CSN complex controls the stability of selected synaptic proteins via a torsinA-dependent process

DYT1 dystonia is caused by an autosomal dominant mutation that leads to a glutamic acid deletion in torsinA (TA), a member of the AAA+ ATPase superfamily. In this study, we identified a novel-binding partner of TA, the subunit 4 (CSN4) of CSN signalosome. TA binds CSN4 and the synaptic regulator snapin in neuroblastoma cells and in brain synaptosomes. CSN4 and TA are required for the stability of both snapin and the synaptotagmin-specific endocytic adaptor stonin 2, as downregulation of CSN4 or TA reduces the levels of both proteins. Snapin is phosphorylated by the CSN-associated kinase protein kinase D (PKD) and its expression is decreased upon PKD inhibition. In contrast, the stability of stonin 2 is regulated by neddylation, another CSN-associated activity. Overexpression of the pathological TA mutant (ΔE-TA) reduces stonin 2 expression, causing the accumulation of the calcium sensor synaptotagmin 1 on the cell surface. Retrieval of surface-stranded synaptotagmin 1 is restored by overexpression of stonin 2 in ΔE-TA-expressing cells, suggesting that the DYT1 mutation compromises the role of TA in protein stabilisation and synaptic vesicle recycling.     

C2B polylysine motif of synaptotagmin facilitates a Ca2+-independent stage of synaptic vesicle priming in vivo

Synaptotagmin I, a synaptic vesicle protein required for efficient synaptic transmission, contains a highly conserved polylysine motif necessary for function. Using Drosophila, we examined in which step of the synaptic vesicle cycle this motif functions. Polylysine motif mutants exhibited an apparent decreased Ca2+ affinity of release, and, at low Ca2+, an increased failure rate, increased facilitation, and increased augmentation, indicative of a decreased release probability. Disruption of Ca2+ binding, however, cannot account for all of the deficits in the mutants; rather, the decreased release probability is probably due to a disruption in the coupling of synaptotagmin to the release machinery. Mutants exhibited a major slowing of recovery from synaptic depression, which suggests that membrane trafficking before fusion is disrupted. The disrupted process is not endocytosis because the rate of FM 1-43 uptake was unchanged in the mutants, and the polylysine motif mutant synaptotagmin was able to rescue the synaptic vesicle depletion normally found in syt(null) mutants. Thus, the polylysine motif functions after endocytosis and before fusion. Finally, mutation of the polylysine motif inhibits the Ca2+-independent ability of synaptotagmin to accelerate SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor)-mediated fusion. Together, our results demonstrate that the polylysine motif is required for efficient Ca2+-independent docking and/or priming of synaptic vesicles in vivo.     

The TRPM7 ion channel functions in cholinergic synaptic vesicles and affects transmitter release

A longstanding hypothesis is that ion channels are present in the membranes of synaptic vesicles and might affect neurotransmitter release. Here we demonstrate that TRPM7, a member of the transient receptor potential (TRP) ion channel family, resides in the membrane of synaptic vesicles of sympathetic neurons, forms molecular complexes with the synaptic vesicle proteins synapsin I and synaptotagmin I, and directly interacts with synaptic vesicular snapin. In sympathetic neurons, changes in TRPM7 levels and channel activity alter acetylcholine release, as measured by EPSP amplitudes and decay times in postsynaptic neurons. TRPM7 affects EPSP quantal size, an intrinsic property of synaptic vesicle release. Targeted peptide interference of TRPM7's interaction with snapin affects the amplitudes and kinetics of postsynaptic EPSPs. Thus, vesicular TRPM7 channel activity is critical to neurotransmitter release in sympathetic neurons.     

Recombinant expression, purification, and comparative characterization of torsinA and its torsion dystonia-associated variant Delta E-torsinA

Early-onset torsion dystonia is an autosomal dominant movement disorder that has been linked to the deletion of one of a pair of glutamic acid residues in the protein torsinA (E(302/303); DeltaE-torsinA). In transfected cells, DeltaE-torsinA exhibits similar biochemical properties to wild type (WT)-torsinA, but displays a distinct subcellular localization. Primary structural analysis of torsinA suggests that this protein is a membrane-associated member of the AAA family of ATP-binding proteins. However, to date, neither WT- nor DeltaE-torsinA has been obtained in sufficient quantity and purity to permit detailed biochemical and biophysical characterization. Here, we report a baculovirus expression system that provides milligram quantities of purified torsin proteins. Recombinant WT- and DeltaE-torsinA were found to be membrane-associated glycoproteins that required detergents for solubilization and purification. Analysis of the biophysical properties of WT- and DeltaE-torsinA indicated that both proteins were folded monomers in solution that exhibited equivalent denaturation behaviors under thermal and chaotropic (guanidinium chloride) stress. Additionally, both forms of torsinA were found to display ATPase activity with similar k(cat) and K(m) values. Collectively, these data reveal that torsinA is a membrane-associated ATPase and indicate that the DeltaE(302/303) dystonia-associated mutation in this protein does not cause gross changes in its catalytic or structural properties. These findings are consistent with a disease mechanism in which DeltaE-torsinA promotes dystonia through a gain rather than loss of function. The recombinant expression system for torsinA proteins described herein should facilitate further biochemical and structural investigations to test this hypothesis.     

Preserved synaptic vesicle recycling in hippocampal neurons in a mouse Alzheimer's disease model

A recently described triple-transgenic mouse model (3xTg, PS1(M146V), APP(Swe), and tau(P301L)) develops a neuropathology similar to the brains of Alzheimer's disease patients including progressive deposits of plaques and tangles [Neuron 39 (2003) 409]. These mice also show age-related deficits in hippocampal synaptic plasticity that occurs before the development of plaques and tangles. Here we report unchanged synaptic vesicle recycling, as measured by FM1-43 release, in the hippocampal neurons of the 3xTg mice. Expression levels of presynaptic protein synaptophysin and of proteins involved in synaptic vesicle recycling including AP180, dynamin I, and synaptotagmin I also remain unaffected. These data suggest that the synaptic deficits observed in the 3xTg neurons may not arise from the preserved synaptic vesicle recycling.     

C-terminal ECFP fusion impairs synaptotagmin 1 function: crowding out synaptotagmin 1

To allow the monitoring of synaptotagmin 1 trafficking in vivo, we generated transgenic mice expressing a synaptotagmin 1-enhanced cyan fluorescent protein (ECFP) fusion protein under control of the Thy1 promoter. Transgenic synaptotagmin 1-ECFP is expressed throughout the brain where it localizes to synapses and marks synapses in vivo. However, when we crossed transgenic synaptotagmin 1-ECFP mice with synaptotagmin 1 knock-out mice, we detected no rescue of survival or function. Furthermore, viral overexpression of synaptotagmin 1-ECFP in synaptotagmin 1-deficient neurons failed to restore normal Ca2+-triggered release, whereas overexpression of wild type synaptotagmin 1 did so efficiently. To determine whether synaptotagmin 1-ECFP is non-functional because the ECFP-fusion interferes with its biochemical activities, we measured Ca2+-independent binding of synaptotagmin 1-ECFP to SNARE complexes, and Ca2+-dependent binding of synaptotagmin 1-ECFP to phospholipids and to itself. Although the apparent Ca2+ affinity of synaptotagmin 1-ECFP was decreased compared with wild type synaptotagmin 1, we observed no major changes in Ca2+-dependent or -independent activities, indicating that the non-functionality of the synaptotagmin 1-ECFP fusion protein was not because of inactivation of its biochemical properties. These data suggest that synaptotagmin 1-ECFP is suitable for monitoring synaptic vesicle traffic in vivo because the synaptotagmin 1-ECFP marks synaptic vesicles without participating in exocytosis. In addition, the data demonstrate that synaptotagmin 1 function requires a free C terminus, possibly because of spatial constraints at the release sites.     

Reinvestigation of the role of snapin in neurotransmitter release

Snapin, a 15-kDa protein, has been identified recently as a binding partner of SNAP-25. Moreover, snapin is regulated by phosphorylation and enhances synaptotagmin binding to SNAREs. Furthermore, snapin and C-terminal snapin fragments have been effective in changing the release properties of neurons and chromaffin cells. Here we have reinvestigated the role of snapin using both biochemical and electrophysiological approaches. Snapin is ubiquitously expressed at low levels with no detectable enrichment in the brain or in synaptic vesicles. Using non-equilibrium and equilibrium assays including pulldown experiments, co-immunoprecipitations, and CD and fluorescence anisotropy spectroscopy, we were unable to detect any specific interaction between snapin and SNAP-25. Similarly, overexpression of a C-terminal snapin fragment in hippocampal neurons failed to influence any of the analyzed parameters of neurotransmitter release. Initial biochemical characterization of recombinant snapin revealed that the protein is a stable dimer with a predominantly alpha-helical secondary structure. We conclude that the postulated role of snapin as a SNARE regulator in neurotransmitter release needs reconsideration, leaving the true function of this evolutionarily conserved protein to be discovered.     

Neurotransmitter vesicle release from human model neurons (NT2) is sensitive to botulinum toxin A

Botulinum neurotoxins (BoNTs) internalize into nerve terminals and block the release of neurotransmitters into the synapse. BoNTs are widely used as a therapeutic agent for treatment of movement disorders and recently gained more attention as a biological weapon. Consequently, there is strong interest to develop a cell-based assay platform to screen the toxicity and bioactivity of the BoNTs. In this study, we present an in vitro screening assay for BoNT/A based on differentiated human embryonal carcinoma stem (NT2) cells. The human NT2 cells fully differentiated into mature neurons that display immunoreactivity to cytoskeletal markers (βIII-tubulin and MAP2) and presynaptic proteins (synapsin and synaptotagmin I). We showed that the human NT2 cells undergo a process of exo-endocytotic synaptic vesicle recycling upon depolarization with high K(+) buffer. By employing an antibody directed against light chain of BoNT/A, we detected internalized toxin as a punctate staining along the neurites of the NT2 neurons. Using well-established methods of synaptic vesicle exocytosis assay (luminal synaptotagmin I and FM1-43 imaging) we show that pre-incubation with BoNT/A resulted in a blockade of vesicle release from human NT2 neurons in a dose-dependent manner. Moreover, this blocking effect of BoNT/A was abolished by pre-adsorbing the toxin with neutralizing antibody. In a proof of principle, we demonstrate that our cell culture assay for vesicle release is sensitive to BoNT/A and the activity of BoNT/A can be blocked by specific neutralizing antibodies. Overall our data suggest that human NT2 neurons are suitable for large scale screening of botulinum bioactivity.     

Mutations in palmitoyl-protein thioesterase 1 alter exocytosis and endocytosis at synapses in Drosophila larvae

Infantile-onset neuronal ceroid lipofuscinosis (INCL) is a severe pediatric neurodegenerative disorder produced by mutations in the gene encoding palmitoyl-protein thioesterase 1 (Ppt1). This enzyme is responsible for the removal of a palmitate group from its substrate proteins, which may include presynaptic proteins like SNAP-25, cysteine string protein (CSP), dynamin, and synaptotagmin. The fruit fly, Drosophila melanogaster, has been a powerful model system for studying the functions of these proteins and the molecular basis of neurological disorders like the NCLs. Genetic modifier screens and tracer uptake studies in Ppt1 mutant larval garland cells have suggested that Ppt1 plays a role in endocytic trafficking. We have extended this analysis to examine the involvement of Ppt1 in synaptic function at the Drosophila larval neuromuscular junction (NMJ). Mutations in Ppt1 genetically interact with temperature sensitive mutations in the Drosophila dynamin gene shibire, accelerating the paralytic behavior of shibire mutants at 27 °C. Electrophysiological work in NMJs of Ppt1-deficient larvae has revealed an increase in miniature excitatory junctional potentials (EJPs) and a significant depression of evoked EJPs in response to repetitive (10 hz) stimulation. Endocytosis was further examined in Ppt1-mutant larvae using FM1–43 uptake assays, demonstrating a significant decrease in FM1–43 uptake at the mutant NMJs. Finally, Ppt1-deficient and Ppt1 point mutant larvae display defects in locomotion that are consistent with alterations in synaptic function. Taken together, our genetic, cellular, and electrophysiological analyses suggest a direct role for Ppt1 in synaptic vesicle exo- and endocytosis at motor nerve terminals of the Drosophila NMJ.     

Direct, Ca2+-dependent interaction between tubulin and synaptotagmin I: a possible mechanism for attaching synaptic vesicles to microtubules

The synaptic vesicle protein synaptotagmin I probably plays important roles in the synaptic vesicle cycle. However, the mechanisms of its action remain unclear. In this study, we have searched for cytoplasmic proteins that interact with synaptotagmin I. We found that the cytoskeletal protein tubulin directly and stoichiometrically bound to recombinant synaptotagmin I. The binding depended on mm Ca(2+), and 1 mol of tubulin dimer bound 2 mol of synaptotagmin I with half-maximal binding at 6.6 microm tubulin. The Ca(2+) dependence mainly resulted from Ca(2+) binding to the Ca(2+) ligands of synaptotagmin I. The C-terminal region of beta-tubulin and both C2 domains of synaptotagmin I were involved in the binding. The YVK motif in the C2 domains of synaptotagmin I was essential for tubulin binding. Tubulin and synaptotagmin I were co-precipitated from the synaptosome extract with monoclonal antibodies to tubulin and SNAP-25 (synaptosome-associated protein of 25 kDa), indicating the presence of tubulin/synaptotagmin I complex and tubulin binding to synaptotagmin I in SNARE (soluble N-ethylmaleimide-sensitive factor attachment protein receptor) complexes. Synaptotagmin I promoted tubulin polymerization and bundled microtubules in the presence of Ca(2+). These results suggest that direct interaction between synaptotagmin I and tubulin provides a mechanism for attaching synaptic vesicles to microtubules in high Ca(2+) concentrations.     

Essential role of endophilin A in synaptic vesicle budding at the Drosophila neuromuscular junction

We characterized Drosophila endophilin A (D-endoA), and generated and analysed D-endoA mutants. Like its mammalian homologue, D-endoA exhibits lysophosphatidic acid acyl transferase activity and contains a functional SH3 domain. D-endoA is recruited to the sites of endocytosis, as revealed by immunocytochemistry of the neuromuscular junction (NMJ) of mutant L3 larvae carrying the temperature-sensitive allele of dynamin, shibire. D-endoA null mutants show severe defects in motility and die at the early L2 larval stage. Mutants with reduced D-endoA levels exhibit a range of defects of synaptic vesicle endocytosis, as observed at L3 larvae NMJs using FM1-43 uptake and electron microscopy. NMJs with an almost complete loss of synaptic vesicles did not show an accumulation of intermediates of the budding process, whereas NMJs with only slightly reduced levels of synaptic vesicles showed a striking increase in early-stage, but not late-stage, budding intermediates at the plasma membrane. Together with results of previous studies, these observations indicate that endophilin A is essential for synaptic vesicle endocytosis, being required from the onset of budding until fission.     

Normal biogenesis and cycling of empty synaptic vesicles in dopamine neurons of vesicular monoamine transporter 2 knockout mice

The neuronal isoform of vesicular monoamine transporter, VMAT2, is responsible for packaging dopamine and other monoamines into synaptic vesicles and thereby plays an essential role in dopamine neurotransmission. Dopamine neurons in mice lacking VMAT2 are unable to store or release dopamine from their synaptic vesicles. To determine how VMAT2-mediated filling influences synaptic vesicle morphology and function, we examined dopamine terminals from VMAT2 knockout mice. In contrast to the abnormalities reported in glutamatergic terminals of mice lacking VGLUT1, the corresponding vesicular transporter for glutamate, we found that the ultrastructure of dopamine terminals and synaptic vesicles in VMAT2 knockout mice were indistinguishable from wild type. Using the activity-dependent dyes FM1-43 and FM2-10, we also found that synaptic vesicles in dopamine neurons lacking VMAT2 undergo endocytosis and exocytosis with kinetics identical to those seen in wild-type neurons. Together, these results demonstrate that dopamine synaptic vesicle biogenesis and cycling are independent of vesicle filling with transmitter. By demonstrating that such empty synaptic vesicles can cycle at the nerve terminal, our study suggests that physiological changes in VMAT2 levels or trafficking at the synapse may regulate dopamine release by altering the ratio of fillable-to-empty synaptic vesicles, as both continue to cycle in response to neural activity.     

The subcellular localizations of atypical synaptotagmins III and VI. Synaptotagmin III is enriched in synapses and synaptic plasma membranes but not in synaptic vesicles.

Multiple synaptotagmins are expressed in brain, but only synaptotagmins I and II have known functions in fast, synchronous Ca2+-triggered neurotransmitter release. Synaptotagmin III was proposed to regulate other aspects of synaptic vesicle exocytosis, particularly its slow component. Such a function predicts that synaptotagmin III should be an obligatory synaptic vesicle protein, as would also be anticipated from its high homology to synaptotagmins I and II. To test this hypothesis, we studied the distribution, developmental expression, and localization of synaptotagmin III and its closest homolog, synaptotagmin VI. We find that synaptotagmins III and VI are present in all brain regions in heterogeneous distributions and that their levels increase during development in parallel with synaptogenesis. Furthermore, we show by immunocytochemistry that synaptotagmin III is concentrated in synapses, as expected. Surprisingly, however, we observed that synaptotagmin III is highly enriched in synaptic plasma membranes but not in synaptic vesicles. Synaptotagmin VI was also found to be relatively excluded from synaptic vesicles. Our data suggest that synaptotagmins III and VI perform roles in neurons that are not linked to synaptic vesicle exocytosis but to other Ca2+-related nerve terminal events, indicating that the functions of synaptotagmins are more diverse than originally thought.     

Kinetics of synaptotagmin responses to Ca2+ and assembly with the core SNARE complex onto membranes

The synaptic vesicle protein synaptotagmin I binds Ca2+ and is required for efficient neurotransmitter release. Here, we measure the response time of the C2 domains of synaptotagmin to determine whether synaptotagmin is fast enough to function as a Ca2+ sensor for rapid exocytosis. We report that synaptotagmin is "tuned" to sense Ca2+ concentrations that trigger neuronal exocytosis. The speed of response is unique to synaptotagmin I and readily satisfies the kinetic constraints of synaptic vesicle membrane fusion. We further demonstrate that Ca2+ triggers penetration of synaptotagmin into membranes and simultaneously drives assembly of synaptotagmin onto the base of the ternary SNARE (soluble N-ethylmaleimide-sensitive fusion protein [NSF] attachment receptor) complex, near the transmembrane anchor of syntaxin. These data support a molecular model in which synaptotagmin triggers exocytosis through its interactions with membranes and the SNARE complex.     

Is synaptotagmin the calcium sensor?

After much debate, recent progress indicates that the synaptic vesicle protein synaptotagmin I probably functions as the calcium sensor for synchronous neurotransmitter release. Following calcium influx into presynaptic terminals, synaptotagmin I rapidly triggers the fusion of synaptic vesicles with the plasma membrane and underlies the fourth-order calcium cooperativity of release. Biochemical and genetic studies suggest that lipid and SNARE interactions underlie synaptotagmin's ability to mediate the incredible speed of vesicle fusion that is the hallmark of fast synaptic transmission.     

Okadaic acid disrupts synaptic vesicle trafficking in a ribbon-type synapse

Protein phosphorylation plays an essential role in regulating synaptic transmission and plasticity. However, regulation of vesicle trafficking towards and away from the plasma membrane is poorly understood. Furthermore, the extent to which phosphorylation modulates ribbon-type synapses is unknown. Using the phosphatase inhibitor okadaic acid (OA), we investigated the influence of persistent phosphorylation on vesicle cycling in goldfish bipolar cells. We followed uptake of FM1-43 during vesicle recycling in control and OA-treated cells. FM1-43 fluorescence spread to the center of control synaptic terminals after depolarization elicited Ca2+ influx. However, OA (1-50 nm) impaired this spatial spread of FM1-43 in a dose-dependent manner. Capacitance measurements revealed that OA (50 nm) did not modify either the amount or kinetics of exocytosis and endocytosis evoked by depolarizing pulses. The extremely low concentrations of OA (1-5 nm) sufficient to observe the inhibition of vesicle mobility implicate phosphatase 2A (PP2A) as a major regulator of vesicle trafficking after endocytosis. These results contrast with those at the neuromuscular junction where OA enhances lateral movement of vesicles between distinct vesicle clusters. Thus, our results suggest that phosphatases regulate vesicle translocation at ribbon synapses in a different manner than conventional active zones.     

Spatial variability in release at the frog neuromuscular junction measured with FM1-43

We quantified the spatial variability in release properties at different synaptic vesicle clusters in frog motor nerve terminals, using a combination of fluorescence and electron microscopy. Individual synaptic vesicle clusters labeled with FM1-43 varied more than 10-fold in initial intensity (integrated FM1-43 fluorescence) and in absolute rate of dye loss during tetanic electrical nerve stimulation. Most of this variability arose because large vesicle clusters spanned more than one presynaptic active zone (inferred from postsynaptic acetylcholine receptor stripes labeled with rhodamine-conjugated alpha-bungarotoxin); when the rate of dye loss was normalized to the length of receptor stripe covered, variability from spot to spot was greatly reduced. In addition, electron microscopic measurements showed that large vesicle clusters (i.e., those spanning multiple active zones) were also thicker, and the increased depth of vesicles led to increased total spot fluorescence without a corresponding increase in the rate of dye loss during stimulation. These results did not reveal the presence of "hot zones" of secretory activity.     

The v-ATPase V0 subunit a1 is required for a late step in synaptic vesicle exocytosis in Drosophila

The V(0) complex forms the proteolipid pore of an ATPase that acidifies vesicles. In addition, an independent function in membrane fusion has been proposed largely based on yeast vacuolar fusion experiments. We have isolated mutations in the largest V(0) component vha100-1 in flies in an unbiased genetic screen for synaptic malfunction. The protein is only required in neurons, colocalizes with markers for synaptic vesicles as well as active zones, and interacts with t-SNAREs. Loss of vha100-1 leads to vesicle accumulation in synaptic terminals, suggesting a deficit in release. The amplitude of spontaneous release events and release with hypertonic stimulation indicate normal levels of neurotransmitter loading, yet mutant embryos display severe defects in evoked synaptic transmission and FM1-43 uptake. Our data suggest that Vha100-1 functions downstream of SNAREs in synaptic vesicle fusion.     

Synaptotagmin in Ca2+ -dependent exocytosis: dynamic action in a flash

Synaptotagmins have been the popular candidates for the Ca2+ sensor that couples local rise in Ca2+ to neurotransmitter release. Studies in worm, fly, and mouse corroborate the likely role for synaptotagmin I, the best-studied synaptotagmin prototype, as a Ca2+ trigger for synaptic vesicle exocytosis. Recent investigations have focused on structural domains of synaptotagmin that are critical for its function. Here we provide a brief overview of synaptotagmin I and discuss recent studies within the framework of neurotransmitter release mechanisms for fast synaptic transmission.