The role of coated vesicles in recycling of synaptic vesicle membrane

The uptake of extracellular tracers into synaptic nerve terminals has been a phenomenon of persistent interest. Uptake is into synaptic vesicles, hence vesicles spend part of their life in continuity with the plasma membrane, as expected if exocytosis underlies the quantal discharge of neurotransmitters. However, exactly how or when synaptic vesicles acquire extracellular tracers has not been unambiguously determined. Two schools of thought have developed, one holding that vesicles acquire tracers directly via a reversible exo/endocytotic sequence in which they consistently maintain their biochemical identity during their transient continuity with the plasma membrane, the other holding that synaptic vesicles acquire tracers indirectly, via the formation of clathrin-coated vesicles which are spatially and temporally separate from exocytosis and reverse a temporary loss of the vesicles' individual identity upon merger with the plasma membrane. Efforts to distinguish between these two alternatives have generated an interesting diversity of electron microscopic experiments, many of which are reviewed here. However, definitive determination of which view is correct may ultimately require direct visualization of synaptic vesicle turnover in living nerve terminals. To this end, we here review the results of visualizing endocytosis in tissue cultured cells, where light microscopy can provide sufficient resolution to reveal membrane dynamics in living cells. This has allowed visual discrimination of two different types of endocytosis, one clathrin-mediated (coated vesicle formation) and the other actin-mediated (macropinocytosis). Current work is also reviewed which aims at determining experimental methods for inhibiting each type of endocytosis selectively. Hypertonicity and severe cytoplasmic acidification turn out to inhibit coated vesicle formation, while cytochalasin D and mild cytoplasmic acidification selectively inhibit macropinocytosis. Applied to nerves, these various treatments affect synaptic vesicle turnover in a manner that supports the notion that synaptic vesicle membrane recycles via the "indirect" route of coated vesicle formation.     

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.     

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.     

The dystonia-associated protein torsinA modulates synaptic vesicle recycling

The loss of a glutamic acid residue in the AAA-ATPase (ATPases associated with diverse cellular activities) torsinA is responsible for most cases of early onset autosomal dominant primary dystonia. In this study, we found that snapin, which binds SNAP-25 (synaptosome-associated protein of 25,000 Da) and enhances the association of the SNARE complex with synaptotagmin, is an interacting partner for both wild type and mutant torsinA. Snapin co-localized with endogenous torsinA on dense core granules in PC12 cells and was recruited to perinuclear inclusions containing mutant DeltaE-torsinA in neuroblastoma SH-SY5Y cells. In view of these observations, synaptic vesicle recycling was analyzed using the lipophilic dye FM1-43 and an antibody directed against an intravesicular epitope of synaptotagmin I. We found that overexpression of wild type torsinA negatively affects synaptic vesicle endocytosis. Conversely, overexpression of DeltaE-torsinA in neuroblastoma cells increases FM1-43 uptake. Knockdown of snapin and/or torsinA using small interfering RNAs had a similar inhibitory effect on the exo-endocytic process. In addition, down-regulation of torsinA causes the persistence of synaptotagmin I on the plasma membrane, which closely resembles the effect observed by the overexpression of the DeltaE-torsinA mutant. Altogether, these findings suggest that torsinA plays a role together with snapin in regulated exocytosis and that DeltaE-torsinA exerts its pathological effects through a loss of function mechanism. This may affect neuronal uptake of neurotransmitters, such as dopamine, playing a role in the development of dystonic movements.     

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.     

Evidence for a primary endocytic vesicle involved in synaptic vesicle biogenesis

The regulated release of neurotransmitters at synapses is mediated by the fusion of neurotransmitter-filled synaptic vesicles with the plasma membrane. Continuous synaptic activity relies on the constant recycling of synaptic vesicle proteins into newly formed synaptic vesicles. At least two different mechanisms are presumed to mediate synaptic vesicle biogenesis at the synapse as follows: direct retrieval of synaptic vesicle proteins and lipids from the plasma membrane, and indirect passage of synaptic vesicle proteins through an endosomal intermediate. We have identified a vesicle population with the characteristics of a primary endocytic vesicle responsible for the recycling of synaptic vesicle proteins through the indirect pathway. We find that synaptic vesicle proteins colocalize in this vesicle with a variety of proteins known to recycle from the plasma membrane through the endocytic pathway, including three different glucose transporters, GLUT1, GLUT3, and GLUT4, and the transferrin receptor. These vesicles differ from "classical" synaptic vesicles in their size and their generic protein content, indicating that they do not discriminate between synaptic vesicle-specific proteins and other recycling proteins. We propose that these vesicles deliver synaptic vesicle proteins that have escaped internalization by the direct pathway to endosomes, where they are sorted from other recycling proteins and packaged into synaptic vesicles.     

Myosin accelerates synaptic vesicle recycling in the motor nerve endings

Neurotransmitter release and exocytosis of synaptic vesicles in the motor nerve endings of the frog cutaneous-pectoris muscle were studied using electrophysiological and optical methods under the conditions of inhibition of the myosin light-chain kinase and non-muscle myosin by the specific inhibitors ML-7 (12 μM) and (–)-blebbistatin (100 μM). At high-frequency stimulation (20 pulses/s), these inhibitors strengthened suppression of transmitter release during the first 20–25 s and slowed down the release of the fluorescent dye FM 1-43. The obtained results indicate that myosin accelerates rapid synaptic vesicle recycling upon high-frequency stimulation.     

Essential role of phosphoinositide metabolism in synaptic vesicle recycling.

Growing evidence suggests that phosphoinositides play an important role in membrane traffic. A polyphosphoinositide phosphatase, synaptojanin 1, was identified as a major presynaptic protein associated with endocytic coated intermediates. We report here that synaptojanin 1-deficient mice exhibit neurological defects and die shortly after birth. In neurons of mutant animals, PI(4,5)P2 levels are increased, and clathrin-coated vesicles accumulate in the cytomatrix-rich area that surrounds the synaptic vesicle cluster in nerve endings. In cell-free assays, reduced phosphoinositide phosphatase activity correlated with increased association of clathrin coats with liposomes. Intracellular recording in hippocampal slices revealed enhanced synaptic depression during prolonged high-frequency stimulation followed by delayed recovery. These results provide genetic evidence for a crucial role of phosphoinositide metabolism in synaptic vesicle recycling.     

Genetic and molecular analysis of synaptic vesicle recycling in Drosophila

Following exocytosis, one of the major presynaptic events is replenishing synaptic vesicles (SVs) to ensure the possibility of continuous synaptic transmission. The nerve terminal is thought to recycle SVs through clathrin-mediated endocytosis and by a clathrin-independent pathway called ‘kiss and run’. This review highlights the use of the genetic model organism, the fruit fly (Drosophila melanogaster), in dissecting the molecular mechanisms of clathrin-mediated endocytosis in recycling SVs at neuromuscular junctions (NMJs). Analyses of endocytotic mutants in Drosophila indicate that clathrin-mediated endocytosis may be essential for SV recycling, including a putative fast recycling mechanism uncovered recently. Further, a rather complex picture begins to emerge suggesting that clathrin-mediated endocytosis involves several sequential steps mediated by a large number of proteins. Finally, these studies also reveal that SV proteins may be selectively retrieved into nascent SVs by clathrin accessory proteins and defects in protein retrieval have significant impacts on synaptic transmission. Following the completion of the Drosophila Genome Project and the development of gene targeting and RNAi approaches, genetic studies in Drosophila have become increasingly efficient. Hence, Drosophila is expected to continue to serve as an important model organism for studies of SV recycling.     

Mechanisms of synaptic vesicle recycling illuminated by fluorescent dyes

The recycling of synaptic vesicles in nerve terminals involves multiple steps, underlies all aspects of synaptic transmission, and is a key to understanding the basis of synaptic plasticity. The development of styryl dyes as fluorescent molecules that label recycling synaptic vesicles has revolutionized the way in which synaptic vesicle recycling can be investigated, by allowing an examination of processes in neurons that have long been inaccessible. In this review, we evaluate the major aspects of synaptic vesicle recycling that have been revealed and advanced by studies with styryl dyes, focussing upon synaptic vesicle fusion, retrieval, and trafficking. The greatest impact of styryl dyes has been to allow the routine visualization of endocytosis in central nerve terminals for the first time. This has revealed the kinetics of endocytosis, its underlying sequential steps, and its regulation by Ca2+. In studies of exocytosis, styryl dyes have helped distinguish between different modes of vesicle fusion, provided direct support for the quantal nature of exocytosis and endocytosis, and revealed how the probability of exocytosis varies enormously from one nerve terminal to another. Synaptic vesicle labelling with styryl dyes has helped our understanding of vesicle trafficking by allowing better understanding of different synaptic vesicle pools within the nerve terminal, vesicle intermixing, and vesicle clustering at release sites. Finally, the dyes are now being used in innovative ways to reveal further insights into synaptic plasticity.     

A surprisingly singular role for endophilin in synaptic vesicle recycling

Synaptic vesicle endocytosis requires membrane curvature, fission, and uncoating. Endophilin has been proposed to play a role in all three steps, but in this issue of Neuron, De Camilli and colleagues show that at mammalian central synapses it is primarily involved in clathrin uncoating.     

Colocalization of synapsin and actin during synaptic vesicle recycling

It has been hypothesized that in the mature nerve terminal, interactions between synapsin and actin regulate the clustering of synaptic vesicles and the availability of vesicles for release during synaptic activity. Here, we have used immunogold electron microscopy to examine the subcellular localization of actin and synapsin in the giant synapse in lamprey at different states of synaptic activity. In agreement with earlier observations, in synapses at rest, synapsin immunoreactivity was preferentially localized to a portion of the vesicle cluster distal to the active zone. During synaptic activity, however, synapsin was detected in the pool of vesicles proximal to the active zone. In addition, actin and synapsin were found colocalized in a dynamic filamentous cytomatrix at the sites of synaptic vesicle recycling, endocytic zones. Synapsin immunolabeling was not associated with clathrin-coated intermediates but was found on vesicles that appeared to be recycling back to the cluster. Disruption of synapsin function by microinjection of antisynapsin antibodies resulted in a prominent reduction of the cytomatrix at endocytic zones of active synapses. Our data suggest that in addition to its known function in clustering of vesicles in the reserve pool, synapsin migrates from the synaptic vesicle cluster and participates in the organization of the actin-rich cytomatrix in the endocytic zone during synaptic activity.     

Entering neurons: botulinum toxins and synaptic vesicle recycling

Botulinum toxins are metalloproteases that act inside nerve terminals and block neurotransmitter release through their cleavage of components of the exocytosis machinery. These toxins are used to treat human diseases that are characterized by hyperfunction of cholinergic terminals. Recently, evidence has accumulated that gangliosides and synaptic vesicle proteins cooperate to mediate toxin binding to the presynaptic terminal. The differential distribution of synaptic vesicle protein receptors, gangliosides and toxin substrates in distinct neuronal populations opens up the possibility of using different serotypes of botulinum toxins for the treatment of central nervous system diseases caused by altered activity of selected neuronal populations.     

Synaptotagmin 7 splice variants differentially regulate synaptic vesicle recycling

The speed of synaptic vesicle recycling determines the efficacy of neurotransmission during repetitive stimulation. Synaptotagmins are synaptic C(2)-domain proteins that are involved in exocytosis, but have also been linked to endocytosis. We now demonstrate that upon expression in transfected neurons, a short splice variant of synaptotagmin 7 that lacks C(2)-domains accelerates endocytic recycling of synaptic vesicles, whereas a longer splice variant that contains C(2)-domains decelerates recycling. These results suggest that alternative splicing of synaptotagmin 7 acts as a molecular switch, which targets vesicles to fast and slow recycling pathways.     

The t-SNAREs syntaxin 1 and SNAP-25 are present on organelles that participate in synaptic vesicle recycling

Syntaxin 1 and synaptosome-associated protein of 25 kD (SNAP-25) are neuronal plasmalemma proteins that appear to be essential for exocytosis of synaptic vesicles (SVs). Both proteins form a complex with synaptobrevin, an intrinsic membrane protein of SVs. This binding is thought to be responsible for vesicle docking and apparently precedes membrane fusion. According to the current concept, syntaxin 1 and SNAP-25 are members of larger protein families, collectively designated as target-SNAP receptors (t-SNAREs), whose specific localization to subcellular membranes define where transport vesicles bind and fuse. Here we demonstrate that major pools of syntaxin 1 and SNAP-25 recycle with SVs. Both proteins cofractionate with SVs and clathrin-coated vesicles upon subcellular fractionation. Using recombinant proteins as standards for quantitation, we found that syntaxin 1 and SNAP-25 each comprise approximately 3% of the total protein in highly purified SVs. Thus, both proteins are significant components of SVs although less abundant than synaptobrevin (8.7% of the total protein). Immunoisolation of vesicles using synaptophysin and syntaxin specific antibodies revealed that most SVs contain syntaxin 1. The widespread distribution of both syntaxin 1 and SNAP-25 on SVs was further confirmed by immunogold electron microscopy. Botulinum neurotoxin C1, a toxin that blocks exocytosis by proteolyzing syntaxin 1, preferentially cleaves vesicular syntaxin 1. We conclude that t- SNAREs participate in SV recycling in what may be functionally distinct forms.     

Newly produced synaptic vesicle proteins are preferentially used in synaptic transmission

Aged proteins can become hazardous to cellular function, by accumulating molecular damage. This implies that cells should preferentially rely on newly produced ones. We tested this hypothesis in cultured hippocampal neurons, focusing on synaptic transmission. We found that newly synthesized vesicle proteins were incorporated in the actively recycling pool of vesicles responsible for all neurotransmitter release during physiological activity. We observed this for the calcium sensor Synaptotagmin 1, for the neurotransmitter transporter VGAT, and for the fusion protein VAMP2 (Synaptobrevin 2). Metabolic labeling of proteins and visualization by secondary ion mass spectrometry enabled us to query the entire protein makeup of the actively recycling vesicles, which we found to be younger than that of non‐recycling vesicles. The young vesicle proteins remained in use for up to ~ 24 h, during which they participated in recycling a few hundred times. They were afterward reluctant to release and were degraded after an additional ~ 24–48 h. We suggest that the recycling pool of synaptic vesicles relies on newly synthesized proteins, while the inactive reserve pool contains older proteins.     

Two mechanisms of synaptic vesicle recycling in rat brain nerve terminals

KCl and 4-aminopyridine (4-AP) evoke glutamate release from rat brain cortical nerve terminals by voltage clamping or by Na(+) channel-generated repetitive action potentials, respectively. Stimulation by 4-AP but not KCl is largely mediated by protein kinase C (PKC). To determine whether KCl and 4-AP utilise the same mechanism to release glutamate, we correlated glutamate release with release of the hydrophobic synaptic vesicle (SV) marker FM2-10. A strong correlation was observed for increasing concentrations of KCl and after application of phorbol 12-myristate 13-acetate (PMA) or staurosporine. The parallel increase in exocytosis measured by two approaches suggested it occurred by a PKC-independent mechanism involving complete fusion of SVs with the plasma membrane. At low concentrations of 4-AP, alone or with staurosporine, glutamate and FM2-10 release also correlated. However, higher concentrations of 4-AP or of 4-AP plus PMA greatly increased glutamate release but did not further increase FM2-10 release. This divergence suggests that 4-AP recruits an additional mechanism of release during strong stimulation that is PKC dependent and is superimposed upon the first mechanism. This second mechanism is characteristic of kiss-and-run, which is not detectable by styryl dyes. Our data suggest that glutamate release in nerve terminals occurs via two mechanisms: (1) complete SV fusion, which is PKC independent; and (2) a kiss-and-run-like mechanism, which is PKC dependent. Recruitment of a second release mechanism may be a widespread means to facilitate neurotransmitter release in central neurons.