Local protein synthesis in axon terminals and dendritic spines differentiates plasticity contexts
Posted on: 27 August 2018
Preprint posted on 5 July 2018
Article now published in Science at http://dx.doi.org/10.1126/science.aau3644
Categories: cell biology, neuroscience
Background / Context
The mammalian brain is made up of millions of interlinked neuronal circuits that form through specialized junctions known as synapses. Localized protein synthesis in neurons is an important mechanism which influences synaptic strength and plasticity. Thus, control of localized protein synthesis and degradation at synapses is an important mechanism underlying memory and learning (1).
The pre-synaptic axonal bouton and the post-synaptic dendritic spine are separated by ~20nm (region known as the ‘synaptic cleft’). Therefore,discriminating between molecules in the pre- and post-synapticcompartments using fluorescence microscopy is challenging. In this preprint, the Schuman lab have attempted to identify and distinguish mRNA molecules and newly synthesized nascent proteins in pre- and post-synapticcompartments using expansion microscopy. Expansion microscopy enlarged both pre- and post-synaptic compartments by an average ~3.5 fold, yielding a clear separation between the two partitions in cultured rat hippocampal neurons.
Key Findings
- Excitatory and Inhibitory axonal boutons contain mature mRNA and ribosomes.
Using fluorescence in situ hybridization (FISH) with poly-dT probes in combination with expansion microscopy, the authors demonstrate that >80% of pre-synaptic inhibitory and excitatory boutons contain poly-adenylated/poly(A) mRNA. The presence of poly(A) mRNA in axon terminals suggests the capacity for protein synthesis. Indeed, the authors also show the presence of the 28S ribosomal RNA (rRNA) and ribosomal protein RPS11 in excitatory and inhibitory axonal boutons, demonstrating the presence of the translational machinery in pre-synaptic nerve terminals.
- Pre-synaptic compartments contain distinct mRNAs for protein synthesis.
The authors used fluorescence-activated synaptosome sorting (FASS) to purify fluorescently labeled synaptosomes from the forebrain of vGLUT1VENUSknock-in mice (2). These synapotosomes contain resealed ‘intact’ presynaptic compartments and RNA sequencing revealed >150 unique mRNA transcripts in vGLUT1+synaptosomes. There was an over representation of genes coding for ribosomal proteins and regulators of translation indicating localized synthesis of translational components is likely in presynaptic terminals. Amongst the most enriched transcripts in the vGLUT1+presynaptic transcriptome were mRNAs for well-known presynaptic proteins, including Bassoon, Rims1 and Rims3.
- Active translation occurs in pre- and post-synaptic compartments
To obtain direct evidence for protein synthesis in synaptic compartments, the authors used the puromycin-based metabolic labeling protocol to tag newly synthesized proteins – a technique which has previously been pioneered by the same group (3). Puromycin is a tRNA analog and is incorporated only into newly synthesized proteins, thus making it possible to detect all newly synthesized proteins using an anti-puromycin antibody. Neurons were incubated with a low dose of puromycin for 5 minutes to detect all newly synthesized proteins within this time frame and then fixed and processed for expansive microscopy (EM). Puromycin labeling demonstrated that ~37% and ~61% percent of excitatory pre- and post-synaptic compartments respectively, underwent active translation in a 5 minute window. Furthermore, they were able to show that some specific candidate mRNAs, such as Bassoon, is synthesized in presynaptic compartments within minutes. Taken together, these data indicate that post-synaptic spines as well as excitatory and inhibitory pre-synaptic boutons exhibit local translation with a relatively high frequency.
- Activity-dependent Increase in local translation
The authors evaluated the pattern of local translation in dendritic spines, excitatory boutons and inhibitory boutons in response to plasticity-induced changes by BDNF, DHPG and ACEA. BDNF increased local translation in all three compartments, DHPG caused an increase in dendritic spines only and ACEA increased translation primarily in inhibitory boutons. These fundamental experiments highlight global translational changes occur during synaptic plasticity.
What I like about the preprint?
This is the first study, to the best of my knowledge, which uses expansion fluorescent microscopy to distinguish between RNA molecules present in pre- and post-synaptic compartments. Furthermore, they utilize state of the art purification methods to isolate synaptosomes and identify RNA species within them. The techniques utilized in this preprint demonstrate the ability to monitor local translation at unprecedented spatial resolution and could revolutionize the way we study protein synthesis in neurons.
Future questions
The elegant methods used in this preprint could be used to answer a plethora of interesting questions, including but not limited to:
1) How does mRNA translation change in response to other plasticity-induced changes?Identifying how mRNA translation (of total mRNAs and of specific mRNAs) in pre- and post-synaptic compartments changes in response to LTP and LTD induced stimuli will provide fascinating insight into how translation dictates plasticity. Furthermore, neurological pathologies are associated with synaptic defects and therefore identifying how the rate of mRNA translation is altered in disease is worth exploring.
2) Do pre-synaptic terminals contain miRNAs? miRNAs are well established for playing fundamental roles in influencing plasticity of dendritic spines. FASS in combination with RNA sequencing chould be used to determine which miRNAs are enriched in pre-synaptic terminals. Also, are components of the RNA Inducing Silencing Complex (RISC) present within pre-synaptic terminals? Are mRNAs in pre-synaptic terminals silenced by miRNAs?
References
1) The Ins and Outs of miRNA-Mediated Gene Silencing during Neuronal Synaptic Plasticity. Rajgor D & Hanley JG. Non-Coding RNA (2016)
2) Proteomic screening of glutamatergic mouse brain synaptosomes isolated by fluorescence activated sorting. Biesemann et. al. The EMBO Journal (2014)
3) Activity-dependent spatially localized miRNA maturation in neuronal dendrites. Sambandan et. al. Science (2017)
doi: https://doi.org/10.1242/prelights.4411
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