Optogenetic control of phosphoinositide at mouse calyx of Held synapse

突触传递依赖于突触前动作电位在一毫秒内有效转导到Ca2+触发的突触囊泡（SV）融合。为了达到这个速度，SVs在形态上附着在质膜上，并在动作电位到达之前准备融合能力。整个过程发生在一个被称为活性区的特殊结构中，该结构由复杂的分子结构和空间组织的脂筏组成，它调节突触传递的速度、精度和可塑性。磷脂酰肌醇4,5-二磷酸（PIP2）是位于活性区膜内小叶的磷脂之一。其携带多个负电荷的独特性质被认为是在胞吐和内吞过程中招募下游效应蛋白的重要因素。然而，尽管多年来对其功能进行了广泛的研究，但我们对PIP2如何调节突触传递的理解仍然非常有限。这主要是由于缺乏时空控制，无法直接控制突触前膜的数量。大多数方法依赖于脂激酶/磷酸酶的药理学或遗传扰动，然而脱靶或长期代偿效应往往使对PIP2作用的解释复杂化。为了克服这一限制，我的目标是在哺乳动物中枢神经突触（称为calyx of Held）上开拓一种新的光遗传pip2缺失系统（CIB1-CRY2）。该工具通过使用蓝光脉冲使磷酸酶瞬时可逆地募集到突触前膜，从而在几秒钟内快速局部消除PIP2。结合双光子PIP2实时成像和突触前后的膜片钳记录，本研究旨在直接探讨PIP2的空间分布与SV融合之间的功能联系。具体来说，本提案将解决三个主要问题：(1)PIP2是否调节突触前末端电压门控Ca2+通道的门控和/或聚集？(2) PIP2-SV的相互作用是否决定了活性区易释放sv的数量？(3) PIP2是否调控突触短期可塑性？这些结果有望为PIP2在突触传递中的功能作用提供明确的见解，并进一步加深我们对哺乳动物中枢神经突触突触可塑性机制的理解。

Synaptic transmission relies on effective transduction of presynaptic action potentials into Ca2+-triggered synaptic vesicle (SV) fusion within a millisecond. To achieve the speed, SVs morphologically attach to the plasma membrane and prepare to be fusion competent before the arrival of action potentials. This entire process occurs in a specialized structure called active zone that constitutes a complex molecular architecture and spatially organized lipid rafts, which mediates the speed, precision and the plasticity of synaptic transmission. Phosphatidylinositol 4,5-bisphosphate (PIP2) is one of the phospholipids that locates at the inner leaflet of the active zone membrane. Its unique property of carrying multiple negative charges is proposed to be important for recruiting downstream effector proteins during exocytosis and endocytosis. However, despite the extensive efforts over the years for studying its function, our understanding of how PIP2 regulates synaptic transmission is still very limited. This is largely due to the lack of spatiotemporal control that allows direct manipulation of its quantity at presynaptic membrane. Most of the methods relied on pharmacological or genetic perturbations of lipid kinase/phosphatase, however off-target or long-term compensatory effects often complicate the interpretation of PIP2’s role. To overcome this limitation, I aim to pioneer a novel optogenetic PIP2-deletion system (CIB1-CRY2) at a mammalian central nervous synapse called calyx of Held. This tool enables transient and reversible recruitment of phosphatase into the presynaptic membrane by using pulses of blue light, which leads to a rapid and local elimination of PIP2 within a few seconds. In combination with two-photon PIP2 live-imaging and pre- and post-synaptic patch-clamp recordings, this proposal aims to directly probe the functional link between the spatial distribution of PIP2 and SV fusion. Specifically, three major questions will be addressed in the present proposal: (1) Whether PIP2 regulates the gating and/or clustering of voltage-gated Ca2+ channel at presynaptic terminal? (2) Do PIP2-SV interactions determine the number of readily-releasable SVs at active zone? (3) Does PIP2 regulate synaptic short-term plasticity? These results are expected to gain unequivocal insights into the functional role of PIP2 in synaptic transmission and further lead weight on our understanding of the mechanisms of synaptic plasticity at mammalian central nervous synapse.