Local protein synthesis in axon terminals and dendritic spines differentiates plasticity contexts

Anne-Sophie Hafner, Paul Donlin-Asp, Beulah Leitch, Etienne Herzog, Erin Margaret Schuman

Preprint posted on July 05, 2018

Expanding ways to monitor local translation in neurons

Selected by Dipen Rajgor

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?



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)



Posted on: 27th August 2018

Read preprint (No Ratings Yet)

  • Have your say

    Your email address will not be published. Required fields are marked *

    This site uses Akismet to reduce spam. Learn how your comment data is processed.

    Sign up to customise the site to your preferences and to receive alerts

    Register here

    Also in the cell biology category:

    The autophagic membrane tether ATG2A transfers lipids between membranes

    Shintaro Maeda, Chinatsu Otomo, Takanori Otomo

    Selected by Sandra Malmgren Hill

    LTK is an ER-resident receptor tyrosine kinase that regulates secretion

    Federica G. Centonze, Veronika Reiterer, Karsten Nalbach, et al.

    Selected by Nicola Stevenson


    Distinct RhoGEFs activate apical and junctional actomyosin contractility under control of G proteins during epithelial morphogenesis

    Alain Garcia De Las Bayonas, Jean-Marc Philippe, Annemarie C. Lellouch, et al.

    Selected by Ivana Viktorinová


    In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

    Jacob P. Keller, Jonathan S. Marvin, Haluk Lacin, et al.

    Selected by Stephan Daetwyler


    The spindle assembly checkpoint functions during early development in non-chordate embryos

    Janet Chenevert, Marianne Roca, Lydia Besnardeau, et al.

    Selected by Maiko Kitaoka

    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray

    Selected by Zheng-Shan Chong

    Mutations in the Insulator Protein Suppressor of Hairy Wing Induce Genome Instability

    Shih-Jui Hsu, Emily C. Stow, James R. Simmons, et al.

    Selected by Maiko Kitaoka


    Multi-immersion open-top light-sheet microscope for high-throughput imaging of cleared tissues

    Adam K. Glaser, Nicholas P. Reder, Ye Chen, et al.

    Selected by Tim Fessenden


    ATAT1-enriched vesicles promote microtubule acetylation via axonal transport

    Aviel Even, Giovanni Morelli, Chiara Scaramuzzino, et al.

    Selected by Stephen Royle


    HIV-1 Gag specifically restricts PI(4,5)P2 and cholesterol mobility in living cells creating a nanodomain platform for virus assembly

    C. Favard, J. Chojnacki, P. Merida, et al.

    Selected by Amberley Stephens

    Hepatocyte-specific deletion of Pparα promotes NASH in the context of obesity

    Marion Regnier, Arnaud Polizzi, Sarra Smati, et al.

    Selected by Pablo Ranea Robles

    Mitochondrial biogenesis is transcriptionally repressed in lysosomal lipid storage diseases

    King Faisal Yambire, Lorena Fernandez-Mosquera, Robert Steinfeld, et al.

    Selected by Sandra Franco Iborra


    Thyroid hormone regulates distinct paths to maturation in pigment cell lineages

    Lauren Saunders, Abhishek Mishra, Andrew J Aman, et al.

    Selected by Hannah Brunsdon


    Kinesin-6 Klp9 plays motor-dependent and -independent roles in collaboration with Kinesin-5 Cut7 and the microtubule crosslinker Ase1 in fission yeast

    Masashi Yukawa, Masaki Okazaki, Yasuhiro Teratani, et al.

    Selected by I. Bouhlel

    A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

    Nicholas P Boyer, Laura E McCormick, Fabio L Urbina, et al.

    Selected by Angika Basant


    SorCS1-mediated Sorting of Neurexin in Dendrites Maintains Presynaptic Function

    Luis Filipe Ribeiro, Ben Verpoort, Julie Nys, et al.

    Selected by Carmen Adriaens


    Also in the neuroscience category:

    The Hunchback temporal transcription factor determines motor neuron axon and dendrite targeting in Drosophila

    Austin Q Seroka, Chris Q Doe

    Selected by Abagael Lasseigne


    Molecular Logic of Spinocerebellar Tract Neuron Diversity and Connectivity

    Myungin Baek, Vilas Menon, Thomas Jessell, et al.

    Selected by Yen-Chung Chen

    In vivo glucose imaging in multiple model organisms with an engineered single-wavelength sensor

    Jacob P. Keller, Jonathan S. Marvin, Haluk Lacin, et al.

    Selected by Stephan Daetwyler


    Blue light induces neuronal-activity-regulated gene expression in the absence of optogenetic proteins

    Kelsey M. Tyssowski, Jesse M. Gray

    Selected by Zheng-Shan Chong

    A pair of E3 ubiquitin ligases compete to regulate filopodial dynamics and axon guidance

    Nicholas P Boyer, Laura E McCormick, Fabio L Urbina, et al.

    Selected by Angika Basant


    Prospective, brain-wide labeling of neuronal subclasses with enhancer-driven AAVs

    Lucas T Graybuck, Adriana Sedeño-Cortés, Thuc Nghi Nguyen, et al.

    Selected by Jesus Victorino

    Multilevel regulation of the glass locus during Drosophila eye development

    Cornelia Fritsch, F. Javier Bernardo-Garcia, Tim Humberg, et al.

    Selected by Gabriel Aughey


    Regulation of modulatory cell activity across olfactory structures in Drosophila melanogaster

    Xiaonan Zhang, Kaylynn Coates, Andrew Dacks, et al.

    Selected by Rudra Nayan Das


    On-site ribosome remodeling by locally synthesized ribosomal proteins in axons

    Toshiaki Shigeoka, Max Koppers, Hovy Ho-Wai Wong, et al.

    Selected by Srivats Venkataramanan

    Single cell RNA-Seq reveals distinct stem cell populations that drive sensory hair cell regeneration in response to loss of Fgf and Notch signaling

    Mark E. Lush, Daniel C. Diaz, Nina Koenecke, et al.


    Distinct progenitor populations mediate regeneration in the zebrafish lateral line.

    Eric D Thomas, David Raible

    Selected by Rudra Nayan Das


    A schizophrenia risk gene, NRGN, bidirectionally modulates synaptic plasticity via regulating the neuronal phosphoproteome

    Hongik Hwang, Matthew J Szucs, Lei J Ding, et al.

    Selected by Laura McCormick

    Actomyosin-II facilitates long-range retrograde transport of large cargoes by controlling axonal radial contractility

    Tong Wang, Wei Li, Sally Martin, et al.

    Selected by Ivana Viktorinová

    Unlimited genetic switches for cell-type specific manipulation

    Jorge Garcia-Marques, Ching-Po Yang, Isabel Espinosa-Medina, et al.

    Selected by Rafael Almeida


    Defining the design requirements for an assistive powered hand exoskeleton

    Quinn A Boser, Michael R Dawson, Jonathon S Schofield, et al.

    Selected by Joanna Cross

    Strong preference for autaptic self-connectivity of neocortical PV interneurons entrains them to γ-oscillations

    Charlotte Deleuze, Gary S Bhumbra, Antonio Pazienti, et al.

    Selected by Mahesh Karnani

    Proteomic Studies reveal Disrupted in Schizophrenia 1 as a key regulator unifying neurodevelopment and synaptic function

    Adriana Ramos, Carmen Rodriguez-Seoane, Isaac Rosa, et al.

    Selected by Yasmin Lau