Regulation of synaptic vesicle recycling by calcineurin in different vesicle pools

The synaptic vesicles keep recycling by the processes of endocytosis and exocytosis to maintain the normal synaptic transmission. The synaptic vesicles are classified as the readily releasable pool (RRP) and the reserve pool (RP). In the endocytosis process, calcineurin (CaN), a Ca2+/calmodulin-dependent protein phosphatase, has been shown to play important roles. However, it is unclear about its roles in different vesicle pools. Here, we investigated the role of CaN in the regulation of vesicle recycling in the RRP and RP. Vesicle recycling was monitored by using fluorescent dyes FM1-43 and FM4-64 in the primary cultures of hippocampal neurons. Inhibition of CaN by FK506 and cyclosporin A suppressed the endocytosis in the RP, but not in the RRP. Inhibition of CaN also restrained the exocytic process triggered by 10 Hz stimulation, but had no effect on 3-5 Hz stimulation-induced exocytosis. FK506 also reduced the total vesicle pool size in the synaptic terminals. A synthesized CaN inhibitory peptide showed the similar effects as FK506 and cyclosporin A. These results revealed a novel mechanism that CaN plays critical roles in the distinct vesicle recycling processes.     

Ca(2+)-dependent regulation of synaptic vesicle endocytosis

Action potentials, when arriving at presynaptic terminals, elicit Ca(2+) influx through voltage-gated Ca(2+) channels. Intracellular [Ca(2+)] elevation around the channels subsequently triggers synaptic vesicle exocytosis and also induces various protein reactions that regulate vesicle endocytosis and recycling to provide for long-term sustainability of synaptic transmission. Recent studies using membrane capacitance measurements, as well as high-resolution optical imaging, have revealed that the dominant type of synaptic vesicle endocytosis at central nervous system synapses is mediated by clathrin and dynamin. Furthermore, Ca(2+)-dependent mechanisms regulating endocytosis may operate in different ways depending on the distance from Ca(2+) channels: (1) intracellular Ca(2+) in the immediate vicinity of a Ca(2+) channel plays an essential role in triggering endocytosis, and (2) intracellular Ca(2+) traveling far from the channels has a modulatory effect on endocytosis at the periactive zone. Here, I integrate the latest progress in this field to propose a compartmental model for regulation of vesicle endocytosis at synapses and discuss the possible roles of presynaptic Ca(2+)-binding proteins including calmodulin, calcineurin and synaptotagmin.     

Differential modulation of short-term synaptic dynamics by long-term potentiation at mouse hippocampal mossy fibre synapses

After exocytosis, synaptic vesicle components are selectively retrieved by clathrin-mediated endocytosis and then re-used in future rounds of transmitter release. Under some conditions, synaptic terminals in addition perform bulk endocytosis of large membranous sacs. Bulk endocytosis is less selective than clathrin-mediated endocytosis and probably internalizes components normally targeted to the plasma membrane. Nonetheless, this process plays a major role in some tonic ribbon-type synapses, which release neurotransmitter for prolonged periods of time. We show here, that large endosomes formed after strong and prolonged stimulation undergo stimulated exocytosis in retinal bipolar neurons. The result suggests how cells might return erroneously internalized components to the plasma membrane, and also demonstrates that synaptic vesicles are not the only neuronal organelle that stains with styryl dyes and undergoes stimulated exocytosis.     

Fast synaptic fatigue in shibire mutants reveals a rapid requirement for dynamin in synaptic vesicle membrane trafficking

The GTPase dynamin is involved in endocytosis in many cell types, as first revealed by temperature-sensitive paralytic mutations in the Drosophila dynamin gene, shibire (shi), which disrupt synaptic vesicle endocytosis and deplete synaptic terminals of vesicles. Here we report that shi synapses exhibit a fast synaptic fatigue phenotype within 20 ms of repetitive stimulation, which cannot be explained by vesicle depletion, as we confirmed by electron microscopy. These results suggest that, in addition to its well-characterized role in synaptic vesicle recycling, dynamin may be required for short-term maintenance of the readily releasable pool of synaptic vesicles.     

Live imaging of bulk endocytosis in frog motor nerve terminals using FM dyes

We observed endocytosis in real time in stimulated frog motor nerve terminals by imaging the growth of large membrane infoldings labeled with a low concentration of FM dye. The spatial and temporal information made available by these experiments allowed us to image several new aspects of this synaptic vesicle recycling pathway. Membrane infoldings appeared near synaptic vesicle clusters and grew rapidly during long-duration, high-frequency stimulation. In some cases, we observed large, elongated infoldings growing laterally into the terminal. We used these observations to calculate infolding growth rates. A decrease in stimulation frequency caused a decrease in growth rates, but the overall length of these structures was unaffected by frequency changes. Attempts to wash the dye from these infoldings after stimulation were unsuccessful, demonstrating that the fluorescent structures had been endocytosed. We also used this technique to trigger and image infoldings during repeated, short trains. We found that membrane uptake occurred repeatedly at individual endocytosis sites, but only during a portion of the total number of trains delivered to the terminal. Finally, we showed that phosphatidylinositol 3-kinase, but not actin, was involved in this endocytosis pathway. The ability to monitor many individual bulk endocytosis sites in real time should allow for new types of endocytosis measurements and could reveal novel and unexpected mechanisms for coordinating membrane recovery during synaptic activity.     

Dynamin-independent synaptic vesicle retrieval?

A new study proposes that synaptic vesicle endocytosis at a large synaptic terminal is partly independent of dynamin and GTP hydrolysis, suggesting a new mechanism leading to vesicle fission and maintenance of neurotransmission.     

Uncoupling the roles of synaptotagmin I during endo- and exocytosis of synaptic vesicles

Synaptotagmin I (syt1) is required for normal rates of synaptic vesicle endo- and exocytosis. However, whether the kinetic defects observed during endocytosis in Syt1 knockout neurons are secondary to defective exocytosis or whether syt1 directly regulates the rate of vesicle retrieval remains unknown. To address this question, we sought to dissociate these two activities. We uncoupled the function of syt1 in exo- and endocytosis in mouse neurons either by re-targeting the protein or via mutagenesis of its tandem C2 domains. The effect of these manipulations on exo- and endocytosis were analyzed using electrophysiology, in conjunction with optical imaging of the vesicle cycle. Our results indicate that syt1 is directly involved in endocytosis. Notably, either of the C2 domains of syt1, C2A or C2B, was able to function as a Ca(2+) sensor for endocytosis. Thus, syt1 functions as a dual Ca(2+) sensor for both endo- and exocytosis, potentially coupling these two components of the vesicle cycle.     

The vesicle cycle in motor nerve endings of the mouse diaphragm

Experiments on the mouse diaphragm muscle using intracellular microelectrode recordings and fluorescence microscopy were performed to study the dynamics of transmitter secretion and synaptic vesicle recycling processes (the exocytosis-endocytosis cycle) in motor nerve endings (NE) during prolonged rhythmic stimulation (20 impulses/sec). During stimulation, there were triphasic changes in the amplitude of endplate potentials (EPP): an initial rapid reduction, followed by prolonged (1–2 min) stabilization of amplitude, i.e., a plateau, and then a further slow decrease. Restoration of EPP amplitude after stimulation for 3 min occurred over a period of several seconds. Loading of synaptic vesicles with the fluorescent endocytic stain FM1-43 showed that rhythmic stimulation led to a gradual (over 5–6 min) decrease in NE fluorescence, demonstrating exocytosis of synaptic vesicles. Quantum analysis of the electrophysiological data and comparison of these data with results from fluorescence studies suggested that mouse NE have a high rate of endocytosis and reutilization of synaptic vesicles (the mean recycling time was about 50 sec), which may support the maintenance of reliable synaptic transmission during prolonged high-frequency activity. The sizes of the release-ready and recycling pools of synaptic vesicles were determined quantitatively. It is suggested that vesicle recycling in mouse NE occurs via a short, rapid pathway with incorporation into the recycling pool. Vesicles of the reserve pool are not used for transmitter secretion in the stimulation conditions used here. Translated from Rossiiskii Fiziologicheskii Zhurnal imeni I. M. Sechenova, Vol. 94, No. 2, pp. 129–141, February, 2008.     

Clathrin-mediated endocytosis: the physiological mechanism of vesicle retrieval at hippocampal synapses

The maintenance of synaptic transmission requires that vesicles are recycled after releasing neurotransmitter. Several modes of retrieval have been proposed to operate at small synaptic terminals of central neurons, but the relative importance of these has been controversial. It is established that synaptic vesicles can collapse on fusion and the machinery for retrieving this membrane by clathrin-mediated endocytosis (CME) is enriched in the presynaptic terminal. But it has also been suggested that the majority of vesicles released by physiological stimulation are recycled by a second, faster mechanism called 'kiss-and-run', which operates in 1 s or less to retrieve a vesicle before it has collapsed. The most recent evidence argues against the occurrence of 'kiss-and-run' in hippocampal synapses. First, an improved fluorescent reporter of exocytosis (sypHy), indicates that only a slow mode of endocytosis  (τ= 15 s)  operates when vesicle fusion is triggered by a single nerve impulse or short burst. Second, this retrieval mechanism is blocked by overexpressing the C-terminal fragment of AP180 or by knockdown of clathrin using RNAi. Third, vesicle fusion is associated with the movement of clathrin and vesicle proteins out of the synapse into the neighbouring axon. These observations indicate that clathrin-mediated endocytosis is the major, if not exclusive, mechanism of retrieval in small hippocampal synapses.     

A readily retrievable pool of synaptic vesicles

Although clathrin-mediated endocytosis is thought to be the predominant mechanism of synaptic vesicle recycling, it seems to be too slow for fast recycling. Therefore, it was suggested that a presorted and preassembled pool of synaptic vesicle proteins on the presynaptic membrane might support a first wave of fast clathrin-mediated endocytosis. In this study we monitored the temporal dynamics of such a 'readily retrievable pool' of synaptic vesicle proteins in rat hippocampal neurons using a new type of probe. By applying cypHer5E, a new cyanine dye-based pH-sensitive exogenous marker, coupled to antibodies to luminal domains of synaptic vesicle proteins, we could reliably monitor synaptic vesicle recycling and demonstrate the preferential recruitment of a surface pool of synaptic vesicle proteins upon stimulated endocytosis. By using fluorescence nanoscopy of surface-labeled synaptotagmin 1, we could resolve the spatial distribution of the surface pool at the periactive zone in hippocampal boutons, which represent putative sites of endocytosis.     

Multiple vesicle recycling pathways in central synapses and their impact on neurotransmission

Short-term synaptic depression during repetitive activity is a common property of most synapses. Multiple mechanisms contribute to this rapid depression in neurotransmission including a decrease in vesicle fusion probability, inactivation of voltage-gated Ca²⁺ channels or use-dependent inhibition of release machinery by presynaptic receptors. In addition, synaptic depression can arise from a rapid reduction in the number of vesicles available for release. This reduction can be countered by two sources. One source is replenishment from a set of reserve vesicles. The second source is the reuse of vesicles that have undergone exocytosis and endocytosis. If the synaptic vesicle reuse is fast enough then it can replenish vesicles during a brief burst of action potentials and play a substantial role in regulating the rate of synaptic depression. In the last 5 years, we have examined the impact of synaptic vesicle reuse on neurotransmission using fluorescence imaging of synaptic vesicle trafficking in combination with electrophysiological detection of short-term synaptic plasticity. These studies have revealed that synaptic vesicle reuse shapes the kinetics of short-term synaptic depression in a frequency-dependent manner. In addition, synaptic vesicle recycling helps maintain the level of neurotransmission at steady state. Moreover, our studies showed that synaptic vesicle reuse is a highly plastic process as it varies widely among synapses and can adapt to changes in chronic activity levels.     

发动蛋白-1的脯氨酸和精氨酸富集域功能域与大鼠神经突触小体相互作用蛋白的筛选及鉴定

目的 筛选及鉴定发动蛋白-1(Dynamin-1)的脯氨酸和精氨酸富集域(PRD)功能域在大鼠神经突触小体中的相互作用蛋白.方法 构建Dynamin-1 PRD功能域的重组原核表达质粒pGEX-4T-2-PRD,通过大肠杆菌表达系统进行诱导表达;谷胱甘肽琼脂糖凝脂柱纯化得到融合蛋白GST-PRD,继而利用谷胱甘肽巯基转移酶沉淀技术,筛选出GST-PRD融合蛋白与分离提取的大鼠神经突触小体的相互作用蛋白,并通过液相质谱技术结合数据库,对上述获得的相互作用蛋白进行分析、鉴定.结果 通过构建pGEX-4T-2-PRD重组质粒及诱导表达,成功纯化出了融合蛋白GST-PRD;同时成功提取了大鼠神经突触小体,经谷胱甘肽巯基转移酶沉淀技术联合液相质谱分析,获得在大鼠神经突触小体中与Dynamin-1的PRD域有相互作用的蛋白共35个,分别属于突触囊泡相关蛋白、细胞骨架蛋白、代谢酶及其他类的蛋白.结论 本研究获得的与Dynamin-1的PRD域有相互作用的蛋白共35个.     

Syndapin I is the phosphorylation-regulated dynamin I partner in synaptic vesicle endocytosis

Dynamin I is dephosphorylated at Ser-774 and Ser-778 during synaptic vesicle endocytosis (SVE) in nerve terminals. Phosphorylation was proposed to regulate the assembly of an endocytic protein complex with amphiphysin or endophilin. Instead, we found it recruits syndapin I for SVE and does not control amphiphysin or endophilin binding in rat synaptosomes. After depolarization, syndapin showed a calcineurin-mediated interaction with dynamin. A peptide mimicking the phosphorylation sites disrupted the dynamin-syndapin complex, not the dynamin-endophilin complex, arrested SVE and produced glutamate release fatigue after repetitive stimulation. Pseudophosphorylation of Ser-774 or Ser-778 inhibited syndapin binding without affecting amphiphysin recruitment. Site mutagenesis to alanine arrested SVE in cultured neurons. The effects of the sites were additive for syndapin I binding and SVE. Thus syndapin I is a central component of the endocytic protein complex for SVE via stimulus-dependent recruitment to dynamin I and has a key role in synaptic transmission.     

Beta-amyloid disrupted synaptic vesicle endocytosis in cultured hippocampal neurons

Neuronal death leading to gross brain atrophy is commonly seen in Alzheimer's disease (AD) patients. Yet, it is becoming increasingly apparent that the pathogenesis of AD involves early and more discrete synaptic changes in affected brain areas. However, the molecular mechanisms that underlie such synaptic dysfunction remain largely unknown. Recently, we have identified dynamin 1, a protein that plays a critical role in synaptic vesicle endocytosis, and hence, in the signaling properties of the synapse, as a potential molecular determinant of such dysfunction in AD. In the present study, we analyzed beta-amyloid (Abeta)-induced changes in synaptic vesicle recycling in rat cultured hippocampal neurons. Our results showed that Abeta, the main component of senile plaques, caused ultrastructural changes indicative of impaired synaptic vesicle endocytosis in cultured hippocampal neurons that have been stimulated by depolarization with high potassium. In addition, Abeta led to the accumulation of amphiphysin in membrane fractions from stimulated hippocampal neurons. Moreover, experiments using FM1-43 showed reduced dye uptake in stimulated hippocampal neurons treated with Abeta when compared with untreated stimulated controls. Similar results were obtained using a dynamin 1 inhibitory peptide suggesting that dynamin 1 depletion caused deficiency in synaptic vesicle recycling not only in Drosophila but also in mammalian neurons. Collectively, these results showed that Abeta caused a disruption of synaptic vesicle endocytosis in cultured hippocampal neurons. Furthermore, we provided evidence suggesting that Abeta-induced dynamin 1 depletion might play an important role in this process.     

Endocytosis at the synaptic terminal

Exocytosis of neurotransmitter from a synaptic vesicle is followed by efficient retrieval of its constituent membrane and proteins. Real-time measurements indicate that fast and slow modes of retrieval operate in parallel at a number of presynaptic terminals. Two mechanisms can be distinguished by electron microscopy: clathrin-mediated retrieval of small vesicles and bulk retrieval of large cisternae. Methods that investigate the behaviour of individual vesicles have recently demonstrated a third route of retrieval: the rapid reversal of a pore-like connection between the vesicle and surface ('kiss-and-run'). Key aims for the future are to identify the molecules underlying different mechanisms of endocytosis at the synapse and the signals that select between them.     

RNA干扰endophilin A2抑制大鼠海马神经元突触囊泡内吞

 目的:探讨内吞蛋白(endophilin)A2对大鼠海马神经元突触囊泡内吞的影响。方法:设计干扰endophilin A2的小分子干扰片段,通过Western blot的方法筛选出针对endophilin A2有效的特异性干扰片段;用免疫荧光的方法验证干扰片段对神经元内源性endophilin A2的干扰效果;利用双全细胞膜片钳的方法,记录不同刺激方案下的兴奋性突触后电流(excitatory postsynaptic currents,EPSCs)的情况,以明确endophilin A2对突触囊泡循环的影响。结果:(1)免疫荧光结果显示endophilin A2的干扰片段能显著减少海马神经元内源性endophilin A2的表达(P<0.05);(2)双全细胞膜片钳结果显示,0.1 Hz的低频刺激下,干扰endophilin A2表达的神经元EPSCs大小与阴性对照组没有明显差异;而无论是单串高频刺激下还是多串高频刺激下,干扰endophilin A2表达的神经元标准化EPSCs的下降程度都明显增强(P<0.05)。结论:(1)成功筛选出特异性干扰endophilin A2的有效干扰片段;(2)干扰endophilin A2的表达不影响海马神经元突触囊泡胞吐;(3)干扰endophilin A2的表达可抑制大鼠海马神经元突触囊泡内吞。     

Effects of wortmannin and latrunculin A on slow endocytosis at the frog neuromuscular junction

Phosphoinositides are key regulators of synaptic vesicle cycling and endocytic traffic; the actin cytoskeleton also seems to be involved in modulating these processes. We investigated the effects of perturbing phosphoinositide signalling and actin dynamics on vesicle cycling in frog motor nerve terminals, using fluorescence and electron microscopy, and electrophysiology. Antibody staining for β-actin revealed that actin surrounds but does not overlap with synaptic vesicle clusters. Latrunculin A, which disrupts actin filaments by binding actin monomers, and wortmannin, an inhibitor of phosphatidyl inositol-3-kinase (PI3-kinase), each disrupted the pattern of presynaptic actin staining, but not vesicle clusters in resting terminals. Latrunculin A, but not wortmannin, also reduced vesicle mobilization and exocytosis. Both drugs inhibited the stimulation-induced uptake of the styryl dye FM1-43 and blocked vesicle reformation from internalized membrane objects after tetanic stimulation. These results are consistent with a role of PI3-kinase and the actin cytoskeleton in the slow pathway of vesicle endocytosis, used primarily by reserve pool vesicles.     

AP180 maintains the distribution of synaptic and vesicle proteins in the nerve terminal and indirectly regulates the efficacy of Ca2+-triggered exocytosis

AP180 plays an important role in clathrin-mediated endocytosis of synaptic vesicles (SVs) and has also been implicated in retrieving SV proteins. In Drosophila, deletion of its homologue, Like-AP180 (LAP), has been shown to increase the size of SVs but decrease the number of SVs and transmitter release. However, it remains elusive whether a reduction in the total vesicle pool directly affects transmitter release. Further, it is unknown whether the lap mutation also affects vesicle protein retrieval and synaptic protein localization and, if so, how it might affect exocytosis. Using a combination of electrophysiology, optical imaging, electron microscopy, and immunocytochemistry, we have further characterized the lap mutant and hereby show that LAP plays additional roles in maintaining both normal synaptic transmission and protein distribution at synapses. While increasing the rate of spontaneous vesicle fusion, the lap mutation dramatically reduces impulse-evoked transmitter release at steps downstream of calcium entry and vesicle docking. Notably, lap mutations disrupt calcium coupling to exocytosis and reduce calcium cooperativity. These results suggest a primary defect in calcium sensors on the vesicles or on the release machinery. Consistent with this hypothesis, three vesicle proteins critical for calcium-mediated exocytosis, synaptotagmin I, cysteine-string protein, and neuronal synaptobrevin, are all mislocalized to the extrasynaptic axonal regions along with Dap160, an active zone marker (nc82), and glutamate receptors in the mutant. These results suggest that AP180 is required for either recycling vesicle proteins and/or maintaining the distribution of both vesicle and synaptic proteins in the nerve terminal.     

Glutamate acts at NMDA receptors on fresh bovine and on cultured human retinal pigment epithelial cells to trigger release of ATP

Efficient vesicle membrane recycling at presynaptic terminals is pivotal for preventing depletion and maintaining high firing rates in neuronal networks. We used a new approach, based on the combination of spectrally different optical probes, to investigate how stimulation determines the fate of synaptic vesicles after endocytosis. We found that in the small central synapses of rat hippocampal neurones low frequency stimulation (40 action potentials at 2 Hz) targets vesicles preferentially to vesicle pools that were kinetically faster. Vesicles taken up during endocytosis triggered by high frequency stimulation (400 action potentials, 20 Hz) were also placed in the back of the release queue. We performed a spatial analysis of the recycled vesicles in living hippocampal boutons using two spectrally different FM-dyes (FM1-43 and FM5-95). By using these consecutively, vesicles endocytosed by either stimulation protocol were labelled with a different colour. This revealed that the kinetic arrangement was also reflected in the spatial organization of vesicles within the bouton. Next, we identified the postsynaptic site of the active zone by transfecting the neurones with postsynaptic density protein PSD-95-CFP. The data from these triple colour experiments suggest that retrieval after low frequency stimulation keeps vesicles in a more confined region closer to the active zone as identified by PSD-95-CFP expression at the postsynaptic site.     

Impairment of synaptic vesicle exocytosis and recycling during neuromuscular weakness produced in mice by 2,4-dithiobiuret

Chronic treatment of rodents with 2,4-dithiobiuret (DTB) induces a neuromuscular syndrome of flaccid muscle weakness that mimics signs seen in several human neuromuscular disorders such as congenital myasthenic syndromes, botulism, and neuroaxonal dystrophy. DTB-induced muscle weakness results from a reduction of acetylcholine (ACh) release by mechanisms that are not yet clear. The objective of this study was to determine if altered release of ACh during DTB-induced muscle weakness was due to impairments of synaptic vesicle exocytosis, endocytosis, or internal vesicular processing. We examined motor nerve terminals in the triangularis sterni muscles of DTB-treated mice at the onset of muscle weakness. Uptake of FM1-43, a fluorescent marker for endocytosis, was reduced to approximately 60% of normal after either high-frequency nerve stimulation or K(+) depolarization. Terminals ranged from those with nearly normal fluorescence ("bright terminals") to terminals that were poorly labeled ("dim terminals"). Ultrastructurally, the number of synaptic vesicles that were labeled with horseradish peroxidase (HRP) was also reduced by DTB to approximately 60%; labeling among terminals was similarly variable. A subset of DTB-treated terminals having abnormal tubulovesicular profiles in their centers did not respond to stimulation with increased uptake of HRP and may correspond to dim terminals. Two findings suggest that posttetanic "slow endocytosis" remained qualitatively normal: the rate of this type of endocytosis as measured with FM1-43 did not differ from normal, and HRP was observed in organelles associated with this pathway- coated vesicles, cisternae, as well as synaptic vesicles but not in the tubulovesicular profiles. In DTB-treated bright terminals, end-plate potential (EPP) amplitudes were decreased, and synaptic depression in response to 15-Hz stimulation was increased compared with those of untreated mice; in dim terminals, EPPs were not observed during block with D-tubocurarine. Nerve-stimulation-induced unloading of FM1-43 was slower and less complete than normal in bright terminals, did not occur in dim terminals, and was not enhanced by alpha-latrotoxin. Collectively, these results indicate that the size of the recycling vesicle pool is reduced in nerve terminals during DTB-induced muscle weakness. The mechanisms by which this reduction occurs are not certain, but accumulated evidence suggests that they may include defects in either or both exocytosis and internal vesicular processing.