Synaptogenic activity of the axon guidance molecule Robo2 is critical for hippocampal circuit function

Abstract

The developmental transition between axon guidance and synapse formation is critical for circuit assembly but still poorly understood at the molecular level. We hypothesized that this key transition could be regulated by axon guidance cues switching their function to regulate synaptogenesis with subcellular specificity. Here, we report evidence for such a functional switch, describing a novel role for the axon guidance molecule Robo2 in excitatory synapse formation onto dendrites of CA1 pyramidal neurons (PNs) in the mouse hippocampus. Cell-autonomous deletion of Robo2 from CA1 PNs leads to a drastic reduction of the number of excitatory synapses specifically in proximal dendritic compartments. At the molecular level, we show that this novel postsynaptic function of Robo2 depends on both its canonical ligand Slit and a novel interaction with presynaptic Neurexins. Biophysical analysis reveals that Robo2 binds directly to Neurexins via itsIg4-5 domains. In vivo 2-photon Ca 2+ imaging of CA1 PNs during spatial navigation in mice shows that sparse deletion of Robo2 during development drastically reduces the likelihood of place cell emergence and alters spatial coding properties of the remaining place cells. Our results identify Robo2 as a novel molecular effector linking synaptic specificity to the acquisition of spatial coding properties characterizing hippocampal circuits.Proper circuit function relies on the establishment of synaptic connections characterized by a high degree of cell type and subcellular specificity. How this striking degree of synaptic specificity is achieved during development remains poorly understood, especially in the complex brains of mammals. Many cell surface molecules that mediate molecular recognition between axons and dendrites during circuit development have been identified 1-4 . These trans-synaptic adhesion molecules are often expressed in a cell-type specific manner 5 thereby determining the pool of a cell's possible synaptic partners. Some classes of these molecules have synaptic organizing properties evidenced by their direct or indirect ability to recruit key pre-and postsynaptic proteins. When axons reach their target areas, a cellular switch from a phase of growth and branching to synaptogenesis is observed. This switch is characterized by dynamic instability of adhesion progressively leading to more stable patterns of synaptic connectivity characterizing adult circuits. One of the foremost outstanding questions in neuroscience is how synapse formation is mechanistically integrated with earlier developmental steps such as axon guidance 3 and branching. One potential mechanism for coordinating this transition relies on the possibility that axon guidance cues acquire synaptogenic functions during circuit wiring. Indeed, evidence for such dual use of developmental molecules has been reported 6 . However, it remains unclear what prompts molecules to switch their function from axon guidance to synaptogenesis.Here we report that the well-studied axon guidance ligand-receptor pair Sl...

Beyond Axon Guidance: Roles of Slit-Robo Signaling in Neocortical Formation

“…Whereas the role of Slit-Robo signaling in axon guidance is conserved from Drosophila to mammals ( Bagri et al, 2002 ; Andrews et al, 2006 ; Fouquet et al, 2007 ; López-Bendito et al, 2007 ; Unni et al, 2012 ), several additional roles of Slit-Robo signaling have been identified in mammals. Studies have shown that Robo-mediated signaling is required for the proliferation of neural progenitor cells, as well as for the migration and morphological differentiation of cortical neurons ( Andrews et al, 2006 , 2008 ; Barber et al, 2009 ; Hernández-Miranda et al, 2011 ; Zheng et al, 2012 ; Borrell et al, 2012 ; Gonda et al, 2013 ; Yeh et al, 2014 ; Cárdenas et al, 2018 ; Blockus et al, 2019 ). These findings support the view that Robo signaling plays important roles in addition to axonal pathfinding in the developing neocortex.…”

“…Recently, the association between Robo and spine formation has been reported. Robo2 is localized at the postsynaptic membrane of hippocampal CA1 pyramidal neurons, and directly binds to presynaptic neurexin irrespective of Slit ( Blockus et al, 2019 ). This binding promotes spine formation and subsequent excitatory synapse formation.…”

Developmental mechanisms underlying circuit wiring: Novel insights and challenges ahead

“…A recent study identified another trans-synaptic complex formed by postsynaptic Robo2, its soluble ligand Slit and presynaptic Neurexins in the formation of CA3 → CA1 connectivity [ 38•• ]. Robo2 has been characterized extensively for its role in axon guidance in the developing brain of many model organisms [ 39 ].…”

“…The results from Sando et al [ 36•• ] and those of Blockus et al [ 38•• ], suggest that deletion of two completely distinct trans-synaptic molecular complexes (Lphn3/Ten/FLRT and Robo2/Slit/Nrxn) both lead to ~40–50% loss of CA3 → CA1 synapses. This poses the question whether Lphn3 and Robo2localize to different subsets of spines and/or receive input from different (previously unknown) subpopulations of axons from CA3 PNs.…”

The RNA-binding protein Musashi controls axon compartment-specific synaptic connectivity through ptp69D mRNA poly(A)-tailing

“…They play instructive roles in axon guidance and in the targeting of neuronal terminals to neuropil structures (Matsuoka et al, 2011;Timofeev et al, 2012;Xie et al, 2017;Yamagata and Sanes, 2008). Cell-surface proteins are also potent inducers of synapse formation in vitro (Biederer et al, 2002;Blockus et al, 2019;Scheiffele et al, 2000), but their role in vivo has not been clarified comprehensively (Sanes and Zipursky, 2020). The cell-type-specific temporal and quantitative transcriptional regulation of these factors is the subject of intense research efforts (Li et al, 2017;Fö ldy et al, 2016).…”