The C-terminal of CASY-1/Calsyntenin regulates GABAergic synaptic transmission at the Caenorhabditis elegans neuromuscular junction

Thapliyal, Shruti; Vasudevan, Amruta; Dong, Yongming; Bai, Jihong; Koushika, Sandhya P.; Babu, Kavita

doi:10.1371/journal.pgen.1007263

Cited by 16 publications

(21 citation statements)

References 95 publications

(99 reference statements)

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“…82 Similar GABAergic neuron-specific effects to those we observed were seen with inhibition of the anaphase promoting complex and EEL-1 ubiquitin ligases, the F-box protein MEC-15 FBXW9, as well as the cell adhesion molecule CASY-1, in prior studies at this synapse. 48,80,81,83 These collective results demonstrate GABAergic neuron-specific regulation is possible, and even common, and that the aldicarb paralysis assay may be particularly sensitive to defects in inhibitory neurons. 50 We found that GABAergic neuron-specific overexpression not only of wild type UBC-9 but also the C93S mutant increased muscle excitation (Figure 6a), suggesting UBC-9's effects may be independent of its catalytic activity; however, because of the overexpression context, we believe this does not necessarily reflect a SUMOylation-independent role for endogenous UBC-9 at this synapse.…”

Section: Tight Regulation Of Ubc-9 Activity Is Required In Gabaergic mentioning

confidence: 72%

UBC-9 Acts in GABA Neurons to Control Neuromuscular Signaling inC. elegans

et al. 2020

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Regulation of excitatory to inhibitory signaling balance is essential to nervous system health and is maintained by numerous enzyme systems that modulate the activity, localization, and abundance of synaptic proteins. SUMOylation is a key post-translational regulator of protein function in diverse cells, including neurons. There, its role in regulating synaptic transmission through pre- and postsynaptic effects has been shown primarily at glutamatergic central nervous system synapses, where the sole SUMO-conjugating enzyme Ubc9 is a critical player. However, whether Ubc9 functions globally at other synapses, including inhibitory synapses, has not been explored. Here, we investigated the role of UBC-9 and the SUMOylation pathway in controlling the balance of excitatory cholinergic and inhibitory GABAergic signaling required for muscle contraction in Caenorhabditis elegans. We found inhibition or overexpression of UBC-9 in neurons modestly increased muscle excitation. Similar and even stronger phenotypes were seen with UBC-9 overexpression specifically in GABAergic neurons, but not in cholinergic neurons. These effects correlated with accumulation of synaptic vesicle-associated proteins at GABAergic presynapses, where UBC-9 and the C. elegans SUMO ortholog SMO-1 localized, and with defects in GABA-dependent behaviors. Experiments involving expression of catalytically inactive UBC-9 [UBC-9(C93S)], as well as co-expression of UBC-9 and SMO-1, suggested wild type UBC-9 overexpressed alone may act via substrate sequestration in the absence of sufficient free SUMO, underscoring the importance of tightly regulated SUMO enzyme function. Similar effects on muscle excitation, GABAergic signaling, and synaptic vesicle localization occurred with overexpression of the SUMO activating enzyme subunit AOS-1. Together, these data support a model in which UBC-9 and the SUMOylation system act at presynaptic sites in inhibitory motor neurons to control synaptic signaling balance in C. elegans. Future studies will be important to define UBC-9 targets at this synapse, as well as mechanisms by which UBC-9 and the SUMO pathway are regulated.

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Section: Tight Regulation Of Ubc-9 Activity Is Required In Gabaergic mentioning

confidence: 72%

UBC-9 Acts in GABA Neurons to Control Neuromuscular Signaling inC. elegans

et al. 2020

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“…In C. elegans , CASY-1A expression is not seen in cholinergic or GABAergic ventral cord motor neurons ( Thapliyal et al 2018 ). However, it is strongly expressed in several sensory neurons and interneurons located in the anterior region of the animal (Figure S1 in File S1 and see Ikeda et al 2008 ).…”

Section: Resultsmentioning

confidence: 99%

“…We have recently shown that CASY-1 is required for regulating GABA release, and, hence, maintaining normal GABAergic neurotransmission at the C. elegans NMJ ( Thapliyal et al 2018 ). In this study, we report a possible mechanism for CASY-1A-mediated regulation of excitatory cholinergic transmission.…”

Section: Discussionmentioning

confidence: 99%

Regulation of Glutamate Signaling in the Sensorimotor Circuit by CASY-1A/Calsyntenin in Caenorhabditis elegans

2018

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Locomotion is one of the most prominent behaviors in the nematode Caenorhabditis elegans. Neuronal circuits that ultimately produce coordinated dorso-ventral sinusoidal bends mediate this behavior. Synchronized locomotion requires an intricate balance between excitation and inhibition at the neuromuscular junctions (NMJ), the complex cellular and molecular mechanisms of which are not fully understood. Here, we describe the role of a cell adhesion molecule CASY-1, which functions to maintain this balance at the NMJ. In this study, we dissect out mechanisms by which the longer CASY-1A isoform could be affecting the excitatory cholinergic signaling at the NMJ by modulating the activity of sensory neurons. Mutants in casy-1 appear to have hyperactive sensory neurons, resulting in accelerated locomotion and motor circuit activity. These sensory neurons mediate increased motor activity via enhanced glutamate release. Using genetic, pharmacological, and optogenetic manipulations, we establish that CASY-1A is required to monitor the activity of these neurons. Our study illustrates a novel neuromodulatory role of CASY-1-mediated signaling in regulating the excitation-inhibition balance of the motor circuit.

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“…Furthermore, rigorous analyses are warranted to address whether and how this circuit specificity is related to the altered anxiety-like behavior observed in Clstn3-cKO mice (Figure 6). A complete understanding of how Clstn3 functions will also require investigation of the potential contributions of other Clstn3 domains (e.g., LNS domain, intracellular region) to the functions of Clstn3, as was similarly shown in C. elegans (Ikeda et al, 2008;Kim and Emmons, 2017;Thapliyal et al, 2018).…”

Section: Discussionmentioning

confidence: 99%

Calsyntenin-3 directly interacts with neurexins to orchestrate excitatory synapse development in the hippocampus

Kim

et al. 2020

Preprint

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Calsyntenin-3 (Clstn3) is a postsynaptic adhesion molecule that induces presynaptic differentiation via presynaptic neurexins (Nrxns), but whether Nrxns directly bind to Clstn3 has been a matter of debate. Here, we show that β-Nrxns directly interact via their LNS domain with Clstn3 and Clstn3 cadherin domains. Expression of splice site 4 (SS4) insert-positive β-Nrxn variants, but not insertnegative variants, reversed the impaired Clstn3 synaptogenic activity observed in Nrxn-deficient neurons. Consistently, Clstn3 selectively formed complexes with SS4-positive Nrxns in vivo. Neuronspecific Clstn3 deletion caused significant reductions in number of excitatory synaptic inputs, and moderate impairment of light-induced anxiety-like behaviors in mice. Moreover, expression of Clstn3 cadherin domains in CA1 neurons of Clstn3 conditional knockout mice rescued structural deficits in excitatory synapses, especially within the stratum radiatum layer. Collectively, our results suggest that Clstn3 links to SS4-positive Nrxns to induce presynaptic differentiation and orchestrate excitatory synapse development in specific hippocampal neural circuits. ResultsClstn3 directly binds β-Nrxns. Our previous cell surface-binding assays used a variety of Nrxn1α deletion variants derived from the bovine Nrxn1α gene or Nrxn1β variants derived from the rat Nrxn1β gene (Um et al., 2014). These vectors have long been used to characterize Nrxn interactions with neuroligins (NLs) and other ligands, including leucine-rich repeat transmembrane neuronal proteins (LRRTMs), neurexophilins, and latrophilin-1 (Boucard et al., 2012;Ko et al., 2009a;Missler et al., 1998). However, using Nrxn vectors constructed from mouse Nrxn genes, Craig and colleagues reported that Nrxn1α, but not Nrxn1β, binds to Clstn3 (Pettem et al., 2013). To resolve this discrepancy, we performed affinity chromatography of solubilized mouse synaptosomes using immobilized recombinant Ig-Clstn3 followed by mass spectrometry (MS). Intriguingly, among the captured proteins was a tryptic peptide unique to Nrxn1β (in addition to Nrxn1α peptides) ( Figures 1A-D; see Table 1). To confirm binding between Clstn3 and Nrxn1β, we engineered mouse Nrxn1β expression constructs and performed cell surface-binding assays. We found robust binding of Ig-Clstn3 to HEK293T cells expressing C-terminally FLAG-tagged mNrxn1β lacking (mNrxn1β -SS4 -FLAG) or containing (mNrxn1β +SS4 -FLAG) the SS4 insert (Figures 1E and 1F). In assays measuring dimeric ligand binding to mNrxn-expressing cell surfaces, Ig-Clstn3 interacted with both mNrxn1β -SS4 and mNrxn1β +SS4 with nanomolar affinity (Figures 1G and 1H). Kd values calculated by Scatchard analyses were 51.39 ± 5.26 nM for Nrxn1β +SS4 and 105.94 ± 7.89 nM for Nrxn1β -SS4 (Figures 1G and 1H). These findings indicate that Clstn3 binds Nrxn1β with high affinity and exhibits a slight preference for SS4 insert-positive splice variants. To investigate differences between bovine and rat Nrxn1 plasmids (bNrxn1α and rNrxn1β) used in our previous studies and t...

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The C-terminal of CASY-1/Calsyntenin regulates GABAergic synaptic transmission at the Caenorhabditis elegans neuromuscular junction

Cited by 16 publications

References 95 publications

UBC-9 Acts in GABA Neurons to Control Neuromuscular Signaling inC. elegans

UBC-9 Acts in GABA Neurons to Control Neuromuscular Signaling inC. elegans

Regulation of Glutamate Signaling in the Sensorimotor Circuit by CASY-1A/Calsyntenin in Caenorhabditis elegans

Calsyntenin-3 directly interacts with neurexins to orchestrate excitatory synapse development in the hippocampus

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