HuD interacts with Bdnf mRNA and is essential for activity-induced BDNF synthesis in dendrites.

Highly specific activity-dependent neuronal responses are necessary for modulating synapses to facilitate learning and memory. We present evidence linking a number of important processes involved in regulating synaptic plasticity, suggesting a mechanistic pathway whereby activity-dependent signaling, likely through protein kinase C (PKC)-mediated phosphorylation of HuD, can relieve basal repression of Bdnf mRNA translation in dendrites, allowing for increased TrkB signaling and synaptic remodeling. We demonstrate that the neuronal ELAV family of RNA binding proteins associates in vivo with several Bdnf mRNA isoforms present in the adult brain in an activity-dependent manner, and that one member, HuD, interacts directly with sequences in the long Bdnf 3' untranslated region (3'UTR) and co-localizes with Bdnf mRNA in dendrites of hippocampal neurons. Activation of PKC leads to increased dendritic translation of mRNAs containing the long Bdnf 3'UTR, a process that is dependent on the presence of HuD and its phosphorylation at threonine residues 149 and/or 165. Thus, we found a direct effect of HuD on regulating translation of dendritic Bdnf mRNAs to mediate local and activity-dependent increases in dendritic BDNF synthesis

Quantitative RT-PCR analysis of <i>in vivo</i> mRNPs containing neuronal Hu proteins.

<p>(A) mRNPs were immunoprecipitated from mouse whole brain lysates using either the HuC/HuD neuronal protein antibody (16A11) or mouse IgG isotype control, and subjected to quantitative RT-PCR analysis using primers specific to <i>Bdnf</i> mRNA isoforms I, II, IV and VI, as well as primers for <i>Rpl10a</i> as reference control. Fold change was calculated as 2^(-ΔΔCt), using values from the mouse IgG IP as control (Mean ± SEM; n = 3; *, p<0.05; **, p<0.01; student's t-test). (B) Schematic showing the drug injection protocol for seizure induction. Atropine MeNO₃ was injected alone or with Ro-32–0432 (systemic PKC inhibitor), followed 30 minutes later by injection of pilocarpine for seizure induction or PBS as a negative control, and mice were euthanized 30 minutes later, collecting forebrain tissue for isolation of mRNPs. (C) mRNPs were immunoprecipitated using lysates from mice injected with either pilocarpine or PBS. Percent recovery versus control was calculated as the ratio of the Ct for the IP sample to the Ct for the pre-IP sample, normalized to the value obtained from the PBS-injected mice (Mean ± SEM; n = 3; *, p<0.05; **, p<0.01; student's t-test). (D) The experiment from (C) was repeated with the modification that all mice were pre-injected with Ro-32–0432, a systemic PKC inhibitor (Mean ± SEM; n = 3; student's t-test).</p

PMA treatment leads to PKC-dependent increases in dendritic translation of local reporter constructs with the long <i>Bdnf</i> 3' UTR.

<p>(A) Schematic representing the constructs used. (B and C) Colorized dendritic GFP fluorescence from dissociated rat hippocampal cultures transfected at 14 DIV with the indicated constructs and on 16 DIV treated for 1 hour with KCl, PMA or vehicle, fixed and then imaged (scale bar, 20 μm). (D and E) Mean ± SEM in 50-μm bins from images represented in (B) and (C) (n = 25; *, p<0.05; **, p<0.01; student's t-test). (F) Colorized dendritic GFP fluorescence from hippocampal cultures transfected at 14 DIV with ExIV A*B and on 16 DIV pre-treated for 30 min with GF109203X, a PKC inhibitor, then treated for 1 hour with KCl or PMA, fixed and then imaged (scale bar, 20 μm). (G) Mean ± SEM in 50-μm bins from images represented in (F) (n = 25; *, p<0.001; student's t-test).</p

PKC-dependent increases in dendritic translation of local reporter constructs with the long <i>Bdnf</i> 3' UTR are mediated by HuD.

<p>(A) Schematic representing the mouse HuD overexpression cassettes with <i>in silico</i>-predicted threonine phosphorylation sites and their scores (RRM1–3, RNA recognition motifs; open triangles, predicted threonine phosphorylation sites; closed triangles, threonine-to-alanine substitutions). (B) Immunocytochemistry of hippocampal cultures transfected with the indicated plasmids on 14 DIV, then fixed, stained with monoclonal anti-HuD antibody and imaged on 16 DIV (Control: ExIV A*B, pSuper, pcDNA; HuD Knockdown: ExIV A*B, rHuD shRNA, pcDNA; mHuD Recovery: ExIV A*B, rHuD shRNA, mHuD; mHuDpd Recovery: ExIV A*B, rHuD shRNA, mHuDpd; scale bar, 20 μm). (C) Mean ± SEM of soma from images represented in (B) (n = 30; *, p<0.05; **, p<0.001; student's t-test). (D) Colorized dendritic GFP fluorescence from hippocampal cultures transfected at 14 DIV with the indicated plasmids, then on 16 DIV treated for 1 hour with PMA or vehicle, fixed and then imaged (scale bar, 20 μm). (E) Mean ± SEM in 50-μm bins from images represented in (D) (n = 40; *, p<0.05; **, p<0.01; ***, p<0.001; student's t-test against control for each bin; †, p<0.001; student's t-test against conditions underneath bar).</p

Changes in HuD expression and short-term PMA treatment do not affect levels of dendritic mRNAs with <i>Bdnf</i> 5' and 3'-UTRs.

<p>(A) Dual <i>Bdnf</i> mRNA FISH and HuD immunocytochemistry on hippocampal cultures transfected with recombinant GFP-mHuD at 14 DIV and fixed, stained and imaged at 16 DIV, using antisense probe against the <i>Bdnf</i> coding sequence for <i>in situ</i> and anti-GFP for immunocytochemistry (n = 30; scale bar, 20 μm). Co-localization data was obtained using the JACoP plugin on ImageJ. (B) Colorized dendritic <i>in situ</i> signal using a probe against the <i>Bdnf</i> mRNA coding sequence in hippocampal cultures transfected with the indicated plasmids at 14 DIV and then fixed at 16 DIV (sense, sense <i>in situ</i> probe; scale bar, 20 μm). (C) Mean ± SEM in 20-μm bins from images represented in (B), normalized to the mean dendritic signal from 20–40 μm away from the soma of untransfected neurons on the same coverslip (n = 30; *, p<0.05; **, p<0.01; student's t-test). (D) Colorized dendritic <i>in situ</i> signal using a probe against the GFP coding sequence in hippocampal cultures transfected with the reporter construct ExIV A*B at 14 DIV and then treated for 1 or 3 hours with PMA or vehicle, then fixed at 16 DIV (sense, sense <i>in situ</i> probe; scale bar, 20 μm). (E) Mean ± SEM in 20-μm bins from images represented in (D) (n = 30; *, p<0.01; **, p<0.001; student's t-test).</p

A model on <i>Bdnf</i> mRNA translation.

<p>The model illustrates the proposed mechanism of the PKC-mediated component of activity-dependent upregulation of local <i>Bdnf</i> mRNA translation in dendrites.</p

HuD transport to dendrites requires phosphorylation at T149 and/or T165.

<p>(A) Colorized dendritic HuD immunocytochemistry signal in hippocampal cultures transfected at 14 DIV with the indicated plasmids, then treated for 1 hour with PMA or vehicle, fixed, stained with monoclonal anti-HuD antibody and imaged at 16 DIV (US, unstimulated; scale bar, 20μm). (B) Mean ± SEM in 20-μm bins from images represented in (A) (n = 30; *, p<0.05; **, p<0.01; student's t-test against control for each bin; †, p<0.05; student's t-test against conditions underneath bar).</p

Effect of <i>Bdnf</i> 5' UTR sequences on local reporter synthesis.

<p>(A) Schematic representing the constructs used. SYN, human synapsin promoter; I, IIc, IV, VI, <i>Bdnf</i> 5' UTR sequences; dEGFP, destabilized EGFP; myr, myristoylation sequence; NLS, nuclear localization sequence; A, short <i>Bdnf</i> 3' UTR; B, long <i>Bdnf</i> 3' UTR; dashed line, non-functional mutated polyadenylation site. (B) Somatic GFP fluorescence from dissociated rat hippocampal cultures transfected at 7 DIV with the indicated constructs and fixed and imaged at 9 DIV (scale bar, 20 μm). (C) Mean ± SEM from images represented in (B) (n = 30; *, p<0.05; **, p<0.01; ***, p<0.001; student's t-test). Intensity of GFP fluorescence is shown in arbitrary unit (AU). (D) Colorized dendritic GFP fluorescence from hippocampal cultures transfected at 7 DIV with the indicated constructs and fixed and imaged at 9 DIV, using min and max pixel values from representative images across all conditions to generate the color table (scale bar, 20 μm). (E) Mean ± SEM in 50-μm bins from images represented in (D) (n = 20; *, p<0.001; student's t-test).</p

HuD directly interacts with sequences in the long <i>Bdnf</i> 3' UTR, and this interaction is not impaired by substitution of PKC phosphorylation sites important for upregulation of its target mRNAs.

<p>(A) Schematic showing the regions of the <i>Bdnf</i> gene covered by the RNA probes used in the REMSA assay. (B) SDS PAGE of the purified recombinant GST-HuD and GST-HuDpd, stained with coomassie brilliant blue to show the extent of enrichment of the overexpressed GST fusion products. (C and E) Biotin-labeled RNA probes corresponding to the <i>Bdnf</i> mRNA regions shown in (A) were incubated with buffer (-) or the indicated purified recombinant GST fusion protein (+) and visualized on non-denaturing PAGE using IRdye-conjugated streptavidin. (D and F) Biotin-labeled RNA probe corresponding to the positive control <i>Nova1</i> 3' UTR sequence was incubated with buffer, the indicated GST fusion protein alone, or the protein pre-incubated with unlabeled competitor RNAs covering the sequences shown in (A) and visualized as in (C) and (E).</p

Hop2/Mnd1 acts on two critical steps in Dmc1-promoted homologous pairing

Meiotic recombination between homologous chromosomes ensures their proper segregation at the first division of meiosis and is the main force shaping genetic variation of genomes. The HOP2 and MND1 genes are essential for this recombination: Their disruption results in severe defects in homologous chromosome synapsis and an early-stage failure in meiotic recombination. The mouse Hop2 and Mnd1 proteins form a stable heterodimer (Hop2/Mnd1) that greatly enhances Dmc1-mediated strand invasion. In order to elucidate the mechanism by which Hop2/Mnd1 stimulates Dmc1, we identify several intermediate steps in the homologous pairing reaction promoted by Dmc1. We show that Hop2/Mnd1 greatly stimulates Dmc1 to promote synaptic complex formation on long duplex DNAs, a step previously revealed only for bacterial homologous recombinases. This synaptic alignment is a consequence of the ability of Hop2/Mnd1 to (1) stabilize Dmc1–single-stranded DNA (ssDNA) nucleoprotein complexes, and (2) facilitate the conjoining of DNA molecules through the capture of double-stranded DNA by the Dmc1–ssDNA nucleoprotein filament. To our knowledge, Hop2/Mnd1 is the first homologous recombinase accessory protein that acts on these two separate and critical steps in mammalian meiotic recombination