Weimer RM et al. (2000) West Coast Worm Meeting "ric-7 encodes a novel presynaptic protein required for neurotransmission"

Weimer RM, Jorgensen EM

[West Coast Worm Meeting, 2000]

Regulated release of neurotransmitter from presynaptic terminals requires the coordinated function of several presynaptic proteins. Our current knowledge of this process has come primarily from biochemical analysis of synaptic proteins. We have taken a genetic approach in Caenorhabditis elegans to complement these studies. Previously, we reported the identification of a novel protein, RIC-7, identified genetically by screens for neurotransmission mutants. We now report that RIC-7 functions in neurons and is localized to presynaptic terminals. Mutations in ric-7 result in a pleiotropic neurotransmission phenotype. ric-7 animals lack enteric muscle contractions during defecation and display a weak shrinker phenotype, indicating of a loss of GABAergic function. In addition, ric-7 animals are resistant to the acetylcholinesterase inhibitor aldicarb, although they remain sensitive to the acetylcholine receptor agonist levamisole indicating a presynaptic cholinergic defect. These phenotypes are not due to defects in neuronal developmental since the overall structure of the nervous system appears normal. This suggests that mutations in ric-7 disrupt a common presynaptic step in chemical neurotransmission. The ric-7 phenotype is rescued by an 18kb PCR fragment that contains a single open reading frame encoding a predicted 694 amino acid protein with no similarities to known proteins or functional motifs. Expression of the reporter gene green fluorescent protein (GFP) from the ric-7 promoter identifies a predominantly neuronal expression pattern. RIC-7 GFP fusion protein is localized to synaptic terminals independent of UNC-104 function. Furthermore, expression of RIC-7 solely in GABAergic neurons rescues all GABA-specific phenotypes. Together, these data suggest that ric-7 encodes a neuronal protein that is localized to and functions at presynaptic terminals. In addition, ultrastructural data indicate that synaptic vesicle biogenesis is not affected in a ric-7 mutant background. Thus, RIC-7 is likely to play a role in synaptic vesicle exocytosis. Ongoing experiments are designed to identify RIC-7 interacting proteins and to determine the molecular function of RIC-7 in neurotransmission.

Weimer RM et al. (1998) West Coast Worm Meeting "CLONING AND CHARACTERIZATION OF ric-7 AND ric-6, TWO NEUROTRANMISSION MUTANTS IN C. elegans"

Jorgensen EM, Weimer RM

[West Coast Worm Meeting, 1998]

Release of neurotransmitter into the synaptic cleft requires the coordinated function of a number of proteins at the presynaptic terminal. Our current understanding of this process has come, primarily, from biochemical analysis. We have taken a genetic approach to identify other proteins required for the release of neurotransmitter. To this end, we are currently in the process of characterizing two genes, ric-6 and ric-7, that were previously identified in a behavioral screen and appear defective in neurotransmission. Mutations in ric-7 disrupt both GABAergic and cholinergic neurotransmission. ric-7 animals lack enteric muscle contractions during defecation and display a weak shrinker phenotype, supporting a role for ric-7 in GABAergic function. Furthermore, ric-7 animals are resistant to aldicarb, an inhibitor of acetylcholinesterase, supporting a role for ric-7 in cholinergic function. By fluorescence microscopy, the organization of ventral cord motor neurons along with the density of neuromuscular junctions appears to be normal in ric-7 animals. This suggests that ric-7 is involved in the function rather than the development of neurons in C. elegans. The ric-7 phenotype is rescued by an 18kb fragment that is predicted to contain a single open reading frame encoding a 681 amino acid protein with no known homology. We are currently in the process of constructing RIC-7::GFP fusion proteins to determine the RIC-7 expression pattern. Less is known about the ric-6 gene. Like ric-7, a mutation in ric-6 result in aldicarb resistant animals that also display a weak shrinker phenotype supporting a role for ric-6 in both cholinergic and GABAergic function, respectively. However, ric-6 animals, unlike ric-7 animals, do not lack enteric muscle contraction during defecation. We have recently rescued the ric-6 phenotype with a pool of three cosmids, and we are in the process of cloning the ric-6 gene.

RM Weimer et al. (1999) International C. elegans Meeting "ric-6 and ric-7, two genes involved in C. elegans neurotransmission"

RM Weimer, EM Jorgensen

[International C. elegans Meeting, 1999]

Release of neurotransmitter into the synaptic cleft requires the coordinated action of numerous proteins at the presynaptic terminal. We have taken a genetic approach to identify proteins required for this process. To this end, we are in the process of characterizing two genes, ric-6 and ric-7 , that were previously identified in a behavioral screen and appear defective in neurotransmission. Mutations in ric-7 disrupt both GABAergic and cholinergic neurotransmission. ric-7 animals lack enteric muscle contractions during defecation and display a weak shrinker phenotype, both features of a loss of GABAergic function. In addition, ric-7 animals are resistant to the acetylcholinesterase inhibitor aldicarb, although they are sensitive to the acetylcholine receptor agonist levamisole indicating a presynaptic cholinergic defect. The organization of ventral cord motor neurons and the density of neuromuscular junctions appear to be normal in ric-7 animals and thus, RIC-7 does not function in neuronal development. Taken together, these phenotypes suggest a role for RIC-7 in the release of neurotransmitter at synapses. The ric-7 phenotype is rescued by an 18kb fragment that is predicted to contain a single open reading frame encoding a 694 amino acid protein with no known homology. Expression of green fluorescent protein (GFP) under the control of the ric-7 promoter identifies a predominately neuronal expression pattern. This data further supports the hypothesis that ric-7 encodes a neuronal protein involved in presynaptic function. We are currently in the process of identifying other loci that genetically interact with ric-7 . ric-6 , like ric-7 , also plays a role in both presynaptic cholinergic and GABAergic function. Mutations in ric-6 result in aldicarb resistant, levamisole sensitive animals that also display a weak shrinker phenotype. The ric-6 phenotype is rescued by a 5kb fragment that encodes the C. elegans homolog of the vacuolar ATPase B subunit. Vacuolar ATPase function is thought to be required for synaptic vesicle acidification, a process that is essential for loading neurotransmitter into vesicles. We will test this model using electrophysiological techniques recently developed in C. elegans .

Jason M McEwen et al. (2005) International Worm Meeting "UNC-18/nSec1 is required for proper trafficking of UNC-64/Syntaxin-1 in C. elegans neurons"

Josh Kaplan, Jason M McEwen

[International Worm Meeting, 2005]

UNC-18/nSec1 appears to have many roles in regulating synaptic vesicle release. Studies in Drosophila and cell culture have shown that UNC-18 may act as an inhibitor of synaptic transmission 2. In C. elegans, unc-18 mutant animals show a decrease in neurotransmitter secretion at the neuromuscular junction and in both worms and mice there is a decrease in the number of docked vesicles 1,4,5. One contributing factor in how UNC-18 positively regulates synaptic release may be the availability of UNC-64/Syntaxin-1 for forming SNARE complexes. If UNC-18/nSec1 is functioning to maintain levels of UNC-64/Syntaxin at the plasma membrane, this may explain some of the synaptic transmission defects seen in unc-18 mutant worms. Studies using non-neuronal vertebrate cell types have shown that UNC-18/nSec1 is required for UNC-64/Syntaxin-1 to be properly trafficked to the plasma membrane 3.To address the role of UNC-18 in regulating anterograde transport in neurons, we looked to see if UNC-64 is properly localized in unc-18 mutant worms. Using both antibodies against UNC-64 and an UNC-64a::GFP fusion protein we showed that a larger fraction of UNC-64 is retained in the cell bodies of neurons in unc-18 animals when compared to wild type. Trafficking of other synaptic proteins such as SNB-1/Synaptobrevin, RIC-4/SNAP-25, UNC-10/RIM-1 and SYD-2 is unaffected in unc-18 mutants, suggesting that this is not a general defect in intracellular transport. Current experiments are testing whether UNC-18 regulates ER or Golgi exit of UNC-64.1. Gengyo-Ando K et al. Neuron 19932. Schulze KL et al. Neuron 19943. Rowe J, et al. J Cell Sci 20014. Voets T, et al. Neuron 20015. Weimer RM, et al. Nature Neuroscience 2003

Michelle Po et al. (2007) International Worm Meeting "An unc-7 suppressor screen to identify novel active zone regulators."

Taizo Kawano, Michelle Po, Mei ZHEN, Magali BOUHOURS

[International Worm Meeting, 2007]

Synapses are asymmetric structures that facilitate the transmission of signals between neurons and their targets. The mature synapse consists of a presynaptic terminal in direct apposition to a postsynaptic terminal. Neurotransmission occurs when neurotransmitter-filled presynaptic vesicles dock and fuse with the plasma membrane. The neurotransmitter contents are released into the synaptic cleft and subsequently bind to receptors at the postsynapse. The active zone is an electron-dense region of the synapse that is required for the docking and fusion events of synaptic transmission. Our lab is interested in identifying genes that regulate the formation of active zones. In our study we are using the GABAergic nervous system of C. elegans as our model system. The active zones in this nervous system are visualized using a unique active zone specific marker SYD-2::GFP that was developed in our laboratory (Yeh et al., 2006). A forward genetic screen isolated an unc-7 loss-of-function mutant that shows a significant decrease in number of SYD-2::GFP puncta. We have determined that the innexin UNC-7 localizes to perisynaptic regions in addition to gap junctions. Subsequently, we showed that loss-of-function unc-9 mutants, which encodes an innexin protein that is 56% identical to UNC-7, shows the same synaptic defects as unc-7 mutants (Yeh and Ng, unpublished), suggesting a novel regulatory role for innexins at chemical synapses. We further investigated the role of innexins at the synapse by performing an unc-7 suppressor screen. We identified 10 dominant alleles of unc-1, a gene that encodes a stomatin homologue, that suppress the kinker behavior as well as the synaptic phenotype of unc-7 loss-of-function mutants. We found that UNC-7 and UNC-1 co-localize at peri-synaptic regions, indicating that the two proteins may functionally interact. We are investigating the physical properties of the potential UNC-7, UNC-9, and UNC-1 channel complexes. Current progress will be presented at the meeting. Yeh E, Kawano T, Weimer RM, Bessereau JL, Zhen M. 2005. Identification of genes involved in synaptogenesis using a fluorescent active zone marker in Caenorhabditis elegans. J Neurosci. 25(15):3833-41.

Richmond JE et al. (2000) West Coast Worm Meeting "The requirement of synaptic vesicle loading for synaptic vesicle exocytosis"

Weimer RM, Richmond JE, Jorgensen EM

[West Coast Worm Meeting, 2000]

Neurotransmitters are released from presynaptic terminals by the fusion of synaptic vesicles with the plasma membrane. The amount of neurotransmitter in a vesicle causes a consistent amount of current, called a quanta, in the postsynaptic cell. Studies have shown that quanta are invariant within a synapse. This observation raises an interesting question. Does synaptic vesicle fusion require the proper loading of neurotransmitter into the vesicle? We have characterized a mutant in C. elegans that will allow us to address this question. Loading of neurotransmitter into synaptic vesicles requires a vesicular neurotransmitter-specific transporter and a driving force. Several studies have shown that the vacuolar H+ ATPase (vATPase) generates the required driving force for loading. Previously, we reported the cloning of the C. elegans homolog of the vacuolar ATPase B subunit, which we now refer to as vha-12 (vacuolar-type H+ ATPase). A hypomorphic allele of vha-12 results in a pleiotropic neurotransmission phenotype which is consistent with the know function of the vATPase in neurotransmission. However, animals homozygous for the hypomorphic allele are not paralyzed. This suggests that neurotransmitter is still released from presynaptic terminals in a vha-12 animal. Two models could account for this observation (1) synaptic vesicles are fusing with the plasma membrane with incomplete loading of neurotransmitter or (2) only completely loaded vesicles are competent to fuse and the proportion of competent vesicles are decreased in a vha-12 mutant background. In order to distinguish between these two models we are characterizing neurotransmitter release events in a vha-12 background by electrophysiologically recording from neuromuscular junctions.

Edward Yeh et al. (2003) International Worm Meeting "A screen to identify factors involved in active zone formation."

Mei ZHEN, Edward YEH

[International Worm Meeting, 2003]

The active zone is a specialized presynaptic structure where vesicles containing neurotransmitters fuse with the plasma membrane, releasing the neurotransmitters to the post-synaptic membrane. At the EM level, electron dense active zones are surrounded by clusters of vesicles. A few components of the active zone have been identified such as UNC-10 (Koushika et al., 2001), UNC-13 (Richmond et al., 1999; Aravamudan et al., 1999) and SYD-2 (Zhen and Jin, 1999) but even fewer have been implicated in active zone establishment, differentiation or maintainance.syd-2 encodes a homolog of liprin- and has been shown to be a component of the active zone. Mutations in syd-2 give rise to larger active zones. At a previous meeting, we reported a SYD-2::GFP marker that could be used to visualize active zones in live animals. The SYD-2::GFP protein marker was placed under the promoter of unc-25 which expresses in GABAergic neurons. We have performed a SYD-2::GFP screen to identify a dditional factors involved in the formation of the active zone. An EMS mutagenesis screen was performed looking for mutations affecting the formation of the active zone. Approximately 2500 haploid genomes were screened and we are currently characterizing 5 isolated mutant alleles. hp77 and hp121 represent two loci on chromosome X, hp102 and hp198 represent two loci on chromosome IV and hp129 is on chromosome V. hp121 and hp102 have behavioural unc phenotypes, hp198 are slightly dumpy while hp77 and hp129 do not appear to have any behaviour or physical phenotypes. Initial characterization and phenotypic analysis will be presented on these mutants. It is our hope that the molecular characterization of these mutants will provide information on factors involved in the establishment, differentiation, and maintenance of active zones. Ultimately, this information will provide insight into the genetic pathways used to establish neural synapses in general.References: Aravamudan B, Fergestad T, Davis WS, Rodesch CK, and Broadie K. (1999) Nature Neuroscience. 2:965; Koushika SP, Richmond JE, Hadwiger G, Weimer RM, Jorgensen EM and Nonet M. (2001) Nature Neuroscience. 4:997-1005; Richmond JE, Davis WS and Jorgensen EM. (1999) Nature Neuroscience. 2:959-64; Zhen M and Jin Y. (1999) Nature. 401:371-5

Klosterman S M et al. (2010) Neuronal Development, Synaptic Function and Behavior, Madison, WI "Regulation of tomosyn (TOM-1) trafficking in C. elegans"

Klosterman S M, Richmond J E

[Neuronal Development, Synaptic Function and Behavior, Madison, WI, 2010]

Regulated exocytosis of secretory vesicles is critically dependent on the assembly of SNARE complexes between the plasma membrane SNAREs, synatxin and SNAP-25 and vesicle-associated synaptobrevin, which render vesicles fusion competent. Tomosyn, a syntaxin-binding protein, forms a complex with syntaxin and SNAP-25 in competition with synaptobrevin predicted to limit fusogenic SNARE complex formation (Fujita et al, 1998). Consistent with these data, the highly conserved C. elegans tomosyn homolog, TOM-1, has been shown to negatively regulate both synaptic and dense-core vesicle release (Gracheva et al., 2006; Gracheva et al.,2007). TOM-1 appears to be associated with both synaptic vesicles (Takamori et al., 2006) and dense core vesicles (Gracheva et al., 2007) via an unknown mechanism. Consequently, the synaptic localization of TOM-1 is dependent on the anterograde vesicle kinesin motor (UNC-104) (McEwen et al., 2006). Given the importance of TOM-1 in the regulation of synaptic strength, we are interested in identifying the mechanism responsible for the synaptic localization of this protein. Neither syntaxin nor SNAP-25, the two established tomosyn-interacting proteins appear to be involved in TOM-1 transport to synapses (McEwen et al., 2006). In order to screen for TOM-1 trafficking defective mutants, we have generated an integrated transgene expressing TOM-1::GFP. We have verified that this protein is expressed at synapses and localizes in an UNC-104-dependent manner. We are currently screening known vesicle-associated candidate proteins by RNAi or by crossing the TOM-1::GFP transgene into reference alleles to examine their potential role in the regulation of tomosyn trafficking. Fujita Y, Shirataki H, Sakisaka T, Asakura T, Ohya T, Kotani H, YokoyamaS, Nishioka H, Matsuura Y, Mizoguchi A, Scheller RH, Takai Y (1998) Tomosyn: asyntaxin-1-binding protein that forms a novel complex in the neurotransmitterrelease process. Neuron 20:905-915. Gracheva EO, Burdina AO, Touroutine D, Berthelot-Grosjean M, Parekh H,Richmond JE (2007) Tomosyn negatively regulates CAPS-dependent peptide releaseat Caenorhabditis elegans synapses. J Neurosci 27:10176-10184. Gracheva EO, Burdina AO, Holgado AM, Berthelot-Grosjean M, Ackley BD,Hadwiger G, Nonet ML, Weimer RM, Richmond JE (2006) Tomosyn inhibits synapticvesicle priming in Caenorhabditis elegans. PLoS Biol 4:e261.McEwen JM, Madison JM, Dybbs M, Kaplan JM (2006) Antagonistic regulationof synaptic vesicle priming by Tomosyn and UNC-13. Neuron 51:303-315. Takamori S et al. (2006) Molecular anatomy of a trafficking organelle.Cell 127:831-846.

Davis WS et al. (2000) West Coast Worm Meeting "Electrophysiological analysis of unc-18 mutants."

Richmond JE, Weimer RM, Davis WS, Jorgensen EM

[West Coast Worm Meeting, 2000]

UNC-18 is a C. elegans neuron-specific protein belonging to a conserved protein family implicated in vesicle fusion. UNC-18 interacts with the SNARE protein syntaxin, which is believed to mediate fusion of synaptic vesicles with the plasma membrane. Mutations in unc-18 result in severe locomotory defects and acetylcholine accumulation, implying a critical role for UNC-18 in synaptic transmission. Three mutants were examined, unc-18(e81 and e234) which have premature stop codons and unc-18(b403) which disrupts binding to syntaxin. The mutant phenotype was not caused by developmental abnormalities since GFP-expression patterns revealed motor neurons had normal morphology and exhibited wild-type distributions of synapses. We have used electrophysiological techniques to analyze synaptic function in these unc-18 mutants. Evoked release at the C. elegans neuromuscular junction was reduced to 5% of wild-type amplitudes (from 1800pA to 100pA) in all three mutants. A similar reduction in endogenous synaptic event frequency (from 44Hz to 5 Hz) was observed. The mutant synaptic event amplitudes were unaffected demonstrating that the reduction in the evoked response was not due to a defect in vesicle neurotransmitter filling or in post-synaptic receptor sensitivity. Preliminary EM data showed an accumulation of vesicles at the neuromuscular synapses of unc-18 mutants consistent with a defect in exocytosis as opposed to defects in vesicle biogenesis or retrieval. In the absence of external Ca2+, the rates of spontaneous fusion events were also reduced, suggesting that either the docking or fusion competence of unc-18 mutants is defective. Given these data, how might UNC-18 function in exocytosis? UNC-18 forms a heterodimer with syntaxin, which is mutually exclusive of SNARE complex formation. Several studies suggest that the interaction of UNC-18 and syntaxin is an essential step in regulated release, and that this interaction may be required to promote a conformational change in syntaxin which facilitates SNARE complex formation. To test this hypothesis, we have engineered a double mutation in syntaxin, which, in vitro eliminates UNC-18 binding, and induces the syntaxin open configuration. We are currently examining the effects of this mutation in wild-type, unc-64 (syntaxin) and unc-18 mutants.

Zhou, Y et al. (2017) International Worm Meeting "Genetic and chemical modulation of C. elegans intestinal antiviral innate immunity."

Zhou, Y, Tao, Y, Bedian, L, Zhong, W

[International Worm Meeting, 2017]

The RNA virus Orsay is the only known natural viral pathogen infecting the intestine cells of the nematode C. elegans. The C. elegans intestine cells resemble human intestinal epithelial cells not only in morphological features such as microvilli, but also in molecular mechanisms such as antibacterial innate immune response. As Orsay was discovered only six years ago, the C. elegans antiviral innate immune system remains largely unknown. Only two pathways, RNAi and ubiquitin-mediated protein degradation, have been discovered to be involved in the C. elegans antiviral innate immunity. To explore the genetic landscape of the C. elegans antiviral innate immunity, we conducted a genome-wide RNAi screen for genes whose inactivation sensitizes worms to Orsay infection. 110 genes were identified to be required for C. elegans antiviral innate immunity. 72 of these genes have human orthologs. Tissue-specific RNAi experiments showed that the majority of these genes function in the intestine cells to modulate antiviral innate immunity. In addition to RNAi and ubiquitin-mediated protein degradation, these genes encompass pathways in autophagy, mitochondrial unfolded protein responses, collagens, cytoskeletal organization, RNA processing, and transcription. To identify the gene products that are druggable, we screened 2000 chemicals for drugs that can alleviate Orsay infection symptoms, and discovered four innate immunity enhancing drugs, resorcinol monoacetate (RM), berberine (BBR), bismuth subsalicylate, 3,3'- diindolymethane (DIM). BBR and bismuth subsalicylate are known antidiarrhea drugs, with possible antiviral functions against human gastrointestinal viruses. Chemical-genetic experiments revealed that these drugs have different mechanism of actions: RM strengthens the collagen barrier for viral entry; BBR and DIM enhances the ubiquitin-mediated protein degradation pathway. Together, these data revealed a multifaceted antiviral innate immune system in the C. elegans intestine. Aspects of genetic and chemical modulation of this system may be conserved in human innate immunity against gastrointestinal viruses.