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.

中文翻译：

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Synaptotagmin-3 驱动 AMPA 受体内吞、突触强度降低和遗忘

遗忘和受体去除AMPA 受体进出突触后膜表面的运输调节突触强度并成为学习和记忆的基础。阿瓦斯蒂等人。发现整合膜蛋白突触结合蛋白 3 (Syt3) 主要存在于神经元的突触后内吞区，在那里它促进 AMPA 受体内化（参见 Mandelberg 和 Tsien 的观点）。在 Syt3 过表达或敲低的神经元中，突触传递和短期可塑性没有改变。然而，在来自 Syt3 基因敲除小鼠的神经元中，突触长期抑制被消除，长期增强持续衰减。在 Syt3 基因敲除小鼠中，空间学习没有改变；然而，这些动物在水迷宫空间记忆任务中表现出遗忘和重新学习受损的迹象。科学，这个问题 p。eaav1483; 另见第。31 在小鼠中，神经元膜运输蛋白突触结合蛋白 3 参与需要遗忘的学习过程。介绍 记忆在大脑中以分子和细胞变化的形式存储。突触是神经元之间的连接节点，可以凭借其调节神经元之间通信效率的能力来存储记忆。突触可塑性的这种特性使大脑能够存储和检索记忆——重放重要事件期间发生的电活动模式。遗忘使记忆潜伏或衰减到任何有用的质量以下，从而导致无法检索记忆。然而，是什么决定了一段记忆是否被遗忘呢？一种机制是调节突触后质膜上的神经递质受体数量。这些受体通过将突触前释放的神经递质转换为电信号来介导突触传递。神经元活动通过插入受体来增强突触，或通过从突触后膜去除受体来削弱突触。受体运输是通过钙流入神经元来控制的；然而，介导这种控制的钙传感器是未知的。基本原理突触结合蛋白感知钙以触发膜融合。刺激神经元培养物引起钙介导的大多数突触结合蛋白同种型外化到质膜中，但突触结合蛋白 3 (Syt3) 从突触后膜内化。刺激 AMPA（α-氨基-3-羟基-5-甲基-4-异恶唑丙酸）或 NMDA（N-甲基 d-天冬氨酸）受体诱导 AMPA 受体（介导大脑中大部分快速突触传递）和 Syt3 的内化. 这提出了一种有趣的可能性，即 Syt3 介导活动诱导的受体内化以削弱突触并导致遗忘。我们使用异构体特异性抗体对 Syt3 成像，使用脑切片和神经元培养物中的电生理学方法测试其在受体运输中的作用，并使用小鼠的空间记忆任务测试其在遗忘中的作用。结果 Syt3 位于内吞区的突触后膜上，这是靠近突触后密度的富含网格蛋白的区域。Syt3 结合 GluA2 AMPA 受体亚基，也结合 AP2 和 BRAG2，两种蛋白质通过网格蛋白介导的内吞作用参与 AMPA 受体的活性依赖性内化。Syt3 不影响基础 AMPA 受体的运输。然而，敲除 Syt3（或表达钙结合缺陷型 Syt3）会消除由 AMPA、NMDA 或长期抑制突触强度的电生理刺激诱导的 AMPA 受体内化。它还阻断了通常会衰减突触强度长期增强的 AMPA 受体内化。这些作用通过急性应用 Tat-GluA2-3Y 肽在野生型背景中得到模拟，该肽竞争性地抑制 Syt3 与 GluA2 细胞质尾部富含酪氨酸 (3Y) 基序的结合。在空间记忆任务中，Syt3 被敲除的小鼠（Syt3 敲除小鼠）正常学习逃逸位置，但坚持先前学习的位置，这可以通过缺乏忘记先前获得的记忆来解释。在野生型小鼠中注射 Tat-GluA2-3Y 肽模拟了空间记忆的缺乏，而这种效果在 Syt3 敲除小鼠中被遮挡。结论 记忆的持久性或退化是由一种知之甚少的分子机制控制的。我们发现了一种独特的突触结合蛋白亚型，可触发钙介导的 AMPA 受体内化，导致小鼠突触传递减弱和空间记忆遗忘。Syt3 基因敲除小鼠不要忘记。野生型小鼠和 Syt3 基因敲除小鼠都可以在水迷宫中学习逃生位置，其中通过AMPA受体的增加相应的突触得到加强。如果不再需要记忆——例如，当学习到一个新的逃逸位置时，这些突触会因受体的移除而减弱。Syt3 基因敲除小鼠无法移除受体，因此无法忘记之前的逃逸位置。遗忘很重要。没有它，在不断变化的环境中获得的记忆的相对重要性就会丢失。我们发现突触结合蛋白 3 (Syt3) 定位于突触后内吞区并响应刺激从突触质膜中去除 AMPA 受体。AMPA 受体内化、长期抑制 (LTD) 和突触强度的长期增强 (LTP) 衰减需要 Syt3 的钙感应，并通过 Syt3 敲除被消除。在空间记忆任务中，Syt3 被敲除的小鼠学习正常，但表现出缺乏遗忘。在野生型背景中破坏 Syt3:GluA2 结合模拟了 LTP 衰减和遗忘的缺乏，而这些影响在 Syt3 敲除背景中被遮挡。我们的研究结果为 Syt3 内化 AMPA 受体以抑制突触强度和促进遗忘的分子机制提供了证据。