Latrophilin GPCR Signaling Mediates Synapse Formation
Curation statements for this article:-
Curated by eLife
Evaluation Summary:
The main finding that GPCR activity is necessary for latrophilins' role in synapse formation is both surprising and important. This work will inspire new research on compartmentalized GPCR signaling at the synapse.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
This article has been Reviewed by the following groups
Discuss this preprint
Start a discussion What are Sciety discussions?Listed in
- Evaluated articles (eLife)
Abstract
Neural circuit assembly in the brain requires precise establishment of synaptic connections, but the mechanisms of synapse assembly remain incompletely understood. Latrophilins are postsynaptic adhesion-GPCRs that engage in trans-synaptic complexes with presynaptic teneurins and FLRTs. In CA1-region neurons, Latrophilin-2 and Latrophilin-3 are essential for formation of entorhinal-cortex-derived and Schaffer-collateral-derived synapses, respectively. However, it is unknown whether latrophilins function as GPCRs in synapse formation. Here, we show that Latrophilin-2 and Latrophilin-3 exhibit constitutive GPCR activity that increases cAMP levels, which was blocked by a mutation interfering with G-protein and arrestin interactions of GPCRs. The same mutation impaired the ability of Latrophilin-2 and Latrophilin-3 to rescue the synapse-loss phenotype in Latrophilin-2 and Latrophilin-3 knockout neurons in vivo . Our results suggest that Latrophilin-2 and Latrophilin-3 require GPCR signaling in synapse formation, indicating that latrophilins promote synapse formation in the hippocampus by activating a classical GPCR-signaling pathway.
Article activity feed
-
Response to Reviewer #3 (Public Review):
We also thank this reviewer for constructive and positive comments. The biological questions raised by the Reviewer, such as synaptic compartmentalization of Lphn signaling, G-protein coupling, and ligand engagement/signaling are incredibly interesting topics for future studies. We also agree that these questions are outside the scope of our current study, which initially establishes Lphns as compartmentalized synaptic GPCRs mediating synapse specificity, but we will pursue these important projects in future experiments.
We thank the reviewers for their valuable comments, and hope to have addressed all of their concerns.
-
Response to Reviewer #2 (Public Review):
We thank the reviewer for their positive and helpful comments. We agree that compartmentalized synaptic GPCR signaling by Adhesion-class GPCRs and other GPCRs is an exciting research area for multiple future studies. We have added additional data regarding cell surface localization of our mutant receptors in neurons and have revised the text according to the reviewer suggestions.
-
Response to Reviewer #1 (Public Review):
We thank the reviewer for finding our study important and interesting. We agree that the biological mechanisms of synaptic specificity require in vivo systems, with implementation of a reduced culture system to examine basic cell biological questions such as mutant receptor expression. We have added an additional control experiment pertaining to our mutant receptor validation.
-
Reviewer #3 (Public Review):
The manuscript by Sando et al. describes experiments directed at unraveling how latrophilins (Lphns) orchestrate synapse formation. Lphns are a unique family of adhesion molecules harboring extensive extracellular N-terminal domains with several known interacting motifs coupled to the classical 7 transmembrane architecture of G-protein coupled receptors. In recently published work from the Sudhof group, Lphns were shown to play a surprising postsynaptic role in synapse formation onto CA1 pyramidal neurons with Lphn2 and 3 important for perforant path and Schaffer collateral synapse formation respectively (Sando et al., Anderson et al). However, it remains unclear whether G-protein signaling through Lphns is important for their role as synapse organizers.
To address this issue, the authors use conditional …
Reviewer #3 (Public Review):
The manuscript by Sando et al. describes experiments directed at unraveling how latrophilins (Lphns) orchestrate synapse formation. Lphns are a unique family of adhesion molecules harboring extensive extracellular N-terminal domains with several known interacting motifs coupled to the classical 7 transmembrane architecture of G-protein coupled receptors. In recently published work from the Sudhof group, Lphns were shown to play a surprising postsynaptic role in synapse formation onto CA1 pyramidal neurons with Lphn2 and 3 important for perforant path and Schaffer collateral synapse formation respectively (Sando et al., Anderson et al). However, it remains unclear whether G-protein signaling through Lphns is important for their role as synapse organizers.
To address this issue, the authors use conditional knockout/rescue approaches to convincingly demonstrate an essential role of the GPCR domain of Lphns 2 and 3 both in vitro and in vivo. Replacing the intracellular 3rd loop of the GPCR domain (which is essential for G-protein activation) of either Lphn2 or 3 fails to rescue reduced synapse number in the knockout background (nor does deleting the entire GPCR domain). Thus it appears that cell adhesion properties alone are not sufficient for Lphn-mediated synapse formation. The experiments appear to be robust and convincing and the conceptual advance of Lphn-mediated GPCR signaling during synapse formation is substantial. I have a few specific points outlined below, but overall the authors use a nice combination of imaging, electrophysiology and rabies virus-based synaptic connectivity measurements to support their conclusions. Naturally, I'd like to know more details about the signaling requirement (e.g. how is Lphn signaling spatially compartmentalized compared to other GPCRs present, which G-protein(s) Lphns couple to, how/when/whether GPCR signaling is regulated by ligand engagement etc.) but these questions seem better suited to a separate study.
-
Reviewer #2 (Public Review):
This manuscript by Sando and Sudhof addresses whether GPCR activity of latrophilin2 and 3 is necessary for the role of these proteins in synapse formation. The key findings are:
— the generation and validation of mutants that lack transmembrane and intracellular domains (but are GPI-anchored instead), the lack only intracellular domains, or that contain all domains but lack GPCR-activity. All mutants work properly in cell aggregation assays and appear to be localized normally when overexpressed in wild type neurons. This also led to the development of an elegant PKA-phosphorylation reporter assay.
— in cultured latrohphilin 3 knockout neurons, latrophilin3 expression restores a decreased synapse density and mini-frequency, but the GPI-anchored, truncated or inactive versions do not restore these parameters.
—…
Reviewer #2 (Public Review):
This manuscript by Sando and Sudhof addresses whether GPCR activity of latrophilin2 and 3 is necessary for the role of these proteins in synapse formation. The key findings are:
— the generation and validation of mutants that lack transmembrane and intracellular domains (but are GPI-anchored instead), the lack only intracellular domains, or that contain all domains but lack GPCR-activity. All mutants work properly in cell aggregation assays and appear to be localized normally when overexpressed in wild type neurons. This also led to the development of an elegant PKA-phosphorylation reporter assay.
— in cultured latrohphilin 3 knockout neurons, latrophilin3 expression restores a decreased synapse density and mini-frequency, but the GPI-anchored, truncated or inactive versions do not restore these parameters.
— in vivo/hippocampal brain slices, latrophilin2 knockout impaired perforant path but not Schaffer collateral transmission onto CA1 neurons, and rescue required latrphilin2 GPCR activity. Conversely, Latrophilin3 knockout impaired Schaffer collateral but not perforant path transmission onto CA1 neurons, and rescue required latrophilin3 GPCR activity.
— finally, monosynaptic tracing confirmed that latrophilin3 knockout reduced inputs onto CA1 starter neurons, and rescue again required GPCR activity.
Altogether, the data are rigorously acquired, the paper is well written, and the finding that GPCR activity is necessary for latrophilins' role is both surprising and important. It is also elegant, as coupling cell-adhesion directly to signal transduction via a single molecule for synapse formation is a compelling way to drive synaptic assemblies. Naturally, the question arises how compartmentalized GPCR-signaling then instructs synapse formation, a topic that will undoubtedly require and attract more research. This is an exciting manuscript that will inspire new research on compartmentalized GPCR signaling at the synapse. Given the central importance of surface trafficking and localization within spines for the conclusions, better description of experimental procedures and quantification, and possibly additional data would clearly strengthen this point.
-
Reviewer #1 (Public Review):
The general thesis of the work, provided by the authors, is the demonstration that latrophilins 2 and 3 function as classical GPCRs at the synapse and that this activity is necessary for synapse formation at a specific synapse within the hippocampus. The topic is interesting and important for several reasons. First, the knowledge of GPCRs at synaptic connections is focused largely on neurotransmitter receptors in the literature – metabotropic GluR and AChR as well as neuromodulatory neurotransmitter receptors (NPY, Seratonin etc). The mechanism demonstrated in this work concerns the function of a GPCR receptor system that could confer specificity to synapse formation.
The effect sizes that are documented throughout this work are large, giving this reviewer confidence that the effects are robust and will be …
Reviewer #1 (Public Review):
The general thesis of the work, provided by the authors, is the demonstration that latrophilins 2 and 3 function as classical GPCRs at the synapse and that this activity is necessary for synapse formation at a specific synapse within the hippocampus. The topic is interesting and important for several reasons. First, the knowledge of GPCRs at synaptic connections is focused largely on neurotransmitter receptors in the literature – metabotropic GluR and AChR as well as neuromodulatory neurotransmitter receptors (NPY, Seratonin etc). The mechanism demonstrated in this work concerns the function of a GPCR receptor system that could confer specificity to synapse formation.
The effect sizes that are documented throughout this work are large, giving this reviewer confidence that the effects are robust and will be reproducible and, more importantly, are indeed a biological mechanism related to synapses.
The other major strength of the work is that the studies in neuronal cell culture are recapitulated in vivo providing additional confidence in the validity and importance of the work. Indeed, the concept of specificity requires this type of in vivo work as the identity of synapses in culture systems can not be readily determined.
A further strength is the rational and implementation of three mutant receptors that are used to dissect the signaling modalities of these receptors, validated for their effects on the protein and then used as rescue constructs in synaptogenesis assays.
-
Evaluation Summary:
The main finding that GPCR activity is necessary for latrophilins' role in synapse formation is both surprising and important. This work will inspire new research on compartmentalized GPCR signaling at the synapse.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1 agreed to share their name with the authors.)
-
