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. 2009 Feb 5;28(6):697–710. doi: 10.1038/emboj.2009.10

Figure 6.

Figure 6

Pum2 is a physiological target of miR-134 during activity-dependent dendritogenesis. (A) Two independent Pum2 shRNAs efficiently downregulate the expression of recombinant Pum2. Western blot analysis of HEK293T cell lysates transfected with the indicated amounts of GFP–Pum2 expression vectors, two Pum2 shRNAs as well as a control shRNA (4 ng each) were analysed by western blot using an anti-Pum2 antibody and an anti-eIF4E antibody as a loading control. (B) Si-Pum2-2 efficiently downregulates the expression of the endogenous Pum2 protein. Hippocampal neurons were transfected at DIV4 with GFP together with either Si-Pum2-2 (left) or a control siRNA (right, 2 ng each). The cells were fixed at DIV7 and analysed by immunocytochemistry using an anti-Pum2 antibody. Arrows point to cell bodies of representative transfected neurons. Note the reduced Pum2 signal in the cell transfected with Si-Pum2-2 (left) compared with the control neuron (right). Scale bar: 10 μm. (C) Pum2 knockdown by siRNA rescues the miR-134 loss-of-function phenotype in membrane-depolarized hippocampal neurons. Hippocampal neurons were transfected with GFP in conjunction with the indicated anti-miRs (50 nM) and shRNA constructs (2 ng). At 7DIV, neurons were incubated with 16 mM KCl for 6 h and dendritic complexity was evaluated 3 days later. Scale bar: 20 μm. (D) Quantitative analysis of the dendritogenesis assay shown in (C). The calculation of dendritic complexity and the data presentation are as in Figure 4B. Here, 10–16 cells for condition were analysed in each experiment. Data represent the mean of three independent experiments+s.d. *P<0.05 (t-test). (E) Overexpression of miR-134 duplex RNA perturbs membrane depolarization-induced dendritic outgrowth. Hippocampal neurons (4DIV) were transfected with GFP together with the indicated duplex RNAs (10 nM), and depolarization was performed at 7DIV followed by the assessment of dendritic complexity at 10DIV, as described in (C). Here, 10–16 cells for condition were analysed in each experiment. Data represent the mean of three independent experiments+s.d. *P<0.05 (t-test). (F) Both knockdown and overexpression of Pum2 perturb membrane depolarization-induced dendritic outgrowth. Hippocampal neurons (4DIV) were transfected with GFP together with the indicated shRNA (2 ng) or Pum2 expression (100 ng) constructs, and depolarization was performed at 7DIV followed by the assessment of dendritic complexity, as described in (C). Here, 10–16 cells for condition were analysed in each experiment. Data represent the mean of three independent experiments+s.d. *P<0.05 (t-test). (G) Schematic diagram of the fine-tuning of Pum2 levels by miR-134 in membrane-depolarized neurons, see main text for details. (H) Model for the miR379–410 activity-dependent dendritogenesis pathway. Neuronal activity activates Mef2 which in turn induces the expression of the miR379–410 cluster. Several members of the cluster are necessary for activity-dependent dendritogenesis. Among them is miR-134, which binds to the Pum2 mRNA and downregulates its translation. Lower levels of the translational repressor Pum2 allow for the translation of a set of so far unknown mRNAs that are likely positive regulators of dendrite outgrowth.