Dendritic glutamate receptor mRNAs show contingent local hotspot-dependent translational dynamics

Abstract

Protein synthesis in neuronal dendrites underlies long-term memory formation in the brain. Local translation of reporter mRNAs has demonstrated translation in dendrites at focal points called translational hotspots. Various reports have shown that hundreds to thousands of mRNAs are localized to dendrites, yet the dynamics of translation of multiple dendritic mRNAs has remained elusive. Here, we show that the protein translational activities of two dendritically localized mRNAs are spatiotemporally complex but constrained by the translational hotspots in which they are colocalized. Cotransfection of glutamate receptor 2 (GluR2) and GluR4 mRNAs (engineered to encode different fluorescent proteins) into rat hippocampal neurons demonstrates a heterogeneous distribution of translational hotspots for the two mRNAs along dendrites. Stimulation with s-3,5-dihydroxy-phenylglycine modifies the translational dynamics of both of these RNAs in a complex saturable manner. These results suggest that the translational hotspot is a primary structural regulator of the simultaneous yet differential translation of multiple mRNAs in the neuronal dendrite.

Figure 1. GluR2-RFP mRNA Shows Faster Translational Progression than GluR4-GFP mRNA in Hippocampal Neurons

(A) Example fluorescence images of neurons cotransfected with GluR2-RFP and GluR4-GFP mRNAs. Scale bar represents 10 μm. (B) Bar graphs display the time-dependent increase of the fluorescent intensities of each mRNA and controls from three independent experiments (117 cells for GluR2-RFP and GluR4-GFP mRNAs, and 94 cells for mock transfections). Error bars are SEM. (C) RT-PCR of a single neuron that was transfected with GluR2-RFP and GluR4-GFP mRNAs shows that comparable amounts of both mRNAs were transfected. Upper panel: M, DNA ladder; lane 1, single neuron without transfection; lane 2, single neuron transfected with GluR2-RFP mRNA and GluR4-GFP mRNA; lane 3, cDNA directly reverse transcribed from GluR2-RFP mRNA and GluR4-GFP mRNA; lane 4, no template. Lower panel: Quantitation of GluR2-RFP and GluR4-GFP PCR products was measured using UltraQuant v6.0 software (Specialty Laboratory). Primers are designed to span GluR ORFs (forward primers) and tomato/wasabi ORFs (reverse primers) and cDNAs are generated using reverse primers on the RNA from a single transfected cell. The control PCRs in lane 3 are generated with equal amounts of starting GluR2-RFP mRNA and GluR4-GFP mRNA in a mixture using the same procedures employed for the single-cell cDNA synthesis.

Figure 2. The Translational Hotspots of Two mRNAs Show Distinct Distribution Patterns by Dendrites, and DHPG Treatment Changes Colocalization Features

(A) Fluorescent images of GluR2-RFP and GluR4-GFP without DHPG treatment. Examples of colocalized hotspots (stars) and offset area (arrowheads) are displayed. (B) Line graphs of normalized fluorescence from fluorescent images (Figure 2A) display the feature of colocalization of hotspots without DHPG treatment. (C) Scatterplot showing colocalized pixels (within dotted oval) between GluR2-RFP and GluR4-GFP. (D) Colocalized translation features are displayed on a scatterplot (see text for details). The same symbols represent the dendrites from the same cell for each treatment condition. Colocalization coordinates are calculated at 10 min after DHPG treatment and at the same time point for no DHPG treatment.

Figure 3. The Protein-Synthesis Activities of GluR2-RFP and GluR4-GFP Are Dynamic

(A) Normalized fluorescence profiles display temporally dynamic changes of protein synthesis. D, distal end of dendrite; P, proximal end of dendrite. (B) Magnified boxes 1 and 2. (C) Scatterplot showing time-dependent changes in the colocalization features of non-DHPG-treated and DHPG-treated dendrites. (D) Average dispersion of the points around the centroid for the coordinates. ***p < 0.001.

Figure 4. The Characteristics of Translational Hotspots Are Heterogeneous along and among Dendrites

(A) Blue bar graphs show significant temporal correlation coefficient values (p < 0.05) at each location along with the estimated translational hotspots of GluR2-RFP (red) and GluR4-GFP (green) shown as thick horizontal bars. (B) The overall temporal correlation of DHPG-treated dendrites shows a lower correlation coefficient than that of non-DHPG-treated dendrites. (C) The percentage of negatively correlated locations is not significantly different between non-DHPG-treated and DHPG-treated dendrites. (D) The mean width of the correlated area is not significantly different between non-DHPG-treated and DHPG-treated dendrites. ***p < 0.001. NS, not significant. Error bars are SEM. (E) Area graph shows the proportion of different dynamical types of translational hotspots along dendrites (the color codes for the types are shown in the inset). The dendrite numbers used in the calculation are shown as black horizontal lines. (F) Bar graphs show the percentages of hotspot types (colors) from each panel in Figure 4F.

Figure 5. Photoconversion of GluR2-Kaede Protein Shows Limited Protein Movement within Hotspots, and Degradation and Synthesis of Proteins at the Hotspots

(A) A fluorescence image shows translational hotspots indicated by green GluR2-Kaede. An arrow indicates a hotspot illuminated with a 405 nm laser (lightning symbol) and an arrowhead indicates a hotspot without 405 nm laser illumination. (B) Left: magnified time-lapse fluorescence images from (A). Right: changes in normalized red fluorescent values over a photoconverted hotspot (arrow). (C) Change in the width of the red fluorescent area over time after photoconversion (n = 10, error bar is ±SEM). (D) Change in the mean fluorescent value of red and green fluorescence after photoconversion. (E) DHPG and translational inhibitors change the new green GluR2-Kaede protein-synthesis rates at the hotspots. DHPG-treated hotspots show increased translational activity (red, n = 7) compared with non-DHPG-treated hotspots (blue, n = 4), whereas addition of translation inhibitors (green, n = 5, anisomycin 50 μM and emetine 5 μM) to DHPG-treated hotspots shows attenuated translation at the hotspots. Error bars are SEM.

Figure 6. Model for Showing Differently Reacting Translational Hotspots in Non-DHPG-Treated and DHPG-Treated Dendrites

(A) Translational hotspots at which translational machinery is clustered produce GluR2-RFP or GluR4-GFP (red circle: GluR2-RFP; green circle: GluR4-GFP). With DHPG stimulation of local translation, the hotspots rapidly increase protein synthesis with different translational rates of particular mRNAs depending on the hotspot kinetics. Meanwhile, hotspots in non-DHPG-treated dendrites produce proteins moderately. (B) The distribution of temporal correlation coefficient values between GluR2-RFP and GluR4-GFP translations (blue, DHPG; red, non-DHPG) shows that DHPG-treated dendrites have less temporally correlated locations than nontreated dendrites. The means of the correlation coefficient of overall dendrites (dashed line) are indicated.