Synaptic Vesicles In Presynaptic Terminals Research Articles

Exploring the Roles of Post-Translational Modifications in the Pathogenesis of Parkinson's Disease Using Synthetic and Semisynthetic Modified α-Synuclein.

Alpha-synuclein (α-syn), a small soluble protein containing 140 amino acids, is associated with the recycling pool of synaptic vesicles in presynaptic terminals. The misfolding and aggregation of α-syn is closely related to a group of neurodegenerative diseases, including Parkinson's disease (PD), which is one of the most common progressive neurodegenerative diseases. Varieties of the post-translational modifications (PTMs) of α-syn, including phosphorylation, ubiquitination, and glycosylation, have been detected in soluble and aggregated α-syn in vivo. These PTMs can have either positive or negative effects on α-syn aggregation and toxicity, which may play critical roles in PD pathogenesis. Herein, we review the advances in synthetic and semisynthetic chemistry to generate homogeneous α-syn variants with site-specific modifications. Using these modified α-syn, we gain insight into the consequences of PTMs on α-syn aggregation and other biophysical properties, which can help elucidate the role of PTMs in the pathogenesis of PD and develop potential therapies to PD.

Time course and temperature dependence of synaptic vesicle endocytosis.

In presynaptic nerve terminals, synaptic vesicles are recycled locally via an evolutionarily conserved process that ensures maintenance of neurotransmission as well as structural integrity of synapses. Temperature is a key environmental factor that impacts critical steps involved in fusion, endocytosis and transport in different vesicle trafficking pathways. In neurons, temperature changes have been shown to impact synaptic vesicle recycling and synaptic efficacy. But contrary to non-neuronal systems, the temperature dependence of the steps involved in fusion, endocytosis and recycling of synaptic vesicles in presynaptic terminals is not completely understood, and the existing data remain highly debated. In this Review, we discuss the implications of biophysical, biochemical and functional findings on temperature dependence of membrane retrieval in multiple systems. We propose that systematic investigation of the temperature dependence of the presynaptic vesicle trafficking process can provide novel insight into poorly understood mechanisms that govern synaptic vesicle trafficking under diverse physiological conditions.

SNAP-25 phosphorylation at Ser187 regulates synaptic facilitation and short-term plasticity in an age-dependent manner

Neurotransmitter release is mediated by the SNARE complex, but the role of its phosphorylation has scarcely been elucidated. Although PKC activators are known to facilitate synaptic transmission, there has been a heated debate on whether PKC mediates facilitation of neurotransmitter release through phosphorylation. One of the SNARE proteins, SNAP-25, is phosphorylated at the residue serine-187 by PKC, but its physiological significance has been unclear. To examine these issues, we analyzed mutant mice lacking the phosphorylation of SNAP-25 serine-187 and found that they exhibited reduced release probability and enhanced presynaptic short-term plasticity, suggesting that not only the release process, but also the dynamics of synaptic vesicles was regulated by the phosphorylation. Furthermore, it has been known that the release probability changes with development, but the precise mechanism has been unclear, and we found that developmental changes in release probability of neurotransmitters were regulated by the phosphorylation. These results indicate that SNAP-25 phosphorylation developmentally facilitates neurotransmitter release but strongly inhibits presynaptic short-term plasticity via modification of the dynamics of synaptic vesicles in presynaptic terminals.

Functional characterization of alpha-synuclein protein with antimicrobial activity

Alpha-synuclein (α-Syn), a small (14 kDa) protein associated with Parkinson's disease, is abundant in human neural tissues. α-Syn plays an important role in maintaining a supply of synaptic vesicles in presynaptic terminals; however, the mechanism by which it performs this function are not well understood. In addition, there is a correlation between α-Syn over-expression and upregulation of an innate immune response. Given the growing body of literature surrounding antimicrobial peptides (AMPs) in the brain, and the similarities between α-Syn and a previously characterized AMP, Amyloid-β, we set out to investigate if α-Syn shares AMP-like properties. Here we demonstrate that α-Syn exhibits antibacterial activity against Escherichia coli and Staphylococcus aureus. In addition, we demonstrate a role for α-Syn in inhibiting various pathogenic fungal strains such as Aspergillus flavus, Aspergillus fumigatus and Rhizoctonia solani. We also analyzed localizations of recombinant α-Syn protein in E. coli and Candida albicans. These results suggest that in addition to α-Syn's role in neurotransmitter release, it appears to be a natural AMP.

Early Exposure to General Anesthesia Disrupts Spatial Organization of Presynaptic Vesicles in Nerve Terminals of the Developing Rat Subiculum.

Exposure to general anesthesia (GA) during critical stages of brain development induces widespread neuronal apoptosis and causes long-lasting behavioral deficits in numerous animal species. Although several studies have focused on the morphological fate of neurons dying acutely by GA-induced developmental neuroapoptosis, the effects of an early exposure to GA on the surviving synapses remain unclear. The aim of this study is to study whether exposure to GA disrupts the fine regulation of the dynamic spatial organization and trafficking of synaptic vesicles in presynaptic terminals. We exposed postnatal day 7 (PND7) rat pups to a clinically relevant anesthetic combination of midazolam, nitrous oxide, and isoflurane and performed a detailed ultrastructural analysis of the synaptic vesicle architecture at presynaptic terminals in the subiculum of rats at PND 12. In addition to a significant decrease in the density of presynaptic vesicles, we observed a reduction of docked vesicles, as well as a reduction of vesicles located within 100 nm from the active zone, in animals 5 days after an initial exposure to GA. We also found that the synaptic vesicles of animals exposed to GA are located more distally with respect to the plasma membrane than those of sham control animals and that the distance between presynaptic vesicles is increased in GA-exposed animals compared to sham controls. We report that exposure of immature rats to GA during critical stages of brain development causes significant disruption of the strategic topography of presynaptic vesicles within the nerve terminals of the subiculum.

Multiple Forms of Activity-Dependent Competition Refine Hippocampal Circuits In Vivo

Efficient memory formation relies on the establishment of functional hippocampal circuits. It has been proposed that synaptic connections are refined by neural activity to form functional brain circuitry. However, it is not known whether and how hippocampal connections are refined by neural activity in vivo. Using a mouse genetic system in which restricted populations of neurons in the hippocampal circuit are inactivated, we show that inactive axons are eliminated after they develop through a competition with active axons. Remarkably, in the dentate gyrus, which undergoes neurogenesis throughout life, axon refinement is achieved by a competition between mature and young neurons. These results demonstrate that activity-dependent competition plays multiple roles in the establishment of functional memory circuits in vivo.

An emerging view of presynaptic structure from electron microscopic studies

In response to calcium influx, some of the synaptic vesicles in presynaptic terminals fuse rapidly with the presynaptic membrane, allowing fast synaptic transmission. The regulated recycling of synaptic vesicles at the terminals is required for a sustained release of neurotransmitters. Localization of 'ready to be released' vesicles in close vicinities to voltage-gated calcium channels enables the rapid release of neurotransmitters. Thus, recycling vesicles must translocate from the sites of endocytosis to these release sites. However, the sub-cellular organization that supports this local vesicular traffic remains poorly understood. We will review the results of various electron microscopy studies, which have begun to unveil the structure of presynaptic terminals.

Brevity of the Ca2+ Microdomain and Active Zone Geometry Prevent Ca2+-Sensor Saturation for Neurotransmitter Release

The brief time course of the calcium (Ca2+) channel opening combined with the molecular-level colocalization of Ca2+ channels and synaptic vesicles in presynaptic terminals predict sub-millisecond calcium concentration ([Ca2+]) transients of > or = 100 microM in the immediate vicinity of the vesicle. This [Ca2+] is much higher than some of the recent estimates for the equilibrium dissociation constant of the Ca2+ sensor(s) that control neurotransmitter release, suggesting release should be close to saturation, yet it is well known that release is highly sensitive to changes in Ca2+ influx. We show that due to the brevity of the Ca2+ influx the binding kinetics of the Ca2+ sensor rather than its equilibrium affinity determine receptor occupancy. For physiologically relevant Ca2+ currents and forward Ca2+ binding rates, the effective affinity of the Ca2+ sensor can be several-fold lower than the equilibrium affinity. Using simple models, we show redundant copies of the binding sites increase effective affinity of the Ca2+ sensor for release. Our results predict that different levels of expression of Ca2+ binding sites could account for apparent differences in Ca2+ sensor affinities between synapses. Using Monte Carlo simulations of Ca2+ dynamics with nanometer resolution, we demonstrate that these kinetic constraints combined with vesicles acting as diffusion barriers can prevent saturation of the Ca2+-sensor(s) for neurotransmitter release. We further show the random positioning of the Ca2+-sensor molecules around the vesicle can result in the emergence of two distinct populations of the vesicles with low and high release probability. These considerations allow experimental evidence for the Ca2+ channel-vesicle colocalization to be reconciled with a high equilibrium affinity for the Ca2+ sensor of the release machinery.

Regulation of neurotransmitter release by synapsin III.

Synapsin III is the most recently identified member of the synapsin family, a group of synaptic vesicle proteins that play essential roles in neurotransmitter release and neurite outgrowth. Here, through the generation and analysis of synapsin III knock-out mice, we demonstrate that synapsin III regulates neurotransmitter release in a manner that is distinct from that of synapsin I or synapsin II. In mice lacking synapsin III, the size of the recycling pool of synaptic vesicles was increased, and synaptic depression was reduced. The number of vesicles that fuse per action potential was similar between synapsin III knock-out and wild-type mice, and there was no change in the quantal content of EPSCs; however, IPSCs were greatly reduced in synapsin III-deficient neurons. The density and distribution of synaptic vesicles in presynaptic terminals did not appear to be different in synapsin III knock-out mice in comparison to wild-type littermates. In addition to the changes in neurotransmitter release, we observed a specific delay in axon outgrowth in cultured hippocampal neurons from synapsin III knock-out mice. Our data indicate that synapsin III plays unique roles both in early axon outgrowth and in the regulation of synaptic vesicle trafficking.

Regulation of Neurotransmitter Release by Synapsin III

Synapsin III is the most recently identified member of the synapsin family, a group of synaptic vesicle proteins that play essential roles in neurotransmitter release and neurite outgrowth. Here, through the generation and analysis of synapsin III knock-out mice, we demonstrate that synapsin III regulates neurotransmitter release in a manner that is distinct from that of synapsin I or synapsin II. In mice lacking synapsin III, the size of the recycling pool of synaptic vesicles was increased, and synaptic depression was reduced. The number of vesicles that fuse per action potential was similar between synapsin III knock-out and wild-type mice, and there was no change in the quantal content of EPSCs; however, IPSCs were greatly reduced in synapsin III-deficient neurons. The density and distribution of synaptic vesicles in presynaptic terminals did not appear to be different in synapsin III knock-out mice in comparison to wild-type littermates. In addition to the changes in neurotransmitter release, we observed a specific delay in axon outgrowth in cultured hippocampal neurons from synapsin III knock-out mice. Our data indicate that synapsin III plays unique roles both in early axon outgrowth and in the regulation of synaptic vesicle trafficking.

Spatial distribution of synaptic vesicles in presynaptic terminals: Computer simulation

A procedure for computer simulation is proposed, which allows one to quantitatively characterize the spatial distribution of synaptic vesicles in presynaptic terminals (PST) using ultrathin sections of such terminals. The procedure includes three stages: simulation, topographical analysis, and comparison. At the first stage, the spatial distribution of vesicles within a PST and the process of random sectioning of it are simulated using the corresponding mathematical model. At the second stage, the topographical distribution of vesicle profiles within the plane of PST section is estimated; three respective approaches have been used: (i) nearest neighbor distance distribution; (ii) minimal spanning tree; and (iii) Voronoi paving. At the third stage, the simulated parameters are compared with the parameters of native terminal sections; when the coincidence of these two parameter groups is satisfactory, we believe that the simulated spatial distribution agrees with the real distribution. The software for the procedure is written in C++ programing langage. The results of a pilot study on ultrathin sections of cultured rat hippocampal neurons showed that the method offers broad possibilities for spatial interpretation and quantitative characterization of distributions of synaptic vesicles.

Neuropathology of ALS: an overview.

ALS is an age-associated neurodegenerative disease that primarily affects the motor neuron system. Despite having been studied for over 100 years, its etiology is still a mystery, and no specific diagnostic laboratory test has been developed. The diagnosis of ALS is therefore based on clinical features and/or neuropathologic findings. The neuropathologic findings on patients with classic ALS are quite distinct, with loss and degeneration of the large anterior horn cells of the spinal cord and lower cranial motor nuclei of the brainstem. The striated muscles display denervation atrophy. Upper motor neurons, such as the Betz cells in the motor cortex, are also affected. The several symposia and workshops on the cytopathology of ALS held within the past 3 years are a reflection of the many new and important developments taking place in this area. [1] Although loss of motor neurons has been known for many years, cytoplasmic alterations of the lower motor neurons have been studied in detail only in recent years by using immunohistochemical and electron microscopic techniques. An overview of some of the novel findings associated with lower motor neuron alterations is presented here. The synapse is the most important site of neuronal function and is unique to the neuron. Despite many years of extensive studies on ALS, data regarding synaptic alterations are limited to a relatively small number of ultrastructural investigations. [2] This paucity of information is due to the inherent difficulties that affect the proper evaluation of synapses in autopsy material. Synaptophysin, a 38-kDa glycoprotein, is a constituent of the membrane of synaptic vesicles in presynaptic terminals. The evaluation of synaptophysin expression in the lumbar spinal cord of ALS patients was apparently presented for the first time by Kawanami et al. [3] at the Annual Meeting of the Canadian Association of Neuropathologists in September 1992. This …

The action of black widow spider venom on cholinergic mechanisms in synaptosomes.

Abstract: Black widow spider venom (BWSV) promoted the massive release of labeled acetylcholine from synaptosomes and in addition, inhibited high‐affinity choline uptake into the preparation. Both activities occurred in the absence of [Ca2+]0. When Na+ in Krebs‐Ringer was replaced isotonically by sucrose, BWSV did not cause any release of [3H]ACh. On the other hand, BWSV was still effective if Na+ was replaced by lithium, glucosamine, or Tris. Tetrodotoxin (10−5 M) failed to prevent the ACh‐releasing action of the venom. The uptake of [3H]norepinephrine and [3H]tyrosine into the P2 fraction was significantly inhibited by BWSV pretreatment. However, the effect of the venom on the uptake of [3H]deoxyglucose was slight. In addition, the venom‐induced release of [3H]norepinephrine was much higher than that of [3H]deoxyglucose. The change in membrane potential of the preparation in duced by BWSV as examined using the voltage‐sensitive fluorescence probe, 3, 3′‐dipentyl‐2, 2′‐oxacarbocyanine. BWSV pretreatment markedly increased the synaptosomal fluorescence, indicating a depolarization of the preparation. This action of the venom was also observed in a Ca2+ ‐free or K+ ‐free medium, but could be blocked by pretreatment with antivenom. Pretreatment of the P2 fraction with concanavalin A completely blocked the action of BWSV. Also, the BWSV failed to promote the release of transmitter if the venom was prein‐cubated with a low concentration of purified gangliosides. Even after prolonged treatment with high concentrations of BWSV, an electron microscopic study showed no depletion of the synaptic vesicles in presynaptic terminals of the cortical P2 preparations or striatal slices. It is suggested that the venom expresses its activity by binding to glycoproteins and/or gangliosides on the synaptic membrane, opening a cation channel. The subsequent depolarization then inhibits uptake processes and promotes transmitter release that is independent of external calcium.