The dynamic membrane recruitment of miRNA processing complexes in neurons controls dendritogenesis (2017)
Figure 5

Figure 5: MiR-16-5p mediates BDNF-induced dendritogenesis of hippocampal neurons.

Schematic of single-cell dual-fluorescence miRNA sensor assay. The control sensor plasmid consists of GFP and dsRed expressed under a separate promoter (top panel). The miR16 sensor includes two miR-16-5p binding sites at the 3'UTR of dsRed. Binding of endogenous miR-16-5p to the dsRed mRNA would inhibit the translation of dsRed but not GFP (bottom panel). B) Endogenous miR-16-5p activity in developing hippocampal neurons. Hippocampal neurons were transfected with 500ng miR16 or control sensor at 4 DIV and with or without LNA control ('LNA Ctrl') or the anti-sense miR-16-5p inhibitor ('LNA-miR16'). Cells were fixed at 7 DIV and the number of neurons expressing GFP and not dsRed ('GFP-only') was compared to the total number of transfected neurons. LNA-miR16 reduces the amount of repression of the miR16 sensor compared to LNA-Ctrl (n=4, p=0.02, t-test type 2, error bars represent s.d.). C) Short-term BDNF stimulation inhibits endogenous miR-16-5p activity. Hippocampal neurons were transfected at 4 DIV and treated with BDNF either 24h or 20 minutes before fixation and neurons were counted as in B. Repression index was obtained by normalizing the miR16 sensor to the control sensor condition in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.). D&E) Inhibition of miR-16-5p increases dendritic complexity and occludes BDNF-induced dendritogenesis. D) Hippocampal neurons were transfected with the control sensor (A), and stimulated with control or BDNF 24 hours before fixation. Pictures were obtained on a confocal microscope and thresholds were set to dsRed signal fluorescence in order to distinguish individual dendrites (scale bars are 10μm). E) To quantify dendritic complexity the total number of dendritic intersections was calculated using Sholl analysis. Values were normalized to LNA-Ctrl and control treatment (n=3, p<0.04, t-test type 3, error bars represent s.d.). F&G) Overexpression of miR-16-5p blocks BDNF-induced dendritogenesis. F) Hippocampal neurons were transfected with control sensor and either control miRNA mimic (miR-Ctrl) or a miR-16-5p mimic (miR16) and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). G) Total number of intersections was quantified in Sholl analysis and values were normalized to miR-Ctrl, control treatment in each experiment (n=4, p=0.02, t-test type 3, error bars; s.d.). H&I) Overexpression of the Dicer-binding deficient deletion mutant of TRBP (GFP-TRBP Δ) occludes BDNF-induced dendritogenesis. H) Hippocampal neurons were transfected with 200ng dsRed and either 50ng GFP or 200ng GFP-TRBP Δ, and treated two days later with BDNF or control vehicle. DsRed signal fluorescence was used to set a threshold on images obtained on a confocal microscope (scale bars; 10μm). I) Total number of intersections was quantified in Sholl analysis and values were normalized to control treatment in each experiment (n=5, p<0.02, t-test type 3, error bars; s.d.).