Brain Cognition and Brain Disease Institute, Shenzhen Institute of Advanced Technology, Chinese Academy of Sciences; CAS Key Laboratory of Brain Connectome and Manipulation; Shenzhen-Hong Kong Institute of Brain Science-Shenzhen Fundamental Research Institutions, Shenzhen, Guangdong 518055, China.
Beijing Institute of Brain Disorders, Laboratory of Brain Disorders, Ministry of Science and Technology, Collaborative Innovation Center for Brain Disorders, Capital Medical University, Beijing, 100069, China.
J Neurosci. 2023 Sep 6;43(36):6230-6248. doi: 10.1523/JNEUROSCI.1348-21.2023. Epub 2023 Jul 20.
Synaptic vesicle (SV) endocytosis is a critical and well-regulated process for the maintenance of neurotransmission. We previously reported that synaptotagmin-11 (Syt11), an essential non-Ca-binding Syt associated with brain diseases, inhibits neuronal endocytosis (Wang et al., 2016). Here, we found that Syt11 deficiency caused accelerated SV endocytosis and vesicle recycling under sustained stimulation and led to the abnormal membrane partition of synaptic proteins in mouse hippocampal boutons of either sex. Furthermore, our study revealed that Syt11 has direct but Ca-independent binding with endophilin A1 (EndoA1), a membrane curvature sensor and endocytic protein recruiter, with high affinity. EndoA1-knockdown significantly reversed Syt11-KO phenotype, identifying EndoA1 as a main inhibitory target of Syt11 during SV endocytosis. The N-terminus of EndoA1 and the C2B domain of Syt11 were responsible for this interaction. A peptide (amino acids 314-336) derived from the Syt11 C2B efficiently blocked Syt11-EndoA1 binding both and Application of this peptide inhibited SV endocytosis in WT hippocampal neurons but not in EndoA1-knockdown neurons. Moreover, intracellular application of this peptide in mouse calyx of Held terminals of either sex effectively hampered both fast and slow SV endocytosis at physiological temperature. We thus propose that Syt11 ensures the precision of protein retrieval during SV endocytosis by inhibiting EndoA1 function at neuronal terminals. Endocytosis is a key stage of synaptic vesicle (SV) recycling. SV endocytosis retrieves vesicular membrane and protein components precisely to support sustained neurotransmission. However, the molecular mechanisms underlying the regulation of SV endocytosis remain elusive. Here, we reported that Syt11-KO accelerated SV endocytosis and impaired membrane partition of synaptic proteins. EndoA1 was identified as a main inhibitory target of Syt11 during SV endocytosis. Our study reveals a novel inhibitory mechanism of SV endocytosis in preventing hyperactivation of endocytosis, potentially safeguarding the recycling of synaptic proteins during sustained neurotransmission.
突触小泡（SV）内吞作用是维持神经递质传递的关键和受调控的过程。我们之前报道过，突触结合蛋白 11（Syt11）是一种与脑部疾病相关的必需非 Ca 结合 Syt，它可抑制神经元内吞作用（Wang 等人，2016）。在此，我们发现 Syt11 缺失会导致持续刺激下 SV 内吞作用和囊泡再循环加速，并导致雄性和雌性小鼠海马触突中突触蛋白的异常膜分区。此外，我们的研究揭示了 Syt11 与内收蛋白 A1（EndoA1）具有直接但 Ca 独立的结合，EndoA1 是一种膜曲率传感器和内吞蛋白招募物，具有高亲和力。EndoA1 敲低显著逆转了 Syt11-KO 表型，确定了 EndoA1 是 Syt11 在 SV 内吞作用过程中的主要抑制靶标。EndoA1 的 N 端和 Syt11 的 C2B 结构域负责这种相互作用。源自 Syt11 C2B 的一个肽（氨基酸 314-336）有效阻断了 Syt11-EndoA1 结合。该肽的应用抑制了 WT 海马神经元中的 SV 内吞作用，但不能抑制 EndoA1 敲低神经元中的 SV 内吞作用。此外，该肽在雄性和雌性小鼠 calyx of Held 终末的细胞内应用有效地阻止了生理温度下快速和慢速 SV 内吞作用。因此，我们提出 Syt11 通过抑制神经元末端的 EndoA1 功能来确保 SV 内吞作用过程中蛋白回收的精确性。内吞作用是突触小泡（SV）再循环的关键阶段。SV 内吞作用精确回收囊泡膜和蛋白成分，以支持持续的神经递质传递。然而，SV 内吞作用调控的分子机制仍不清楚。在此，我们报道了 Syt11-KO 加速了 SV 内吞作用，并损害了突触蛋白的膜分区。EndoA1 被鉴定为 SV 内吞作用过程中 Syt11 的主要抑制靶标。我们的研究揭示了 SV 内吞作用的一种新的抑制机制，可防止内吞作用过度激活，从而在持续神经递质传递过程中保护突触蛋白的再循环。
