Dendritic trafficking of brain-derived neurotrophic factor mRNA: regulation by translin-dependent and -independent mechanisms

Abstract

Dendritic trafficking and translation of brain-derived neurotrophic factor (BDNF) transcripts play a key role in mediating synaptic plasticity. Recently, we demonstrated that siRNA-mediated knockdown of translin, an RNA-binding protein, impairs KCl-induced dendritic trafficking of BDNF mRNA in cultured hippocampal neurons. We have now assessed whether translin deletion impairs dendritic trafficking of BDNF mRNA in hippocampal neurons in vivo. We have found that translin and its partner protein, trax, undergo dendritic translocation in response to treatment with pilocarpine, a pro-convulsant muscarinic agonist that increases dendritic trafficking of BDNF mRNA in hippocampal neurons. In translin knockout mice, the basal level of dendritic BDNF mRNA is decreased in CA1 pyramidal neurons. However, translin deletion does not block pilocarpine's ability to increase dendritic trafficking of BDNF mRNA indicating that the requirement for translin in this process varies with the stimulus employed to drive it. Consistent with this inference, we found that dendritic trafficking of BDNF mRNA induced by bath application of recombinant BDNF in cultured hippocampal neurons, is not blocked by siRNA-mediated knockdown of translin. Taken together, these in vivo and in vitro findings indicate that dendritic trafficking of BDNF mRNA can be mediated by both translin-dependent and -independent mechanisms.

Figure 1. Specificity of translin and trax immunostaining in brain

A) Immunoblots of forebrain extracts harvested from adult wild-type (+/+), heterozygous (+/-), or translin knockout (-/-) mice show that translin and trax protein levels are reduced below wild type levels in heterozygous samples and absent in knockout samples. Tubulin blot shown as loading control. B) The translin/trax gel-shift complex (indicated by arrow at left of blot) is absent in cerebellar (Cb) and hippocampal (Hip) extracts harvested from translin knockout mice. C and D) Low power images of cortex (top panels) and hippocampus (bottom panels) show immunostaining for both translin and trax in sections taken from wild type mice (wt). In contrast, only negligible staining is detected in corresponding sections taken from knockout (ko) mice. Note that while some residual translin staining is detected in the dentate gyrus and the overlying stratum lacunosum-moleculare, the CA1 region is devoid of staining.

Figure 2. Dendritic localization of translin and trax

(A) Panels show images taken of the hippocampal CA1 region. In control mice (Ctrl), translin and trax staining is restricted to the cell bodies of CA1 pyramidal neurons but translocate out into apical dendrites in the stratum radiatum in mice treated with pilocarpine (Pilo) 3 hours prior to perfusion. As shown in the right hand panels, BDNF mRNA is restricted to the cell bodies of pyramidal neurons of CA1 in control mice but undergoes translocation into apical dendrites following pilocarpine treatment. (B) In contrast to their localization pattern in hippocampus, both translin and trax, as well as BDNF mRNA, are present in dendrites of cortical pyramidal neurons in control mice (arrows). These panels show layer V pyramidal neurons in the barrel field of the somatosensory cortex (S1BF).

Figure 3. Association of translin and trax

(A) Co-immunoprecipitation of translin and trax from mouse forebrain extracts. Extracts were incubated with or without trax antibody and then aliquots of the starting lysate, supernatant or pellet were processed for immunoblotting with either trax (left blot) or translin (right blot). The lanes located at the right of each blot contain the pellets obtained without (-) or with (+) trax antibody. (The additional bands present in the rightmost lane of the trax blot reflect heavy and light chains of the trax antibody, since the same guinea pig antibody was used for both the i.p. and blotting steps.) The lanes located at the extreme left of both blots contain aliquots of the starting lysate or “offered” (Off). The lanes labeled (S+) contain the supernatant from the tubes that were incubated with trax antibody and show depletion of both trax and translin relative to the levels found in the offered sample. B) Co-localization of recombinant translin and trax in mouse hippocampal neurons. Mouse cultures were transfected at 7 DIV with both mCherry Translin and Trax GFP plasmids and then fixed the next day. Low power images shown in top row illustrate co-localization of these constructs throughout the cell. Staining with MAP2 antibody indicates that both proteins localize to MAP2-positive, dendritic processes as well as a MAP2-negative process that extends from the top right corner of the cell body, which appears to be an axonal process. Bottom panels show high power images of boxed area in top left panel. We have also detected endogenous trax puncta in MAP2-negative, presumed axonal processes indicating that localization to axons is not an artifact of overexpression of these recombinant constructs (data not shown). Co-localization of recombinant translin and trax was also observed following co-transfection of rat hippocampal cultures. C) Co-localization of endogenous trax puncta with recombinant translin. Top panels show low power images of a rat hippocampal neuron (8DIV) that was transfected with a myc-His translin construct (green) and stained for endogenous trax (red). Bottom panels show high power images of boxed area in top left panel. Arrowheads indicate discrete puncta that are stained for both myc-His translin and endogenous trax.

Figure 4. Effect of translin deletion on the pattern of BDNF mRNA localization

A) Low power images of cortex (top panels) or hippocampus (bottom panels) show staining obtained by processing brain sections from wild type (wt) and translin knockout (ko) mice for in situ hybridization with a BDNF mRNA probe targeting the coding region. The overall hybridization pattern displayed by wt and ko mice with the anti-sense probe is similar. The hybridization signal was absent in adjacent sections from wt mice that were processed with the corresponding sense probe (right panels). B) Top panels show BDNF mRNA hybridization signal in the CA1 region of control wild type (left panel) and translin knockout (right panel) mice. Bottom panels show increased dendritic localization (arrows) following pilocarpine treatment. High magnification images correspond to areas indicated by arrows in the panels directly to their left.

Figure 5. Effect of translin deletion on dendritic localization of BDNF mRNA in CA1 region in control and pilocarpine-treated mice

Bar graph shown presents quantification of hybridization signal in dendrites by treatment (control and pilocarpine) and genotype (wt and ko). For each of the distances shown from the edge of the CA1 cell body layer, the dendritic trafficking index was determined by dividing the average signal intensity for that 15 μm interval in the stratum radiatum by the average staining intensity of the overlying cell body layer. Error bars indicate standard errors. Two-way ANOVA analysis at each interval, except the 50 to 65 μm distance, revealed significant main effects of both genotype and treatment (p<0.005). At the longest distance, 50 to 65 μm, there is a significant effect of pilocarpine (p<0.0005) but not of genotype. There was no significant interaction effect at any of these intervals (p>0.5).

Figure 6. Differential effect of translin siRNA on dendritic trafficking of BDNF mRNA induced by BDNF or KCl

Treatment of rat hippocampal cultures with translin siRNA markedly decreases the ability of KCl to stimulate dendritic trafficking of BDNF mRNA, but does not impair the response to BDNF (50ng/ml). The effects of these stimuli are plotted as the fold increase in the relative dendritic filling value determined for each experimental condition. The specificity of the BDNF response is confirmed by its blockade by pre-treatment with K252a (30nM, 30 minutes), a selective antagonist of TrkB tyrosine kinase activity. Asterisks indicate significant difference (p<0.001) compared to control values.