Figure 3. BDNF Increases Dicer Levels and the Biogenesis of Mature miRNAs.

(A) (Left) Percentage of miRNAs from TaqMan miRNA array with levels decreased over 50% (open bar) or increased over 2-fold (black bar) by BDNF (30 min, plus Actinomycin-D). (Right) Scatter plot of relative quantities (RQ) of individual miRNA species (solid circles) following BDNF relative to mock-stimulation. Red line = 1.0 or no change; each dot above the line represents a miRNA species increased by BDNF, each dot below the line represents a miRNA species decreased by BDNF. Normalization is to averaged reference RNAs U6snRNA, and snoRNA202, which are unchanged by BDNF; n = 3 separate miRNA array pairs for mock and BDNF conditions.

(B) Immunoblot of cultured hippocampal neurons stimulated with BDNF for indicated min in the presence of Actinomycin-D. Dicer peaks near 20 and declines by 60 min.

(C) (Top) BDNF enhances TRBP and ERK phosphorylation as shown by immunoblot for TRBP and phospho-Erk. Cultured hippocampal neurons were stimulated with BDNF for indicated min in the presence of Actinomycin-D. (Bottom) Lysates incubated with λ-phosphatase (λ-phos) as indicated demonstrate loss of phosphorylated TRBP (upper band).

(D) P body appearance in dendrites of hippocampal pyramidal neurons expressing control (sh-control-2, top) or Dicer-targeting shRNA (DicerKD, bottom) following BDNF, t = min poststimulation.

(E) Quantification and time course of P body numbers in Dicer-deficient (DicerKD, boxes) or control (sh-control-2, circles) expressing hippocampal neurons following mock (open shapes) or BDNF stimulation (closed shapes).

(F) Quantification and time course of P body numbers in hippocampal pyramidal neurons treated with enoxacin (15 µM, closed circles) or oxolinic acid (15 µM, open circles).

(G) The effect of Dicer loss on BDNF-regulated protein synthesis. (Top) Immunoblotting for BDNF target proteins in Dicer-wild-type (Dicer^flox/flox) or Dicer-deficient (Dicer^−/−) neurons. Cre^ERT2-expressing cells were treated with 4-hydroxy tamoxifen (800 nM) to induce recombination for 2.5 days before BDNF stimulation.

Asterisk indicates nonspecific band. (Bottom) Densitometric quantification of immunoblots.

All error bars represent SEM. *p < 0.05 by unpaired Student’s t test. See also Figure S4.