Palmitoylation regulates neuropilin-2 localization and function in cortical neurons and conveys specificity to semaphorin signaling via palmitoyl acyltransferases

The central nervous system (CNS) consists of numerous disparate classes of neurons with remarkably distinct morphologies and functions. This is particularly prominent in laminated structures, including the cerebral cortex, where pyramidal neurons occupying different cortical layers acquire distinct morphologies and their processes exhibit subcellular compartmentalization that mediates distinct functions (Spruston, 2008). For example, dendritic spines, which receive the vast majority of excitatory inputs, have specific subcellular distributions that directly impact electrical properties of neurons and subsequently the activity within neuronal circuits. Numerous studies highlight the physiological importance of dendritic spines, linking alterations in spine number and morphology to various neuropsychiatric disorders (Penzes et al., 2011).

One class of proteins implicated in nervous system development are semaphorins (Koropouli and Kolodkin, 2014). Class 3 secreted semaphorins semaphorin 3F (Sema3F) and semaphorin 3A (Sema3A) play critical roles in several aspects of neural development and function, including axon guidance, axon pruning, dendritic arborization, dendritic spine distribution, synaptic transmission, and homeostatic synaptic plasticity (Danelon et al., 2020; Demyanenko et al., 2014; Gu et al., 2003; Koropouli and Kolodkin, 2014; Li et al., 2022; Riccomagno et al., 2012; Sahay et al., 2005; Tran et al., 2009; Wang et al., 2017). In mammalian cortical neurons, Sema3F mediates pruning of excess dendritic spines along the apical dendrite of layer V cortical pyramidal neurons, whereas Sema3A promotes the elaboration of basal dendrites in the same neurons (Tran et al., 2009). Sema3F and Sema3A bind distinct holoreceptor complexes that include neuropilin (Nrp) and plexin (Plex) transmembrane proteins; Sema3F exerts many of its effects via a holoreceptor complex that includes Nrp2/PlexA3, whereas Sema3A acts through a holoreceptor complex that includes Nrp1/PlexA4 (Yaron et al., 2005). Recent work provides insight into the signaling pathways that mediate Sema3F/Nrp2-dependent cytoskeletal rearrangements resulting in dendritic spine pruning in cortical neurons, including contributions by specific immunoglobin superfamily transmembrane proteins that mediate select responses to secreted semaphorins and proteins that regulate actin cytoskeleton dynamics (Demyanenko et al., 2014; Duncan et al., 2021). However, molecular mechanisms that regulate neuropilin subcellular localization and underlie divergent Sema3F and Sema3A functions in cortical neurons remain largely unknown.

The attachment of the fatty acid palmitate on thiol groups of cysteine residues, known as S-palmitoylation (hereafter referred to as palmitoylation), is a reversible posttranslational modification that dynamically regulates protein localization and function of a vast protein repertoire in different tissues (Linder and Deschenes, 2007; Salaun et al., 2010). In the nervous system, palmitoylation is critically involved in all aspects of neural development and function (Fukata and Fukata, 2010; Kang et al., 2008), including axon outgrowth (Tortosa et al., 2017), dendritic arborization (Takemoto-Kimura et al., 2007), spine formation (George et al., 2015; Kang et al., 2008; Kutzleb et al., 1998), synapse assembly, synaptic transmission (El-Husseini et al., 2002; Hayashi et al., 2005; Keith et al., 2012; Lin et al., 2009; Sanders et al., 2020; Thomas et al., 2012), and synaptic plasticity (Brigidi et al., 2014). Strikingly, there is an apparent interaction between palmitoylation and synaptic transmission since neuronal activity can regulate protein palmitoylation (Brigidi et al., 2014; Hayashi et al., 2005; Kang et al., 2008). Palmitoylation is catalyzed by palmitoyl acyltransferases (PATs), enzymes that harbor the catalytically active Asp-His-His-Cys (DHHC) signature motif (thereby, also referred to as DHHCs). Originally discovered in the yeast Saccharomyces cerevisiae (Lobo et al., 2002; Roth et al., 2002), thus far 23 DHHC enzymes are predicted to exist in humans and mice. PATs exhibit overlapping, yet distinct, specificities for their substrates (Huang et al., 2004; Roth et al., 2006). Despite systematic and extensive efforts (Roth et al., 2006), our knowledge of DHHC enzyme substrates remains very limited, hindering our understanding of the roles that PATs play in neuronal development and function.

Here, we show that both Nrp2 and Nrp1 are palmitoylated in cortical neurons in vitro and in vivo, and that palmitoylation of select Nrp2 cysteine residues is required for correct Nrp2 subcellular localization, cell surface clustering, and for Sema3F/Nrp2-dependent dendritic spine pruning both in vitro and in vivo. Our findings also reveal that the PAT ZDHHC15 is required for proper Nrp2 palmitoylation and Sema3F/Nrp2-dependent dendritic spine pruning in layer V cortical pyramidal neurons, but not for Nrp1 palmitoylation or for Sema3A/Nrp1-dependent basal dendrite elaboration in these same neurons. These results highlight the importance of guidance cue receptor posttranslational lipid modifications to regulate distinct aspects of cortical neuron morphology.

We show here that Nrp2 and Nrp1 are novel neuronal palmitoylation substrates and that select palmitoyl acceptor Nrp2 cysteines are critical for Nrp2 protein subcellular localization, for plasma membrane clustering and for Sema3F/Nrp2-dependent apical dendritic spine pruning in deep layer cortical pyramidal neurons in vitro and in vivo (Figure 7E and F). Importantly, a comparative analysis between Nrp2 and Nrp1 shows that PAT-substrate specificity contributes to the functional divergence and specification of Sema3F/Nrp2 as compared to Sema3A/Nrp1 signaling pathways in the CNS.

The discovery and functional characterization of neuropilin palmitoylation opens new avenues for investigating how these multifaceted neuronal cue receptors sculpt neuronal circuits. Despite Nrp2 and Nrp1 displaying similar palmitoylation patterns in their transmembrane and juxtamembrane segments, they exhibit critical differences with regard to their subcellular localization and function, and these can be partially ascribed to palmitoylation. Specifically, palmitoylation conveys upon Nrp2, a highly clustered localization pattern on the cell surface, whereas Nrp1 is diffusely localized on the plasma membrane regardless of its palmitoylation state. Further, our biochemical and phenotypic analyses of Sema3F/Nrp2-dependent cortical neuron dendritic spine pruning and Sema3A/Nrp1-dependent cortical neuron dendritic elaboration reveal that the PAT ZDHHC15 is required for Sema3F/Nrp2 function in cortical neurons, whereas it is not required for Sema3A/Nrp1 function in this same class of neuron. These observations show that palmitoylation endows Nrp2 and Nrp1 with distinct functional properties that allow these receptors to exert their well-defined and distinct effects on cortical layer V pyramidal neuron morphogenesis. This may be explained by palmitoylation events mediated by distinct PATs and/or palmitoylation at distinct subcellular sites being differentially important for protein localization and function. For example, palmitoylation close to the plasma membrane might regulate the targeting of a protein to dendritic spines and/or synapses, and these events may also include activity-dependent or ligand-dependent effects on protein trafficking (Brigidi et al., 2014; Noritake et al., 2009). Additional factors that may interact with palmitoylation signaling, including other posttranslational modifications (Lin et al., 2009; Salaun et al., 2010), sorting signals, and protein-protein interactions, may endow Nrp2 and Nrp1 with additional protein-specific localizing and functional properties.

Our finding that Nrp2 cysteines critically control Nrp2 localization, homo-multimerization, and function in cortical neurons raises the question as to whether these effects are mediated by palmitoylation per se, or they result from cysteine residue-dependent structural effects. Several lines of evidence presented here, including robust Nrp2 palmitoylation, the effect of 2-bromopalmitate on Nrp2 cell surface localization, the Sema3F-induced enhancement of Nrp2 palmitoylation, and the critical role of ZDHHC15 in Nrp2 function, provide strong support for a palmitoylation-dependent role for these cysteines. Interestingly, cysteine palmitoylation itself has been shown to impact protein dimerization (Bhattacharyya et al., 2016). Given its reversibility, palmitoylation could give rise to bidirectional transformation between a palmitoylated state and a non-palmitoylated state that takes part in disulfide-linked dimer formation. In addition, given the critical role of neuron-glial related cell adhesion molecule (NrCAM) in Sema3F-dependent dendritic spine pruning in cortical neurons (Demyanenko et al., 2014; Duncan et al., 2021), it will be of interest to investigate the role of Nrp2 palmitoylation in the physical and functional interactions between the Nrp2/PlexA3 holoreceptor complex and NrCAM.

We find that the C-terminal Nrp2 di-cysteine motif CCXXX_* (‘_*’ denotes a stop codon) does not appear to play essential roles in Nrp2 localization on Golgi membranes, cell surface compartmentalization, or dendritic spine pruning, though minor effects of these cysteines can be observed in certain localization assays. This C-terminal motif is also present in paralemmin (Kutzleb et al., 1998) and RhoB (Adamson et al., 1992), proteins in which the upstream cysteine is palmitoylated and the downstream cysteine is prenylated (Adamson et al., 1992; Fukata and Fukata, 2010; Kutzleb et al., 1998). It is likely that the Nrp2 C-terminal di-cysteine motif is important for other aspects of Nrp2 function such as axon guidance or axon pruning. Further, the similar cell surface distribution patterns of Nrp2^TCS and Nrp2^{Full CS} suggest that the juxtamembrane cysteines predominate over the C-terminal cysteines in regulating Nrp2 subcellular localization. Even within the juxtamembrane cysteine cluster there appears to be synergistic and/or dominant effects among the three cysteines, as revealed by the non-additive effects of different cysteine mutants on overall Nrp2 palmitoylation and by the ability of these different Nrp2 proteins to regulate dendritic spine pruning. However, with regard to effects on overall protein clustering, more extensive Nrp2 CS mutants (e.g. Nrp2^TCS, Nrp2^{Full CS}) display more severe deficits in protein localization as compared to single CS mutants (Figure 3—figure supplement 1 and data not shown). Overall, our observations favor a model whereby there is functional segregation within the Nrp2 transmembrane and cytoplasmic domains that is attributable in part to distinct roles in Nrp2 localization and function being imparted by distinct cysteine clusters, consistent with previous observations on palmitoylation effects on ionotropic receptors (Hayashi et al., 2009; Hayashi et al., 2005).

Our observation that Nrp2 distribution includes localization in the Golgi apparatus suggests that bulk Nrp2 palmitoylation occurs on Golgi membranes, and that palmitoylated Nrp2 enters the post-Golgi secretory pathway to undergo anterograde trafficking toward the plasma membrane. Interestingly, a significant fraction of Nrp2 protein is associated with Golgi membranes in dendrites of cortical neurons (Figure 3C and D and Figure 3—figure supplement 3), known as Golgi outposts (Horton et al., 2005). This suggests that Nrp2 may also be palmitoylated locally in response to stimuli including its ligand Sema3F, other signaling effectors or even neuronal activity, and that Nrp2 is then delivered to dendritic spines and synapses in response to these cues, as has been observed for other proteins (Noritake et al., 2009). Therefore, the enhancement we observe of Nrp2 palmitoylation by exposure to exogenous Sema3F (Figure 4E and F) may lead to rapid and/or controlled delivery of Nrp2 to synaptic sites. Moreover, Nrp2 and its ligand Sema3F are dynamically regulated in response to alterations in neuronal activity (Lee et al., 2012; Wang et al., 2017); in the case of Nrp2 this may in part be controlled by rapid cycles of Nrp2 palmitoylation-depalmitoylation in the vicinity of individual dendritic spines, a mechanism that can confer both spatial and temporal precision of protein localization and function (Fivaz and Meyer, 2003; Fukata et al., 2004). This idea is in line with studies demonstrating activity-dependent palmitoylation of synapse-associated proteins in neural tissue (Brigidi et al., 2014; Hayashi et al., 2005; Noritake et al., 2009).

The spatial and temporal organization of the palmitoylation network is largely governed by the precise spatiotemporal regulation of PATs (Greaves and Chamberlain, 2011; Noritake et al., 2009; Ohno et al., 2006), rendering substrate specificity a major determinant of the functional specification of cues that can regulate neuronal architecture and polarity. However, the landscape of protein palmitoylation is further enriched by modulators of DHHC activity and the concurrent activity of depalmitoylating enzymes (Salaun et al., 2020; Tortosa et al., 2017; Yokoi et al., 2016). Of note, there is increasing evidence that palmitoylation deficits lead to altered neuronal excitability and aberrant neuronal phenotypes linked to neural diseases (Mansouri et al., 2005; Milnerwood et al., 2013; Mukai et al., 2015; Mukai et al., 2008; Mukai et al., 2004; Pinner et al., 2016; Raymond et al., 2007; Singaraja et al., 2011; Sutton et al., 2013). In particular ZDHHC15, shown here to play essential roles in Nrp2 palmitoylation and function, has been implicated in X-linked intellectual disability (Lewis et al., 2021; Mansouri et al., 2005), impairments in learning and memory (Wang et al., 2015), hyperactivity associated with a novel environment and sensitivity to psychostimulants (Mejias et al., 2021), and also dendritic outgrowth and formation of mature spines in hippocampal neurons in vitro (Shah et al., 2019). Intriguingly, Sema3F/Nrp2 signaling has been shown to play important roles in hippocampal circuit function, and its dysregulation may cause epileptic activity in mice (Eisenberg et al., 2021; Li et al., 2022; Sahay et al., 2005). Moreover, neuropilins are key players in cancer growth and progression and response to antineoplastic therapies (Napolitano and Tamagnone, 2019; Rizzolio and Tamagnone, 2011), and they also play important roles in the function of the immune system (Kumanogoh and Kikutani, 2013) and the vascular system (Gu et al., 2003; Simons et al., 2016). Our experiments identify neuropilins as new members of the neuronal palmitoyl proteome, reveal new DHHC enzymatic substrates in the CNS, and define DHHC enzyme-substrate specificity as a novel mechanism specifying the functional identity of neuronal substrates. These results advance our understanding of CNS development and function and may prove invaluable for the development of targeted therapeutic approaches directed toward amelioration of neural disorders associated with aberrant function of palmitoylation signaling pathways.

All data quantifications, western blots, and images that are presented in this study and for which links are provided in the manuscript as available on the eLife web site, in addition to all data relating to every graph, western blot and all quantifications, have been uploaded in order to be made easily available. Long-term archiving of data, as described above, that are part of this published study in eLife, along with all supporting data that contributed to each and every published file, will be managed by Johns Hopkins Data Services (JHUDS) using the Johns Hopkins Research Data Repository. This repository provides public access to data through an established platform supported by storage and preservation practices that follow the Open Archival Information System reference model. Deposited data has been given standard data citations and persistent identifiers (DOIs). Data are archived under a memorandum of understanding renewed every 5 years with the PI's consent. The DOI for these data is https://doi.org/10.7281/T1/OY2X8T.

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   (2023) Johns Hopkins Research Data Repository

   Data associated with the publication: Palmitoylation regulates neuropilin-2 localization and function in cortical neurons and conveys specificity to semaphorin signaling via palmitoyl acyltransferases.

   https://doi.org/10.7281/T1/OY2X8T

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Author details

1. Eleftheria Koropouli

   The Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States

   Present address

   First Department of Neurology, Aiginition Hospital, National and Kapodistrian University of Athens School of Medicine, Athens, Greece

   Contribution

   Conceptualization, Formal analysis, Funding acquisition, Validation, Investigation, Methodology, Writing – original draft, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-8537-4471

2. Qiang Wang

   The Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States

   Present address

   Department of Neurology, Zhongshan Hospital, Fudan University, Shanghai, China

   Contribution

   Formal analysis, Validation, Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

3. Rebeca Mejías

   Department of Physiology,University of Seville, Seville, Spain

   Contribution

   Investigation, Writing – review and editing

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0003-1936-7219

4. Randal Hand

   The Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States

   Present address

   Prilenia Therapeutics Development LTD, Herzliya, Israel

   Contribution

   Formal analysis, Validation, Investigation, Writing – review and editing

   Competing interests

   Randal Hand is affiliated with Prilenia Therapeutics Development LTD. The author has no financial interests to declare

5. Tao Wang

   McKusick-Nathans Institute of Genetic Medicine and Department of Pediatrics, The Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Supervision, Writing – review and editing

   Competing interests

   No competing interests declared

6. David D Ginty

   Department of Neurobiology, Howard Hughes Medical Institute, Harvard Medical School, Boston, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Writing – review and editing

   Competing interests

   Reviewing editor, eLife

   "This ORCID iD identifies the author of this article:" 0000-0001-9723-8530

7. Alex L Kolodkin

   The Solomon H Snyder Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, United States

   Contribution

   Conceptualization, Supervision, Funding acquisition, Validation, Methodology, Writing – original draft, Writing – review and editing

   For correspondence

   kolodkin@jhmi.edu

   Competing interests

   No competing interests declared

   "This ORCID iD identifies the author of this article:" 0000-0001-7562-5513

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