Convergence of adenosine and GABA signaling for synapse stabilization during development

Description Synapse stabilization Early in brain development, neurons connect to each other enthusiastically. With development, an overabundance of synapses is winnowed down to refine efficiently connected circuits. Inactive synapses are prime targets for elimination, whereas active synapses tend to be retained. Gomez-Castro et al. took a closer look at how those choices are made (see the Perspective by Blum and Lopes). When postsynaptic adenosine receptors are muted or do not find enough extracellular adenosine, synapses get eliminated. Neurotransmitter-dependent signaling pathways drive protein kinase A to phosphorylate the postsynaptic scaffolding molecule gephyrin. Together with a partner synaptogenic membrane protein, gephyrin is required for the stabilization of γ-aminobutyric acid receptors. Adenosine receptors thus detect synaptic activity and in turn drive the stabilization of synapses that produce such activity. —PJH Detection of synaptic neurotransmitter activity is translated into stabilization of active synapses. INTRODUCTION During development, brain circuits go through phases of synapse formation, stabilization, or elimination. γ-aminobutyric acid–mediated (GABAergic) synapse formation depends mainly on cell adhesion molecules, such as neuroligins and leucine-rich repeat transmembrane proteins, that interact with presynaptic neurexins and Slit- and Trk-like family proteins that bind to presynaptic protein tyrosine phosphatases. GABA and GABA type A (GABAA) receptors are involved in an activity-dependent manner in the maturation and pruning of synapses. Adenosine triphosphate (ATP) and adenosine can be coreleased with GABA at synapses to be perceived by adenosine A2A receptors. We tested the role of adenosine signaling in the stabilization and elimination of GABAergic synapses. RATIONALE A2A receptors control migration speed, axonal elongation, and dendrite branching. Whether A2A receptors control synapse formation, stabilization, or elimination in the brain is not known. In the adult brain, A2A receptors are mostly expressed on presynaptic terminals, where they control the probability of synaptic vesicle release. The amount, location, and function of A2A receptors at neural synapses during early brain development has been unclear. RESULTS During synaptogenesis in the developing mouse hippocampus, between postnatal days P5 and P16, the density of A2A receptors increases transiently around the postsynaptic density. Activity-dependent release of its endogenous ligand, adenosine, increases as well. A2A receptors control the fate of GABAergic synapses. Suppression of A2A receptors, their pharmacological blockade, or the removal of adenosine results in the destabilization of pre- and postsynaptic sites in vivo, ex vivo, and in vitro. If A2A receptors remain inactive for >20 min, synapse destabilization is irreversible. We found that A2A receptor activation is necessary and sufficient for GABAergic synapse stabilization, whereas GABAA receptor activation is not necessary as long as A2A receptors remain activated. We studied the molecular mechanism at play. A2A receptor and GABAA receptor signaling pathways converge onto calcium-calmodulin–sensitive adenylyl cyclases to produce adenosine 3′,5′-monophosphate (cAMP). The resulting activation of protein kinase A leads to phosphorylation of the postsynaptic scaffolding molecule gephyrin at the protein kinase A–sensitive serine residue 303 site. Expression of the gephyrin mutant mimicking this phosphorylation state prevents synapse loss upon the removal of extracellular adenosine. Phosphorylated gephyrin can be coimmunoprecipitated with the postsynaptic transmembrane Slit- and Trk-like family protein 3 that binds in the synaptic cleft to presynaptic protein tyrosine phosphatase σ to organize inhibitory synapses. The contribution of Slit- and Trk-like family protein 3 in stabilizing GABAergic synapses through adenosine signaling is demonstrated with a short hairpin RNA (shRNA) approach or after the expression of a mutant. Finally, antagonizing A2A receptors during synaptogenesis in vivo results in the loss of GABAergic synapses during development and cognitive deficits when animals reach adulthood. CONCLUSION A2A receptors regulate the elimination of certain GABAergic synapses when they become inactive. A2A receptors are poised to detect active presynaptic terminals and trigger synapse removal after a defined period of synaptic inactivity. Adenosine signaling stabilizes nascent GABAergic synapses. (Left) Active synapse: Corelease of adenosine, ATP, and GABA activates A2A receptors and GABAA receptors, whose signaling pathways converge on Ca2+-calmodulin–dependent adenylyl cyclases and cAMP production, which in turn may stabilize the nascent synapse through recruitment of the Slitrk3-PTPσ transsynaptic organizers by gephyrin phosphorylated at a protein kinase A (PKA) site. (Right) Inactive synapse: In the absence of adenosine, ATP, and GABA release at inactive synapses, this pathway is not activated, and the synapse is eliminated. ADO, adenosine; A2AR, adenosine type 2A receptor; GABAAR, GABAA receptor; VDCC, voltage-dependent calcium channel; AC1 or AC8, adenylyl cyclase 1 or 8; PTPδ, protein tyrosine phosphatase δ; Slitrk3, Slit- and Trk-like family member 3; CaM, calmodulin. During development, neural circuit formation requires the stabilization of active γ-aminobutyric acid–mediated (GABAergic) synapses and the elimination of inactive ones. Here, we demonstrate that, although the activation of postsynaptic GABA type A receptors (GABAARs) and adenosine A2A receptors (A2ARs) stabilizes GABAergic synapses, only A2AR activation is sufficient. Both GABAAR- and A2AR-dependent signaling pathways act synergistically to produce adenosine 3′,5′-monophosphate through the recruitment of the calcium–calmodulin–adenylyl cyclase pathway. Protein kinase A, thus activated, phosphorylates gephyrin on serine residue 303, which is required for GABAAR stabilization. Finally, the stabilization of pre- and postsynaptic GABAergic elements involves the interaction between gephyrin and the synaptogenic membrane protein Slitrk3. We propose that A2ARs act as detectors of active GABAergic synapses releasing GABA, adenosine triphosphate, and adenosine to regulate their fate toward stabilization or elimination.