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.     

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.     

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.     

UNC-41/stonin functions with AP2 to recycle synaptic vesicles in Caenorhabditis elegans

The recycling of synaptic vesicles requires the recovery of vesicle proteins and membrane. Members of the stonin protein family (Drosophila Stoned B, mammalian stonin 2) have been shown to link the synaptic vesicle protein synaptotagmin to the endocytic machinery. Here we characterize the unc-41 gene, which encodes the stonin ortholog in the nematode Caenorhabditis elegans. Transgenic expression of Drosophila stonedB rescues unc-41 mutant phenotypes, demonstrating that UNC-41 is a bona fide member of the stonin family. In unc-41 mutants, synaptotagmin is present in axons, but is mislocalized and diffuse. In contrast, UNC-41 is localized normally in synaptotagmin mutants, demonstrating a unidirectional relationship for localization. The phenotype of snt-1 unc-41 double mutants is stronger than snt-1 mutants, suggesting that UNC-41 may have additional, synaptotagmin-independent functions. We also show that unc-41 mutants have defects in synaptic vesicle membrane endocytosis, including a ～50% reduction of vesicles in both acetylcholine and GABA motor neurons. These endocytic defects are similar to those observed in apm-2 mutants, which lack the μ2 subunit of the AP2 adaptor complex. However, no further reduction in synaptic vesicles was observed in unc-41 apm-2 double mutants, suggesting that UNC-41 acts in the same endocytic pathway as μ2 adaptin.     

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.     

Synaptojanin 2 functions at an early step of clathrin-mediated endocytosis

Synaptojanin 2 is a ubiquitously expressed polyphosphoinositide phosphatase that displays a high degree of homology in its catalytic domains with synaptojanin 1 [1,2]. Neurons of synaptojanin 1-deficient mice display an increase in clathrin-coated vesicles and delayed reentry of recycling vesicles into the fusion-competent vesicle pool, but no defects in early steps of endocytosis [3,4]. Here we show that inhibition of synaptojanin 2 expression via small interfering (si) RNA causes a strong defect in clathrin-mediated receptor internalization in a lung carcinoma cell line. This inhibitory phenotype is rescued by overexpression of wild-type synaptojanin 2, but not of wild-type synaptojanin 1 or mutant synaptojanin 2 that is deficient in 5'-phosphatase activity. In addition, electron-microscopic analysis shows that synaptojanin 2 depletion causes a decrease in clathrin-coated pits and vesicles. These results suggest a role for synaptojanin 2 in clathrin-coated pit formation and imply that lipid hydrolysis is required at an early stage of clathrin-mediated endocytosis. Taken together, our results also indicate that synaptojanin 2 is functionally distinct from synaptojanin 1.     

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.     

Mutations in the unc-41 Gene Cause Elevation of Acetylcholine Levels

Abstract: Mutations in the Caenorhabditis elegans unc-41 gene result in an allele-dependent elevation of acetylcholine content. Eight recessive alleles ( cn252, e268, e399, e650, e1175, e1199, e1294, and e870 ) lead to phenotypes including uncoordinated locomotion, slow growth, a small mature body, and resistance to the acetylcholinesterase inhibitors as well as the elevation of acetylcholine content. The remaining two alleles, e554 and e1162 , exhibit normal acetylcholine levels but display the short-body phenotype in a semidominant way. To determine the localization of the elevated acetylcholine content, a method for the isolation of synaptic vesicles from C. elegans was established. The elevation of acetylcholine content in the unc-41 mutants is accompanied by the accumulation of synaptic vesicles. We propose that at least one function of the unc-41 gene relates to the release of neurotransmitters.     

Recruitment of an alternatively spliced form of synaptojanin 2 to mitochondria by the interaction with the PDZ domain of a mitochondrial outer membrane protein.

Synaptojanin 1 is an inositol 5'-phosphatase highly enriched in nerve terminals with a putative role in recycling of synaptic vesicles. We have previously described synaptojanin 2, which is more broadly expressed as multiple alternatively spliced forms. Here we have identified and characterized a novel mitochondrial outer membrane protein, OMP25, with a single PDZ domain that specifically binds to a unique motif in the C-terminus of synaptojanin 2A. This motif is encoded by the exon sequence specific to synaptojanin 2A. OMP25 mRNA is widely expressed in rat tissues. OMP25 is localized to the mitochondrial outer membrane via the C-terminal transmembrane region, with the PDZ domain facing the cytoplasm. Overexpression of OMP25 results in perinuclear clustering of mitochondria in transfected cells. This effect is mimicked by enforced expression of synaptojanin 2A on the mitochondrial outer membrane, but not by the synaptojanin 2A mutants lacking the inositol 5'-phosphatase domain. Our findings provide evidence that OMP25 mediates recruitment of synaptojanin 2A to mitochondria and that modulation of inositol phospholipids by synaptojanin 2A may play a role in maintenance of the intracellular distribution of mitochondria.     

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.     

UNC-16, a JNK-signaling scaffold protein, regulates vesicle transport in C. elegans.

Transport of synaptic components is a regulated process. Loss-of-function mutations in the C. elegans unc-16 gene result in the mislocalization of synaptic vesicle and glutamate receptor markers. unc-16 encodes a homolog of mouse JSAP1/JIP3 and Drosophila Sunday Driver. Like JSAP1/JIP3, UNC-16 physically interacts with JNK and JNK kinases. Deletion mutations in Caenorhabditis elegans JNK and JNK kinases result in similar mislocalization of synaptic vesicle markers and enhance weak unc-16 mutant phenotypes. unc-116 kinesin heavy chain mutants also mislocalize synaptic vesicle markers, as well as a functional UNC-16::GFP. Intriguingly, unc-16 mutations partially suppress the vesicle retention defect in unc-104 KIF1A kinesin mutants. Our results suggest that UNC-16 may regulate the localization of vesicular cargo by integrating JNK signaling and kinesin-1 transport.     

Identification and characterization of a synaptojanin 2 splice isoform predominantly expressed in nerve terminals

We have previously identified synaptojanin 1, a phosphoinositide phosphatase predominantly expressed in the nervous system, and synaptojanin 2, a broadly expressed isoform. Synaptojanin 1 is concentrated in nerve terminals, where it has been implicated in synaptic vesicle recycling and actin function. Synaptojanin 2A is targeted to mitochondria via a PDZ domain-mediated interaction. We have now characterized an alternatively spliced form of synaptojanin 2 that shares several properties with synaptojanin 1. This isoform, synaptojanin 2B, undergoes further alternative splicing to generate synaptojanin 2B1 and 2B2. Both amphiphysin and endophilin, two partners synaptojanin 1, bind synaptojanin 2B2, whereas only amphiphysin binds synaptojanin 2B1. Sequence similar to the endophilin-binding site in synaptojanin 1 is present only in synaptojanin 2B2, and this sequence was capable of affinity purifying endophilin from rat brain. The Sac1 domain of synaptojanin 2 exhibited phosphoinositide phosphatase activity very similar to that of the Sac1 domain of synaptojanin 1. Site-directed mutagenesis further illustrated its functional similarity to the catalytic domain of Sac1 proteins. Antibodies raised against the synaptojanin 2B-specific carboxyl-terminal region identified a 160-kDa protein in brain and testis. Immunofluorescence showed that synaptojanin 2B is localized at nerve terminals in brain and at the spermatid manchette in testis. Active Rac1 GTPase affects the intracellular localization of synaptojanin 2, but not of synaptojanin 1. These results suggest that synaptojanin 2B has a partially overlapping function with synaptojanin 1 in nerve terminals, with additional roles in neurons and other cells including spermatids.     

UNC-13 interaction with syntaxin is required for synaptic transmission

Neurotransmitter secretion at synapses is controlled by several processes-morphological docking of vesicles at release sites, priming of docked vesicles to make them fusion competent, and calcium-dependent fusion of vesicles with the plasma membrane . In worms, flies, and mice, mutants lacking UNC-13 have defects in vesicle priming . Current models propose that UNC-13 primes vesicles by stabilizing Syntaxin's "open" conformation by directly interacting with its amino-terminal regulatory domain . However, the functional significance of the UNC-13/Syntaxin interaction has not been tested directly. A truncated protein containing the Munc homology domains (MHD1 and MHD2) and the carboxy-terminal C2 domain partially rescued both the behavioral and secretion defects of unc-13 mutants in C. elegans. A double mutation in MHD2 (F1000A/K1002A) disrupts the UNC-13/Syntaxin interaction. The rate of endogenous synaptic events and the amplitude of nerve-evoked excitatory post-synaptic currents (EPSCs) were both significantly reduced in UNC-13S(F1000A/K1002A). However, the pool of primed (i.e., fusion-competent) vesicles was normal. These results suggest that the UNC-13/Syntaxin interaction is conserved in C. elegans and that, contrary to current models, the UNC-13/Syntaxin interaction is required for nerve-evoked vesicle fusion rather than synaptic-vesicle priming. Thus, UNC-13 may regulate multiple steps of the synaptic-vesicle cycle.     

Synaptic polarity depends on phosphatidylinositol signaling regulated by myo-inositol monophosphatase in Caenorhabditis elegans

Although neurons are highly polarized, how neuronal polarity is generated remains poorly understood. An evolutionarily conserved inositol-producing enzyme myo-inositol monophosphatase (IMPase) is essential for polarized localization of synaptic molecules in Caenorhabditis elegans and can be inhibited by lithium, a drug for bipolar disorder. The synaptic defect of IMPase mutants causes defects in sensory behaviors including thermotaxis. Here we show that the abnormalities of IMPase mutants can be suppressed by mutations in two enzymes, phospholipase Cβ or synaptojanin, which presumably reduce the level of membrane phosphatidylinositol 4,5-bisphosphate (PIP(2)). We also found that mutations in phospholipase Cβ conferred resistance to lithium treatment. Our results suggest that reduction of PIP(2) on plasma membrane is a major cause of abnormal synaptic polarity in IMPase mutants and provide the first in vivo evidence that lithium impairs neuronal PIP(2) synthesis through inhibition of IMPase. We propose that the PIP(2) signaling regulated by IMPase plays a novel and fundamental role in the synaptic polarity.     

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.     

A putative cation channel, NCA-1, and a novel protein, UNC-80, transmit neuronal activity in C. elegans

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the α1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.     

A Putative Cation Channel, NCA-1, and a Novel Protein, UNC-80, Transmit Neuronal Activity in C. elegans

Voltage-gated cation channels regulate neuronal excitability through selective ion flux. NALCN, a member of a protein family that is structurally related to the α1 subunits of voltage-gated sodium/calcium channels, was recently shown to regulate the resting membrane potentials by mediating sodium leak and the firing of mouse neurons. We identified a role for the Caenorhabditis elegans NALCN homologues NCA-1 and NCA-2 in the propagation of neuronal activity from cell bodies to synapses. Loss of NCA activities leads to reduced synaptic transmission at neuromuscular junctions and frequent halting in locomotion. In vivo calcium imaging experiments further indicate that while calcium influx in the cell bodies of egg-laying motorneurons is unaffected by altered NCA activity, synaptic calcium transients are significantly reduced in nca loss-of-function mutants and increased in nca gain-of-function mutants. NCA-1 localizes along axons and is enriched at nonsynaptic regions. Its localization and function depend on UNC-79, and UNC-80, a novel conserved protein that is also enriched at nonsynaptic regions. We propose that NCA-1 and UNC-80 regulate neuronal activity at least in part by transmitting depolarization signals to synapses in C. elegans neurons.     

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.     

The SAC1 domain in synaptojanin is required for autophagosome maturation at presynaptic terminals

Presynaptic terminals are metabolically active and accrue damage through continuous vesicle cycling. How synapses locally regulate protein homeostasis is poorly understood. We show that the presynaptic lipid phosphatase synaptojanin is required for macroautophagy, and this role is inhibited by the Parkinson's disease mutation R258Q. Synaptojanin drives synaptic endocytosis by dephosphorylating PI(4,5)P₂, but this function appears normal in Synaptojanin^RQ knock‐in flies. Instead, R258Q affects the synaptojanin SAC1 domain that dephosphorylates PI(3)P and PI(3,5)P₂, two lipids found in autophagosomal membranes. Using advanced imaging, we show that Synaptojanin^RQ mutants accumulate the PI(3)P/PI(3,5)P₂‐binding protein Atg18a on nascent synaptic autophagosomes, blocking autophagosome maturation at fly synapses and in neurites of human patient induced pluripotent stem cell‐derived neurons. Additionally, we observe neurodegeneration, including dopaminergic neuron loss, in Synaptojanin^RQ flies. Thus, synaptojanin is essential for macroautophagy within presynaptic terminals, coupling protein turnover with synaptic vesicle cycling and linking presynaptic‐specific autophagy defects to Parkinson's disease.     

ITSN-1 controls vesicle recycling at the neuromuscular junction and functions in parallel with DAB-1

Intersectins (Itsn) are conserved EH and SH3 domain containing adaptor proteins. In Drosophila melanogaster, ITSN is required to regulate synaptic morphology, to facilitate efficient synaptic vesicle recycling and for viability. Here, we report our genetic analysis of Caenorhabditis elegans intersectin. In contrast to Drosophila , C. elegans itsn-1 protein null mutants are viable and display grossly normal locomotion and development. However, motor neurons in these mutants show a dramatic increase in large irregular vesicles and accumulate membrane-associated vesicles at putative endocytic hotspots, approximately 300 nm from the presynaptic density. This defect occurs precisely where endogenous ITSN-1 protein localizes in wild-type animals and is associated with a significant reduction in synaptic vesicle number and reduced frequency of endogenous synaptic events at neuromuscular junctions (NMJs). ITSN-1 forms a stable complex with EHS-1 (Eps15) and is expressed at reduced levels in ehs-1 mutants. Thus, ITSN-1 together with EHS-1, coordinate vesicle recycling at C. elegans NMJs. We also found that both itsn-1 and ehs-1 mutants show poor viability and growth in a Disabled (dab-1) null mutant background. These results show for the first time that intersectin and Eps15 proteins function in the same genetic pathway, and appear to function synergistically with the clathrin-coat-associated sorting protein, Disabled, for viability.