Sema4D localizes to synapses and regulates GABAergic synapse development as a membrane-bound molecule in the mammalian hippocampus

Abstract

While numerous recent advances have contributed to our understanding of excitatory synapse formation, the processes that mediate inhibitory synapse formation remain poorly defined. Previously, we discovered that RNAi-mediated knockdown of a Class 4 Semaphorin, Sema4D, led to a decrease in the density of inhibitory synapses without an apparent effect on excitatory synapse formation. Our current work has led us to new insights about the molecular mechanisms by which Sema4D regulates GABAergic synapse development. Specifically, we report that the extracellular domain of Sema4D is proteolytically cleaved from the surface of neurons. However, despite this cleavage event, Sema4D signals through its extracellular domain as a membrane-bound, synaptically localized protein required in the postsynaptic membrane for proper GABAergic synapse formation. Thus, as Sema4D is one of only a few molecules identified thus far that preferentially regulates GABAergic synapse formation, these findings have important implications for our mechanistic understanding of this process.

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.