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Syntaxin 4: Biological Overview | References

BIOLOGICAL OVERVIEW

Postsynaptic cells can induce synaptic plasticity through the release of activity-dependent retrograde signals. A Ca(2+)-dependent retrograde signaling pathway mediated by postsynaptic Synaptotagmin 4 (Syt4) has been previously described in this context. To identify proteins involved in postsynaptic exocytosis, this study conducted a screen for candidates that disrupt trafficking of a pHluorin-tagged Syt4 at Drosophila neuromuscular junctions (NMJs). The study further characterized one candidate, the postsynaptic t-SNARE Syntaxin 4 (Syx4). Analysis of Syx4 mutants reveals that Syx4 mediates retrograde signaling, modulating the membrane levels of Syt4 and the transsynaptic adhesion protein Neuroligin 1 (Nlg1). Syx4-dependent trafficking regulates synaptic development, including controlling synaptic bouton number and the ability to bud new varicosities in response to acute neuronal stimulation. Genetic interaction experiments demonstrate Syx4, Syt4, and Nlg1 regulate synaptic growth and plasticity through both shared and parallel signaling pathways. These findings suggest a conserved postsynaptic SNARE machinery controls multiple aspects of retrograde signaling and cargo trafficking within the postsynaptic compartment (Harris, 2016).

Synaptic connections form and mature through signaling events in both pre- and postsynaptic cells. The release of signaling molecules into the synaptic cleft depends on SNARE proteins that drive membrane fusion. This machinery is well understood for neurotransmitter release from the presynaptic cell: in response to an action potential, a v-SNARE in the synaptic vesicle membrane (Synpatobrevin/VAMP) engages t-SNARES in the presynaptic membrane (Syx1 and SNAP-25), forming a four-helix structure that brings the membranes into close proximity and initiates fusion. Although SNARE-dependent fusion drives membrane dynamics in all cell types, it is specialized in the presynaptic terminal to be Ca²⁺-dependent, employing Ca²⁺ sensors like Synaptotagmin 1 (Syt1) to link synaptic vesicle fusion to Ca²⁺ influx following an action potential (Harris, 2016).

The postsynaptic cell also exhibits activity-dependent exocytosis. Altering the composition of the postsynaptic membrane, including regulated trafficking of neurotransmitter receptors, is an important plastic response to neural activity (Chater, 2014). The postsynaptic cell also releases retrograde signals into the synaptic cleft to modulate synaptic growth and function. These retrograde messengers include lipid-derived molecules like endocannabinoids, gases like nitric oxide, neurotransmitters, neurotrophins, and other signaling factors like TGF-β and Wnt. Adhesion complexes that provide direct contacts across the synaptic cleft also participate in retrograde signaling (Harris, 2016).

Although retrograde signaling is a key modulator of synaptic function, little is known about how postsynaptic exocytosis is regulated and coordinated. Components of a postsynaptic SNARE complex have been recently identified in mammalian dendrites. The t-SNAREs Syntaxin 3 (Stx3) and SNAP-47 are required for regulated AMPA receptor exocytosis during long term potentiation, while the v-SNARE synaptobrevin-2 regulates both activity-dependent and constitutive AMPAR trafficking (Jurado, 2013). Stx4 has also been implicated in activity-dependent AMPAR exocytosis (Harris, 2016).

In Drosophila, a Ca²⁺-dependent retrograde signaling pathway relies on the postsynaptic Ca²⁺ sensor Syt4. Syt4 vesicles fuse with the postsynaptic membrane in an activity-dependent fashion (Yoshihara, 2005), and loss of Syt4 leads to abnormal development and function of the NMJ. Syt4 null animals have smaller synaptic arbors, indicating a defect in synaptic growth, and also fail to exhibit several forms of synaptic plasticity seen in control animals, including robust enhancement of presynaptic release in response to high frequency stimulation, and rapid budding of synaptic boutons in response to strong neuronal stimulation (Barber, 2009; Korkut, 2013; Piccioli, 2014; Yoshihara, 2005). However, a detailed understanding of how the postsynaptic cell regulates constitutive and activity-dependent signaling of multiple retrograde pathways is lacking. In addition to exocytosis, it is likely that many cellular processes including vesicle trafficking and polarized transport of protein and transcript are specialized to facilitate postsynaptic signaling. Identifying such regulatory mechanisms is crucial for understanding synaptic development and function (Harris, 2016).

This study carried out a candidate-based transgenic RNAi screen to identify regulators of postsynaptic exocytosis at the Drosophila NMJ, a model for studying glutamatergic synapse growth and plasticit. Using a fluorescently tagged form of the postsynaptic Ca²⁺ sensor Syt4, candidate gene products were screened that disrupted the localization of Syt4 at the postsynaptic membrane. This study describes characterization of one candidate from this screen, Syntaxin 4 (Syx4). Drosophila Syx4 is the sole homolog of the mammalian Stx 3/4 family of plasma membrane t-SNAREs that also includes Syntaxin 1. The mammalian Stx3 and Stx4 homologs regulate activity-dependent AMPA receptor trafficking in mammalian neurons (Jurado, 2013; Kennedy, 2010), while Stx4 also participates in regulated secretory events in several other mammalian cell types, including insulin-stimulated delivery of the glucose transporter to the plasma membrane in adipocytes and glucose-stimulated insulin secretion from pancreatic beta cells (reviewed by Jewell, 2010). The results demonstrate that the Drosophila Syx4 homolog is essential for retrograde signaling, regulating the membrane delivery of both Syt4 and Neuroligin (Nlg1), a transsynaptic adhesion protein that plays important roles in synapse formation and function, and is linked to autism spectrum disorder (ASD). Through genetic interaction experiments, this study defined functions of the Syx4, Syt4, and Nlg1 pathway in regulating multiple aspects of synaptic growth and plasticity within the postsynaptic compartment (Harris, 2016).

To identify regulators of postsynaptic exocytosis, a screen was conducted for gene products regulating Syt4 plasma membrane accumulation, resulting in the identification of the plasma membrane t-SNARE Syx4. Analysis of a Syx4 null mutant indicates that Syx4 is essential for development of the Drosophila NMJ and regulates the membrane delivery of at least two proteins that are important for synaptic growth and plasticity: the postsynaptic Ca²⁺ sensor Syt4 and the transsynaptic adhesion protein Nlg1 (Harris, 2016).

The screen identified 15 candidate gene products that altered the localization of Syt4-pH. In addition to Syx4, several other candidates motivate interesting hypotheses about regulatory pathways for postsynaptic exocytosis. MyoV is a Ca²⁺-sensitive unconventional myosin that regulates polarized traffic. Thus, MyoV could play a role linking Ca²⁺ influx to vesicle delivery or release at the synapse. Indeed, MyoV homologs have been implicated in regulated AMPA trafficking in mammalian dendrites. Two Rab regulators (Gdi and Rabex) suggest that key vesicle trafficking steps en route to the synapse are modulated by Rab activation states. Also, two cell adhesion molecules (Neuroglian and Contactin) indicate potential transsynaptic mechanisms regulating retrograde signaling. Neuroglian has been shown to be required for synaptic stability and it is possible that Syt4-mediated retrograde signaling plays some role in this process (Harris, 2016).

Syt4 has also been shown to be transferred transsynaptically from the presynaptic terminal to the postsynaptic terminal on exosomes (Korkut, 2013). Thus, the approach of expressing Syt4-pH postsynaptically may not reveal components for the biosynthetic synthesis and transport of presynaptic Syt4. Nevertheless, the requirement for Syt4 in the postsynaptic cell for retrograde signaling is clear, and the results of the screen highlight regulators of Syt4 trafficking to and from the postsynaptic membrane where Syt4 vesicles fuse in an activity-dependent manner. The observation that endogenously expressed Syt4-GFP (Syt4^GFP-2M) shows a similar distribution to Syt4-pH supports the biological relevance of the screen data for identifying regulators of Syt4 trafficking in the postsynaptic cell (Harris, 2016).

The Syx4 null allele phenocopies the Syx4-RNAi knockdown, reducing the delivery of Syt4-pH to the postsynaptic membrane. Consistent with this finding, loss of Syx4 produces similar phenotypes to loss of Syt4. Both null mutants exhibit a reduction in the total number of boutons at the NMJ, indicating a defect in synaptic growth. Moreover, genetic interaction experiments clearly indicate that Syx4 and Syt4 interact with respect to synaptic growth. A strong genetic interaction between Syx4 and Syt4 is also evident at the level of lethality, as double mutant animals are lethal at a much earlier stage than either single mutant alone. Thus, even though Syx4 affects the localization of Syt4, suggesting they act in the same pathway, the genetic interaction data do not support a simple epistatic relationship. The difference in phenotypic severity, with the Syx4 bouton number defect being significantly stronger than the Syt4 defect, also points to Syt4 not being absolutely required for Syx4 signaling. A similar phenomenon is observed presynaptically where the t-SNARE Syx1 is indispensible for synaptic vesicle fusion, while fusion is only reduced in the absence of the synaptic vesicle Ca²⁺ sensor Syt1. Taken together, it is hypothesized that (1) Syx4 and Syt4 act together in a single pathway where Syx4 regulates the exocytosis of vesicles containing Syt4, and (2) Syx4 and Syt4 also act in divergent pathways, where Syt4 cooperates with other t-SNARES, and Syx4 mediates the exocytosis of vesicles in a Syt4-independent manner. This model allows for multiple possible postsynaptic SNARE complexes, regulating distinct release events. Dissecting the other components of these fusion machineries, and distinguishing activity-dependent from constitutive release events, will be important to build understanding of how retrograde signaling is regulated (Harris, 2016).

In addition to affecting the localization of Syt4, Syx4 mutants also exhibit a decrease in the amount of Nlg1 at the postsynaptic membrane. Nlg1 has several functions at the synapse, along with its presynaptic binding partner Nrx-1. Together they regulate bouton number as well as the size and spacing of active zones and glutamate receptors, though some aspects of Nlg1 signaling appear to be independent of Nrx-1. Mutations in Nrx and Nlg family genes are also linked to autism spectrum disorder (ASD), highlighting the importance of Nrx-Nlg signaling in neuronal development. Consistent with a reduction of Nlg1 levels at the synapse, strong genetic interactions were observed between Syx4, Nlg1 and Nrx-1 with respect to bouton number. However, the prominent AZ/GluR defects seen in Nlg1 and Nrx-1 mutants were not observed in Syx4 mutants, and heterozygous combinations did not produce these defects. It is likely that Syx4 mutants exhibit a partial loss of function of Nlg1, and that bouton number is sensitive to this loss while AZ/GluR organization can be maintained with low levels of Nlg1 (Harris, 2016).

A dramatic change in distribution of Nlg1^Δcyto is observed in the Syx4 mutant background, providing further evidence that Syx4 regulates the localization of Nlg1. The redistribution of Nlg1^Δcyto to large accumulations is striking compared to full-length Nlg1, which is simply reduced at the synapse in the Syx4 mutant background. This observation points to complex Syx4-dependent regulation of Nlg1 localization. One model is that trafficking of Nlg1 involves both a Syx4-dependent pathway and a second pathway that depends on an interaction with the Nlg1 C-terminus, which includes a PDZ-domain-binding motif. In this scenario, a severe Nlg1 trafficking defect is revealed only when both pathways are compromised. A second possibility is that in the absence of Syx4, a portion of the Nlg1 content in the cell is degraded, but that this degradation step depends on the presence of the Nlg1 cytoplasmic tail, leading to the observed aggregation of Nlg1^Δcyto in Syx4 mutants (Harris, 2016).

Analysis of Nlg1 trafficking in live animals reveals that Nlg1 is strikingly stable, in both control and Syx4 mutant backgrounds. A motivation in performing these experiments was to test possible mechanisms underlying the decrease in Nlg1 levels in Syx4 mutants. It is possible that some Nlg1 mobility would be observed over a longer time course. Mammalian Nlg has been shown to undergo significant turnover at postsynaptic sites under LTP conditions in neuronal cell culture. Also, synaptic activity has been shown to induce cleavage of Nlg and the subsequent destabilization of the Nrx-Nlg complex. Thus, it remains a possibility that Nlg1 would be mobilized in response to activity in the preparation; however, no increased mobility was observed in response to high K⁺ incubations in preliminary tests. The data are most consistent with Syx4 regulating Nlg1 over a developmental time course. A detailed examination of the relationship between Syx4 and Nlg1 dynamics will be crucial to understand how Syx4 contributes to this important pathway in synaptic development (Harris, 2016).

A strong suppression of acute structural plasticity was observed in null mutants of Syx4, Syt4 and Nlg1. Double heterozygous combinations also indicated strong genetic interactions between all three of these genes with respect to plasticity. GB budding is regulated by both acute and developmental signaling. Because Syt4 postsynaptic vesicles fuse in an activity-dependent manner, it is possible that Syt4-dependent signaling releases an acute instructive cue for GB budding. Thus, one attractive model is that Nlg1 is delivered to the membrane in response to stimulation, depending on the Ca²⁺ sensitivity of Syt4 and the presence of the t-SNARE Syx4 at the membrane. It is also possible that Syx4-Syt4-Nlg1 signaling is required throughout development to potentiate the synapse to respond to strong neuronal stimulation. In conclusion, Syx4, Syt4, and Nlg1 interact to regulate several aspects of synaptic biology. The data support multiple overlapping signaling pathways regulated by these proteins, reflecting a complex modulation of retrograde signaling to control synaptic growth and plasticity at the Drosophila NMJ (Harris, 2016).

Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release

Signaling from the postsynaptic compartment regulates multiple aspects of synaptic development and function. Syntaxin 4 (Syx4) is a plasma membrane t-SNARE that promotes the growth and plasticity of Drosophila neuromuscular junctions (NMJs) by regulating the localization of key synaptic proteins in the postsynaptic compartment. This study describes electrophysiological analyses and reports that loss of Syx4 leads to enhanced neurotransmitter release, despite a decrease in the number of active zones. A requirement is described for postsynaptic Syx4 in regulating several presynaptic parameters, including Ca(2+) cooperativity and the abundance of the presynaptic calcium channel Cacophony (Cac) at active zones. These findings indicate Syx4 negatively regulates presynaptic neurotransmitter release through a retrograde signaling mechanism from the postsynaptic compartment (Harris, 2018).

Syntaxin 4 regulates multiple aspects of synaptic biology. This study reports that loss of Syx4 leads to synaptic enhancement, a surprising finding give that Syx4 mutant synapses have significantly fewer active zones than the control animals. An increase in evoked release and a reduction in paired-pulse facilitation were observed in Syx4 mutants. Two mechanisms were identified that are likely to contribute to the increase in neurotransmission: an increase in the levels of the presynaptic Ca2+ channel Cac at individual active zones, and a decrease in Ca2+ cooperativity. These two potentiation mechanisms could be linked – for example, an increase in Cac channels, leading to changes in Ca2+ influx and the spatial arrangement of the channels, could contribute to changes in the sensitivity of the exocytotic machinery to Ca2+. However, the possibility cannot be ruled out that they are distinct phenomena. As all of these phenotypes are rescued by postsynaptic, but not presynaptic, expression of Syx4, the data indicate a retrograde signaling mechanism by which Syx4 regulates active zones (Harris, 2018).

Cac clustering at active zones is regulated by components of the active zone cytomatrix, including Brp. Of these, Brp has the largest effect, with an approximate 50% reduction in Cac levels at active zones of brp null mutants. Although Syx4 mutants have no obvious defects in the size or intensity of Brp clusters, the distribution other cytomatrix proteins has not yet been examined. One mechanism for Cac regulation downstream of Syx4 signaling could be through changes in the levels of other active zone cytomatrix components (Harris, 2018).

One possible explanation for the potentiation observed in Syx4 mutants is that it is the result of homeostatic compensation. Many studies have described homeostatic mechanisms of potentiation and depression at the fly NMJ. In presynaptic homeostatic potentiation (PHP), perturbations that inhibit the function of postsynaptic glutamate receptors by acute pharmacological blockade or genetic loss, though most studies have not reported Cac levels. Presynaptic homeostatic depression (PHD) is a distinct phenomenon in which overexpression of the vesicular glutamate transporter, resulting in more glutamate packaged per synaptic vesicle, is offset by compensatory decreases in neurotransmitter release. PHD has been shown to involve a decrease in presynaptic Ca2+ influx and a decrease in Cac levels at active zones. Thus, the synapse employs multiple mechanisms during homeostatic plasticity, including regulation of Cac channels and Ca2+ influx (Harris, 2018).

An important distinction is that during homeostatic compensation the compensatory changes typically restore muscle depolarization precisely, whereas in Syx4 mutants a significant enhancement of neurotransmission is seen, well beyond control levels. Nevertheless, it may be interesting to investigate whether any of the known homeostatic pathways are required for potentiation in Syx4 mutants. It is also possible that Syx4 itself is engaged in homeostatic mechanisms. For example, if Syx4 is involved in downregulating Cac channels, it could potentially participate in a previously described PHD mechanism, which would therefore be impaired in Syx4 mutant animals. Syx4 could also interact with other retrograde pathways that affect presynaptic release probability, such as signaling through the importin Imp13, which functions postsynaptically to regulate release probability and presynaptic intracellular Ca2+ (Harris, 2018).

One intriguing observation from the current study is that paired-pulse facilitation is enhanced, compared to controls, when Syx4 is expressed postsynaptically in Syx4 mutants. This is surprising since simple overexpression of Syx4 in a wildtype animal does not affect facilitation. While it is not yet understood how this enhanced facilitation arises, one possibility could be differential expression of Syx4 isoforms. All of the overexpression and rescue experiments described in this study were conducted using a full-length Syx4 cDNA (Syx4A). However, there is a second isoform of Syx4 (Syx4B), encoding a shorter N-terminus, which is redundant with the A isoform with respect to all other phenotypes characterized for Syx4 to date. However no evidence was found that Syt4 or Nlg participate with Syx4 to regulate the Syx4 presynaptic enhancement phenotypes. Thus, it is likely that Syx4, as a postsynaptic t-SNARE, mediates the release of additional retrograde signals, and that multiple overlapping Syx4-dependent pathways are involved in establishing normal synaptic morphology, plasticity, and function (Harris, 2018).

This study has described electrophysiological analysis of animals lacking the postsynaptic t-SNARE Syx4. Syx4 mutants exhibit synaptic enhancement accompanied by presynaptic changes at active zones, including an increase in presynaptic Ca2+ channels at active zones and a decrease in Ca2+ cooperativity. All of these features are rescued by restoring postsynaptic Syx4. It is concluded that retrograde pathways regulated by Syx4 inhibit active zone potentiation, and that Syx4 modulates multiple postsynaptic signaling pathways with overlapping function (Harris, 2018).

Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development

A central function of the Drosophila salivary glands (SGs), historically known for their polytene chromosomes, is to produce and then release during pupariation the secretory glue used to affix a newly formed puparium to a substrate. This essential event in the life history of Drosophila is regulated by the steroid hormone ecdysone in the late-larval period. Ecdysone triggers a cascade of sequential gene activation that leads to glue secretion and initiates the developmentally-regulated programmed cell death (PCD) of the larval salivary glands, which culminates 16 h after puparium formation (APF). This study demonstrates that, even after the larval salivary glands have completed what is perceived to be one of their major biological functions -- glue secretion during pupariation -- they remain dynamic and physiologically active up until the execution phase of PCD. This study used specific metabolic inhibitors and genetic tools, including mutations or transgenes for shi, Rab5, Rab11, vha55, vha68-2, vha36-1, syx1A, syx4, and Vps35 to characterize the dramatic series of cellular changes occurring in the SG cells between pupariation and 7-8 h APF. Early in the prepupal period, they are remarkably active in endocytosis, forming acidic vacuoles. Midway through the prepupal period, there is abundant late endosomal trafficking and vacuole growth, which is followed later by vacuole neutralization and disappearance via membrane consolidation. This work provides new insights into the function of Drosophila SGs during the early- to mid-prepupal period (Farkas, 2015).

Search PubMed for articles about Drosophila

Barber, C. F., Jorquera, R. A., Melom, J. E. and Littleton, J. T. (2009). Postsynaptic regulation of synaptic plasticity by synaptotagmin 4 requires both C2 domains. J Cell Biol 187: 295-310. PubMed ID: 19822673

Chater, T. E. and Goda, Y. (2014). The role of AMPA receptors in postsynaptic mechanisms of synaptic plasticity. Front Cell Neurosci 8: 401. PubMed ID: 25505875

Farkas, R., Benova-Liszekova, D., Mentelova, L., Mahmood, S., Datkova, Z., Beno, M., Pecenova, L., Raska, O., Smigova, J., Chase, B. A., Raska, I. and Mechler, B. M. (2015). Vacuole dynamics in the salivary glands of Drosophila melanogaster during prepupal development. Dev Growth Differ 57: 74-96. PubMed ID: 25611296

Harris, K.P., Zhang, Y.V., Piccioli, Z.D., Perrimon, N. and Littleton, J.T. (2016). The postsynaptic t-SNARE Syntaxin 4 controls traffic of Neuroligin 1 and Synaptotagmin 4 to regulate retrograde signaling. Elife 5. PubMed ID: 27223326

Harris, K. P., Littleton, J. T. and Stewart, B. A. (2018). Postsynaptic Syntaxin 4 negatively regulates the efficiency of neurotransmitter release. J Neurogenet 32(3): 221-229. PubMed ID: 30175640

Jewell, J. L., Oh, E. and Thurmond, D. C. (2010). Exocytosis mechanisms underlying insulin release and glucose uptake: conserved roles for Munc18c and syntaxin 4. Am J Physiol Regul Integr Comp Physiol 298: R517-531. PubMed ID: 20053958

Jurado, S., Goswami, D., Zhang, Y., Molina, A. J., Sudhof, T. C. and Malenka, R. C. (2013). LTP requires a unique postsynaptic SNARE fusion machinery. Neuron 77: 542-558. PubMed ID: 23395379

Kennedy, M. J., Davison, I. G., Robinson, C. G. and Ehlers, M. D. (2010). Syntaxin-4 defines a domain for activity-dependent exocytosis in dendritic spines. Cell 141(3): 524-535. PubMed ID: 20434989

Korkut, C., Li, Y., Koles, K., Brewer, C., Ashley, J., Yoshihara, M., Budnik, V. (2013). Regulation of postsynaptic retrograde signaling by presynaptic exosome release. Neuron 77: 1039-1046. PubMed ID: 23522040

Piccioli, Z. D. and Littleton, J. T. (2014). Retrograde BMP signaling modulates rapid activity-dependent synaptic growth via presynaptic LIM kinase regulation of cofilin. J Neurosci 34: 4371-4381. PubMed ID: 24647957

Yoshihara, M., Adolfsen, B., Galle, K. T. and Littleton, J. T. (2005). Retrograde signaling by Syt 4 induces presynaptic release and synapse-specific growth. Science 310: 858-863. PubMed ID: 16272123

date revised: 5 August 2023

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