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. 2016 May;64(5):323-33.
doi: 10.1369/0022155416641604. Epub 2016 Mar 29.

Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging

Aron Kos  1   2 , Kai A Wanke  2   3 , Anthony Gioio  4 , Gerard J Martens  1   5 , Barry B Kaplan  1   4 , Armaz Aschrafi  4
Affiliations

Monitoring mRNA Translation in Neuronal Processes Using Fluorescent Non-Canonical Amino Acid Tagging

Aron Kos et al. J Histochem Cytochem. 2016 May.

Abstract

A steady accumulation of experimental data argues that protein synthesis in neurons is not merely restricted to the somatic compartment, but also occurs in several discrete cellular micro-domains. Local protein synthesis is critical for the establishment of synaptic plasticity in mature dendrites and in directing the growth cones of immature axons, and has been associated with cognitive impairment in mice and humans. Although in recent years a number of important mechanisms governing this process have been described, it remains technically challenging to precisely monitor local protein synthesis in individual neuronal cell parts independent from the soma. This report presents the utility of employing microfluidic chambers for the isolation and treatment of single neuronal cellular compartments. Furthermore, it is demonstrated that a protein synthesis assay, based on fluorescent non-canonical amino acid tagging (FUNCAT), can be combined with this cell culture system to label nascent proteins within a discrete structural and functional domain of the neuron. Together, these techniques could be employed for the detection of protein synthesis within developing and mature neurites, offering an effective approach to elucidate novel mechanisms controlling synaptic maintenance and plasticity.

Keywords: FUNCAT; axon; click assay; local protein synthesis; microfluidic chambers; neurons; synapse.

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Conflict of interest statement

Competing Interests: The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

Figures

Figure 1.
Figure 1.
Protein labeling using the click reaction. (A) A scheme showing the copper-catalyzed click reaction in which two molecules containing either an alkyne (blue) or azide (red) group are covalently bound to form a stable triazole conjugate. (B) Click reaction workflow for labeling nascent proteins in neuronal cultures. First, cells are depleted of methionine (brown dots) and the medium replaced with HBSS buffer. Homopropargylglycine (HPG) is added to the cells, where it will be incorporated into newly synthesized proteins (blue hexagons). The incorporated HPG is then labeled with Alexa 594 containing an azide moiety (red hexagons). Thereafter, cells are subjected to immunostaining to label specific neuronal components.
Figure 2.
Figure 2.
HPG incorporation into neurons is dependent on translational activity. (A) Representative images of HPG signal (inverted greyscale and red) in DIV 14 cortical neurons. Translation was stimulated by KCl-mediated neuronal potentiation and reduced by pre-incubation with the translation inhibitor cycloheximide (CHX). Immunostaining with anti-Tau (green) and anti-MAP2 (blue) distinguishes between axons and dendrites, respectively. (B) Quantification of the relative changes in somatic HPG signal intensity after treatment with CHX, KCl or CHX and KCl. (C-D) Shows the relative intensity of HPG in the (C) MAP2 positive dendrites and (D) Tau positive axons after treatment with CHX, KCl or CHX and KCl. Data represent the mean ± SEM and single values shown for n=11 to 15 samples collected from three independent experiments. DIV, days in vitro; HPG, homopropargylglycine; MAP2, microtubule-associated protein 2. One-way ANOVA with Bonferroni multiple comparison test; **p<0.01; ***p<0.001. Scale, 50 µm.
Figure 3.
Figure 3.
Nascent protein tagging in axons of cortical neurons growing in microfluidic chambers. (A) Schematic representation of a compartmentalized microfluidic chamber with the grey channel holding the cells from which the axons will originate. These will grow through the middle microgrooves towards the axon chamber (green). Right: A representative micrograph of growing axons from DIV 7 cortical neurons stained with phalloidin (green) and DAPI (blue). The white insert denotes the typical area in which the axonal HPG signal is measured. (B) Representative images of axons treated with HPG alone or combined with the translation inhibitor CHX; these compounds were added to the axon compartment and fluidically isolated. Neurons were stained for Tau (green) and HPG incorporation (inverted greyscale and red) and the overlay of the two fluorophores. (C) Quantification of the HPG signal in immature axons relative to axons treated with CHX. (D) Agarose gel electropherogram of the soma-enriched genes Scn3b and Glrb after RT-PCR amplification of RNA samples isolated from the soma or axon compartment including a H2O-negative RT-PCR control. (E) Representative images of cells within the soma compartment of the axon chamber in which only the axon compartment was treated with HPG. Neurons were stained for Tau (green) and HPG incorporation (inverted greyscale and red). (F) Quantification of the HPG signal in the soma compartment compared with cells treated with CHX. Data represent the mean ± SEM and single values shown for n=6 to 9 chambers collected from three independent experiments. CHX, cyclohexamide; DIV, days in vitro; HPG, homopropargylglycine. p-values are determined by two-tailed unpaired Students t-test. **p<0.01. Scale (A, B, E) 50 µm.
Figure 4.
Figure 4.
Imaging of protein synthesis in neurons grown in microfluidic perfusion chambers. (A) Schematic overview of a microfluidic perfusion chamber with one region (green square) enlarged (inset of the perfusion channel on the right). On the left side of the chamber, there are three reservoirs. The two outer green wells contain a buffer to hold the perfusate inside the perfusion chamber preventing it from entering the microgrooves. The middle red well contains the desired perfusate. In the enlargement image, the red cells (lower) generate the axons growing towards the perfusion channel (presynaptic compartment), while the blue cells (upper) generate dendrites (postsynaptic compartment). (B) Representative confocal images of the HPG signal (red and greyscale) and Tau (blue) within the perfusion channel of chambers in which mature cortical neurons (DIV 14–16) were cultured. The neurons were treated locally within the perfusion channel with HPG alone or in combination with CHX. (C) Magnifications of the white square inserts depicted in (B) with the HPG shown in red and inverted greyscale. (D) Quantification of the relative intensity of the HPG signal inside the perfusion channel compared with neurons treated with CHX. (E) Representative confocal images of cells on the dendrite side (postsynaptic compartment) of the perfusion chamber with Tau (blue) and the HPG signal (greyscale). (F) Quantification of the relative intensity of the HPG signal of cells cultured on the dendrite side of the perfusion chamber. Data represent the mean ± SEM and single values shown for n=4 independent experiments. p-values are determined by two-tailed unpaired Students t-test. p<0.01. CHX, cyclohexamide; DIV, days in vitro; HPG, homopropargylglycine. **Scale (B and E) 50 µm; (C) 5 µm.
Figure 5.
Figure 5.
Visualization of nascent protein synthesis in postsynaptic densities from neurons grown in microfluidic perfusion chambers. (A) Representative confocal images of the perfusion channel from microfluidic perfusion chambers in which DIV 14-16 primary cortical neurons were grown. Neurons were treated with HPG alone or in combination with CHX and stained for HPG incorporation (inverted greyscale and red) or PSD-95 (green), the overlay shows the overlap between these two signals. (B) Quantified relative HPG signals inside PSD-95 puncta of neurons treated HPG alone or in which translation was inhibited using CHX, normalized for the total selected PSD-95-positive-area. Data represent the mean ± SEM and single values shown for n=10 to 11 culture chambers collected from three independent experiments. CHX, cyclohexamide; DIV, days in vitro; HPG, homopropargylglycine; PSD-95, postsynaptic density protein 95. p-values are determined by two-tailed unpaired Students t-test. ***p<0.0001. Scale, 5 µm.
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