Synaptotagmin-3 drives AMPA receptor endocytosis, depression of synapse strength, and forgetting

Forgetting and receptor removal The trafficking of AMPA receptors to and from the surface of postsynaptic membranes regulates synaptic strength and underlies learning and memory. Awasthi et al. found that the integral membrane protein synaptotagmin-3 (Syt3) is predominantly found on postsynaptic endocytic zones of neurons, where it promotes AMPA receptor internalization (see the Perspective by Mandelberg and Tsien). In Syt3 overexpressing or knockdown neurons, synaptic transmission and short-term plasticity were unchanged. However, in neurons from Syt3 knockout mice, synaptic long-term depression was abolished and decaying long-term potentiation endured. In Syt3 knockout mice, spatial learning was unaltered; however, these animals showed signs of impaired forgetting and relearning during the water maze spatial memory task. Science, this issue p. eaav1483; see also p. 31 In mice, the neuronal membrane trafficking protein synaptotagmin-3 is involved in learning processes that require forgetting. INTRODUCTION Memories are stored as molecular and cellular changes in the brain. Synapses, the nodes of connection between neurons, can store memories by virtue of their ability to tune the efficacy of communication between neurons. This property of synaptic plasticity makes it possible for the brain to store and retrieve memories—to replay patterns of electrical activity that occurred during an important event. Forgetting leads to the inability to retrieve memories by making them latent or decaying them below any useful quality. However, what determines whether a memory is forgotten? A mechanism is the regulation of neurotransmitter receptor numbers on the postsynaptic plasma membrane. These receptors mediate synaptic transmission by transducing presynaptically released neurotransmitters into an electrical signal. Neuronal activity strengthens synapses by inserting receptors or weakens synapses by removing receptors from the postsynaptic membrane. Receptor trafficking is controlled through calcium influx into the neuron; however, the calcium sensors mediating this control are not known. RATIONALE Synaptotagmin proteins sense calcium to trigger membrane fusion. Stimulating neuronal cultures elicits a calcium-mediated externalization of most synaptotagmin isoforms into plasma membranes, but synaptotagmin-3 (Syt3) internalizes from postsynaptic membranes. Stimulating AMPA (α-amino-3-hydroxy-5-methyl-4-isoxazolepropionic) or NMDA (N-methyl d-aspartate) receptors induces internalization of AMPA receptors (which mediate most of the fast synaptic transmission in the brain) and Syt3. This raised the intriguing possibility that Syt3 mediates activity-induced internalization of receptors to weaken synapses and cause forgetting. We imaged Syt3 using an isoform-specific antibody, tested its role in receptor trafficking using electrophysiological methods in brain slices and neuronal cultures, and tested its role in forgetting using spatial memory tasks in mice. RESULTS Syt3 is on postsynaptic membranes at endocytic zones, which are clathrin-rich regions close to the postsynaptic density. Syt3 binds the GluA2 AMPA receptor subunit and also binds AP2 and BRAG2, two proteins implicated in activity-dependent internalization of AMPA receptors via clathrin-mediated endocytosis. Syt3 does not affect basal AMPA receptor trafficking. However, knocking out Syt3—or expressing calcium-binding– deficient Syt3—abolishes AMPA receptor internalization induced by AMPA, NMDA, or electrophysiological stimulation of long-term depression of synaptic strength. It also blocks the AMPA receptor internalization that normally decays long-term potentiation of synaptic strength. These effects are mimicked in a wild-type background through acute application of the Tat-GluA2-3Y peptide, which competitively inhibits binding of Syt3 to a tyrosine-rich (3Y) motif on the cytoplasmic tail of GluA2. In spatial memory tasks, mice in which Syt3 was knocked out (Syt3 knockout mice) learn escape positions normally but persevere to previously learned positions, which can be explained by a lack of forgetting previously acquired memories. Injecting the Tat-GluA2-3Y peptide in wild-type mice mimics the lack of forgetting of spatial memories, and this effect is occluded in Syt3 knockout mice. CONCLUSION The persistence or degradation of memories is governed by a poorly understood molecular machinery. We have discovered a distinct synaptotagmin isoform that triggers calcium-mediated internalization of AMPA receptors, resulting in a weakening of synaptic transmission and forgetting of spatial memories in mice. Syt3 knockout mice do not forget. Both wild-type mice and Syt3 knockout mice can learn an escape position in the water maze, in which corresponding synapses are strengthened through the increase of AMPA receptors. These synapses are weakened by the removal of receptors if the memory is no longer needed—for example, when a new escape position is learned. Syt3 knockout mice cannot remove receptors and therefore cannot forget previous escape positions. Forgetting is important. Without it, the relative importance of acquired memories in a changing environment is lost. We discovered that synaptotagmin-3 (Syt3) localizes to postsynaptic endocytic zones and removes AMPA receptors from synaptic plasma membranes in response to stimulation. AMPA receptor internalization, long-term depression (LTD), and decay of long-term potentiation (LTP) of synaptic strength required calcium-sensing by Syt3 and were abolished through Syt3 knockout. In spatial memory tasks, mice in which Syt3 was knocked out learned normally but exhibited a lack of forgetting. Disrupting Syt3:GluA2 binding in a wild-type background mimicked the lack of LTP decay and lack of forgetting, and these effects were occluded in the Syt3 knockout background. Our findings provide evidence for a molecular mechanism in which Syt3 internalizes AMPA receptors to depress synaptic strength and promote forgetting.