Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus
Introduction
The formation of synapses, which are sites of cell–cell communication between neurons, is a complex process that is regulated by a number of transmembrane and membrane-associated proteins (Dalva et al., 2007). The majority of studies on mammalian synapse development thus far have focused on the hippocampus, where glutamate is the major excitatory neurotransmitter and γ-aminobutyric acid (GABA) is the major inhibitory neurotransmitter. Historically, greater attention has been paid to the molecules that direct formation of glutamatergic synapses, although recently, we and others have identified proteins important for GABAergic synapse formation (Chih, 2005, Paradis et al., 2007, Pietro Fazzari et al., 2010, Shin et al., 2003, Terauchi et al., 2010, Vicario-Abejón et al., 1998). The Semaphorins are one such family of proteins that have emerged as important regulators of synapse development throughout the mammalian nervous system. In addition to our studies implicating Semaphorin signaling in GABAergic synapse formation (Paradis et al., 2007), studies have implicated other Semaphorin family members in glutamatergic synapse formation or elimination (Morita et al., 2006, O'Connor et al., 2009, Paradis et al., 2007, Tran et al., 2009, Yamashita et al., 2007).
Semaphorins are a large family of transmembrane and secreted glycoproteins that were originally identified as axon guidance molecules (Kolodkin et al., 1993, Tran et al., 2007). Of the eight total Semaphorin subclasses, five are expressed in mammals and all share a conserved extracellular Sema domain (Tran et al., 2007, Love et al., 2003). The Class 4 Semaphorins in particular have been shown to play roles in the immune response as well as neural development (Kumanogoh et al., 2002, Oinuma et al., 2010, Paradis et al., 2007, Shi et al., 2000, Zhu et al., 2007). Our previous work demonstrated that the Class 4 Semaphorin Sema4D mediates GABAergic synaptic development both in vitro and in vivo without an apparent effect on glutamatergic synapse development (Kuzirian et al., 2013, Paradis et al., 2007). In addition, the Class 4 Semaphorin Sema4B mediates both GABAergic and glutamatergic synapse development, suggesting a conserved role for Class 4 Semaphorins in the regulation of mammalian CNS synaptogenesis (Paradis et al., 2007). The molecular mechanisms by which Semaphorins regulate synaptogenesis, and the subcellular localization of these ligands in the nervous system, are not well understood.
The Sema4D protein consists of a short cytoplasmic tail, transmembrane domain, and extracellular Ig and Sema domains (Furuyama et al., 1996, Hall et al., 1996, Shi et al., 2000). Cleavage of Sema4D in non-neuronal cells occurs at the cell surface, putatively between the transmembrane domain and Ig domain (Elhabazi et al., 2001), resulting in an extracellular soluble fragment and an intracellular C-terminal fragment (Basile et al., 2007, Zhu et al., 2007). Although a recent study demonstrated that the extracellular domain of Sema4D is sufficient to drive functional GABAergic synapse formation (Kuzirian et al., 2013), whether cleavage of the Sema4D extracellular domain occurs from the neuronal cell surface, and its implication for signal transduction in the nervous system, has not been addressed until now. In addition, the C-terminal, cytoplasmic domain of Sema4D has no known function and thus it remains an open question as to whether or not signaling through the intracellular domain of Sema4D also influences synapse development.
As a means to gain insight into the molecular mechanisms that instruct GABAergic synaptic development in the rodent hippocampus, we investigated the domains within Sema4D that are required to mediate GABAergic synapse development. We constructed multiple chimeras of Sema4D by replacing different domains of Sema4D with that of the transmembrane protein CD4, a small single pass protein involved in T-cell activation. Using this approach we discovered that Sema4D signaling through its N-terminal extracellular domain is absolutely required to promote GABAergic synapse formation. In addition, we observed that while Sema4D is proteolytically cleaved in the mammalian brain, this event is not required for Sema4D to regulate synaptogenesis, suggesting that it signals as a membrane-bound molecule. Consistent with this model, we demonstrate that Sema4D is localized to the synaptic membrane in the mammalian hippocampus. Taken together, our data establish that Sema4D is a synaptic protein that can regulate GABAergic synaptogenesis exclusively through its extracellular domain and as a membrane-bound molecule.
Section snippets
The extracellular, N-terminal domain of Sema4D is required for GABAergic synapse formation
In order to determine the signal transduction mechanism(s) by which Sema4D regulates synapse development, we asked if the intracellular domain of Sema4D was required to mediate GABAergic synapse formation. To address this question, we generated epitope-tagged, RNAi-resistant Sema4D cDNA constructs harboring either a deletion or a swap between regions of Sema4D and the transmembrane immune receptor CD4 (Fig. 1A). To assess the role of the intracellular domain of Sema4D in GABAergic synapse
Discussion
Our study provides novel insight into the molecular mechanism by which Sema4D regulates GABAergic synapse development in the mammalian hippocampus: Sema4D signals as a synaptically localized, membrane-bound molecule through its extracellular N-terminus to promote synapse development. First, we show that a Sema4D protein which lacks a C-terminus is sufficient to rescue Sema4D RNAi-mediated decrease in GABAergic synapse density. In addition, a Sema4D protein which lacks its N-terminus fails to
Sequence alignment
Amino acid sequences comprising the extracellular domain of each mammalian Class 4 Semaphorin were aligned compared, and percent identity determined using the ClustalW alignment tool in MegaAlign software (DNAstar). Percent homology was directly calculated from the alignment. Percent identity between Sema4D and Sema5A was calculated by a pairwise comparison in MegaAlign. Protein Dendogram was generated directly from alignment in MegaAlign software.
Viral transduction of neuronal cultures
Neurons were isolated from Long Evans embryonic
Acknowledgments
We thank Dr. Michael Marr 2nd at Brandeis University for technical assistance, assistance with data interpretation, and critical reading of the manuscript. We also thank Dr. Atsushi Kumanogoh from Osaka University for generously providing us with Sema4D and Sema4A cDNA constructs. This work was supported by the National Institutes of Health grant (to S. Paradis) R01NS065856, Research grant no. 5-FY09-125 from the March of Dimes Foundation (S.P), P30NS45713 for Core Facilities for Neurobiology
References (47)
MT1-MMP controls tumor-induced angiogenesis through the release of semaphorin 4D
J. Biol. Chem.
(2007)EphB receptors interact with NMDA receptors and regulate excitatory synapse formation
Cell
(2000)Identification of a novel transmembrane semaphorin expressed on lymphocytes
J. Biol. Chem.
(1996)- et al.
The semaphorin genes encode a family of transmembrane and secreted growth cone guidance molecules
Cell
(1993) - et al.
Emerging themes in GABAergic synapse development
Prog. Neurobiol.
(2011) Ephrin-B2-induced cleavage of EphB2 receptor is mediated by matrix metalloproteinases to trigger cell repulsion
J. Biol. Chem.
(2008)Semaphorin 4C and 4G are ligands of Plexin-B2 required in cerebellar development
Mol. Cell. Neurosci.
(2011)Semaphorin 4D/Plexin-B1 stimulates PTEN activity through R-Ras GTPase-activating protein activity, inducing growth cone collapse in hippocampal neurons
J. Biol. Chem.
(2010)An RNAi-based approach identifies molecules required for glutamatergic and GABAergic synapse development
Neuron
(2007)SynCAM 1 adhesion dynamically regulates synapse number and impacts plasticity and learning
Neuron
(2010)
The class IV semaphorin CD100 plays nonredundant roles in the immune system: defective B and T cell activation in CD100-deficient mice
Immunity
Association of the kinesin motor KIF1A with the multimodular protein liprin-alpha
J. Biol. Chem.
Plexin-B1 directly interacts with PDZ-RhoGEF/LARG to regulate RhoA and growth cone morphology
Neuron
Plexins are a large family of receptors for transmembrane, secreted, and GPI-anchored semaphorins in vertebrates
Cell
Functional soluble CD100/Sema4D released from activated lymphocytes: possible role in normal and pathologic immune responses
Blood
Class IV semaphorins promote angiogenesis by stimulating Rho-initiated pathways through plexin-B
Cancer Res.
GABA: a pioneer transmitter that excites immature neurons and generates primitive oscillations
Physiol. Rev.
Sarm1, a negative regulator of innate immunity, interacts with syndecan-2 and regulates neuronal morphology
J. Cell Biol.
Control of excitatory and inhibitory synapse formation by neuroligins
Science
Cell adhesion molecules: signalling functions at the synapse
Nat. Rev. Neurosci.
Dopamine D1 receptor-dependent trafficking of striatal NMDA glutamate receptors to the postsynaptic membrane
J. Neurosci.
Biological activity of soluble CD100. I. The extracellular region of CD100 is released from the surface of T lymphocytes by regulated proteolysis
J. Immunol.
Function of an axonal chemoattractant modulated by metalloprotease activity
Science (New York, N.Y.)
Cited by (24)
A proteolytic C-terminal fragment of Nogo-A (reticulon-4A) is released in exosomes and potently inhibits axon regeneration
2020, Journal of Biological ChemistryCitation Excerpt :After spinal cord trauma, damaged axons are frequently demyelinated, so axonal cell surface contact with membrane-associated MAIs may be limited. Another injury-induced transmembrane MAI, Semaphorin 4D, is proteolyzed, and soluble protein fragments are released from the cell membrane after SCI (19–21). There are no direct mechanistic data regarding the release of Nogo-A, myelin-associated glycoprotein, or oligodendrocyte myelin glycoprotein, the MAIs secreted after injury.
Activity-dependent development of GABAergic synapses
2019, Brain ResearchClass 4 Semaphorins and Plexin-B receptors regulate GABAergic and glutamatergic synapse development in the mammalian hippocampus
2018, Molecular and Cellular NeuroscienceCitation Excerpt :Nonetheless our findings demonstrate that Plxnb1, Plxnb2, Sema4a, and Sema4d mRNAs are also expressed in neurons, thus we sought to determine if and how these genes function in specific neuronal sub-types to regulate synapse development. Previously, we demonstrated that Sema4D is enriched at synapses and is required in postsynaptic excitatory neurons to regulate GABAergic synapse development (Paradis et al., 2007; Raissi et al., 2013). We hypothesized that Sema4D expression in excitatory neurons could also be sufficient to promote organization of these opposing presynaptic GABAergic terminals.
Regulation of GABAergic synapse development by postsynaptic membrane proteins
2017, Brain Research Bulletin