‐plot presentation with mean relative protein levels (miR‐129‐5p/control) ± s.e.m. (Atp2b4, Dcx n = 3; actin n = 5; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Atp2b4 *P = 0.0437, Dcx *P = 0.0236, actin P = 0.4222; ns: not significant).

Luciferase reporter gene assay in miR‐129‐5p or control duplex RNA‐transfected cortical neurons co‐transfected with the indicated 3′UTR reporter constructs containing wild‐type (wt) or mutated (129mut) miR‐129‐5p binding sites. Dot‐plot presentation with mean relative luciferase activity (miR‐129‐5p/control) ± s.e.m. Effect of miR‐129‐5p was tested by one‐sample t‐test (n = 4; two‐tailed, heteroscedastic variance; Atp2b4‐luc wt # P = 0.027, Atp2b4‐luc 129mut P = 0.151, Dcx‐luc wt # P = 0.022, Dcx‐luc 129mut P = 0.082) or two‐sample t‐test (two‐tailed, homoscedastic variance; Atp2b4 wt vs. 129mut **P = 0.004; Dcx wt vs. 129mut P = 0.302).

Luciferase reporter gene assay in vehicle (Veh)‐ or PTX‐treated hippocampal neurons transfected with the Atp2b4 3′UTR and Dcx 3′UTR reporter construct containing either wt or 129mut binding sites (see panel D). Dot‐plot presentation with mean relative luciferase activity (PTX/Veh) ± s.e.m. pGl4 (PTX/Veh) was set to 1. Effect of PTX was tested by one‐sample t‐test (Atp2b4‐luc n = 5; Dcx‐luc n = 4; two‐tailed, heteroscedastic variance; Atp2b4‐luc wt # P = 0.003, 129mut P = 0.117, Dcx‐luc wt # P = 0.004, 129mut P = 0.213) or two‐sample t‐test (two‐tailed, homoscedastic variance; Atp2b4‐luc wt vs. Mut **P = 0.008; Dcx‐luc wt vs. Mut P = 0.190, ns: not significant).

Western blot analysis of Atp2b4 and Dcx protein expression in PTX‐ or mock‐treated hippocampal neurons that were transfected with the indicated anti‐miRs. Left: Representative Western blot. Tubulin was used as a loading control. Right: Quantification of three independent experiments. Dot‐plot presentation with mean relative protein levels (PTX/Veh) ± s.e.m. (n = 3; one‐way ANOVA; Atp2b4: F (2,6) = 10.323, P = 0.011; groups were compared by Bonferroni post hoc test; anti‐miR‐129‐5p vs. anti‐miR Cont *P = 0.044; anti‐miR‐129‐5p vs. Empty *P = 0.015; Dcx: one‐way ANOVA; F (2,6) = 0.243; P = 0.791).

Correlation of miR‐129‐5p and Atp2b4 (G) or Dcx (H) mRNA expression levels in the hippocampus of human TLE patients. Data are presented as rel. RNA or miRNA levels in log2 scale (Atp2b4: n = 15, Pearson's r = −0.706; P = 0.003; Dcx: n = 8, Pearson's r = −0.745; P = 0.034).

Source data are available online for this figure.
Figure 6
Figure 6. Downregulation of Atp2b4 is required for synaptic downscaling in vitro

Analysis of spine size when Atp2b4 or Dcx were overexpressed during PTX application. Upper panel: Representative confocal microscopy images of dendritic segments from PTX‐ or vehicle (Veh)‐treated hippocampal neurons transfected with indicated overexpression vectors and eGFP (control = Creb‐dMVP16, scale bar = 5 μm). Bottom panel: Quantification of dendritic spine size from four independent experiments. Dot‐plot presentation with mean relative spine size (PTX/Veh) ± s.e.m. (n = 4; 8 neurons, ˜200 spines/neuron per experimental condition; one‐way ANOVA; F (2,9) = 39.376, P < 0.001; groups were compared by Bonferroni post hoc test; Contr vs. Atp2b4 ***P < 0.001; Contr vs. Dcx ***P = 0.001).

Upper panel: Representative traces of mEPSC recordings from bicuculline (Bic)‐ or vehicle‐treated neurons transfected with Atp2b4 or control expression vector. Bottom panel: Quantification of mEPSC amplitudes. Data are presented as box plots (control Veh/Bic n = 14/16; Atp2b4 Veh/Bic n = 13/16; GLM model; activity P = 0.001; overexpression × activity P = 0.048; estimated means of specific experimental conditions were compared by pairwise comparison in SPSS; Control protein Veh vs. Bic ***P < 0.001, Control protein Bic vs. Atp2b4 Bic *P = 0.022, Veh Control protein vs. Atp2b4 P = 0.561, Atp2b4 Veh vs. Bic P = 0.348).

Figure 7
Figure 7. Rbfox1 is a positive regulator of Atp2b4 and Dcx expression

Bioinformatic analysis of published CLIP experiments for indicated RBPs performed in mouse brains. Average number of RBP CLIP tags in 3′UTRs of genes corresponding to proteins whose synthesis is increased or decreased in PTX‐treated neurons (Mann–Whitney U‐test; *P = 0.016 (Ago); **P = 0.004 (nElavl); *P = 0.010 (Ptbp2); **P = 0.005 (TDP‐43); ***P < 0.001).

Abundance of Rbfox binding motifs (GCAUG/UGCAUG) in 3′UTRs corresponding to genes with PTX‐decreased (D) or PTX‐increased (I) protein synthesis (Fig 4D) or mRNA levels (Fig 4A) according to TargetScan‐Human version 6.2. Seeds were classified as poorly conserved (≤ 3 species), conserved (4–9 species), or highly conserved (≥ 10 species). Bars represent total number of binding motifs per group.

Localization of Rbfox binding motifs in the 3′UTRs of Atp2b4 and Dcx. Motifs were classified as poorly conserved, conserved, or highly conserved as described in (B).

Western blot analysis of Atp2b4, Dcx, and Rbfox1 protein expression in hippocampal neurons transfected with siRbfox1 or siControl. Left: Representative Western blot. Tubulin was used as a loading control. Right: Quantification of four independent experiments. Dot‐plot presentation with mean relative protein levels (siRbfox1/siControl) normalized to tubulin ± s.e.m. (Atp2b4, Dcx n = 4; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Atp2b4 **P = 0.005, Dcx *P = 0.013).

Luciferase reporter gene assay in shRNA‐Rbfox1‐ or shRNA‐Control‐transfected hippocampal neurons co‐transfected with the Dcx or Atp2b4 3′UTR reporter constructs (Fig 5D). Dot‐plot presentation with mean relative luciferase activity (shRbfox1/shControl) ± s.e.m. pGl4 (shRbfox1/shControl) was set to 1 (n = 3 for Dcx‐luc; n = 4 for Atp2b4‐luc; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Atp2b4‐luc *P = 0.017, Dcx‐luc *P = 0.015).

Source data are available online for this figure.
Figure 8
Figure 8. Rbfox1/3 proteins are downregulated by PTX and miR‐129‐5p

Western blot analysis of Rbfox1 and Rbfox3 expression in lysates from vehicle (Veh)‐ and PTX (48 h)‐treated hippocampal neurons. Left: Representative Western blot. Tubulin was used as a loading control. Right: Quantification of multiple independent experiments. Dot‐plot presentation with mean relative protein levels (PTX/Veh) normalized to tubulin ± s.e.m. (Rbfox1 n = 6; Rbfox3 n = 3; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Rbfox1 **P = 0.006, Rbfox3 **P = 0.005).

Localization of mir‐129‐5p binding sites in the 3′UTRs of Rbfox1/3 based on TargetScan‐Human version 6.0. Color code indicates different degree of conservation as described in Fig 4H.

Western blot analysis of Rbfox1 and Rbfox3 protein expression in hippocampal neurons transfected with miR‐129‐5p or control duplex RNA. Left: Representative Western blot. Tubulin was used as a loading control. Right: Quantification of multiple independent experiments. Dot‐plot presentation with mean relative protein levels (miR‐129‐5p/control) normalized to tubulin ± s.e.m. (Rbfox1 n = 4; Rbfox3 n = 6; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Rbfox1 **P = 0.002, Rbfox3 *P = 0.025).

Luciferase reporter gene assay in miR‐129‐5p‐ or control duplex RNA‐transfected hippocampal neurons co‐transfected with the Rbfox1 3′UTR reporter construct (Rbfox1‐luc) containing either wt or 129mut (2‐highly conserved sites) binding sites. Dot‐plot presentation with mean relative luciferase activity (miR‐129‐5p/control) normalized to pGL4 empty vector ± s.e.m. (n = 4; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Rbfox1‐luc wt *P = 0.0476, Rbfox1‐luc 129mut P = 0.4428).

Luciferase reporter gene assay in vehicle (Veh)‐ or PTX‐treated hippocampal neurons transfected with the Rbfox1‐luc containing either wt or 129mut (2‐highly conserved sites) binding sites. Dot‐plot presentation with mean luciferase activity (PTX/Veh) ± s.e.m. pGl4 (PTX/Veh) was set to 1 (n = 4; independent one‐sample t‐test, two‐tailed, heteroscedastic variance; Rbfox1‐luc wt **P = 0.00185, Rbfox1‐luc 129mut P = 0.0812).

Western blot analysis of Rbfox1 protein expression in PTX‐ or mock‐treated hippocampal neurons that were transfected with the indicated anti‐miRs. Upper panel: Representative Western blot. Tubulin was used as a loading control. Bottom panel: Quantification of three independent experiments. Dot‐plot presentation with mean relative protein levels (PTX/Veh) ± s.e.m. (n = 3; one‐way ANOVA; F (2,6) = 10.956, P = 0.010; groups were compared by Bonferroni post hoc test; anti‐miR‐129‐5p vs. anti‐miR Cont *P = 0.026; anti‐miR‐129‐5p vs. Empty *P = 0.016).

Overlap between targets that contain miR‐129‐5p and Rbfox binding motifs. Selected targets are indicated.

Source data are available online for this figure.

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