Posttranscriptional regulation of gene expression in learning by the neuronal ELAV-like mRNA-stabilizing proteins

Abstract

The view that memory is encoded by variations in the strength of synapses implies that long-term biochemical changes take place within subcellular microdomains of neurons. These changes are thought ultimately to be an effect of transcriptional regulation of specific genes. Localized changes, however, cannot be fully explained by a purely transcriptional control of gene expression. The neuron-specific ELAV-like HuB, HuC, and HuD RNA-binding proteins act posttranscriptionally by binding to adenine- and uridine-rich elements (AREs) in the 3' untranslated region of a set of target mRNAs, and by increasing mRNA cytoplasmic stability and/or rate of translation. Here we show that neuronal ELAV-like genes undergo a sustained up-regulation in hippocampal pyramidal cells only of mice and rats that have learned a spatial discrimination paradigm. This learning-specific increase of ELAV-like proteins was localized within cytoplasmic compartments of the somata and proximal dendrites and was associated with the cytoskeleton. This increase was also accompanied by enhanced expression of the GAP-43 gene, known to be regulated mainly posttranscriptionally and whose mRNA is demonstrated here to be an in vivo ELAV-like target. Antisense-mediated knockdown of HuC impaired spatial learning performance in mice and induced a concomitant down-regulation of GAP-43 expression. Neuronal ELAV-like proteins could exert learning-induced posttranscriptional control of an array of target genes uniquely suited to subserve substrates of memory storage.

Figure 1

Up-regulation of neuronal ELAV-like gene expression in mouse hippocampi after radial arm maze training. (A) (Upper) Representative Western blots showing whole-cell lysate immunoreactivity of the ELAV-like and BRUNO-like proteins (using the 16A11 and 3B1 mAbs, respectively) in the passive control (PC), active control (AC), and trained (TR) mouse groups. (Lower) Average results (means ± SEM) for ELAV-like proteins normalized to α-tubulin (n = 5 for each group; ***, P < 0.005, post hoc analysis between AC and TR mice). (B) Determination of the steady-state levels of HuB, HuC, and HuD mRNA by external standard-based, real-time quantitative RT-PCR. The values obtained from hippocampal RNA preparations of the three groups of mice (white bar, PC; light gray bar, AC; black bar, TR) have been normalized to the level of GAPDH mRNA and expressed as means ± SEM (n = 6 for each group; ****, P < 0.001, post hoc analysis between AC and TR mice). (C) Representative images showing the distribution of ELAV-like immunostaining for AC and TR animals in the pyramidal layer of the CA3 hippocampal subregion. (Scale bar, 25 μm.) (D) Representative Western blots (Upper) and average ELAV-like protein levels (Lower) after tissue fractionation. α-Tubulin levels are shown as a control of the nucleocytoplasmic separation, and ELAV-like protein levels are reported as means ± SEM (n = 6 for each group, ***, P < 0.005; ****, P < 0.001, Student's t test). All experiments were repeated at least three times for each hippocampal tissue sample. For statistical analysis, values were subjected to one-way ANOVA and a post hoc Tukey's test unless stated otherwise.

Figure 2

Up-regulation of HuC gene expression in rat hippocampi after Morris water maze training. Representative in situ hybridization results are reported above for both the HuC and the BRUNOL3 genes in naïve (NV), swimming control (SW), and trained (TR) animals. Below, means ± SEM of the densitometric determinations of the mRNA signal in the CA1, CA3, and dentate gyrus (DG) subfields for six animals in each group (white bar, NV; light gray bar, SW; black bar, TR; ****, P < 0.001, post hoc analysis between SW and TR rats).

Figure 3

In vivo binding of GAP-43 mRNA by neuronal ELAV-like proteins and enhancement of GAP-43 gene expression by learning. (a) (Lower) Magnetic bead-mediated coseparation of ELAV-like proteins and GAP-43 mRNA with the use of an RNA oligonucleotide reproducing the GAP-43 ELAV-like binding ARE (Left) and the 16A11 mAb (Right). (Upper) Schematic representation of the strategy used in either case. The presence of the protein and of different mRNAs in the bead-associated and the free fractions is assessed respectively by Western blotting with the 16A11 mAb and by qualitative RT-PCR with specific primer pairs. Negative controls for both experiments are the absence of the bead-linked ligand (RNA oligonucleotide or 16A11 mAb) and a nonligand (fully degenerate RNA oligonucleotide, DN RNA, or a mAb of the same 16A11 isotype, Is). (b) Representative in situ hybridization histochemistry obtained with a GAP-43 mRNA probe in brain slices of AC and TR mice. Note the absence of GAP-43 expression in the dentate gyrus. (c) Representative Western blot (Upper) and related average results normalized to α-tubulin immunoreactivity (Lower; means ± SEM) of GAP-43 protein levels in hippocampi of PC, AC, and TR mice (n = 6 for each group; ****, P < 0.001, post hoc analysis between AC and TR mice).

Figure 4

Behavioral and biochemical effects of antisense-mediated knockdown of HuC gene expression in mouse brain. (a) Performance in the probe session of the radial maze task in sham controls (TR) and in mice daily infused during the training with fully sequence-degenerate (TR + DN) or anti-HuC antisense (TR + AS) oligonucleotides (n = 9 for TR animals and n = 8 for TR + DN and TR + AS animals). Data are expressed as means ± SEM (**, P < 0.01, post hoc analysis between DN-treated and AS-treated mice). (b) Effect of the anti-HuC antisense treatment on HuC gene expression. (Left) Real-time quantitative RT-PCR determination of HuC mRNA steady-state levels normalized to GAPDH levels. (Right) (Upper) Representative Western blot of the overall ELAV-like immunoreactivity in the three groups of mice compared with α-tubulin. (Lower) Means ± SEM, n = 6 each group for both mRNA and protein determinations. ****, P < 0.001, post hoc analysis between DN-treated and AS-treated mice. (c) Sequence specificity of the antisense-mediated HuC gene expression down-regulation. Representative in situ hybridizations showing hippocampal mRNA levels for the HuC and for the phylogenetically related BRUNOL3 genes in the AS ODN-treated and the two control mice groups. (d) Effect of the anti-HuC antisense treatment on GAP-43 gene expression. (Left) Real-time quantitative RT-PCR determination of GAP-43 mRNA levels normalized to GAPDH levels in the hippocampi of the three groups of mice (means ± SEM, n = 6 for each group; **, P < 0.01, post hoc comparison between DN-treated and AS-treated mice). (Right) Densitometric analysis of GAP-43 mRNA levels in the CA1 and CA3 hippocampal subfields from in situ hybridization performed on brain slices of the same mice (means ± SEM, data collected from five to seven mice from each group; *, P < 0.05; ***, P < 0.005).