MicroRNA regulation of homeostatic synaptic plasticity

Abstract

Homeostatic mechanisms are required to control formation and maintenance of synaptic connections to maintain the general level of neural impulse activity within normal limits. How genes controlling these processes are co-coordinately regulated during homeostatic synaptic plasticity is unknown. MicroRNAs (miRNAs) exert regulatory control over mRNA stability and translation and may contribute to local and activity-dependent posttranscriptional control of synapse-associated mRNAs. However, identifying miRNAs that function through posttranscriptional gene silencing at synapses has remained elusive. Using a bioinformatics screen to identify sequence motifs enriched in the 3'UTR of rapidly destabilized mRNAs, we identified a developmentally and activity-regulated miRNA (miR-485) that controls dendritic spine number and synapse formation in an activity-dependent homeostatic manner. We find that many plasticity-associated genes contain predicted miR-485 binding sites and further identify the presynaptic protein SV2A as a target of miR-485. miR-485 negatively regulated dendritic spine density, postsynaptic density 95 (PSD-95) clustering, and surface expression of GluR2. Furthermore, miR-485 overexpression reduced spontaneous synaptic responses and transmitter release, as measured by miniature excitatory postsynaptic current (EPSC) analysis and FM 1-43 staining. SV2A knockdown mimicked the effects of miR-485, and these effects were reversed by SV2A overexpression. Moreover, 5 d of increased synaptic activity induced homeostatic changes in synaptic specializations that were blocked by a miR-485 inhibitor. Our findings reveal a role for this previously uncharacterized miRNA and the presynaptic protein SV2A in homeostatic plasticity and nervous system development, with possible implications in neurological disorders (e.g., Huntington and Alzheimer's disease), where miR-485 has been found to be dysregulated.

Fig. 1.

miR-485 is expressed in hippocampal neurons and developmentally regulated. (A and B) Expression of miR-485 was detected by FISH and RT-PCR. U6 snRNA and scrambled probe are shown as positive and negative controls, respectively. scr-miR, scrambled miR probe. Dendrites (red) were identified by MAP2 immunoreactivity, and nuclei (blue) were stained with Hoechst 33342. (Scale bar = 25 μm.) (B) RT-PCR was performed on neuronal processes growing through 3-μm pores in transwell inserts to isolate from cell soma in the upper compartment. P, process; S, soma + process. Mature miR-485 (based on product size) was present in both fractions. U6 (U6 snRNA) and γ-actin served as reference genes. β-actin was approximately twofold higher in processes, indicating enrichment of neuronal processes. (C) miR-485 transcript is developmentally regulated in the hippocampus. The increase in mature miR-485 (n = 3) at each time point was first normalized to the reference gene U6 snRNA and the abundance expressed relative to embryonic day 18.5. E, embryonic day.

Fig. 2.

miR-485 decreases SV2A expression in hippocampal neurons. (A) SV2A is a putative target of miR-485. Luciferase assays to validate SV2A as a target of miR-485 were performed on constructs containing either the full-length 3′UTR (SV2A-3′UTR), distal 3′UTR containing two miR-485 binding sites (SV2A), or mutated sites in the 5′-seed of MRE1 and MRE2 (SV2A MUT) as controls (Fig. S2B). Translational suppression by miR-485 was normalized to empty psiCHECK-2 plasmid. Treatment with the miR-M decreased SV2A transcript abundance (B) and protein levels (C) significantly without affecting SYP38 or PSD-95. Treatment with negative controls for either the mimic or inhibitor did not alter transcript or protein levels for SV2A, SYP38, or PSD-95. -co. control. (D) miR-485 overexpression reduced SV2A expression in hippocampal neuron synapses. Cultures were cotransfected with DsRed and miRNAs at 7 days in vitro (DIV) and analyzed by immunocytochemistry at 12 DIV for SV2A and SYP38 expression. Representative examples of untreated (control) and neurons transfected with the miR-M or miR-I showing that SV2A expression is reduced in individual presynaptic terminals. (Lower) Higher magnification is shown. Treatment with negative controls did not alter SV2A or SYP38 expression. (Scale bars: Upper, 10 μm; Lower, 2.5 μm.) (E) Quantitative analysis of randomly chosen dendritic fields showing that the miRNA specifically reduced colocalization of SV2A and SYP38.

Fig. 3.

miR-485 negatively regulates spine density, PSD-95 clustering, and surface GluR2 expression. miR-485 reduces spine density (A), PSD-95 clustering (B), and surface GluR2 expression (C) along dendrites. Spine density, PSD-95, and SEP-GluR2 were 12.7 ± 0.4 spines per 20 μm (n = 18), 9.4 ± 0.3 PSD-95–GFP⁺ puncta per 20 μm (n = 34), and 6.9 ± 0.3 SEP-GluR2 puncta per 20 μm (n = 16) in untreated controls, respectively. These values were not affected by transfection with negative controls for the mimic [12.4 ± 0.3 spines per 20 μm (n = 20), 9.1 ± 0.2 PSD-95–GFP⁺ puncta per 20 μm (n = 36), and 7.3 ± 0.3 SEP-GluR2 puncta per 20 μm (n = 16)] or inhibitor [12.6 ± 0.4 spines per 20 μm (n = 19), 9.3 ± 0.2 PSD-95–GFP⁺ puncta per 20 μm (n = 35), and 7.3 ± 0.2 SEP-GluR2 puncta per 20 μm (n = 17)]. (D) Endogenous PSD-95 clustering (green) in hippocampal cultures was decreased by transfection with the miR-M and increased by the miR-I. (Scale bar = 10 μm.) (E) Representative examples of neurons transfected with DsRed, PSD-95 (PSD-95–GFP), or surface GluR2 (SEP-GluR2). (Scale bar = 2.5 μm.)

Fig. 4.

miR-485 reduces spontaneous synaptic activity in hippocampal neurons. (A) Representative traces of spontaneous mEPSCs recorded from cultured hippocampal neurons transfected with negative control for the miR-M (a), miR-M (b), negative control for miR-I (c), and miR-I (d) showing a reduction in spontaneous mEPSC frequency after transfection with the mimic (b) and an increase following transfection with the inhibitor (d). (B) Quantitative analysis showing the transfection significantly changed mEPSP frequency [χ²(3,313) = 191.984, P < 0.001]. (C) Synaptic vesicle release (FM 1-43 destaining kinetics) was not affected by miR-485, but the number of recycled vesicles (amount of destaining) was reduced by miR-485 (yellow) and increased by the inhibitor (red) (P < 0.05). Destaining curves for negative controls (n = 8 coverslips with at least 60 boutons each) and for cultures treated with the miR-M (n = 8) and miR-I (n = 11) are shown.

Fig. 5.

SV2A siRNA-mediated knockdown mimics the effects of miR-485 overexpression on reducing dendritic spine density, PSD-95 clustering, and surface GluR2 expression. SV2A siRNA reduces spine density (A), PSD-95 clustering (B), and surface GluR2 expression (C) along dendrites. Spine density, PSD-95, and SEP-GluR2 were 10.4 ± 0.3 spines per 20 μm (n = 33), 10.3 ± 0.4 PSD-95–GFP⁺ puncta per 20 μm (n = 20), and 6.9 ± 0.3 SEP-GluR2 puncta per 20 μm (n = 16) in untreated controls, respectively. These values were not affected by transfection with negative controls for the siRNA [10.5 ± 0.3 spines per 20 μm (n = 23), 10.3 ± 0.3 PSD-95–GFP⁺ puncta per 20 μm (n = 27), and 7.3 ± 0.3 SEP-GluR2 puncta per 20 μm (n = 16)] or miRNA mimic [10.5 ± 0.3 spines per 20 μm (n = 23), 9.8 ± 0.3 PSD-95–GFP⁺ puncta per 20 μm (n = 20), and 7.3 ± 0.3 SEP-GluR2 puncta per 20 μm (n = 16)]. (D) Representative examples for A–C. (Scale bar = 2.5 μm.)

Fig. 6.

BiC/4-AP for 5 days in vitro (DIV) induces a homeostatic reduction in synaptic connectivity that is reversed by blocking endogenous miR-485. Hippocampal neurons transfected with a miR-I and constructs to visualize changes in synaptic properties were treated with 50 μm BiC plus 500 μm 4-AP for 5 d. BiC/4-AP reduces spine density (A), PSD-95 clustering (B), and surface GluR2 expression (C) along dendrites that are reversed by the miR-I. Spine density, PSD-95, and SEP-GluR2 were 10.8 ± 0.4 spines per 20 μm (n = 35), 9.3 ± 0.3 PSD-95–GFP⁺ puncta per 20 μm (n = 29), and 6.9 ± 0.3 SEP-GluR2 puncta per 20 μm (n = 16) in untreated controls. These values were not affected by transfection with negative controls for the miRNA inhibitor [11.2 ± 0.4 spines per 20 μm (n = 30), 9.1 ± 0.2 PSD-95–GFP⁺ puncta per 20 μm (n = 32), and 7.3 ± 0.2 SEP-GluR2 puncta per 20 μm (n = 17)]. (D) Representative examples for A–C. (Scale bar = 2.5 μm.)