(Middle) The degree of colocalization within a given P body is not changed by BDNF stimulation. The percent of the total fluorescence that colocalized was significantly increased for both YFP-Pat1b and BFP-Dcp1a by BDNF (hatched bars) but not mock (checkered bars) stimulation. The fraction of colocalized fluorescence within a dendritic segment was calculated for each P body component by first summing the aggregate fluorescence values that colocalized with the other P body marker, then dividing this quantity by the value of the total fluorescence intensity within the dendrite for that channel, and multiplying by 100. Fontbase without a virus software#(Top) The fraction of total fluorescence of two coexpressed P body markers, YFP-Pat1b and BFP-Dcp1a, that colocalized was quantified from hippocampal dendrites imaged before and after BDNF stimulation using the spots function and colocalize spots tool in Imaris software (Bitplane). (I) BDNF rapidly enhances the colocalization of P body components by increasing their recruitment to P bodies, rather than by altering their synthesis. Collectively, these data show that the formation of P bodies, containing nontranslating RNA targeted for repression or degradation, is increased by BDNF, a stimulus known to enhance the activity of general translation factors and total cellular translation. Exclusion of ribosomal protein S6 (RPS6) was used to corroborate immunopurification purity ( Figure 1E). Immunoprecipitation of GW182 demonstrated that BDNF increased the association of P body components Argonaute 2 (Ago2) and Dcp1a with GW182 ( Figures 1E and 1F) and, as anticipated because P bodies require RNA for formation, BDNF induced a more than 2-fold increase in the total coimmunoprecipitated RNA ( Figure 1G). BDNF induces P body complex formation rather than synthesis of components because protein levels of endogenous Dcp1a or GW182, or GFP-Dcp1a were not altered by BDNF ( Figure S1H), and BDNF enhanced the total colocalization of two tagged P body components, Dcp1a and Pat1b, without altering their expression ( Figures S1F and S1I). Neurons were preincubated and imaged in the presence of the transcription inhibitor, Actinomycin-D, indicating that the rapid increase in P bodies can be mediated posttranscriptionally. Fontbase without a virus movie#Time-Lapse Movie of P Body Response in Soma and Dendrites of BDNF-Stimulated Hippocampal Neuron, Related to Figure 1) or endogenous staining ( Figure S1G). Time-Lapse Movie of P Body Response in Dendrites of a Mock-Stimulated Hippocampal Pyramidal Neuron, Related to Figure 1, Movie S3. Time-Lapse Movie of P Body Response in Dendrites of a BDNF-Stimulated Hippocampal Pyramidal Neuron, Related to Figure 1, Movie S2. BDNF-stimulated hippocampal pyramidal neurons responded with a rapid and robust increase in the number of both dendritic and somatic P bodies, compared to mock-stimulated neurons, as assessed by live imaging of GFP-Dcp1a ( Figures 1B–1D Movie S1. ) that colocalized with endogenous Dcp1a ( Figure S1A available online) and other P body components, including the RNA-binding protein GW182 (neuronal dendrites, Figures 1A and S1A–S1F). Our data establish that specificity in BDNF-regulated translation depends upon a two-part posttranscriptional control of miRNA biogenesis that generally enhances mRNA repression in association with GW182 while selectively derepressing and increasing translation of specific mRNAs. Lin28 deficiency, or expression of a Lin28-resistant Let-7 precursor miRNA, inhibits BDNF translation specificity and BDNF-dependent dendrite arborization. Binding sites for Lin28-regulated miRNAs are necessary and sufficient to confer BDNF responsiveness to a transcript. BDNF also rapidly induces Lin28, causing selective loss of Lin28-regulated miRNAs and a corresponding upregulation in translation of their target mRNAs. We report that BDNF rapidly elevates Dicer, increasing mature miRNA levels and inducing RNA processing bodies in neurons. The induction of protein synthesis by brain-derived neurotrophic factor (BDNF) critically contributes to enduring modifications of synaptic function, but how BDNF selectively affects only a minority of expressed mRNAs is poorly understood. Control of translation is a fundamental source of regulation in gene expression.
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