Stable and Flexible Synaptic Transmission Controlled by the Active Zone Protein Interactions

An action potential triggers neurotransmitter release from synaptic vesicles docking to a specialized release site of the presynaptic plasma membrane, the active zone. The active zone is a highly organized structure with proteins that serves as a platform for synaptic vesicle exocytosis, mediated by SNAREs complex and Ca²⁺ sensor proteins, within a sub-millisecond opening of nearby Ca²⁺ channels with the membrane depolarization. In response to incoming neuronal signals, each active zone protein plays a role in the release-ready site replenishment with synaptic vesicles for sustainable synaptic transmission. The active zone release apparatus provides a possible link between neuronal activity and plasticity. This review summarizes the mostly physiological role of active zone protein interactions that control synaptic strength, presynaptic short-term plasticity, and homeostatic synaptic plasticity.     

DMSO Chemically Alters Cell Membranes to Slow Exocytosis and Increase the Fraction of Partial Transmitter Released

Dimethyl sulfoxide (DMSO) is frequently used as a solvent in biological studies and as a vehicle for drug therapy; but the side effects of DMSO, especially on the cell environment, are not well understood, and controls with DMSO are not neutral at higher concentrations. Herein, electrochemical measurement techniques are applied to show that DMSO increases exocytotic neurotransmitter release, while leaving vesicular contents unchanged. In addition, the kinetics of release from DMSO‐treated cells are faster than that of untreated ones. The results suggest that DMSO has a significant influence on the chemistry of the cell membrane, leading to alteration of exocytosis. A speculative chemical mechanism of the effect on the fusion pore during exocytosis is presented.     

Vesicle Fusion as a Target Process for the Action of Sphingosine and Its Derived Drugs

The fusion of membranes is a central part of the physiological processes involving the intracellular transport and maturation of vesicles and the final release of their contents, such as neurotransmitters and hormones, by exocytosis. Traditionally, in this process, proteins, such SNAREs have been considered the essential components of the fusion molecular machinery, while lipids have been seen as merely structural elements. Nevertheless, sphingosine, an intracellular signalling lipid, greatly increases the release of neurotransmitters in neuronal and neuroendocrine cells, affecting the exocytotic fusion mode through the direct interaction with SNAREs. Moreover, recent studies suggest that FTY-720 (Fingolimod), a sphingosine structural analogue used in the treatment of multiple sclerosis, simulates sphingosine in the promotion of exocytosis. Furthermore, this drug also induces the intracellular fusion of organelles such as dense vesicles and mitochondria causing cell death in neuroendocrine cells. Therefore, the effect of sphingosine and synthetic derivatives on the heterologous and homologous fusion of organelles can be considered as a new mechanism of action of sphingolipids influencing important physiological processes, which could underlie therapeutic uses of sphingosine derived lipids in the treatment of neurodegenerative disorders and cancers of neuronal origin such neuroblastoma.     

The Thermodynamically Expensive Contribution of Three Calcium Sources to Somatic Release of Serotonin

The soma, dendrites and axon of neurons may display calcium-dependent release of transmitters and peptides. Such release is named extrasynaptic for occurring in absence of synaptic structures. This review describes the cooperative actions of three calcium sources on somatic exocytosis. Emphasis is given to the somatic release of serotonin by the classical leech Retzius neuron, which has allowed detailed studies on the fine steps from excitation to exocytosis. Trains of action potentials induce transmembrane calcium entry through L-type channels. For action potential frequencies above 5 Hz, summation of calcium transients on individual action potentials activates the second calcium source: ryanodine receptors produce calcium-induced calcium release. The resulting calcium tsunami activates mitochondrial ATP synthesis to fuel transport of vesicles to the plasma membrane. Serotonin that is released maintains a large-scale exocytosis by activating the third calcium source: serotonin autoreceptors coupled to phospholipase C promote IP3 production. Activated IP3 receptors in peripheral endoplasmic reticulum release calcium that promotes vesicle fusion. The Swiss-clock workings of the machinery for somatic exocytosis has a striking disadvantage. The essential calcium-releasing endoplasmic reticulum near the plasma membrane hinders the vesicle transport, drastically reducing the thermodynamic efficiency of the ATP expenses and elevating the energy cost of release.     

Secretome and Tunneling Nanotubes: A Multilevel Network for Long Range Intercellular Communication between Endothelial Cells and Distant Cells

As a cellular interface between the blood and tissues, the endothelial cell (EC) monolayer is involved in the control of key functions including vascular tone, permeability and homeostasis, leucocyte trafficking and hemostasis. EC regulatory functions require long-distance communications between ECs, circulating hematopoietic cells and other vascular cells for efficient adjusting thrombosis, angiogenesis, inflammation, infection and immunity. This intercellular crosstalk operates through the extracellular space and is orchestrated in part by the secretory pathway and the exocytosis of Weibel Palade Bodies (WPBs), secretory granules and extracellular vesicles (EVs). WPBs and secretory granules allow both immediate release and regulated exocytosis of messengers such as cytokines, chemokines, extracellular membrane proteins, coagulation or growth factors. The ectodomain shedding of transmembrane protein further provide the release of both receptor and ligands with key regulatory activities on target cells. Thin tubular membranous channels termed tunneling nanotubes (TNTs) may also connect EC with distant cells. EVs, in particular exosomes, and TNTs may contain and transfer different biomolecules (e.g., signaling mediators, proteins, lipids, and microRNAs) or pathogens and have emerged as a major triggers of horizontal intercellular transfer of information.     

Neurotransmitter Release Site Replenishment and Presynaptic Plasticity

An action potential (AP) triggers neurotransmitter release from synaptic vesicles (SVs) docking to a specialized release site of presynaptic plasma membrane, the active zone (AZ). The AP simultaneously controls the release site replenishment with SV for sustainable synaptic transmission in response to incoming neuronal signals. Although many studies have suggested that the replenishment time is relatively slow, recent studies exploring high speed resolution have revealed SV dynamics with milliseconds timescale after an AP. Accurate regulation is conferred by proteins sensing Ca²⁺ entering through voltage-gated Ca²⁺ channels opened by an AP. This review summarizes how millisecond Ca²⁺ dynamics activate multiple protein cascades for control of the release site replenishment with release-ready SVs that underlie presynaptic short-term plasticity.     

Extracellular Vesicles: Interplay with the Extracellular Matrix and Modulated Cell Responses

The discovery that cells secrete extracellular vesicles (EVs), which carry a variety of regulatory proteins, nucleic acids, and lipids, has shed light on the sophisticated manner by which cells can communicate and accordingly function. The bioactivity of EVs is not only defined by their internal content, but also through their surface associated molecules, and the linked downstream signaling effects they elicit in target cells. The extracellular matrix (ECM) contains signaling and structural molecules that are central to tissue maintenance and repair. Recently, a subset of EVs residing within the extracellular matrix has been identified. Although some roles have been proposed for matrix-bound vesicles, their role as signaling molecules within the ECM is yet to be explored. Given the close association of EVs and the ECM, it is not surprising that EVs partly mediate repair and regeneration by modulating matrix deposition and degradation through their cellular targets. This review addresses unique EV features that allow them to interact with and navigate through the ECM, describes how their release and content is influenced by the ECM, and emphasizes the emerging role of stem-cell derived EVs in tissue repair and regeneration through their matrix-modulating properties.     

Efficacious Doxorubicin Delivery Using Glutathione-Responsive Hollow Non-phospholipid Vesicles Bearing Lipoyl Cholesterols

In this study, we developed redox-sensitive vesicles using synthesised lipoyl cholesterol derivatives, a non-ionic surfactant and an optimum level of free cholesterol. Interestingly, concentration-dependent self-assembly was observed by scanning electron microscopy, wherein vesicles manifested as hollow spherical (at 0.15 mm ) and triangular (0.50 mm ). The redoxresponsive characteristics of the vesicles was probed in the presence of dithiothreitol; they underwent a clear increase in size as observed by dynamic light scattering measurements. These vesicles could easily encapsulate an anticancer drug, doxorubicin, and were observed to be stable in the presence of serum. They showed substantial release of the drug in response to biologically relevant stimulus, that is, glutathione. A toxicity assessment on HeLa and HepG2 cancer cells demonstrated activities of the drug-loaded vesicles comparable to that of free drug, whereas significantly enhanced toxicity and apoptotic induction were observed against drug-resistant HeLa cells, which was determined by studying the cellular internalisation of doxorubicin.     

Rab27 GTPases Distribute Extracellular Nanomaps for Invasive Growth and Metastasis: Implications for Prognosis and Treatment

The Rab27 family of small GTPases regulates exocytosis of distinct vesicle types including multivesicular endosomes, which results in the release of exosomes. Exosomes are nanometer-sized membrane vesicles that enclose soluble factors such as proteins and nucleic acids within a lipid bilayer and can travel toward distant tissues to influence multiple aspects of cell behavior. In our view that tumors are endocrine organs producing exosomes, Rab27 GTPases and their effector proteins are critical determinants for invasive growth and metastasis. Rab27 proteins and their effectors may serve as prognostic biomarkers or as targets for patient-tailored therapy.     

Biodegradable vesicular nanocarriers based on poly(?-caprolactone)-block-poly(ethyl ethylene phosphate) for drug delivery

Biodegradable polymer vesicle for drug delivery is reported. Poly(?-caprolactone)-block-poly(ethyl ethylene phosphate) with well-defined structure (PCL₁₅₀-b-PEEP₃₀) has been prepared by ring-opening polymerization. It forms vesicles in aqueous solution using the thin-film hydration method and further exclusion of the as-formed vesicles results in vesicles at nano-size, demonstrated by confocal laser scanning microscope (CLSM) and transmission electron microscopy observations. Doxorubicin (DOX) has been loaded into the vesicles with a loading content of 4.38% using an acid gradient method. The release of DOX from the vesicles is accelerated in the presence of an enzyme phosphodiesterase I that is known to catalyze the degradation of polyphosphoester, achieving 83.8% release of total loaded DOX in 140 h. The DOX-loaded vesicles can be successfully internalized by A549 cells, and it results in enhanced inhibition to A549 cell proliferation, likely owning to the sustained intracellular release of DOX as observed by CLSM. With these properties, the vesicles based on the block copolymer of PCL and PEEP are attractive as drug carriers for pharmaceutical application.     

Bioelectric Responses of Conjunctival Goblet Cells to Dry Eye: Impact of Ion Channels on Exocytotic Function and Viability

How ion channels impact the response of the ocular surface to dry eye is only beginning to be explored. Here, we review recent progress and provide new experimental data clarifying the exocytosis-altering actions of ion channels in conjunctival goblet cells whose release of tear-stabilizing mucin is a key adaptive response to the pre-ocular hyperosmolarity that characterizes dry eye. Patch-clamp recordings of goblet cells located in freshly excised rat conjunctiva reveal that these mucin-releasing cells respond to sustained hyperosmolarity by sequentially activating their ATP-sensitive potassium (K_ATP), nonspecific cation (NSC), voltage-gated calcium (VGCC), and P2X₇ channels; each of which modulates exocytosis. Based on these and other new findings, we now identify four stages in the bioelectric response of conjunctival goblet cells to extracellular hyperosmolarity. To better characterize these stages, we report that high-resolution membrane capacitance (Cm) measurements of the exocytotic activity of single goblet cells demonstrate that the replenishment of mucin-filled granules after neural-evoked exocytosis is a multi-hour process, which VGCCs markedly accelerate. Yet, we also discovered that VGCC activation is high-risk since hyperosmotic-induced goblet cell death is boosted. With dry eye treatments being far from optimal, elucidating the physiologic and pathobiologic impact of the K_ATP/NSC/VGCC/P2X₇ pathway provides a new opportunity to identify novel therapeutic strategies.     

Hofmeister Series: From Aqueous Solution of Biomolecules to Single Cells and Nanovesicles

Hofmeister effects play a critical role in numerous physicochemical and biological phenomena, including the solubility and/or accumulation of proteins, the activities of enzymes, ion transport in biochannels, the structure of lipid bilayers, and the dynamics of vesicle opening and exocytosis. This minireview focuses on how ionic specificity affects the physicochemical properties of biomolecules to regulate cellular exocytosis, vesicular content, and nanovesicle opening. We summarize recent progress in further understanding Hofmeister effects on biomacromolecules and their applications in biological systems. These important steps have increased our understanding of the Hofmeister effects on cellular exocytosis, vesicular content, and nanovesicle opening. Increasing evidence is firmly establishing that the ions along the Hofmeister series play an important role in living organisms that has often been ignored.     

Alpha-Synuclein as a Prominent Actor in the Inflammatory Synaptopathy of Parkinson’s Disease

Parkinson’s disease (PD) is considered the most common disorder of synucleinopathy, which is characterised by intracellular inclusions of aggregated and misfolded α-synuclein (α-syn) protein in various brain regions, and the loss of dopaminergic neurons. During the early prodromal phase of PD, synaptic alterations happen before cell death, which is linked to the synaptic accumulation of toxic α-syn specifically in the presynaptic terminals, affecting neurotransmitter release. The oligomers and protofibrils of α-syn are the most toxic species, and their overexpression impairs the distribution and activation of synaptic proteins, such as the SNARE complex, preventing neurotransmitter exocytosis and neuronal synaptic communication. In the last few years, the role of the immune system in PD has been increasingly considered. Microglial and astrocyte activation, the gene expression of proinflammatory factors, and the infiltration of immune cells from the periphery to the central nervous system (CNS) represent the main features of the inflammatory response. One of the actors of these processes is α-syn accumulation. In light of this, here, we provide a systematic review of PD-related α-syn and inflammation inter-players.     

Proteomic Profiling of Extracellular Vesicles Released by Leptin-Treated Breast Cancer Cells: A Potential Role in Cancer Metabolism

Tumor extracellular vesicles (EVs), as endocytic vesicles able to transport nucleic acids, proteins, and metabolites in recipient cells, have been recognized fundamental mediators of cell-to-cell communication in breast cancer. The biogenesis and release of EVs are highly regulated processes and both the quantity of EVs and their molecular cargo might reflect the metabolic state of the producing cells. We recently demonstrated that the adipokine leptin, whose circulating levels correlate with adipose tissue expansion, is an inducer of EV release from breast cancer cells. Here, we show a specific proteomic signature of EVs released by MCF-7 breast cancer cells grown in the presence of leptin (Lep-EVs), in attempt to find additional molecular effectors linking obesity to breast cancer biology. An analysis of the proteomic profile of Lep-EVs by LC-MS/MS revealed a significant enrichment in biological processes, molecular functions, and cellular components mainly related to mitochondrial machineries and activity, compared to protein content of EVs from untreated breast cancer cells. Metabolic investigations, carried out to assess the autocrine effects of these vesicles on breast cancer cells, revealed that Lep-EVs were able to increase ATP levels in breast cancer cells. This result is associated with increased mitochondrial respiration evaluated by Seahorse analyzer, supporting the concept that Lep-EVs can modulate MCF-7 breast cancer cell oxidative metabolism. Moreover, taking into account the relevance of tumor immune cell crosstalk in the tumor microenvironment (TME), we analyzed the impact of these vesicles on macrophage polarization, the most abundant immune component in the breast TME. We found that tumor-derived Lep-EVs sustain the polarization of M0 macrophages, derived from the human THP-1 monocytic cells, into M2-like tumor-associated macrophages, in terms of metabolic features, phagocytic activity, and increased expression of CD206-positive population. Overall, our results indicate that leptin by inducing the release of EV-enriched in mitochondrial proteins may control the metabolism of MCF-7 breast cancer cells as well as that of macrophages. Characterization of tumor-derived EV protein cargo in an obesity-associated milieu, such as in the presence of elevated leptin levels, might allow identifying unique features and specific metabolic mechanisms useful to develop novel therapeutic approaches for treatment of breast cancer, especially in obese patients.     

Regulation of exocytosis in chromaffin cells by trans-insertion of lysophosphatidylcholine and arachidonic acid into the outer leaflet of the cell membrane

Vesicular exocytosis is an important complex process in the communication between cells in organisms. It controls the release of chemical and biochemical messengers stored in an emitting cell. In this report, exocytosis is studied amperometrically (at carbon fiber ultramicroelectrodes) at adrenal chromaffin cells, which release catecholamines after appropriate stimulation, while testing the effects due to trans-insertion of two exogenous compounds (lysophosphatidylcholine (LPC) and arachidonic acid (AA)) on the kinetics of exocytotic events. Amperometric analyses showed that, under the present conditions (short incubation times and micromolar LPC or AA solutions), LPC favors catecholamine release (rate, event frequency, charge released) while AA disfavors the exocytotic processes. The observed kinetic features are rationalized quantitatively by considering a stalk model, for the fusion pore formation, and the physical constraints applied to the cell membrane by the presence of small fractions of LPC and AA diluted in its external leaflet (trans-insertion). We also observed that the detected amount of neurotransmitters in the presence of LPC was larger than under control conditions, while the opposite trend is observed with AA.     

The Effect of Isosaponarin Derived from Wasabi Leaves on Glutamate Release in Rat Synaptosomes and Its Underlying Mechanism

Excessive glutamate release is known to be involved in the pathogenesis of neurological diseases, and suppression of glutamate release from nerve terminals is considered to be a treatment strategy. In this study, we investigated whether isosaponarin, a flavone glycoside isolated from wasabi leaves, could affect glutamate release in rat cerebral cortex nerve terminals (synaptosomes). The release of glutamate was evoked by the K⁺ channel blocker 4-aminopyridine (4-AP) and measured by an online enzyme-coupled fluorimetric assay. Isosaponarin produced a concentration-dependent inhibition of 4-AP-evoked glutamate release with a half-maximum inhibition of release value of 22 μM. The inhibition caused by isosaponarin was prevented by eliminating extracellular Ca²⁺ or by using bafilomycin A1, an inhibitor of synaptic vesicle exocytosis. Isosaponarin decreased intrasynaptosomal rises in Ca²⁺ levels that were induced by 4-AP, without affecting the synaptosomal membrane potential. The isosaponarin-induced inhibition of glutamate release was significantly prevented in synaptosomes that were pretreated with a combination of the calcium channel blockers ω-conotoxin GVIA (N-type) and ω-agatoxin IVA (P/Q-types). The protein kinase C (PKC) pan-inhibitor GF109203X and the Ca²⁺-dependent PKC inhibitor Go6976 abolished the inhibition of glutamate release by isosaponarin, while the Ca²⁺-independent PKC inhibitor rottlerin did not show any effect. The results from immunoblotting assays also showed that isosaponarin lowered PKC, PKCα, synaptosomal-associated protein of 25 kDa (SNAP-25), and myristoylated alanine-rich C-kinase substrate (MARCKS) phosphorylation induced by 4-AP. In addition, FM1-43-labeled synaptic vesicles in synaptosomes showed that treatment with isosaponarin resulted in an attenuation of the 4-AP-induced decrease in fluorescence intensity that is consistent with glutamate release. Transmission electron microscopy of synaptosomes also provided evidence that isosaponarin altered the number of synaptic vesicles. These results indicate that isosaponarin suppresses the Ca²⁺-dependent PKC/SNAP-25 and MARCKS pathways in synaptosomes, causing a decrease in the number of available synaptic vesicles, which inhibits vesicular glutamate release from synaptosomes.     

Partial protection against Botulinum B neurotoxin-induced blocking of exocytosis by a potent inhibitor of its metallopeptidase activity

Clostridium botulinum neurotoxins (BoNTs) cause botulism, which is characterized by a flaccid paralysis, through inhibition of acetylcholine release by peripheral cholinergic nerve terminals. This is due to the zinc metallopeptidase activity of the neurotoxin, cleaving one component (synaptobrevin for BoNT/B) of the exocytosis machinery. Yet, there are no specific agents able to control the peptidase-related effects of BoNT/B. We recently developed the first compounds to inhibit this enzymatic activity in the nanomolar range. Here we report that two of our best inhibitors prevent the BoNT/B-induced cleavage of native synaptobrevin on synaptic vesicles, and partially inhibit the suppression of [3H]noradrenaline release from synaptosomes that is caused by BoNT/B. These results were obtained at micromolar concentrations, consistent with the measured inhibitory potency of these inhibitors on the native toxin. These compounds provide a new way to possibly prevent and/or to control the neurotoxin effects of botulinum.     

Freeze-drying of liposomal nanohybrid cerasomes with cryoprotectants: In vitro and in vivo evaluation

Liposomal nanohybrid cerasomes as a new type of drug carrier has been widely applied in the controlled release of drugs. However, such vesicles containing polysiloxane networks on the surface were liable to aggregate during the process of freeze-drying for routine storage. To solve this problem, several previously reported cryoprotectants including sucrose, mannitol, sodium alginate, and glucose were chosen for protecting the 10-hydroxycamptothecin-loaded liposomal nanohybrid cerasomes (The drug encapsulation efficiency was of 67.31?±?7.42% and the drug loading content was of 6.73?±?0.74%.) in freeze-drying process. It was found that liposomal nanohybrid cerasomes treated with mannitol can be well dispersed, and the results of particle size and zeta potential showed that the drug-loaded vesicles were well protected with mannitol. In vitro drug release and in vivo pharmacokinetic study showed that the optimal addition amount of mannitol was twice the mass of the drug-loaded vesicles, whereby the cumulative release profile and the pharmacokinetic parameters were similar in the freeze-dried and fresh vesicles.     

Conventional and Unconventional Mechanisms by which Exocytosis Proteins Oversee β-cell Function and Protection

Type 2 diabetes (T2D) is one of the prominent causes of morbidity and mortality in the United States and beyond, reaching global pandemic proportions. One hallmark of T2D is dysfunctional glucose-stimulated insulin secretion from the pancreatic β-cell. Insulin is secreted via the recruitment of insulin secretory granules to the plasma membrane, where the soluble N-ethylmaleimide-sensitive factor attachment protein receptors (SNAREs) and SNARE regulators work together to dock the secretory granules and release insulin into the circulation. SNARE proteins and their regulators include the Syntaxins, SNAPs, Sec1/Munc18, VAMPs, and double C2-domain proteins. Recent studies using genomics, proteomics, and biochemical approaches have linked deficiencies of exocytosis proteins with the onset and progression of T2D. Promising results are also emerging wherein restoration or enhancement of certain exocytosis proteins to β-cells improves whole-body glucose homeostasis, enhances β-cell function, and surprisingly, protection of β-cell mass. Intriguingly, overexpression and knockout studies have revealed novel functions of certain exocytosis proteins, like Syntaxin 4, suggesting that exocytosis proteins can impact a variety of pathways, including inflammatory signaling and aging. In this review, we present the conventional and unconventional functions of β-cell exocytosis proteins in normal physiology and T2D and describe how these insights might improve clinical care for T2D.     

N,N-双烷基壳聚糖结构对其自组装泡囊性质的影响

合成了不同主链相对分子质量的N,N-双辛烷基壳聚糖,N,N-双癸烷基壳聚糖及N,N-双十二烷基壳聚糖,并制备了相应的自组装泡囊,比较研究了侧链长度和主链相对分子质量对载药泡囊药物释放行为的影响。结果表明这类N,N-双长链烷基壳聚糖可形成粒径为100～150 nm的泡囊。原子力显微镜表明,侧链长度对N,N-双烷基壳聚糖泡囊的曲率有较明显影响,主链相对分子质量对泡囊的粒径影响较大。药物释放行为表明,侧链长度或主链相对分子质量的增大会导致药物释放速率的降低,达到平衡时的药物释放率也较低,而其相应自组装泡囊的载药量和包埋率却有所提高。