Modulation of dendritic AMPA receptor mRNA trafficking by RNA splicing and editing

Abstract

RNA trafficking to dendrites and local translation are crucial processes for superior neuronal functions. To date, several α-amino-3-hydroxy-5-methyl-4-isoxazolepropionate receptor (AMPAR) mRNAs have been detected in dendrites and are subject to local protein synthesis. Here, we report the presence of all AMPAR GluA1-4 mRNAs in hippocampal and cortical rat synaptic spines by synaptoneurosomes analysis. In particular, we showed that dendritic AMPAR mRNAs are present in the Flip versions in the cortex and hippocampus. To further confirm these data, we demonstrate, using in situ hybridization, the dendritic localization of the GluA2 Flip isoform in vitro and in vivo, whereas the Flop variant is restricted mainly to the soma. In addition, we report that dendritic AMPA mRNAs are edited at low levels at their R/G sites; this result was also supported with transfection experiments using chimeric GluA2 DNA vectors, showing that transcripts carrying an unedited nucleotide at the R/G site, in combination with the Flip exon, are more efficiently targeted to dendrites when compared with the edited-Flip versions. Our data show that post-transcriptional regulations such as RNA splicing, editing and trafficking might be mutually coordinated and that the localization of different AMPAR isoforms in dendrites might play a functional role in the regulation of neuronal transmission.

Figure 1.

Fluorescence in situ hybridization of GluA1, GluA2 and β3-Tub mRNAs in primary cortical neurons. The right panel shows the hybridization signal (red) of the different mRNAs. In the left panel, the figures are merged with the immunostaining for MAP2. Under each panel, high magnification of the boxed dendritic regions are reported. Scale bar is 10 µm.

Figure 2.

Subcellular distribution of GluA1, GluA2 and β3-Tub mRNAs in the Cx (left panel) and rat Hc (right panel). In situ hybridization with alkaline phosphatase detection was used to visualize target mRNAs in coronal sections of the Cx and CA1 region of the Hc. Scale bar is 50 µm.

Figure 3.

RIP analysis of FMRP and CPEB3 protein from the rat Cx. The RT–PCR products for FMR1, αCamKII, GluA1, GluA2, TF2b and β3-Tub mRNAs are shown.

Figure 4.

Fluorescence in situ analysis of GluA1 (A), GluA2 (B) and β3-Tub (C) mRNA (red—left panel) is reported together with immunostaining with FMRP and CPEB3 (green—middle panel). The merged images are reported in the right panel. Magnifications of boxed dendritic regions are reported under each figure; the artificial white color shows co-localization of the mRNA and protein signals. Scale bar is 10 µm.

Figure 5.

(A) RT–PCR amplification products of GluA1 (lane 1), GluA2 (lane 2), GluA3 (lane 3) and GluA4 (lane 4) mRNAs in hippocampal total and SNS preparations. β3-Tub mRNA (lane 5), a soma-localized mRNA, was used as negative control and was not detected in SNS samples. (B) The relative expression of Flip isoform for GluA1–4 mRNAs in total and SNS preparations of Cx and Hc are reported. Bars represent Flip isoform expression (reported as a percentage of total transcript expression) in the different preparations. Data are presented as means ± SEM (n ≥ 5). Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. SNS versus Total *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 6.

Fluorescent in situ hybridization of GluA2 mRNA in Flip and Flop splicing variants in (A) CA1 hippocampal and (B) cortical coronal sections. (C) The sense probe was used as negative control. Flip Flop hybridizations were performed on consecutive sections. (D) In situ detection of individual transcripts with padlock probes and target-primed RCA. After in situ reverse transcription and RNA degradation, the cDNA was probed using a Padlock probe. The probes were ligated and subject to RCA. RCPs were identified through hybridization with fluorescent detection probes. Figure reports the double in situ detection of GluA2 Flip mRNA (red) and GluA2 Flop mRNA (green). Scale bar is 10 µm.

Figure 7.

RNA editing analysis of the GluA2 Q/R and GluA2-4 R/G sites in total and SNS preparations derived from the rat Cx and Hc. Bars represent the editing efficiency (percentage of edited transcripts) in the different preparations for the Q/R and R/G sites. Data are presented as means ± SEM (n ≥ 5). Statistical analysis was performed using one-way ANOVA followed by Bonferroni post hoc test. SNS versus Total *P < 0.05, **P < 0.01, ***P < 0.001.

Figure 8.

(A) Scheme of the chimeric mRNAs (GluA2-A-Flip; GluA2-G-Flip, MAP2-DTE) used for cortical neurons (DIV12) transfection. (B) In situ hybridization of chimeric mRNAs; mRNAs were detected using a DIG-labeled antisense EGFP riboprobe (red signal in top panels) in tranfected neurons (green signal in bottom panels). Magnifications of the boxed dendritic regions are reported above each image. Scale bar is 10 µm.

Figure 9.

(A) Histogram showing the quantification of chimeric transcripts dendritic localization. The mean fluorescence intensity in dendrites up to 100 µm from the cell soma has been reported for the different chimeric mRNAs (n ≥ 20). (B) Histogram showing the mean fluorescence intensity in dendritic regions of 10 µm in length, up to 100 µm from the cell soma (n ≥ 20).