Synaptogenic activity of the axon guidance molecule Robo2 is critical for hippocampal circuit function
Posted on: 2 January 2020 , updated on: 3 January 2020
Preprint posted on 13 November 2019
Two for the price of one! A new role for the axon guidance molecule Robo2 in hippocampal circuit function
Selected by Ana Dorrego-Rivas
Categories: cell biology, developmental biology, neuroscience
Background
One of the main questions of brain circuit assembly is how the transition between axon guidance and synapse formation takes place. This crucial step could be done by axon guidance molecules that switch their function to specifically regulate synaptogenesis. This study provides evidence for a functional switch: Robo2, an axon guidance molecule, has a new role in excitatory synapse formation onto dendrites of CA1 pyramidal neurons (PNs) in the mouse hippocampus. Concretely, Robo2 localises to the postsynaptic compartment and induces excitatory synapse formation specifically in the proximal dendrites. This new function of Robo2 is dependent on its ligand Slit and on a novel interaction with Neurexins. Moreover, in vivo 2-photon Ca2+ imaging of CA1 PNs during spatial navigation in mice show that the deletion of Robo2 during development reduces the number of place cells and alters the response properties of the remaining. These striking results show a new function for the axon guidance molecule Robo2 in connecting specific synaptogenesis and spatial coding properties in hippocampal circuits.
Main findings
By using publicly available data (Allen Brain Atlas), the authors first checked Robo2 mRNA expression in mice in 3 different stages: P4, P14 and P28. While almost absent at P4, Robo2 strongly accumulates in the Cornu Ammonis (CA) 1 and 3 and in the dentate gyrus of the mature hippocampus (P14 and P28). Interestingly, the expression of the other isoform Robo1 is already strong at P4 and widely distributed in the hippocampus during development until its confinement to the CA3 at P28. The expression of Slit1 and Slit2 follows those of their partners, Robo2 and Robo1, respectively.
Moreover, Robo2 was found at two particular compartments of the CA1, the stratum oriens (SO) and the stratum radiatum (SR). The synaptic connection between the axons from CA3 and CA2 pyramidal neurons (PNs) and the dendrites of CA1 PNs takes place at these areas, showing the specific localisation of Robo2 to these synapses. To get more details about the subcellular localisation of Robo2, the authors performed ex utero electroporation experiments to express a pHluorin-tagged Robo2 construct in CA1 pyramidal neurons. Cultured neurons from electroporated E15 embryos were observed at DIV14 (days in vitro), the time when the peak of synaptogenesis occurs. The Robo2 construct was found to colocalise with the excitatory post-synaptic marker Homer1c in the spines. In addition, biochemical synaptic fractionation experiments showed that Robo2 is enriched in postsynaptic membranes, while its partner Slit2 accumulates in the presynaptic compartment.
Given that Robo2 localises to excitatory synapses and is enriched postsynaptically, the authors wanted to know if it was necessary for excitatory synapse development in CA1 PNs in vivo. Therefore, they performed in utero electroporation into the CA1 progenitors of wild type and Robo2-floxed mice with two specific constructs: the cre-recombinase and a cre-dependent reporter plasmid (flex-tdTomato). The deletion of Robo2 promoted a decrease of the spine density in the proximal dendritic compartments, but not in the distal apical tuft dendrites, while Robo2-dependent growth remained unaffected. This data shows that Robo2 is required for excitatory synapse development in CA1 PNs in a cell-autonomous manner. The absence of Robo2 had also a physiological impact: by performing whole-cell current-clamp recordings of spontaneous excitatory postsynaptic potentials (sESP) in Robo2 KO (tdTomato-expressing) and neighboring WT CA1 PNs, the frequency of the sESPs in the Robo2-null neurons was found to be reduced when compared to the control PNs.
The data shows that Robo2 is necessary for excitatory synapse development in CA1 PNs, but, is it directly involved in synapse formation? To answer this question, the authors used an in vitro hemisynapse assay to determine whether Robo proteins expressed on the surface of HEK293 cells could induce formation of presynaptic boutons from axons of co-cultured primary neurons. This experiment had 3 conditions/constructs: CD8, a negative control; Neuroligin1 (NLG1), a positive control known to induce the formation of excitatory and inhibitory synapses; and Robo1 or Robo2. The expression of Robo1 and Robo2 in these cells promoted a strong clustering of axonal Vglut1 around the cell perimeter. However, the expression of Robo2 did not produce the accumulation of Vgat, showing that Robo proteins specifically induce the formation of excitatory synapses. Interestingly, this Vglut1 aggregation was not found when using Robo3, an isoform that does not bind the Slit proteins. This led to the next question of the study: is this Vglut1 accumulation Slit-dependent? The expression of a Robo2 construct with no Slit-binding domain in HEK293 cells led to the loss of Vglut1 clustering in the presynaptic compartment. This data shows that Slit proteins are necessary for Robo1 and Robo2 to specifically induce excitatory synapses.
The authors next hypothesised, based on previous literature, that Robo2 promotes synapse formation through a transynaptic complex. To test this, they first addressed if the axonal/presynaptic localisation of Robo1 and Robo2 was needed for their synaptogenic activity. The chosen strategy consisted of a mixed culture with neurons from Robo1 KO mice, Robo2-floxed mice neurons infected with a cre-recombinase carrying virus at DIV0 and HEK293 cells expressing Robo2. Surprisingly, the Robo1/2 deficient axons were still able to gather Vglut1 at their boutons due to the exogenous Robo2 of the HEK293 cells. With this, neither Robo2 nor Robo1 localisations to the presynaptic terminals are needed for excitatory synapse formation.
Considering these findings, a third partner, together with Robo and Slit, must functionally complete the complex. The authors used a recently published experimental-based proteomics pipeline to describe the proteome of Robo and Slit in the synapse. P21 rat brains were used to purify the synaptosomes and those were processed for immunoprecipitation using a recombinant Slit2-Fc protein. By using mass spectrometry with Slit2-Fc, some partners were identified: Neurexin 1/2/3 were the most detected, followed by Robo2, Glypican1 and PlexinA1. The authors focused on Neurexin proteins for their next step, as they are present at the presynaptic compartment and are known to form trans-synapse complexes with postsynaptic proteins.
To assess how the deletion of Neurexin would impact synapse formation, the authors used an shRNA for all the isoforms (3 isoforms, each of them generating two isoforms, α and β) to infect primary neurons and, after, HEK293 cells expressing Robo2 were added to the culture. The synaptogenic activity promoted by Robo2 expressed in the cells was abolished in the absence of Neurexin, showing these proteins as the trans-synaptic piece that launches the synaptogenic activity of the Robo/Slit pair.
Finally, the study shows the function of Robo2 in the spatial coding in the CA1 PNs: a subpopulation of these neurons exhibit spatially tuned firing when the animal is exploring an environment. The authors assessed CA1 PN place cell properties at the population level using in vivo two-photon (2p) microscopy-based Ca2+ imaging in head-fixed, awake behaving mice. The experimental strategy was the following: 1) in utero electroporation of hippocampal CA1 PNs of a cre-recombinase and a cre-dependent mCherry plasmids, plus 2) an stereotaxic injection in the CA1 with the genetically-encoded Ca2+ indicator GCamP6f. Mice were placed on a treadmill belt and trained to run for randomly delivered water rewards, while having spatial cues and navigational landmarks. The authors found the Robo2 KO neurons to have reduced spike frequency during running. The number of place cells, which are the neurons that fire when the animal occupies a different location, were also highly reduced in the kO versus the control, and the remaining place cells had altered responsive properties. Altogether, this data shows that Robo2-dependent alteration in excitatory synapse development has a significant impact on in vivo coding properties of hippocampal CA1 PNs.
Questions for the authors
– You show a novel and very specific function for Robo2: the excitatory synaptic induction in the CA1 PNs. Since Robo2 is also expressed in CA3 and DG, do you think this role could apply also to the neurons of these areas? Including its function on spatial coding?
– Related to the previous question: you express Robo1 in HEK293 cells for your hemisynapse studies in vitro, and, like Robo2, also promotes the gathering of Vglut1 in the surrounding neurons. Do you plan to investigate this function in the areas where Robo1 is more expressed, like the CA3? Is there anything known about it?
Why did I choose this preprint?
I highlighted this preprint for the following reasons:
1) It touches one of the main mysteries on brain circuit development: the transition from axon guidance to specific synaptic development
2) It solves the mystery showing a dual function for a single molecule (Robo2), which is one of the beauties (in my opinion) of biology: one protein adapting its function to different contexts
3) The line of the story, going from the molecule (localisation, in the brain, in the neuron, function characterisation in vitro and in vivo) to the hippocampal circuitry functionalities (the role of Robo2 in spatial coding).
References
1. de Wit, J. & Ghosh, A. Specification of synaptic connectivity by cell surface interactions. Nat Rev Neurosci 17, 22-35, doi:10.1038/nrn.2015.3 (2016).
2. Sudhof, T. C. Towards an Understanding of Synapse Formation. Neuron 100, 276-293, doi:10.1016/j.neuron.2018.09.040 (2018).
3. Shen, K. & Cowan, C. W. Guidance molecules in synapse formation and plasticity. Cold Spring Harb Perspect Biol 2, a001842, doi:10.1101/cshperspect.a001842 (2010)
4. Blockus, H. & Chedotal, A. Slit-Robo signaling. Development 143, 3037-3044, doi:10.1242/dev.132829 (2016)
doi: https://doi.org/10.1242/prelights.16008
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