synaptojanin1 Is Required for Temporal Fidelity of Synaptic Transmission in Hair Cells

To faithfully encode mechanosensory information, auditory/vestibular hair cells utilize graded synaptic vesicle (SV) release at specialized ribbon synapses. The molecular basis of SV release and consequent recycling of membrane in hair cells has not been fully explored. Here, we report that comet, a gene identified in an ENU mutagenesis screen for zebrafish larvae with vestibular defects, encodes the lipid phosphatase Synaptojanin 1 (Synj1). Examination of mutant synj1 hair cells revealed basal blebbing near ribbons that was dependent on Cav1.3 calcium channel activity but not mechanotransduction. Synaptojanin has been previously implicated in SV recycling; therefore, we tested synaptic transmission at hair-cell synapses. Recordings of post-synaptic activity in synj1 mutants showed relatively normal spike rates when hair cells were mechanically stimulated for a short period of time at 20 Hz. In contrast, a sharp decline in the rate of firing occurred during prolonged stimulation at 20 Hz or stimulation at a higher frequency of 60 Hz. The decline in spike rate suggested that fewer vesicles were available for release. Consistent with this result, we observed that stimulated mutant hair cells had decreased numbers of tethered and reserve-pool vesicles in comparison to wild-type hair cells. Furthermore, stimulation at 60 Hz impaired phase locking of the postsynaptic activity to the mechanical stimulus. Following prolonged stimulation at 60 Hz, we also found that mutant synj1 hair cells displayed a striking delay in the recovery of spontaneous activity. Collectively, the data suggest that Synj1 is critical for retrieval of membrane in order to maintain the quantity, timing of fusion, and spontaneous release properties of SVs at hair-cell ribbon synapses.     

Drosophila CAPS is an essential gene that regulates dense-core vesicle release and synaptic vesicle fusion

Calcium-activated protein for secretion (CAPS) is proposed to play an essential role in Ca2+-regulated dense-core vesicle exocytosis in vertebrate neuroendocrine cells. Here we report the cloning, mutation, and characterization of the Drosophila ortholog (dCAPS). Null dCAPS mutants display locomotory deficits and complete embryonic lethality. The mutant NMJ reveals a 50% loss in evoked glutamatergic transmission, and an accumulation of synaptic vesicles at active zones. Importantly, dCAPS mutants display a highly specific 3-fold accumulation of dense-core vesicles in synaptic terminals, which was not observed in mutants that completely arrest synaptic vesicle exocytosis. Targeted transgenic CAPS expression in identified motoneurons fails to rescue dCAPS neurotransmission defects, demonstrating a cell nonautonomous role in synaptic vesicle fusion. We conclude that dCAPS is required for dense-core vesicle release and that a dCAPS-dependent mechanism modulates synaptic vesicle release at glutamatergic synapses.     

Polyunsaturated fatty acids influence synaptojanin localization to regulate synaptic vesicle recycling

The lipid polyunsaturated fatty acids are highly enriched in synaptic membranes, including synaptic vesicles, but their precise function there is unknown. Caenorhabditis elegans fat-3 mutants lack long-chain polyunsaturated fatty acids (LC-PUFAs); they release abnormally low levels of serotonin and acetylcholine and are depleted of synaptic vesicles, but the mechanistic basis of these defects is unclear. Here we demonstrate that synaptic vesicle endocytosis is impaired in the mutants: the synaptic vesicle protein synaptobrevin is not efficiently retrieved after synaptic vesicles fuse with the presynaptic membrane, and the presynaptic terminals contain abnormally large endosomal-like compartments and synaptic vesicles. Moreover, the mutants have abnormally low levels of the phosphoinositide phosphatase synaptojanin at release sites and accumulate the main synaptojanin substrate phosphatidylinositol 4,5-bisphosphate at these sites. Both synaptobrevin and synaptojanin mislocalization can be rescued by providing exogenous arachidonic acid, an LC-PUFA, suggesting that the endocytosis defect is caused by LC-PUFA depletion. By showing that the genes fat-3 and synaptojanin act in the same endocytic pathway at synapses, our findings suggest that LC-PUFAs are required for efficient synaptic vesicle recycling, probably by modulating synaptojanin localization at synapses.     

Recruitment of endophilin to clathrin-coated pit necks is required for efficient vesicle uncoating after fission

Endophilin is a membrane-binding protein with curvature-generating and -sensing properties that participates in clathrin-dependent endocytosis of synaptic vesicle membranes. Endophilin also binds the GTPase dynamin and the phosphoinositide phosphatase synaptojanin and is thought to coordinate constriction of coated pits with membrane fission (via dynamin) and subsequent uncoating (via synaptojanin). We show that although synaptojanin is recruited by endophilin at bud necks before fission, the knockout of all three mouse endophilins results in the accumulation of clathrin-coated vesicles, but not of clathrin-coated pits, at synapses. The absence of endophilin impairs but does not abolish synaptic transmission and results in perinatal lethality, whereas partial endophilin absence causes severe neurological defects, including epilepsy and neurodegeneration. Our data support a model in which endophilin recruitment to coated pit necks, because of its curvature-sensing properties, primes vesicle buds for subsequent uncoating after membrane fission, without being critically required for the fission reaction itself.     

Cellular bases of activity-dependent paralysis in Drosophila stress-sensitive mutants

Stress-sensitive mutants in Drosophila have been shown to exhibit activity-dependent defects in neurotransmission. Using the neuromuscular junction (NMJ), this study investigates synaptic function more specifically in two stress-sensitive mutants: stress-sensitive B (sesB), which encodes a mitochondrial ADP/ATP translocase (ANT); and Atpalpha(2206), a conditional mutant of the Na+/K+ ATPase alpha-subunit. Mechanical shock induces a period of brief paralysis in both homozygous and double heterozygous mutants, but further analysis revealed distinct activity-dependent neurotransmission lesions in each mutant. Basal neurotransmission appeared similar to wild-type controls in both mutants under low frequency stimulation. High frequency stimulation, however, caused pronounced synaptic fatigue as well as slow and incomplete synaptic recovery in sesB mutants while Atpalpha(2206) mutants displayed an increase (25-fold) in synaptic failures. Perhaps to compensate for these activity dependent defects, the neuromuscular synapse was found to be overgrown in both mutants. Passive electrotonic stimulation, which initiates synaptic transmission independent of action potentials, ameliorated synaptic failures and resulted in increased neurotransmission amplitude in Atpalpha(2206) mutants. In addition, spontaneous synaptic vesicle fusion rates were increased in Atpalpha(2206) mutants, suggesting that, in the absence of action potential requirements, these synaptic terminals are healthy, if not hyperactive. Dye labeling studies revealed aberrant synaptic vesicle cycling in sesB mutants indicating a reduction of functional synaptic vesicles. We therefore postulate that both stress-sensitive mutants harbor unique neurotransmission defects: Atpalpha(2206) mutants are unable to maintain ionic gradients required during repetitive action potential propagation, and sesB mutants cannot maintain synaptic vesicle cycling during periods of high demand.     

Endophilin is required for synaptic vesicle endocytosis by localizing synaptojanin

Endophilin is a membrane-associated protein required for endocytosis of synaptic vesicles. Two models have been proposed for endophilin: that it alters lipid composition in order to shape membranes during endocytosis, or that it binds the polyphosphoinositide phosphatase synaptojanin and recruits this phosphatase to membranes. In this study, we demonstrate that the unc-57 gene encodes the Caenorhabditis elegans ortholog of endophilin A. We demonstrate that endophilin is required in C. elegans for synaptic vesicle recycling. Furthermore, the defects observed in endophilin mutants closely resemble those observed in synaptojanin mutants. The electrophysiological phenotype of endophilin and synaptojanin double mutants are virtually identical to the single mutants, demonstrating that endophilin and synaptojanin function in the same pathway. Finally, endophilin is required to stabilize expression of synaptojanin at the synapse. These data suggest that endophilin is an adaptor protein required to localize and stabilize synaptojanin at membranes during synaptic vesicle recycling.     

Endophilin mutations block clathrin-mediated endocytosis but not neurotransmitter release

We have identified mutations in Drosophila endophilin to study its function in vivo. Endophilin is required presynaptically at the neuromuscular junction, and absence of Endophilin dramatically impairs endocytosis in vivo. Mutant larvae that lack Endophilin fail to take up FM1-43 dye in synaptic boutons, indicating an inability to retrieve synaptic membrane. This defect is accompanied by an expansion of the presynaptic membrane, and a depletion of vesicles from the bouton lumen. Interestingly, mutant larvae are still able to sustain release at 15%-20% of the normal rate during high-frequency stimulation. We propose that kiss-and-run maintains neurotransmission at active zones of the larval NMJ in endophilin animals.     

N-cofilin can compensate for the loss of ADF in excitatory synapses

Actin plays important roles in a number of synaptic processes, including synaptic vesicle organization and exocytosis, mobility of postsynaptic receptors, and synaptic plasticity. However, little is known about the mechanisms that control actin at synapses. Actin dynamics crucially depend on LIM kinase 1 (LIMK1) that controls the activity of the actin depolymerizing proteins of the ADF/cofilin family. While analyses of mouse mutants revealed the importance of LIMK1 for both pre- and postsynaptic mechanisms, the ADF/cofilin family member n-cofilin appears to be relevant merely for postsynaptic plasticity, and not for presynaptic physiology. By means of immunogold electron microscopy and immunocytochemistry, we here demonstrate the presence of ADF (actin depolymerizing factor), a close homolog of n-cofilin, in excitatory synapses, where it is particularly enriched in presynaptic terminals. Surprisingly, genetic ablation of ADF in mice had no adverse effects on synapse structure or density as assessed by electron microscopy and by the morphological analysis of Golgi-stained hippocampal pyramidal cells. Moreover, a series of electrophysiological recordings in acute hippocampal slices revealed that presynaptic recruitment and exocytosis of synaptic vesicles as well as postsynaptic plasticity were unchanged in ADF mutant mice. The lack of synaptic defects may be explained by the elevated n-cofilin levels observed in synaptic structures of ADF mutants. Indeed, synaptic actin regulation was impaired in compound mutants lacking both ADF and n-cofilin, but not in ADF single mutants. From our results we conclude that n-cofilin can compensate for the loss of ADF in excitatory synapses. Further, our data suggest that ADF and n-cofilin cooperate in controlling synaptic actin content.     

UNC-11, a Caenorhabditis elegans AP180 Homologue, Regulates the Size and Protein Composition of Synaptic Vesicles

The unc-11 gene of Caenorhabditis elegans encodes multiple isoforms of a protein homologous to the mammalian brain-specific clathrin-adaptor protein AP180. The UNC-11 protein is expressed at high levels in the nervous system and at lower levels in other tissues. In neurons, UNC-11 is enriched at presynaptic terminals but is also present in cell bodies. unc-11 mutants are defective in two aspects of synaptic vesicle biogenesis. First, the SNARE protein synaptobrevin is mislocalized, no longer being exclusively localized to synaptic vesicles. The reduction of synaptobrevin at synaptic vesicles is the probable cause of the reduced neurotransmitter release observed in these mutants. Second, unc-11 mutants accumulate large vesicles at synapses. We propose that the UNC-11 protein mediates two functions during synaptic vesicle biogenesis: it recruits synaptobrevin to synaptic vesicle membranes and it regulates the size of the budded vesicle during clathrin coat assembly.     

A structural model for phosphorylation control of Dictyostelium myosin II thick filament assembly

Deletion of the synapsin I genes, encoding one of the major groups of proteins on synaptic vesicles, in mice causes late onset epileptic seizures and enhanced experimental temporal lobe epilepsy. However, mice lacking synapsin I maintain normal excitatory synaptic transmission and modulation but for an enhancement of paired-pulse facilitation. To elucidate the cellular basis for epilepsy in mutants, we examined whether the inhibitory synapses in the hippocampus from mutant mice are intact by electrophysiological and morphological means. In the cultured hippocampal synapses from mutant mice, repeated application of a hypertonic solution significantly suppressed the subsequent transmitter release, associated with an accelerated vesicle replenishing time at the inhibitory synapses, compared with the excitatory synapses. In the mutants, morphologically identifiable synaptic vesicles failed to accumulate after application of a hypertonic solution at the inhibitory preterminals but not at the excitatory preterminals. In the CA3 pyramidal cells in hippocampal slices from mutant mice, inhibitory postsynaptic currents evoked by direct electrical stimulation of the interneuron in the striatum oriens were characterized by reduced quantal content compared with those in wild type. We conclude that synapsin I contributes to the anchoring of synaptic vesicles, thereby minimizing transmitter depletion at the inhibitory synapses. This may explain, at least in part, the epileptic seizures occurring in the synapsin I mutant mice.     

The dual phosphatase activity of synaptojanin1 is required for both efficient synaptic vesicle endocytosis and reavailability at nerve terminals

Phosphoinositides have been implicated in synaptic vesicle recycling largely based on studies of enzymes that regulate phosphoinositide synthesis and hydrolysis. One such enzyme is synaptojanin1, a multifunctional protein conserved from yeast to humans, which contains two phosphoinositol phosphatase domains and a proline-rich domain. Genetic ablation of synaptojanin1 leads to pleiotropic defects in presynaptic function, including accumulation of free clathrin-coated vesicles and delayed vesicle reavailability, implicating this enzyme in postendocytic uncoating of vesicles. To further elucidate the role of synaptojanin1 at nerve terminals, we performed quantitative synaptic vesicle recycling assays in synj1(-/-) neurons. Our studies show that synaptojanin1 is also required for normal vesicle endocytosis. Defects in both endocytosis and postendocytic vesicle reavailability can be fully restored upon reintroduction of synaptojanin1. However, expression of synaptojanin1 with mutations abolishing catalytic activity of each phosphatase domain reveals that the dual action of both domains is required for normal synaptic vesicle internalization and reavailability.     

Dap160/intersectin acts as a stabilizing scaffold required for synaptic development and vesicle endocytosis

We describe the isolation of mutations in dynamin-associated protein 160 kDa (dap160), the Drosophila homolog of intersectin, a putative adaptor for proteins involved in endocytosis, cytoskeletal regulation, and signaling. We show that partial loss-of-function mutants display temperature-sensitive (ts) paralysis, whereas null mutants show ts defects in endocytosis. Loss-of-function mutants exhibit bouton overgrowth at larval neuromuscular junctions (NMJs), but evoked neurotransmission is normal. Mutant NMJs show a mild endocytic defect at 22 degrees C, which is strongly enhanced at 34 degrees C. The levels of dynamin, synaptojanin and endophilin are severely reduced in dap160 mutant NMJs, suggesting that Dap160 serves to stabilize an endocytic macromolecular complex. Electron microscopy reveals fewer vesicles, aberrant large vesicles, and an accumulation of endocytic intermediates at active and periactive zones in mutant terminals. Our data suggest that Dap160, like dynamin, is involved in synaptic vesicle retrieval at active and periactive zones.     

Altered synaptic development and active zone spacing in endocytosis mutants

Many types of synapses have highly characteristic shapes and tightly regulated distributions of active zones, parameters that are important to the function of neuronal circuits. The development of terminal arborizations must therefore include mechanisms to regulate the spacing of terminals, the frequency of branching, and the distribution and density of release sites. At present, however, the mechanisms that control these features remain obscure. Here, we report the development of supernumerary or "satellite" boutons in a variety of endocytic mutants at the Drosophila neuromuscular junction. Mutants in endophilin, synaptojanin, dynamin, AP180, and synaptotagmin all show increases in supernumerary bouton structures. These satellite boutons contain releasable vesicles and normal complements of synaptic proteins that are correctly localized within terminals. Interestingly, however, synaptojanin terminals have more active zones per unit of surface area and more dense bodies (T-bars) within these active zones, which may in part compensate for reduced transmission per active zone. The altered structural development of the synapse is selectively encountered in endocytosis mutants and is not observed when synaptic transmission is reduced by mutations in glutamate receptors or when synaptic transmission is blocked by tetanus toxin. We propose that endocytosis plays a critical role in sculpting the structure of synapses, perhaps through the endocytosis of unknown regulatory signals that organize morphogenesis at synaptic terminals.     

Tomosyn Inhibits Synaptic Vesicle Priming in Caenorhabditis elegans

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.     

Axonal transport of mitochondria to synapses depends on milton,a novel Drosophila protein

A protein required to localize mitochondria to Drosophila nerve terminals has been identified genetically. Photoreceptors mutant for milton show aberrant synaptic transmission despite normal phototransduction. Without Milton, synaptic terminals and axons lack mitochondria, although mitochondria are numerous in neuronal cell bodies. In contrast, synaptic vesicles continue to be transported to and concentrated at synapses. Milton protein is associated with mitochondria and is present primarily in axons and synapses. A likely explanation of the apparent trafficking defect is offered by the coimmunoprecipitation of Milton and kinesin heavy chain. Transfected into HEK293T cells, Milton induces a redistribution of mitochondria within the cell. We propose that Milton is a mitochondria-associated protein required for kinesin-mediated transport of mitochondria to nerve terminals.     

Tomosyn Inhibits Synaptic Vesicle Priming in Caenorhabditis elegans

Caenorhabditis elegans TOM-1 is orthologous to vertebrate tomosyn, a cytosolic syntaxin-binding protein implicated in the modulation of both constitutive and regulated exocytosis. To investigate how TOM-1 regulates exocytosis of synaptic vesicles in vivo, we analyzed C. elegans tom-1 mutants. Our electrophysiological analysis indicates that evoked postsynaptic responses at tom-1 mutant synapses are prolonged leading to a two-fold increase in total charge transfer. The enhanced response in tom-1 mutants is not associated with any detectable changes in postsynaptic response kinetics, neuronal outgrowth, or synaptogenesis. However, at the ultrastructural level, we observe a concomitant increase in the number of plasma membrane-contacting vesicles in tom-1 mutant synapses, a phenotype reversed by neuronal expression of TOM-1. Priming defective unc-13 mutants show a dramatic reduction in plasma membrane-contacting vesicles, suggesting these vesicles largely represent the primed vesicle pool at the C. elegans neuromuscular junction. Consistent with this conclusion, hyperosmotic responses in tom-1 mutants are enhanced, indicating the primed vesicle pool is enhanced. Furthermore, the synaptic defects of unc-13 mutants are partially suppressed in tom-1 unc-13 double mutants. These data indicate that in the intact nervous system, TOM-1 negatively regulates synaptic vesicle priming.     

Differential expression of endophilin 1 and 2 dimers at central nervous system synapses

Endophilin 1 is proposed to participate in synaptic vesicle biogenesis through SH3 domain-mediated interactions with the polyphosphoinositide phosphatase synaptojanin and the GTPase dynamin. Endophilin family members have also been identified as binding partners for a number of diverse cellular proteins. We define here the endophilin 1-binding site within synaptojanin 1 and show that this sequence independently and selectively purifies from brain extracts endophilin 1 and a closely related protein, endophilin 2. Endophilin 2, like endophilin 1, is highly expressed in brain, concentrated in nerve terminals, and found in complexes with synaptojanin and dynamin. Although a fraction of endophilins 1 and 2 coexist in the same complex, the distribution of these endophilin isoforms among central synapses only partially overlaps. Endophilins 1 and 2 are found predominantly as stable dimers through a predicted coiled-coil domain in their conserved NH2-terminal moiety. Dimerization may allow endophilins to link a number of different cellular targets to the endocytic machinery.     

VGLUT1 and VGAT are sorted to the same population of synaptic vesicles in subsets of cortical axon terminals

Glutamate and GABA mediate most of the excitatory and inhibitory synaptic transmission; they are taken up and accumulated in synaptic vesicles by specific vesicular transporters named VGLUT1-3 and VGAT, respectively. Recent studies show that VGLUT2 and VGLUT3 are co-expressed with VGAT. Because of the relevance this information has for our understanding of synaptic physiology and plasticity, we investigated whether VGLUT1 and VGAT are co-expressed in rat cortical neurons. In cortical cultures and layer V cortical terminals we observed a population of terminals expressing VGLUT1 and VGAT. Post-embedding immunogold studies showed that VGLUT1+/VGAT+ terminals formed both symmetric and asymmetric synapses. Triple-labeling studies revealed GABAergic synapses expressing VGLUT1 and glutamatergic synapses expressing VGAT. Immunoisolation studies showed that anti-VGAT immunoisolated vesicles contained VGLUT1 and anti-VGLUT1 immunoisolated vesicles contained VGAT. Finally, vesicles containing VGAT resident in glutamatergic terminals undergo active recycling. In conclusion, we demonstrate that in neocortex VGLUT1 and VGAT are co-expressed in a subset of axon terminals forming both symmetric and asymmetric synapses, that VGLUT1 and VGAT are sorted to the same vesicles and that vesicles at synapses expressing the vesicular heterotransporter participate in the exo-endocytotic cycle.     

Activity-independent prespecification of synaptic partners in the visual map of Drosophila

Specifying synaptic partners and regulating synaptic numbers are at least partly activity-dependent processes during visual map formation in all systems investigated to date . In Drosophila, six photoreceptors that view the same point in visual space have to be sorted into synaptic modules called cartridges in order to form a visuotopically correct map . Synapse numbers per photoreceptor terminal and cartridge are both precisely regulated . However, it is unknown whether an activity-dependent mechanism or a genetically encoded developmental program regulates synapse numbers. We performed a large-scale quantitative ultrastructural analysis of photoreceptor synapses in mutants affecting the generation of electrical potentials (norpA, trp;trpl), neurotransmitter release (hdc, syt), vesicle endocytosis (synj), the trafficking of specific guidance molecules during photoreceptor targeting (sec15), a specific guidance receptor required for visual map formation (Dlar), and 57 other novel synaptic mutants affecting 43 genes. Remarkably, in all these mutants, individual photoreceptors form the correct number of synapses per presynaptic terminal independently of cartridge composition. Hence, our data show that each photoreceptor forms a precise and constant number of afferent synapses independently of neuronal activity and partner accuracy. Our data suggest cell-autonomous control of synapse numbers as part of a developmental program of activity-independent steps that lead to a "hard-wired" visual map in the fly brain.     

A Polychromatic Stain for Use in Parasitology

In sensory systems, insight into synaptic arrangements on cells of known physiological response properties has helped our understanding of the structural basis for these properties. To carry out these types of studies, however, synaptic types in the region of interest must be defined. Unfortunately, defining synaptic types in the brainstem has proved to be a challenging enterprise. Our study was done to classify synapses in the gustatory part of the nucleus solitarius using objective quantitative criteria and a cluster analysis procedure. Cluster analysis allows classification of a population of objects, such as synaptic terminals, into groups that exhibit similar characteristics. Six terminal types were identified using cluster analysis and subsequent analyses of variance and post hoc tests. Unlike classification schemes used for the cerebral cortex, where synaptic apposition density thickness and shape of vesicles is useful (Gray's Type I and II synapses), the concentration of vesicles in a terminal was a more useful measurement with which to classify terminals in the nucleus solitarius. To validate that vesicle density (vesicles/μm²) is a useful defining characteristic to classify terminals in the nucleus solitarius, terminals of a known type were used. GABAergic terminals were identified using postembedding immunohistochemical techniques, and their vesicle density was determined. GABAergic terminals fall into the range of two of the terminal types defined by the cluster analysis and, based on vesicle density, two types of GABAergic terminals were identified. We conclude that vesicle density is a helpful means to identify synapses in this brainstem nucleus.