Palmitoylation regulates neuropilin-2 localization and function in cortical neurons and conveys specificity to semaphorin signaling via palmitoyl acyltransferases
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Signaling mediated by Semaphorins and their receptors Nrp1 and Nrp2 is crucial for regulating the morphology of dendritic spines and dendritic arborization during development. In this manuscript, the authors found that the post-translational modification of S-palmitoylation dictates the subcellular localization and trafficking of Nrp2, but not Nrp1, and is required for Sema3F-dependent pruning of spines on the apical dendrites of layer V cortical neurons. The study provides important insights into how semaphorin signaling achieves spatial specificity on diverse downstream cellular events.
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Abstract
Secreted semaphorin 3F (Sema3F) and semaphorin 3A (Sema3A) exhibit remarkably distinct effects on deep layer excitatory cortical pyramidal neurons; Sema3F mediates dendritic spine pruning, whereas Sema3A promotes the elaboration of basal dendrites. Sema3F and Sema3A signal through distinct holoreceptors that include neuropilin-2 (Nrp2)/plexinA3 (PlexA3) and neuropilin-1 (Nrp1)/PlexA4, respectively. We find that Nrp2 and Nrp1 are S-palmitoylated in cortical neurons and that palmitoylation of select Nrp2 cysteines is required for its proper subcellular localization, cell surface clustering, and also for Sema3F/Nrp2-dependent dendritic spine pruning in cortical neurons, both in vitro and in vivo. Moreover, we show that the palmitoyl acyltransferase ZDHHC15 is required for Nrp2 palmitoylation and Sema3F/Nrp2-dependent dendritic spine pruning, but it is dispensable for Nrp1 palmitoylation and Sema3A/Nrp1-dependent basal dendritic elaboration. Therefore, palmitoyl acyltransferase-substrate specificity is essential for establishing compartmentalized neuronal structure and functional responses to extrinsic guidance cues.
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Author Response
Reviewer #3 (Public Review):
Although initially discovered as axon guidance molecules in the nervous system, Semaphorins, signaling through their receptors the Neuropilins and Plexins, regulate a variety of cell-cell signaling events in a variety of cell types. In addition, cells often express multiple Semas and receptors. Thus, one important question that has yet to be adequately understood about these important signaling proteins is: how does specificity of function arise from a ubiquitously expressed signaling family?
This study addresses that important question by investigating the role of cysteine palmitoylation on the localization and function of the Neuropilin-2 (Nrp-2) receptor. It was already known that Sema3F signaling through a complex of Nrp-2 and Plexin-A3 regulates pruning of dendritic spines in …
Author Response
Reviewer #3 (Public Review):
Although initially discovered as axon guidance molecules in the nervous system, Semaphorins, signaling through their receptors the Neuropilins and Plexins, regulate a variety of cell-cell signaling events in a variety of cell types. In addition, cells often express multiple Semas and receptors. Thus, one important question that has yet to be adequately understood about these important signaling proteins is: how does specificity of function arise from a ubiquitously expressed signaling family?
This study addresses that important question by investigating the role of cysteine palmitoylation on the localization and function of the Neuropilin-2 (Nrp-2) receptor. It was already known that Sema3F signaling through a complex of Nrp-2 and Plexin-A3 regulates pruning of dendritic spines in cortical neurons while Sema3A signals through Nrp-1/PlexA4 to regulate dendritic arborization. The major finding of this study which is well-supported by the data is that palmitoylation of Nrp-2 regulates its cell surface clustering and dendritic spine pruning activity in cortical neurons. Interestingly, palmitoylation of Nrp-1 at homologous residue does not appear to regulate its localization or known neuronal function.
A clear strength of this manuscript is the many techniques that are utilized to examine the question: this study represents a tour de force of biochemical, molecular, genetic, pharmacological and cell biological assays performed both in vitro and in vivo. The authors carefully dissect the function of distinct palmitoylated cysteine residues on Nrp-2 localization and function, concluding that palmitoylation of juxtamembrane cysteines predominates over C-terminal palmityolyation for the Nrp-2 dependent processes assayed in this study. The authors also demonstrate that a specific palmityl transferase (DHHC15) acts on Nrp-2 but not Nrp-1 and is required for Nrp-2 clustering and dendritic spine pruning. These findings are important because they demonstrate one mechanism by which different signaling pathways, even from a related family of proteins, can achieve signaling specificity in the cell.
A minor weakness of the paper is that one would like to see a connection between palmitoylation-dependent cell membrane clustering of Nrp-2 on the cell surface and Nrp-2 regulation of dendritic spine pruning. Although the two phenotypes frequently correlate in the data presented, there are a few notable exceptions: e.g. Nrp-2TCS forms larger clusters in cortical neurons while Nrp-2FullCS is diffuse on the cell surface; both mutants affect spine pruning. In the future, it would also be interesting to know if increased clustering of Nrp-2 was observed at spines that were eliminated, for example. Nonetheless this manuscript represents an important advance in our understanding of synaptic pruning and cellular mechanisms that constrain protein surface localization and signaling pathways.
We agree that the reviewer’s comment on the need to show a direct association between palmitoylation-dependent Nrp-2 clustering on the cell surface and Nrp-2 regulation of dendritic spine pruning is very important. This underscores the need to develop new robust tools that can directly and specifically address the effects of palmitoylation on protein localization and neuronal morphology. For example, an antibody that is specific for palmitoylated Nrp-2, perhaps including site-specific Nrp-2 palmitoylation, would allow for direct visualization of palmitoylated protein localization at subcellular resolution, and if coupled with in vivo imaging, could help address questions related to spine dynamics with respect to Nrp-2 expression and palmitoylation. However, at present we consider this approach an important future direction.
Regarding the Nrp-2 mutants TCS and Full CS, our experiments suggest the existence of a threshold for protein mislocalization beyond which Nrp-2 loses its function. In other words, the defect in protein localization imparted by the mutation of the three juxtamembrane cysteines (TCS Nrp-2 mutant) seems to be sufficient to cause Nrp-2 dysfunction. In addition, as noted above (Reviewer #1), the protein clustering assay is a useful but a more general localization assay; more sophisticated assays need to be developed to investigate palmitoylated proteins when they are mislocalized upon site-specific depalmitoylation, which could provide a more accurate association between a protein’s localization and function.
The reviewer’s idea to look at the localization of Nrp-2 at dendritic spines and correlate this with the fate of spines during postnatal development, including relating to spine maintenance vs elimination, is an excellent suggestion that could link directly Nrp-2 to spine dynamics. To address this, however, again new assays with exogenous Nrp-2 expression will need to be developed, but with very low levels of protein expression to avoid saturation of spines with exogenous tagged-Nrp-2 protein and preserve functional specificity for spine regulation. Alternatively, robust in vivo tagging of ndogenous Nrp-2 protein using CRISPR approaches also provide another avenue to achieve this goal—of note, we are trying this approach but, thus far, we have not been successful in achieving labeling that is robust enough for such experiments.
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eLife assessment
Signaling mediated by Semaphorins and their receptors Nrp1 and Nrp2 is crucial for regulating the morphology of dendritic spines and dendritic arborization during development. In this manuscript, the authors found that the post-translational modification of S-palmitoylation dictates the subcellular localization and trafficking of Nrp2, but not Nrp1, and is required for Sema3F-dependent pruning of spines on the apical dendrites of layer V cortical neurons. The study provides important insights into how semaphorin signaling achieves spatial specificity on diverse downstream cellular events.
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Reviewer #1 (Public Review):
This manuscript by Koropouli et al. is a much-needed study that provides novel mechanistic insight of how signaling receptors can be targeted to distinct subcellular domains or membrane locations that, in part, confer their functional specificity. It is well-established that members of the class 3 secreted semaphorins guidance cues can bind to the receptors the neuropilins (Nrp1 and Nrp2) to elicit numerous cellular processes important for circuit assembly. Previously, it was demonstrated that Sema3F signaling with Nrp2 and its co-receptor Plexin-A3 is required for the removal of excess excitatory synaptic spines on the apical dendrite of layer V cortical neurons, while the closely related member Sema3A signaling with Nrp1/Plexin-A4 promotes the elaboration of the basal dendritic arbor on the same neuron. …
Reviewer #1 (Public Review):
This manuscript by Koropouli et al. is a much-needed study that provides novel mechanistic insight of how signaling receptors can be targeted to distinct subcellular domains or membrane locations that, in part, confer their functional specificity. It is well-established that members of the class 3 secreted semaphorins guidance cues can bind to the receptors the neuropilins (Nrp1 and Nrp2) to elicit numerous cellular processes important for circuit assembly. Previously, it was demonstrated that Sema3F signaling with Nrp2 and its co-receptor Plexin-A3 is required for the removal of excess excitatory synaptic spines on the apical dendrite of layer V cortical neurons, while the closely related member Sema3A signaling with Nrp1/Plexin-A4 promotes the elaboration of the basal dendritic arbor on the same neuron. The question is then how do the two different signaling pathways convey such precise and opposite cellular function of eliminating spines and promoting dendritic elaboration in distinct subcellular compartments of the same neuron? While some hints were provided that the Nrp2 receptor is localized to the apical dendrite and Nrp1 is distributed widely along all dendrites on the same cortical neuron in vitro, this has not been shown in vivo and the mechanism of such targeted subcellular localization is not known. In the current study, the authors used biochemical, cellular, and molecular assays in combination with mouse genetics and live-cell imaging to demonstrate that the post-translational modification of S-palmitoylation dictates the proper subcellular localization and trafficking of Nrp2, but not Nrp1, and is required for Sema3F-dependent pruning of spines on the apical dendrites of layer V cortical neurons. The following are the strength and novel findings of this study.
1. This study confirms previous findings and adds new information by mapping the specific locations of the cysteine amino acid residues to the transmembrane/juxtamembrane region of neuropilin receptors for palmitoylation, which confers the subcellular localization specificity for Nrp2 but not Nrp1, in cortical neurons and non-neuronal cells.
2. The study also found that select cysteine residues on Nrp2 are palmitoylated by the palmitoyltransferase DHHC15, and palmitoylation of these sites are required for the homo-oligomerization of the Nrp2 receptor but not for the association with the co-receptor Plexin-A3.
3. The authors demonstrated that Sema3F signaling itself seems to enhance the level of Nrp2 palmitoylation in some sort of positive feedback loop. It would be interesting for future experiments to determine how Sema3F signaling promotes this palmitoylation.Although most of the key claims are supported by data presented in the paper, clarification of the following concerns would further strengthen the overall conclusion of the study.
1. While some of the qualitative micrograph images are very convincingly in illustrating the drastic difference in Nrp2 versus Nrp1 expression patterns/cell-surface localization, such as Fig. 1A and 1D, many of the quantitative analyses have a low n number and/or low sample size, with only 2 replicate experiments or only 2 brains/animals per genotype analyzed. To increase the rigor of this study, the authors should add a few more replicates to the experiments with low n numbers.
2. The substitutions of C878, C885, and C887 to serines caused an ~80%, ~50%, and ~60% reduction, respectively, in Nrp2 palmitoylation compared to WT neuroblastoma-2a cells (as show in Fig. 2D and 2E). However, when mutating all three of these cysteine sites (the TCS plasmid), there is only ~80% total reduction in Nrp2 palmitoylation (Fig. 2F and 2G), just about equal to the C878S substitution alone. One would expect that the reduction in palmitoylation to be more severe with the TCS plasmid, but might this be due to the low n number in quantifications shown in Fig. 2E and 2G. It would add substantially to support the specificity of these cysteine residues' function if the single C878 was demonstrated to be required for either subcellular localization of Nrp2 leading to the rescue of the dendritic spine phenotype in Nrp2-/- primary neurons or in an in utero experiment. -
Reviewer #2 (Public Review):
The study of Koropouli is a tour the force investigation of the Semaphorins receptor, Neuropilin-2, modification by Palmitoylation. The work consists of biochemical, cellular and in vivo experiments and overall underscore an interesting layer of regulation of axonal guidance receptors membrane localization and function by lipid modification.
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Reviewer #3 (Public Review):
Although initially discovered as axon guidance molecules in the nervous system, Semaphorins, signaling through their receptors the Neuropilins and Plexins, regulate a variety of cell-cell signaling events in a variety of cell types. In addition, cells often express multiple Semas and receptors. Thus, one important question that has yet to be adequately understood about these important signaling proteins is: how does specificity of function arise from a ubiquitously expressed signaling family?
This study addresses that important question by investigating the role of cysteine palmitoylation on the localization and function of the Neuropilin-2 (Nrp-2) receptor. It was already known that Sema3F signaling through a complex of Nrp-2 and Plexin-A3 regulates pruning of dendritic spines in cortical neurons while …
Reviewer #3 (Public Review):
Although initially discovered as axon guidance molecules in the nervous system, Semaphorins, signaling through their receptors the Neuropilins and Plexins, regulate a variety of cell-cell signaling events in a variety of cell types. In addition, cells often express multiple Semas and receptors. Thus, one important question that has yet to be adequately understood about these important signaling proteins is: how does specificity of function arise from a ubiquitously expressed signaling family?
This study addresses that important question by investigating the role of cysteine palmitoylation on the localization and function of the Neuropilin-2 (Nrp-2) receptor. It was already known that Sema3F signaling through a complex of Nrp-2 and Plexin-A3 regulates pruning of dendritic spines in cortical neurons while Sema3A signals through Nrp-1/PlexA4 to regulate dendritic arborization. The major finding of this study which is well-supported by the data is that palmitoylation of Nrp-2 regulates its cell surface clustering and dendritic spine pruning activity in cortical neurons. Interestingly, palmitoylation of Nrp-1 at homologous residue does not appear to regulate its localization or known neuronal function.
A clear strength of this manuscript is the many techniques that are utilized to examine the question: this study represents a tour de force of biochemical, molecular, genetic, pharmacological and cell biological assays performed both in vitro and in vivo. The authors carefully dissect the function of distinct palmitoylated cysteine residues on Nrp-2 localization and function, concluding that palmitoylation of juxtamembrane cysteines predominates over C-terminal palmityolyation for the Nrp-2 dependent processes assayed in this study. The authors also demonstrate that a specific palmityl transferase (DHHC15) acts on Nrp-2 but not Nrp-1 and is required for Nrp-2 clustering and dendritic spine pruning. These findings are important because they demonstrate one mechanism by which different signaling pathways, even from a related family of proteins, can achieve signaling specificity in the cell.
A minor weakness of the paper is that one would like to see a connection between palmitoylation-dependent cell membrane clustering of Nrp-2 on the cell surface and Nrp-2 regulation of dendritic spine pruning. Although the two phenotypes frequently correlate in the data presented, there are a few notable exceptions: e.g. Nrp-2TCS forms larger clusters in cortical neurons while Nrp-2FullCS is diffuse on the cell surface; both mutants affect spine pruning. In the future, it would also be interesting to know if increased clustering of Nrp-2 was observed at spines that were eliminated, for example. Nonetheless this manuscript represents an important advance in our understanding of synaptic pruning and cellular mechanisms that constrain protein surface localization and signaling pathways.
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