Central nervous system (CNS) synapses are complex cell-cell adhesions between neurons. Their establishment requires an interaction between axons and dendrites, accompanied by the appositional organization of pre and postsynaptic specializations. Several neuronal cell surface molecules and secreted signals have been shown to be involved in processes that lead to synaptic organization and maturation (Fox and Umemori, 2006
), but molecules that regulate the formation of initial synaptic adhesions remain poorly understood. Accumulating evidence from our lab and others has shown that astrocytes play active roles in the formation of synapses (Eroglu et al., 2008
). We have previously identified thrombospondins (TSP) as a necessary and sufficient synaptogenic signal secreted by astrocytes that increases synapse number (Christopherson et al., 2005
). TSP is present in astrocyte-conditioned media (ACM), and is responsible for the ability of astrocytes to increase synapse number in vitro
(Christopherson et al., 2005
). TSPs are also important for synapse formation in vivo
. TSP1/2 deficient mice have a significant decrease in the number of excitatory synapses. TSP1 and 2 are expressed during early postnatal ages when the majority of synapses are forming, and these proteins are absent from the adult brain when the amount of excitatory synaptogenesis is significantly reduced (Christopherson et al., 2005
). Upon injury to the CNS TSP1/2 levels are upregulated, and lack of TSP1/2 impairs synaptic and functional recovery from stroke (Liauw et al., 2008
TSP is able to promote synaptic adhesion and initiate the events that lead to the establishment of pre and postsynaptic specializations. Interestingly, these TSP-induced synapses are ultrastructurally identical to fully developed synapses and are presynaptically active but postsynaptically silent due to the lack of surface AMPA receptors. Astrocytes secrete a second unrelated signal that is able to convert these silent synapses into fully active ones ((Christopherson et al., 2005
) and N. A. and B. A. B. unpublished data).
TSPs are large oligomeric, multidomain, extracellular matrix proteins that have been previously shown to play important roles in cell attachment, cell migration, cytoskeletal dynamics and angiogenesis (Bornstein et al., 2004
). TSP mediates these functions via its interaction with various cell surface receptors through specific domains (Adams and Lawler, 2004
). We hypothesized that TSPs induce synapse formation by interacting with a neuronal cell surface receptor. Here we show that TSPs mediate synaptogenesis through their epidermal growth factor (EGF) -like domains, common to all TSP isoforms. Using this domain information, we identified the gabapentin receptor α2δ-1 as the TSP receptor involved in synapse formation.
α2δ-1 (cacna2d1) was originally isolated as a non-essential subunit of the L-type calcium channel complex from skeletal muscle (Arikkath and Campbell, 2003
) and also binds to other proteins (Kaltenbach et al., 2007
). α2δ-1 is ubiquitously expressed in many tissues and is highly expressed by many CNS neurons (Cole et al., 2005
) including retinal ganglion cells (RGCs). α2δ-1 is translated from a single gene product, which gets post-translationally cleaved into α2 and δ parts that remain associated via disulfide bridges. The α2 part of the protein (~950 amino acids) is entirely extracellular while the δ part has a small extracellular part that is attached to α2, and a transmembrane domain with a very short cytoplasmic tail that tethers the protein to the membrane (Davies et al., 2007
Much research on α2δ-1 has focused on its role in the regulation of calcium channel function and trafficking. However, the presence of a large extracellular region containing a well-known protein-protein interaction fold, the Von Willebrand Factor A (VWF-A) domain, suggests that this protein could serve as a receptor for extracellular ligands. A recent study on skeletal muscle cells, which express high levels of α2δ-1, described such a role for α2δ-1 in myoblast attachment and extracellular signaling that is independent of calcium channel function (Garcia et al., 2007
α2δ-1 is the high affinity receptor for two commonly prescribed anti-epileptic, anti-neuropathic pain medications gabapentin (GBP, Neurontin) and pregabalin (Lyrica) (Gee et al., 1996
). GBP and pregabalin were initially designed as hydrophobic gamma amino butyric acid (GABA) analogs that could cross the blood brain barrier. Further studies have shown that even though they posses anti-convulsant properties, they do not bind to GABA receptors or transporters. A recent study using a knock-in mouse that expresses a mutant α2δ-1 which cannot bind GBP or pregabalin has shown that α2δ-1 is the in vivo
target for these drugs, and that these drugs mediate their therapeutic action through binding to α2δ-1 (Field et al., 2006
). GBP or pregabalin do not affect the single channel kinetics of calcium channels and has only modest effects on neurotransmission (Dooley et al., 2007
). Thus the cellular mechanisms underlying the mode of action of these drugs are unclear.
In this study, we show that EGF-like domains of TSP directly bind to α2δ-1 and mediate its synapse-inducing activity via this receptor. These findings identify α2δ–1 as a neuronal TSP receptor that is required for CNS synapse formation. This function of α2δ-1 is independent of calcium channel function. We also show that GBP is a potent inhibitor of TSP/astrocyte-induced excitatory synapse formation in vitro and in vivo. This function of GBP may be a central part of its mechanism of action.