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.     

Key role of clathrin-mediated endocytosis in synaptic vesicle recycling

Using novel fluorescent markers, virus-induced modulation of amphiphysin 1 expression, and electron microscopy, we demonstrated that clathrin-mediated endocytosis is the main mechanism of synaptic vesicle retrieval; a hypothesis on the role of a fast “kiss-and-run” mechanism has not been supported. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 388–389, July–October, 2007.     

Stonin 2: an adaptor-like protein that interacts with components of the endocytic machinery

Endocytosis of cell surface proteins is mediated by a complex molecular machinery that assembles on the inner surface of the plasma membrane. Here, we report the identification of two ubiquitously expressed human proteins, stonin 1 and stonin 2, related to components of the endocytic machinery. The human stonins are homologous to the Drosophila melanogaster stoned B protein and exhibit a modular structure consisting of an NH(2)-terminal proline-rich domain, a central region of homology specific to the stonins, and a COOH-terminal region homologous to the mu subunits of adaptor protein (AP) complexes. Stonin 2, but not stonin 1, interacts with the endocytic machinery proteins Eps15, Eps15R, and intersectin 1. These interactions occur via two NPF motifs in the proline-rich domain of stonin 2 and Eps15 homology domains of Eps15, Eps15R, and intersectin 1. Stonin 2 also interacts indirectly with the adaptor protein complex, AP-2. In addition, stonin 2 binds to the C2B domains of synaptotagmins I and II. Overexpression of GFP-stonin 2 interferes with recruitment of AP-2 to the plasma membrane and impairs internalization of the transferrin, epidermal growth factor, and low density lipoprotein receptors. These observations suggest that stonin 2 is a novel component of the general endocytic machinery.     

Disturbance of synaptic vesicle recycling resulting from deletion of a mammalian actin-binding protein, mAbp1

Physiological and ultrastructural studies of synapses between hippocampal neurons of animals with knock-out of a mammalian actin-binding protein, mAbp1, demonstrated that recycling of synaptic vesicles undergoes, in this case, significant modifications. Thus, mAbp1 is rather important from this aspect, which can be related to the noticeable role of actin in clathrin-mediated endocytosis of synaptic vesicles. Neirofiziologiya/Neurophysiology, Vol. 39, Nos. 4/5, pp. 390–391, July–October, 2007.     

Amphiphysin is a component of clathrin coats formed during synaptic vesicle recycling at the lamprey giant synapse

Amphiphysin is a protein enriched at mammalian synapses thought to function as a clathrin accessory factor in synaptic vesicle endocytosis. Here we examine the involvement of amphiphysin in synaptic vesicle recycling at the giant synapse in the lamprey. We show that amphiphysin resides in the synaptic vesicle cluster at rest and relocates to sites of endocytosis during synaptic activity. It accumulates at coated pits where its SH3 domain, but not its central clathrin/AP-2-binding (CLAP) region, is accessible for antibody binding. Microinjection of antibodies specifically directed against the CLAP region inhibited recycling of synaptic vesicles and caused accumulation of clathrin-coated intermediates with distorted morphology, including flat patches of coated presynaptic membrane. Our data provide evidence for an activity-dependent redistribution of amphiphysin in intact nerve terminals and show that amphiphysin is a component of presynaptic clathrin-coated intermediates formed during synaptic vesicle recycling.     

Synaptic vesicle recycling: steps and principles

Synaptic vesicle recycling is one of the best‐studied cellular pathways. Many of the proteins involved are known, and their interactions are becoming increasingly clear. However, as for many other pathways, it is still difficult to understand synaptic vesicle recycling as a whole. While it is generally possible to point out how synaptic reactions take place, it is not always easy to understand what triggers or controls them. Also, it is often difficult to understand how the availability of the reaction partners is controlled: how the reaction partners manage to find each other in the right place, at the right time. I present here an overview of synaptic vesicle recycling, discussing the mechanisms that trigger different reactions, and those that ensure the availability of reaction partners. A central argument is that synaptic vesicles bind soluble cofactor proteins, with low affinity, and thus control their availability in the synapse, forming a buffer for cofactor proteins. The availability of cofactor proteins, in turn, regulates the different synaptic reactions. Similar mechanisms, in which one of the reaction partners buffers another, may apply to many other processes, from the biogenesis to the degradation of the synaptic vesicle.     

Mutations in dynamin-related protein result in gross changes in mitochondrial morphology and affect synaptic vesicle recycling at the Drosophila neuromuscular junction

Mitochondria are the primary source of ATP needed for the steps of the synaptic vesicle cycle. Dynamin-related protein (DRP) is involved in the fission of mitochondria and peroxisomes. To assess the role of mitochondria in synaptic function, we characterized a Drosophila DRP mutant combination that shows an acute temperature-sensitive paralysis. Sequencing of the mutant reveals a single amino acid change in the guanosine triphosphate hydrolysing domain (GTPase domain) of DRP. The synaptic mitochondria in these mutants are remarkably elongated, suggesting a role for DRP in mitochondrial fission in Drosophila. There is a loss of neuronal transmission at restrictive temperatures in electroretinogram (ERG) recordings. Like stress-sensitive B (sesB), a mitochondrial adenosine triphosphate (ATP) translocase mutant we studied earlier for its effects on synaptic vesicle recycling, an allele-specific reduction in the temperature of paralysis of Drosophila synaptic vesicle recycling mutant shibire was seen in the DRP mutant background. These data, in addition to depletion of vesicles observed in electron microscopic sections of photoreceptor synapses at restrictive temperatures, suggest a block in synaptic vesicle recycling due to reduced mitochondrial function.     

Studies of synaptic vesicle endocytosis in the nematode C. elegans

After synaptic vesicle exocytosis, synaptic vesicle proteins must be retrieved from the plasma membrane, sorted away from other membrane proteins, and reconstituted into a functional synaptic vesicle. The nematode Caenorhabditis elegans is an organism well suited for a genetic analysis of this process. In particular, three types of genetic studies have contributed to our understanding of synaptic vesicle endocytosis. First, screens for mutants defective in synaptic vesicle recycling have identified new proteins that function specifically in neurons. Second, RNA interference has been used to quickly confirm the roles of known proteins in endocytosis. Third, gene targeting techniques have elucidated the roles of genes thought to play modulatory or subtle roles in synaptic vesicle recycling. We describe a molecular model for synaptic vesicle recycling and discuss how protein disruption experiments in C. elegans have contributed to this model.     

Endosomal phosphatidylinositol 3‐phosphate controls synaptic vesicle cycling and neurotransmission

Neural circuit function requires mechanisms for controlling neurotransmitter release and the activity of neuronal networks, including modulation by synaptic contacts, synaptic plasticity, and homeostatic scaling. However, how neurons intrinsically monitor and feedback control presynaptic neurotransmitter release and synaptic vesicle (SV) recycling to restrict neuronal network activity remains poorly understood at the molecular level. Here, we investigated the reciprocal interplay between neuronal endosomes, organelles of central importance for the function of synapses, and synaptic activity. We show that elevated neuronal activity represses the synthesis of endosomal lipid phosphatidylinositol 3‐phosphate [PI(3)P] by the lipid kinase VPS34. Neuronal activity in turn is regulated by endosomal PI(3)P, the depletion of which reduces neurotransmission as a consequence of perturbed SV endocytosis. We find that this mechanism involves Calpain 2‐mediated hyperactivation of Cdk5 downstream of receptor‐ and activity‐dependent calcium influx. Our results unravel an unexpected function for PI(3)P‐containing neuronal endosomes in the control of presynaptic vesicle cycling and neurotransmission, which may explain the involvement of the PI(3)P‐producing VPS34 kinase in neurological disease and neurodegeneration.     

Drosophila homologue of Eps15 is essential for synaptic vesicle recycling

The mammalian protein Eps15 is phosphorylated by EGF receptor tyrosine kinase and has been shown to interact with several components of the endocytic machinery. We have identified a hypomorphic Eps15 mutant in Drosophila which shows reversible paralysis and an altered physiology at restrictive temperatures. In addition, the temperature-sensitive paralytic defect of shibire mutant is enhanced by this mutant. Eps15 is enriched in the larval neuromuscular junction in endocytic 'hot spots' in a pattern similar to Dynamin. Eps15 mutants show a decrease in the alpha-Adaptin levels at the larval neuromuscular junction synapse. Genetic and biochemical studies of interactions with components of the endocytic machinery suggest that Eps15 has an important role in synaptic vesicle recycling and regulates recruitment of alpha-Adaptin.     

Alpha-synuclein and polyunsaturated fatty acids promote clathrin-mediated endocytosis and synaptic vesicle recycling

α-Synuclein (αS) is an abundant neuronal cytoplasmic protein implicated in Parkinson's disease (PD), but its physiological function remains unknown. Consistent with its having structural motifs shared with class A1 apolipoproteins, αS can reversibly associate with membranes and help regulate membrane fatty acid composition. We previously observed that variations in αS expression level in dopaminergic cultured cells or brains are associated with changes in polyunsaturated fatty acid (PUFA) levels and altered membrane fluidity. We now report that αS acts with PUFAs to enhance the internalization of the membrane-binding dye, FM 1-43. Specifically, αS expression coupled with exposure to physiological levels of certain PUFAs enhanced clathrin-mediated endocytosis in neuronal and non-neuronal cultured cells. Moreover, αS expression and PUFA-enhanced basal and -evoked synaptic vesicle (SV) endocytosis in primary hippocampal cultures of wild type (wt) and genetically depleted αS mouse brains. We suggest that αS and PUFAs normally function in endocytic mechanisms and are specifically involved in SV recycling upon neuronal stimulation.     

神经肌肉接头研究世纪回顾

神经肌肉接头作为突触解剖、生理和发育的模型已被研究了一个多世纪。成像技术提供了诸多关于神经肌肉接头的信息,其中一些技术是专为观察该突触的结构和功能而发展起来的。本文回顾了神经肌肉接头研究中几个重要方面的发展史,包括其结构、N型乙酰胆碱受体的分布、突触小泡释放,以及神经肌肉接头的发育。     

Developmental change in the calcium sensor for synaptic vesicle endocytosis in central nerve terminals

Synaptic vesicle endocytosis is stimulated by calcium influx in mature central nerve terminals via activation of the calcium-dependent protein phosphatase, calcineurin. However, in different neuronal preparations calcineurin activity is either inhibitory, stimulatory or irrelevant to the process. We addressed this inconsistency by investigating the requirement for calcineurin activity in synaptic vesicle endocytosis during development, using vesicle recycling assays in isolated nerve terminals. We show that endocytosis occurs independently of calcineurin activity in immature nerve terminals, and that a calcineurin requirement develops 2-4 weeks after birth. Calcineurin-independent endocytosis is not due to the absence of calcineurin activity, since calcineurin is present in immature nerve terminals and its substrate, dynamin I, is dephosphorylated on depolarization. Calcineurin-independent endocytosis is calcium-dependent, since substitution of the divalent cation, barium, inhibits the process. Finally, we demonstrated that in primary neuronal cultures derived from neonatal rats, endocytosis that was initially calcineurin-independent developed a calcineurin requirement on maturation in culture. Our data account for the apparent inconsistencies regarding the role of calcineurin in synaptic vesicle endocytosis, and we propose that an unidentified calcium sensor exists to couple calcium influx to endocytosis in immature nerve terminals.     

Vesicle uncoating regulated by SH3-SH3 domain-mediated complex formation between endophilin and intersectin at synapses

Neurotransmission involves the exo-endocytic cycling of synaptic vesicle (SV) membranes. Endocytic membrane retrieval and clathrin-mediated SV reformation require curvature-sensing and membrane-bending BAR domain proteins such as endophilin A. While their ability to sense and stabilize curved membranes facilitates membrane recruitment of BAR domain proteins, the precise mechanisms by which they are targeted to specific sites of SV recycling has remained unclear. Here, we demonstrate that the multi-domain scaffold intersectin 1 directly associates with endophilin A to facilitate vesicle uncoating at synapses. Knockout mice deficient in intersectin 1 accumulate clathrin-coated vesicles at synapses, a phenotype akin to loss of endophilin function. Intersectin 1/endophilin A1 complex formation is mediated by direct binding of the SH3B domain of intersectin to a non-canonical site on the SH3 domain of endophilin A1. Consistent with this, intersectin-binding defective mutant endophilin A1 fails to rescue clathrin accumulation at neuronal synapses derived from endophilin A1-3 triple knockout (TKO) mice. Our data support a model in which intersectin aids endophilin A recruitment to sites of clathrin-mediated SV recycling, thereby facilitating vesicle uncoating.     

Clathrin‐Mediated Endocytosis at the Synaptic Terminal: Bridging the Gap Between Physiology and Molecules

It has long been known that the maintenance of fast communication between neurons requires that presynaptic terminals recycle the small vesicles from which neurotransmitter is released. But the mechanisms that retrieve vesicles from the cell surface are still not understood. Although we have a wealth of information about the molecular details of endocytosis in non‐neuronal cells, it is clear that endocytosis at the synapse is faster and regulated in distinct ways. A satisfying understanding of these processes will require molecular events to be manipulated while observing endocytosis in living synapses. Here, we review recent work that seeks to bridge the gap between physiology and molecules to unravel the endocytic machinery operating at the synaptic terminal.     

Mutations in synaptojanin disrupt synaptic vesicle recycling

Synaptojanin is a polyphosphoinositide phosphatase that is found at synapses and binds to proteins implicated in endocytosis. For these reasons, it has been proposed that synaptojanin is involved in the recycling of synaptic vesicles. Here, we demonstrate that the unc-26 gene encodes the Caenorhabditis elegans ortholog of synaptojanin. unc-26 mutants exhibit defects in vesicle trafficking in several tissues, but most defects are found at synaptic termini. Specifically, we observed defects in the budding of synaptic vesicles from the plasma membrane, in the uncoating of vesicles after fission, in the recovery of vesicles from endosomes, and in the tethering of vesicles to the cytoskeleton. Thus, these results confirm studies of the mouse synaptojanin 1 mutants, which exhibit defects in the uncoating of synaptic vesicles (Cremona, O., G. Di Paolo, M.R. Wenk, A. Luthi, W.T. Kim, K. Takei, L. Daniell, Y. Nemoto, S.B. Shears, R.A. Flavell, D.A. McCormick, and P. De Camilli. 1999. Cell. 99:179-188), and further demonstrate that synaptojanin facilitates multiple steps of synaptic vesicle recycling.     

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.     

Unique biochemical and behavioral alterations in drosophilashibirets1 mutants imply a conformational state affecting dynamin subcellular distribution and synaptic vesicle cycling

ABSTRACT Dynamin is a GTPase protein that is essential for clathrin‐mediated endocytosis of synaptic vesicle membranes. The Drosophila dynamin mutation shi^ts1 changes a single residue (G273D) at the boundary of the GTPase domain. In cell fractionation of homogenized fly heads without monovalent cations, all dynamin was in pellet fractions and was minimally susceptible to Triton‐X extraction. Addition of Na⁺ or K⁺ can extract dynamin to the cytosolic (supernatant) fraction. The shi^ts1 mutation reduced the sensitivity of dynamin to salt extraction compared with other temperature‐sensitive alleles or wild type. Sensitivity to salt extraction in shi^ts1 was enhanced by GTP and nonhydrolyzable GTP‐γS. The shi^ts1 mutation may therefore induce a conformational change, involving the GTP binding site, that affects dynamin aggregation. Temperature‐sensitive shibire mutations are known to arrest endocytosis at restrictive temperatures, with concomitant accumulation of presynaptic collared pits. Consistent with an effect upon dynamin aggregation, intact shi^ts1 flies recovered much more slowly from heat‐induced paralysis than did other temperature‐sensitive shibire mutants. Moreover, a genetic mutation that lowers GTP abundance (awd^msf15), which reduces the paralytic temperature threshold of other temperature‐sensitive shibire mutations that lie closer to consensus GTPase motifs, did not reduce the paralytic threshold of shi^ts1. Taken together, the results may link the GTPase domain to conformational shifts that influence aggregation in vitro and endocytosis in vivo, and provide an unexpected point of entry to link the biophysical properties of dynamin to physiological processes at synapses. © 2002 Wiley Periodicals, Inc. J Neurobiol 53: 319–329, 2002     

Bassoon controls synaptic vesicle release via regulation of presynaptic phosphorylation and cAMP

Neuronal presynaptic terminals contain hundreds of neurotransmitter‐filled synaptic vesicles (SVs). The morphologically uniform SVs differ in their release competence segregating into functional pools that differentially contribute to neurotransmission. The presynaptic scaffold bassoon is required for neurotransmission, but the underlying molecular mechanisms are unknown. We report that glutamatergic synapses lacking bassoon feature decreased SV release competence and increased resting pool of SVs as assessed by imaging of SV release in cultured neurons. CDK5/calcineurin and cAMP/PKA presynaptic signalling are dysregulated, resulting in an aberrant phosphorylation of their downstream effectors synapsin1 and SNAP25, well‐known regulators of SV release competence. An acute pharmacological restoration of physiological CDK5 and cAMP/PKA activity fully normalises the SV pools in neurons lacking bassoon. Finally, we demonstrate that CDK5‐dependent regulation of PDE4 activity interacts with cAMP/PKA signalling and thereby controls SV release competence. These data reveal that bassoon organises SV pools in glutamatergic synapses via regulation of presynaptic phosphorylation and cAMP homeostasis and indicate a role of CDK5/PDE4/cAMP axis in the control of neurotransmitter release.