Department of Neuroscience and Howard Hughes Medical Institute, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
Medical Scientist Training Program, University of Wisconsin School of Medicine and Public Health, Madison, WI, USA.
J Physiol. 2022 Feb;600(3):645-654. doi: 10.1113/JP282601. Epub 2022 Jan 13.
SV2A, an essential transporter-like synaptic vesicle protein, is a major target for antiepileptic drugs and a receptor for clostridial neurotoxins including Botox. While SV2A is required for normal levels of evoked neurotransmitter release, the mechanism underlying this role remains unclear. Here, we introduce a new chemogenetic approach for all-optical monitoring of excitation-secretion coupling, and we demonstrate its use in characterizing the SV2A knockout (KO) phenotype in cultured hippocampal neurons. This method employs the HaloTag system to target a robust small-molecule Ca indicator, JF -BAPTA, to the presynaptic compartment. The far-red fluorescence of this indicator enables multiplexing with the fluorescent glutamate sensor iGluSnFR for detection of presynaptic Ca influx and glutamate release at the same axonal boutons. Evoked glutamate release probability was reduced in SV2A KO neurons without a change in presynaptic Ca entry, suggesting that SV2A supports vesicle fusion by increasing the functional availability, or efficiency, of the Ca -regulated membrane fusion machinery. KEY POINTS: One of the most prescribed antiepileptic medications, levetiracetam, acts by binding a protein of uncertain molecular function. This transporter-like protein, SV2A, is trafficked to synaptic vesicles and acts to support neurotransmitter release, but the mechanism underlying this function has not been determined In this study, we sought to establish whether SV2A changes Ca signalling at nerve terminals, which is a key regulatory system for synaptic vesicle exocytosis. To do so, we adapted new chemogenetic tools to perform all-optical measurements of presynaptic Ca and glutamate release in neurons lacking SV2A. Our measurements showed that loss of SV2A reduces glutamate release without reducing Ca influx at hippocampal nerve terminals, demonstrating that SV2A increases the likelihood that Ca will trigger synaptic vesicle fusion.
SV2A，一种必需的转运体样突触囊泡蛋白，是抗癫痫药物的主要靶点，也是包括肉毒杆菌毒素在内的梭状芽胞杆菌神经毒素的受体。虽然 SV2A 是正常诱发神经递质释放所必需的，但这一作用的机制仍不清楚。在这里，我们引入了一种新的化学遗传学方法来进行全光学监测兴奋-分泌偶联，并用其来描述培养的海马神经元中 SV2A 敲除 (KO) 表型。这种方法利用 HaloTag 系统将一种强大的小分子 Ca 指示剂 JF -BAPTA 靶向到突触前区室。该指示剂的远红荧光使其能够与荧光谷氨酸传感器 iGluSnFR 进行多路复用，以检测同一轴突末梢的突触前 Ca 内流和谷氨酸释放。SV2A KO 神经元中的诱发谷氨酸释放概率降低，但突触前 Ca 内流没有变化，这表明 SV2A 通过增加 Ca 调节的膜融合机制的功能可用性或效率来支持囊泡融合。关键点：最常开的抗癫痫药物之一，左乙拉西坦，通过结合一种功能未知的蛋白质起作用。这种转运体样蛋白 SV2A 被运输到突触囊泡，并作用于支持神经递质释放，但这一功能的机制尚未确定。在这项研究中，我们试图确定 SV2A 是否改变了神经末梢的 Ca 信号，这是突触囊泡胞吐作用的关键调节系统。为此，我们采用了新的化学遗传学工具，在缺乏 SV2A 的神经元中进行全光学测量突触前 Ca 和谷氨酸释放。我们的测量结果表明，SV2A 的缺失减少了谷氨酸释放，但不减少海马神经末梢的 Ca 内流，这表明 SV2A 增加了 Ca 触发突触囊泡融合的可能性。
