Synapse type-specific proteomic dissection identifies IgSF8 as a hippocampal CA3 microcircuit organizer

Abstract

Synaptic diversity is a key feature of neural circuits. Its underlying molecular basis is largely unknown, due to the challenge of analyzing the protein composition of specific synapse types.Here, we isolate the hippocampal mossy fiber (MF) synapse, taking advantage of its unique size and architecture, and dissect its proteome. We identify a rich cell-surface repertoire that includes 5 adhesion proteins, guidance cue receptors, extracellular matrix (ECM) proteins, and proteins of unknown function. Among the latter, we find IgSF8, a previously uncharacterized neuronal receptor, and uncover its role in regulating MF synapse architecture and feedforward inhibition on CA3 pyramidal neurons. Our findings reveal a diverse MF synapse surface proteome and highlight the role of neuronal surface-ECM interactions in the specification of synapse identity and circuit 10 formation. One Sentence Summary:Proteomic dissection of a specific synapse 15 3 Main Text:Neural circuits are composed of distinct neuronal cell types connected in highly specific patterns.Establishing these patterns of connectivity critically relies on cell-surface proteins (CSPs) expressed in cell type-specific combinations. CSPs, including transmembrane, membraneanchored, and secreted proteins, engage in networks of interactions that control neurite guidance, 5 target selection, and synapse development required for the formation of functional circuits (1).Single-cell RNA sequencing has enabled the characterization of cell type-specific CSP repertoires (2-6), but determining how these dictate complex patterns of connectivity (7, 8) poses a major challenge.This challenge is exemplified by pyramidal neurons, which receive different types of 10 synapses on their dendritic arbor, each with a distinct architecture, subcellular location, and functional properties. This synaptic diversity is essential for information processing in pyramidal neurons (9). Recent studies reveal a synapse type-specific localization and function of postsynaptic adhesion molecules in hippocampal pyramidal neuron dendrites (10-12), suggesting that compartmentalized distributions of CSPs contribute to the specification of synaptic structure and 15 function. Analogous to single-cell sequencing, probing the mechanisms underlying synaptic diversity requires dissecting the molecular composition of specific synapse types. This has remained challenging, as microdissection or chemical labeling strategies combined with mass spectrometry (MS) (13-15) average different synapse types, and affinity purification of synapse type-specific protein complexes (16) requires genetically engineered mice. Here, we isolate the 20 hippocampal mossy fiber (MF) synapse, a large and morphologically complex excitatory synapse (17) connecting dentate granule cell axons (mossy fibers) and CA3 pyramidal neuron dendrites in stratum lucidum (SL) (Fig. 1A, B), from wild-type (WT) tissue and map its CSP landscape. 4To isolate a specific synapse type from the hippocampus, we started with a previously published approach (18) ...

Synaptomic analysis of dopaminergic inputs reveal hub synapses in the mouse striatum

“…Future developments will allow a more thorough multi-omics (Hafner et al, 2019;Poulopoulos et al, 2019) analysis of dopaminergic samples prone to unravelling important key molecular signatures of dopamine hub synapses. More generally, results from our study and the work of Apostolo and colleagues (Apóstolo et al, 2019) on mossy fibre terminals of the hippocampus show that FASS synaptomics is a powerful tool for the exploration of projection-specific synaptomes (Grant, 2019;Zhu et al, 2018).…”

Persistence of Cajal-Retzius cells in the postnatal hippocampus is required for development of dendritic spines of CA1 pyramidal cells

“…To understand if Lphn2 is topographically downregulated, CA1 SLM was microdissected from Pde1c-Cre -/- ;DTA and Pde1c-Cre +/- ;DTA groups at P15 and P30. Total RNA was extracted from the tissue and qRT-PCR analysis was performed targeting genes specific for CA1 synapses ( Lphn2 , (Donohue et al, 2021) Robo2 (Blockus et al, 2021) or CA3 synapses ( Igsf8, Neo1 (Apóstolo et al, 2020)) to assess specificity of changes (Figure 4A). qRT-PCR analysis revealed that Lphn2 and Robo2 were both significantly upregulated at P15 Pde1c-Cre +/- ;DTA compared to Pde1c-Cre -/- ;DTA and conversely, were significantly downregulated at P30 Pde1c-Cre -/- ;DTA compared to Pde1c-Cre -/- ;DTA (Figure 4B).…”

Dynamic proteomic and phosphoproteomic atlas of corticostriatal axon neurodevelopment

“…Profiling the axonal proteome or transcriptome is challenging, particularly from complex tissues composed of multiple intermingled cell types. Canonical approaches utilize cell culture models in compartmentalized devices to ensure directional growth (Cagnetta et al, 2018; Chuang et al, 2018), laser capture microdissection of axon terminals (Zivraj et al, 2010), manual dissection of nerve segments (Michaelevski et al, 2010), synaptosome sorting from hippocampal mossy fibers (Apóstolo et al, 2020), or growth cone (GC) sorting from developing brain (Chauhan et al, 2020; Poulopoulos et al, 2019). However, no single approach can flexibly measure the overall axonal proteome across a wide age range, with high efficiency, genetic targeting, and pre-synaptic specificity.…”