槲皮素抑制中枢神经突触前兴奋性依赖的胞吞动力学

目的 槲皮素是一种广泛分布于药用植物中的黄酮类化合物,传统被认为具有神经保护作用。本研究利用位于大鼠脑干花萼状突触的突触前神经末梢进行膜片钳记录,研究槲皮素调控突触传递和可塑性的突触前机制。方法 利用全细胞膜片钳结合膜电容记录,在突触后记录微小兴奋性突触后电流（m EPSC）,在突触前神经末梢记录钙內流和神经囊泡的释放、回收以及可立即释放库（RRP）的恢复动力学。并且利用纤维刺激在轴突给予5～200 Hz的刺激,诱发突触后EPSC,记录突触后短时程抑制（STD）。结果 100μmol/L槲皮素不影响突触后m EPSC的振幅、频率以及AMPA受体的动力学特征。在突触前神经末梢,槲皮素不改变钙内流或囊泡的释放,但显著抑制胞吐后网格蛋白依赖的慢速胞吞。抑制胞吞会导致突触前囊泡动员的减慢,降低RRP的补充速率,并且增强高频刺激下的短时程可塑性STD。结论 本研究为槲皮素调控中枢神经突触传递提供全新的突触前神经机制,槲皮素有助于抑制中枢神经过度兴奋,进而发挥神经保护作用。     

v-SNAREs在血小板胞吞胞吐中的作用

胞吞胞吐参与了血小板相关物质的摄取、分泌,在血小板止血、血栓形成等过程中发挥着重要作用。近年来研究发现,囊泡可溶性N-乙基马来酰亚胺敏感因子附着蛋白受体家族(vesicle-soluble NSF attachment protein receptors,v-SNAREs)在哺乳动物细胞中高度保守,尤其在胞吞胞吐中扮演了关键角色,发挥着重要作用。本文就v-SNAREs在血小板病理生理过程中的作用机制,及其对血栓形成、冠心病等相关疾病的影响进行了综述,以期为心血管疾病的防治提供新的视野。     

短时程突触可塑性研究概况

突触可塑性是神经系统所具有的重要特征,也是神经系统实现其功能的重要保障。按照持续的时间划分,突触可塑性可分为短时程突触可塑性和长时程突触可塑性。短时程突触可塑性包括短时程增强和短时程压抑两种类型。与长时程突触可塑性不同,短时程突触可塑性的产生主要依赖于神经递质释放概率的变化,其往往决定神经回路的信息处理和反应模式,不仅直接参与了对输入信号的识别和处理,而且还可对长时程突触可塑性的表达产生重要影响。     

PC12细胞中Synaptotagmin Ⅶ 的沉默抑制了致密核心大囊泡的分泌

Syt Ⅶ, 因其具有比Syt Ⅰ和Syt Ⅸ更高的钙离子亲和力, 以及在与脂质结合后表现出更慢的解离动力学过程, 被认为在致密核心大囊泡分泌的慢速动力学过程中起着钙离子感受器的作用, 而并不参与突触囊泡的快速分泌过程. 然而迄今为止, Syt Ⅶ的亚细胞定位和其在胞内具体的生理学功能尚未完全清楚. 在本研究中, 我们首先应用全内反射荧光显微镜技术表明, 在PC12细胞中Syt Ⅶ定位于致密核心囊泡. 通过综合运用高时间分辨率的细胞膜电容测量和碳纤维微电极安培检测, 确定了单独沉默内源性的Syt Ⅶ就可以明显减少PC12细胞中钙触发的致密核心囊泡和质膜的融合和抑制了融合孔的开放, 特别是减少了致密核心大囊泡胞吐触发相的幅值, 而对于其持续成分的速率则几乎没有影响. 这些发现提示我们Syt Ⅶ在PC12细胞致密核心囊泡融合机制中作为钙感受器, 对可释放致密核心大囊泡库的形成和维持起着非常重要的作用.     

槲皮素通过FOXO1介导自噬抑制ox-LDL诱导泡沫细胞脂滴形成

目的 探讨槲皮素对氧化低密度脂蛋白（ox-LDL）诱导的泡沫细胞脂滴形成的影响及作用机制。方法 用50 mg/L的ox-LDL诱导小鼠RAW264.7细胞构建泡沫细胞模型,分别用不同浓度的槲皮素处理不同时间后,通过CCK8筛选槲皮素最佳作用浓度和时间。基于构建的泡沫细胞模型,在添加或不添加AS1842856（FOXO1抑制剂）的情况下,用槲皮素处理后,通过油红O染色观察脂滴形成,通过流式细胞术检测细胞凋亡;通过免疫印迹法（Western blot）检测各组细胞FOXO1蛋白表达水平;通过吖啶橙染色观察自噬小体形成情况;通过实时荧光定量PCR（qRT-PCR）和Western blot检测各组细胞Beclin1、LC3II和P62的mRNA和蛋白质表达水平。结果 100 μmol/L槲皮素干预12 h后,泡沫细胞脂滴形成和细胞凋亡被显著抑制（P<0.05）。与对照组相比,模型组细胞脂滴形成和细胞凋亡增加（P<0.05）,自噬小体减少（P<0.05）,FOXO1蛋白表达减少（P<0.05）,Beclin1和LC3II蛋白的mRNA和蛋白质表达水平均显著下降（P<0.05）,P62的mRNA和蛋白质表达水平显著增加（P<0.05）。与模型组相比,槲皮素处理上调FOXO1蛋白表达（P<0.05）,诱导自噬小体形成（P<0.05）,促进Beclin1和LC3II的蛋白质和mRNA表达水平（P<0.05）,抑制P62的蛋白质和mRNA表达水平（P<0.05）。而FOXO1抑制剂会逆转槲皮素对ox-LDL诱导的泡沫细胞的作用效果。结论 槲皮素通过上调FOXO1的表达诱导自噬,抑制ox-LDL诱导的脂滴形成。     

Visualizing recycling of synaptic vesicle proteins

The use of modern techniques (in particular, novel fluorescence markers of a few molecular participants of the exo-and endocytotic processes, including pH-sensitive agents, immuno-electron and laser-scanning microscopy) allows experimenters to visualize different stages of recycling of synaptic vesicle proteins. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 350–351, July–October, 2007.     

Trafficking of the vesicular acetylcholine transporter in SN56 cells: a dynamin-sensitive step and interaction with the AP-2 adaptor complex

The pathways by which synaptic vesicle proteins reach their destination are not completely defined. Here we investigated the traffic of a green fluorescent protein (GFP)-tagged version of the vesicular acetylcholine transporter (VAChT) in cholinergic SN56 cells, a model system for neuronal processing of this cargo. GFP-VAChT accumulates in small vesicular compartments in varicosities, but perturbation of endocytosis with a dominant negative mutant of dynamin I-K44A impaired GFP-VAChT trafficking to these processes. The protein in this condition accumulated in the cell body plasma membrane and in large vesicular patches therein. A VAChT endocytic mutant (L485A/L486A) was also located at the plasma membrane, however, the protein was not sorted to dynamin I-K44A generated vesicles. A fusion protein containing the VAChT C-terminal tail precipitated the AP-2 adaptor protein complex from rat brain, suggesting that VAChT directly interacts with the endocytic complex. In addition, yeast two hybrid experiments indicated that the C-terminal tail of VAChT interacts with the micro subunit of AP-2 in a di-leucine (L485A/L486A) dependent fashion. These observations suggest that the di-leucine motif regulates sorting of VAChT from the soma plasma membrane through a clathrin dependent mechanism prior to the targeting of the transporter to varicosities.     

Kiss-and-run and full-collapse fusion as modes of exo-endocytosis in neurosecretion

Neurotransmitters and hormones are released from neurosecretory cells by exocytosis (fusion) of synaptic vesicles, large dense-core vesicles and other types of vesicles or granules. The exocytosis is terminated and followed by endocytosis (retrieval). More than fifty years of research have established full-collapse fusion and clathrin-mediated endocytosis as essential modes of exo-endocytosis. Kiss-and-run and vesicle reuse represent alternative modes, but their prevalence and importance have yet to be elucidated, especially in neurons of the mammalian CNS. Here we examine various modes of exo-endocytosis across a wide range of neurosecretory systems. Full-collapse fusion and kiss-and-run coexist in many systems and play active roles in exocytotic events. In small nerve terminals of CNS, kiss-and-run has an additional role of enabling nerve terminals to conserve scarce vesicular resources and respond to high-frequency inputs. Full-collapse fusion and kiss-and-run will each contribute to maintaining cellular communication over a wide range of frequencies.     

Exocytosis in plants

Exocytosis is the final event in the secretory pathway and requires the fusion of the secretory vesicle membrane with the plasma membrane. It results in the release to the outside of vesicle cargo from the cell interior and also the delivery of vesicle membrane and proteins to the plasma membrane. An electrophysiological assay that measures changes in membrane capacitance has recently been used to monitor exocytosis in plants. This complements information derived from earlier light and electron microscope studies, and allows both transient and irreversible fusion of single exocytotic vesicles to be followed with high resolution in protoplasts. It also provides a tool to investigate bulk exocytotic activity in single protoplasts under the influence of cytoplasmic modulators. This research highlights the role of intracellular Ca²⁺, GTP and pressure in the control of exocytosis in plants.In parallel to these functional studies, plant proteins with the potential to regulate exocytosis are being identified by molecular analysis. In this review we describe these electrophysiological and molecular advances, and emphasise the need for parallel biochemical work to provide a complete picture of the mechanisms controlling vesicle fusion at the plasma membrane of plant cells.