Regulation of different phases of AMPA receptor intracellular transport by 4.1N and SAP97
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eLife assessment
This important study by Bonnet et al addresses the question of how AMPA receptor numbers at the synapse are regulated during basal conditions and during chemically induced Long Term Potentiation. Specifically, the study aims to determine the molecular mechanisms that contribute to the intracellular trafficking of AMPA receptors and determine their insertion into the synaptic plasma membrane. Using compelling methodology, the authors dissect the distinct roles of two proteins that bind to the C-terminal domain of the AMPA receptor subunit GluA1: 4.1N and SAP97. The findings will be of interest to anyone trying to understand molecular events contributing to synaptic plasticity in health and disease, and more broadly, the method could be adapted for tracking intracellular movements of a wide range of proteins.
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Abstract
Changes in the number of synaptic AMPA receptors underlie many forms of synaptic plasticity. These variations are controlled by an interplay between their intracellular transport (IT), export to the plasma membrane (PM), stabilization at synapses, and recycling. The cytosolic C-terminal domain of the AMPAR GluA1 subunit is specifically associated with 4.1 N and SAP97. We analyze how interactions between GluA1 and 4.1N or SAP97 regulate IT and exocytosis in basal conditions and after cLTP induction. The down-regulation of 4.1N or SAP97 decreases GluA1 IT properties and export to the PM. The total deletion of its C-terminal fully suppresses its IT. Our results demonstrate that during basal transmission, the binding of 4.1N to GluA1 allows their exocytosis whereas the interaction with SAP97 is essential for GluA1 IT. During cLTP, the interaction of 4.1N with GluA1 allows its IT and exocytosis. Our results identify the differential roles of 4.1N and SAP97 in the control of various phases of GluA1 IT.
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eLife assessment
This important study by Bonnet et al addresses the question of how AMPA receptor numbers at the synapse are regulated during basal conditions and during chemically induced Long Term Potentiation. Specifically, the study aims to determine the molecular mechanisms that contribute to the intracellular trafficking of AMPA receptors and determine their insertion into the synaptic plasma membrane. Using compelling methodology, the authors dissect the distinct roles of two proteins that bind to the C-terminal domain of the AMPA receptor subunit GluA1: 4.1N and SAP97. The findings will be of interest to anyone trying to understand molecular events contributing to synaptic plasticity in health and disease, and more broadly, the method could be adapted for tracking intracellular movements of a wide range of proteins.
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Reviewer #1 (Public Review):
This important study by Bonnet et al addresses the question of how AMPA receptor numbers at the synapse are regulated during basal conditions and during chemically induced Long Term Potentiation (cLTP). Specifically, the study aims to determine which molecular mechanisms contribute to export from Golgi/the ER, intracellular trafficking of AMPA receptors, and insertion into the synaptic plasma membrane, respectively. The authors had previously established an approach to separately measure these distinct events: to enable a high-fidelity measurement of the Golgi/ER release and subsequent speed of GluA1-containing vesicles, the release of vesicles is synchronized. Finally, the insertion into the plasma membrane is measured by immunolabelling.
The authors set out to specifically understand the contributions of …Reviewer #1 (Public Review):
This important study by Bonnet et al addresses the question of how AMPA receptor numbers at the synapse are regulated during basal conditions and during chemically induced Long Term Potentiation (cLTP). Specifically, the study aims to determine which molecular mechanisms contribute to export from Golgi/the ER, intracellular trafficking of AMPA receptors, and insertion into the synaptic plasma membrane, respectively. The authors had previously established an approach to separately measure these distinct events: to enable a high-fidelity measurement of the Golgi/ER release and subsequent speed of GluA1-containing vesicles, the release of vesicles is synchronized. Finally, the insertion into the plasma membrane is measured by immunolabelling.
The authors set out to specifically understand the contributions of two auxiliary proteins in AMPA receptor expression: 4.1N and SAP97. Namely, the authors find that under basal conditions, binding of SAP97 to GluA1 is necessary for the GluA1 release from the Golgi/ER and intracellular trafficking. In turn, binding of 4.1N to GluA1 is necessary for the exocytosis of the receptor at the plasma membrane at basal conditions. Following induction of cLTP, the authors find that the role of SAP97 remains similar to that observed under basal conditions but, interestingly, 4.1N significantly grows in influence and is required for all stages of GluA1 expression - from release from the Golgi/ER to exocytosis and insertion into the plasma membrane.In summary, using convincing methodology, the authors are able to dissect the distinct roles of two proteins that bind to the C-terminal domain of the AMPA receptor subunit GluA1: 4.1N and SAP97.
The scientific rigor is high in this work. For example, the question of whether the expression of GluA1 depends on physical interaction with 4.1N and/or SAP97 is nicely addressed by several, well-considered experiments. Overall, the authors' claims are well justified by the data presented.I did not find any major scientific weaknesses in this manuscript. The approach developed by the group appears to be a good tool for studying the molecular choreography at the synapse under different conditions and the results will be of interest to a wide range of neuroscientists.
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Reviewer #2 (Public Review):
The study of Bonnet et al. focuses on how proteins 4.1N and SAP97 affect intracellular trafficking (IT) and externalisation of AMPA receptors (AMPARs) in cultured rat hippocampal neurons. To specifically look at IT, the authors combine the so-called Ariad approach with confocal spinning disc microscopy and photobleaching of dendritic regions, developed in their previous paper (Hangen et al., 2018). This allowed them to synchronously release newly synthesized AMPARs from the ER (upon addition of a synthetic ligand) and measure the number of vesicles carrying AMPARs, their velocity as well as time spent moving and pausing. To detect the insertion of AMPARs at the plasma membrane, live immunolabelling was used. Using RNA-based knock-outs of 4.1N and SAP97 proteins as well as mutants of the AMPAR C-terminus …
Reviewer #2 (Public Review):
The study of Bonnet et al. focuses on how proteins 4.1N and SAP97 affect intracellular trafficking (IT) and externalisation of AMPA receptors (AMPARs) in cultured rat hippocampal neurons. To specifically look at IT, the authors combine the so-called Ariad approach with confocal spinning disc microscopy and photobleaching of dendritic regions, developed in their previous paper (Hangen et al., 2018). This allowed them to synchronously release newly synthesized AMPARs from the ER (upon addition of a synthetic ligand) and measure the number of vesicles carrying AMPARs, their velocity as well as time spent moving and pausing. To detect the insertion of AMPARs at the plasma membrane, live immunolabelling was used. Using RNA-based knock-outs of 4.1N and SAP97 proteins as well as mutants of the AMPAR C-terminus which mediates interactions with these two proteins, in basal conditions and during chemically induced long-term potentiation (cLTP), they could show that the two proteins play different roles in AMPAR trafficking, with SAP97 more profoundly affecting IT compared to 4.1N in basal conditions.
The unique approach allowing observation of IT of AMPARs and a series of tested mutants in basal and cLTP conditions are the main strengths of the paper and also result in the main new finding which is differential regulation of AMPAR IT by 4.1N and SAP97. The measurements of IT parameters and externalisation of unmodified AMPARs across different conditions (and the previous publication) are very reproducible and that makes the whole approach very reassuring.
However, a few points regarding the methodology and analysis remained after reading the manuscript:
Due to the tested mutants, I find the data for the 4.1N-AMPAR interaction particularly strong, but less so for SAP97. For SAP97, sh-RNA experiments are performed and the delta7 mutant is tested. In the case of 4.1N, sh-RNA knockouts were found to be affected by interactions other than AMPAR-4.1N, so the same might be the case for SAP97. Delta4.1N mutant was found to be less reliable than the S816A S818A mutant, in which the AMPAR C-terminus length was retained and 4.1N binding abolished via two mutations. Although only 4 amino acids were removed in the delta7 mutant, this still changes the length of the AMPAR C-terminus. It would be good to acknowledge these aspects of SAP97 experiments.As there is a number of conditions tested in the paper and to make the conclusions clearer, it might be useful to provide a summary table. It seems to me there are conditions where IT parameters remain unchanged, but no condition where externalisation is not reduced compared to the relevant control condition. Hence, I would agree that 4.1N might be less relevant than SAP97 for IT, but I am not sure it is clear that 4.1N plays a bigger role in externalisation than SAP97, which is what the conclusion figure (Fig. 7) seems to be implying.
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Reviewer #3 (Public Review):
This manuscript uses novel techniques to examine the intracellular trafficking and membrane insertion of AMPA receptors to dissect the molecular mechanism involved in regulating these processes in neuronal cultures under basal conditions and during the induction of a chemical form of long-term potentiation (LTP). Specifically, they examine the role of the interaction of the GluA1 subunit with two neuronal proteins SAP97 and 4.1N. The manuscript uses a novel approach to synchronize and temporally control the release of GluA1-containing receptors from the ER and examine its trafficking through the Golgi and dendrites to the plasma membrane. This assay can measure the number of GluA1-containing intracellular vesicles, their speed of trafficking, and the delivery of newly synthesized GluA1 to the surface.
First, …
Reviewer #3 (Public Review):
This manuscript uses novel techniques to examine the intracellular trafficking and membrane insertion of AMPA receptors to dissect the molecular mechanism involved in regulating these processes in neuronal cultures under basal conditions and during the induction of a chemical form of long-term potentiation (LTP). Specifically, they examine the role of the interaction of the GluA1 subunit with two neuronal proteins SAP97 and 4.1N. The manuscript uses a novel approach to synchronize and temporally control the release of GluA1-containing receptors from the ER and examine its trafficking through the Golgi and dendrites to the plasma membrane. This assay can measure the number of GluA1-containing intracellular vesicles, their speed of trafficking, and the delivery of newly synthesized GluA1 to the surface.
First, the authors use shRNA knockdown (KD) techniques to decrease the expression of SAP97 and 4.1 and found dramatic effects on the number of GluA1-containing vesicles and plasma membrane insertion of GluA1. SAP97 had a larger effect on trafficking while 4.1N had a larger effect on plasma membrane insertion. The authors then went on to use mutants of GluA1 that lack the whole C-terminal domain or mutations in the SAP97 and 4.1N biding sites in GluA1 C-termini and examine the trafficking of these mutants. These mutations decreased the intracellular trafficking and the membrane insertion of GluA1. In addition, the authors mutated phosphorylation sites that have been reported to regulate the interaction of GluA1 with 4.1N. Mutations in these sites that eliminated phosphorylation inhibits membrane insertion while the phosphomimetic mutations did not affect membrane insertion. Finally, mutations in the SAP97 and 4.1N binding sites including mutations in the phosphorylation sites also inhibited chemical-induced LTP increases in the regulation of GluA1 ER-Golgi exit, intracellular transport, and membrane insertion.
These studies are well done and novel and provide support for the role of the GluA1 C-termini and its protein interactors in the trafficking of the AMPA receptor under basal and plasticity conditions. This contributes new data using a novel approach to the controversy over the role of the C-termini of AMPA receptors in the regulation of AMPA receptor function. It supports the role of these interactions in AMPA receptor function.
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