Biomedical subjects

Joshua M Kaplan

Publications and source records attributed to Joshua M Kaplan.

13 recordsLinked to original sources

PubMed2006

PKC-1 regulates secretion of neuropeptides.

The secretion of neurotransmitters and neuropeptides is mediated by distinct organelles-synaptic vesicles (SVs) and dense-core vesicles (DCVs), respectively. Relatively little is known about the factors that differentially regulate SV and DCV secretion. Here we show that protein kinase C-1 (PKC-1), which is most similar to the vertebrate PKC eta and epsilon isoforms, regulates exocytosis of DCVs in Caenorhabditis elegans motor neurons. Mutants lacking PCK-1 activity had delayed paralysis induced by the acetylcholinesterase inhibitor aldicarb, whereas mutants with increased PKC-1 activity had more rapid aldicarb-induced paralysis. Imaging and electrophysiological assays indicated that SV release occurred normally in pkc-1 mutants. By contrast, genetic analysis of aldicarb responses and imaging of fluorescently tagged neuropeptides indicated that mutants lacking PKC-1 had reduced neuropeptide secretion. Similar neuropeptide secretion defects were found in mutants lacking unc-31 (encoding the protein CAPS) or unc-13 (encoding Munc13). These results suggest that PKC-1 selectively regulates DCV release from neurons.

Aldicarb↗

PubMed2006

Antagonistic regulation of synaptic vesicle priming by Tomosyn and UNC-13.

Priming of synaptic vesicles (SVs) is essential for synaptic transmission. UNC-13 proteins are required for priming. Current models propose that UNC-13 stabilizes the open conformation of Syntaxin, in which the SNARE helix is available for interactions with Synaptobrevin and SNAP-25. Here we show that Tomosyn inhibits SV priming. Tomosyn contains a SNARE motif, which forms an inhibitory SNARE complex with Syntaxin and SNAP-25. Mutants lacking Tomosyn have increased synaptic transmission, an increased pool of primed vesicles, and increased abundance of UNC-13 at synapses. Behavioral, imaging, and electrophysiological studies suggest that SV priming was reconstituted in unc-13 mutants by expressing a constitutively open mutant Syntaxin, or by mutations eliminating Tomosyn. Thus, priming is modulated by the balance between Tomosyn and UNC-13, perhaps by regulating the availability of open-Syntaxin. Even when priming was restored, synaptic transmission remained defective in unc-13 mutants, suggesting that UNC-13 is also required for other aspects of secretion.

Animals↗

PubMed2006

Factors regulating the abundance and localization of synaptobrevin in the plasma membrane.

After synaptic vesicle fusion, vesicle proteins must be segregated from plasma membrane proteins and recycled to maintain a functional vesicle pool. We monitored the distribution of synaptobrevin, a vesicle protein required for exocytosis, in Caenorhabditis elegans motor neurons by using a pH-sensitive synaptobrevin GFP fusion protein, synaptopHluorin. We estimated that 30% of synaptobrevin was present in the plasma membrane. By using a panel of endocytosis and exocytosis mutants, we found that the majority of surface synaptobrevin derives from fusion of synaptic vesicles and that, in steady state, synaptobrevin equilibrates throughout the axon. The surface synaptobrevin was enriched near active zones, and its spatial extent was regulated by the clathrin adaptin AP180. These results suggest that there is a plasma membrane reservoir of synaptobrevin that is supplied by the synaptic vesicle cycle and available for retrieval throughout the axon. The size of the reservoir is set by the relative rates of exo- and endocytosis.

Animals↗

PubMed2005

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.

Amino Acid Sequence↗

PubMed2005

Systematic analysis of genes required for synapse structure and function.

Chemical synapses are complex structures that mediate rapid intercellular signalling in the nervous system. Proteomic studies suggest that several hundred proteins will be found at synaptic specializations. Here we describe a systematic screen to identify genes required for the function or development of Caenorhabditis elegans neuromuscular junctions. A total of 185 genes were identified in an RNA interference screen for decreased acetylcholine secretion; 132 of these genes had not previously been implicated in synaptic transmission. Functional profiles for these genes were determined by comparing secretion defects observed after RNA interference under a variety of conditions. Hierarchical clustering identified groups of functionally related genes, including those involved in the synaptic vesicle cycle, neuropeptide signalling and responsiveness to phorbol esters. Twenty-four genes encoded proteins that were localized to presynaptic specializations. Loss-of-function mutations in 12 genes caused defects in presynaptic structure.

Aldicarb↗

PubMed2005

Using microarrays to facilitate positional cloning: identification of tomosyn as an inhibitor of neurosecretion.

Forward genetic screens have been used as a powerful strategy to dissect complex biological pathways in many model systems. A significant limitation of this approach has been the time-consuming and costly process of positional cloning and molecular characterization of the mutations isolated in these screens. Here, the authors describe a strategy using microarray hybridizations to facilitate positional cloning. This method relies on the fact that premature stop codons (i.e., nonsense mutations) constitute a frequent class of mutations isolated in screens and that nonsense mutant messenger RNAs are efficiently degraded by the conserved nonsense-mediated decay pathway. They validate this strategy by identifying two previously uncharacterized mutations: (1) tom-1, a mutation found in a forward genetic screen for enhanced acetylcholine secretion in Caenorhabditis elegans, and (2) an apparently spontaneous mutation in the hif-1 transcription factor gene. They further demonstrate the broad applicability of this strategy using other known mutants in C. elegans,Arabidopsis, and mouse. Characterization of tom-1 mutants suggests that TOM-1, the C. elegans ortholog of mammalian tomosyn, functions as an endogenous inhibitor of neurotransmitter secretion. These results also suggest that microarray hybridizations have the potential to significantly reduce the time and effort required for positional cloning.

Journal Article↗

PubMed2005

LIN-23-mediated degradation of beta-catenin regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans.

Ubiquitin-mediated protein degradation has been proposed to play an important role in regulating synaptic transmission. Here we show that LIN-23, the substrate binding subunit of a Skp1/Cullin/F Box (SCF) ubiquitin ligase, regulates the abundance of the glutamate receptor GLR-1 in the ventral nerve cord of C. elegans. Mutants lacking lin-23 had an increased abundance of GLR-1 in the ventral cord. The increase of GLR-1 was not caused by changes in the ubiquitination of GLR-1. Instead, SCF(LIN-23) regulates GLR-1 through the beta-catenin homolog BAR-1 and the TCF/Lef transcription factor homolog POP-1. We hypothesize that LIN-23-mediated degradation of BAR-1 beta-catenin regulates the transcription of Wnt target genes, which in turn alter postsynaptic properties.

Animals↗

PubMed2005

Functional genomic analysis of RNA interference in C. elegans.

RNA interference (RNAi) of target genes is triggered by double-stranded RNAs (dsRNAs) processed by conserved nucleases and accessory factors. To identify the genetic components required for RNAi, we performed a genome-wide screen using an engineered RNAi sensor strain of Caenorhabditis elegans. The RNAi screen identified 90 genes. These included Piwi/PAZ proteins, DEAH helicases, RNA binding/processing factors, chromatin-associated factors, DNA recombination proteins, nuclear import/export factors, and 11 known components of the RNAi machinery. We demonstrate that some of these genes are also required for germline and somatic transgene silencing. Moreover, the physical interactions among these potential RNAi factors suggest links to other RNA-dependent gene regulatory pathways.

Amino Acid Motifs↗

PubMed2004

The anaphase-promoting complex regulates the abundance of GLR-1 glutamate receptors in the ventral nerve cord of C. elegans.

The anaphase-promoting complex (APC) is a multisubunit E3 ubiquitin ligase that targets key cell cycle regulatory proteins for degradation. Blockade of APC activity causes mitotic arrest. Recent evidence suggests that the APC may have roles outside the cell cycle. Several studies indicate that ubiquitin plays an important role in regulating synaptic strength. We previously showed that ubiquitin is directly conjugated to GLR-1, a C. elegans non-NMDA (N-methyl-D-aspartate) class glutamate receptor (GluR), resulting in its removal from synapses. By contrast, endocytosis of rodent AMPA GluRs is apparently regulated by ubiquitination of associated scaffolding proteins. Relatively little is known about the E3 ligases that mediate these effects. We examined the effects of perturbing APC function on postmitotic neurons in the nematode C. elegans. Temperature-sensitive mutations in APC subunits increased the abundance of GLR-1 in the ventral nerve cord. Mutations that block clathrin-mediated endocytosis blocked the effects of the APC mutations, suggesting that the APC regulates some aspect of GLR-1 recycling. Overexpression of ubiquitin decreased the density of GLR-1-containing synapses, and APC mutations blunted this effect. APC mutants had locomotion defects consistent with increased synaptic strength. This study defines a novel function for the APC in postmitotic neurons.

Anaphase-Promoting Complex-Cyclosome↗

PubMed2004

Clathrin-mediated endocytosis is required for compensatory regulation of GLR-1 glutamate receptors after activity blockade.

Chronic changes in neural activity trigger a variety of compensatory homeostatic mechanisms by which neurons maintain a normal level of synaptic input. Here we show that chronic activity blockade triggers a compensatory change in the abundance of GLR-1, a Caenorhabditis elegans glutamate receptor. In mutants lacking a voltage-dependent calcium channel (unc-2) or a vesicular glutamate transporter (VGLUT; eat-4), the abundance of GLR-1 in the ventral nerve cord was increased. Similarly, the amplitude of glutamate-evoked currents in ventral cord interneurons was increased in eat-4 VGLUT mutants compared with wild-type controls. The effects of eat-4 VGLUT mutations on GLR-1 abundance in the ventral cord were eliminated in double mutants lacking both the clathrin adaptin protein unc-11 AP180 and eat-4 VGLUT. In contrast, mutations that decreased ubiquitination of GLR-1 did not prevent increased ventral cord abundance of GLR-1 in eat-4 VGLUT mutants. Taken together, our results suggest that GLR-1 is regulated in a homeostatic manner and that this effect depends on clathrin-mediated endocytosis but does not require ubiquitination of GLR-1.

Animals↗

PubMed2003

The EGL-21 carboxypeptidase E facilitates acetylcholine release at Caenorhabditis elegans neuromuscular junctions.

Proneuropeptides are packaged into dense-core vesicles in which they are processed into active peptides by copackaged enzymes. Proprotein convertases (PCs) cleave precursors after dibasic residues, and carboxypeptidases remove basic residues from the C terminals. We show here that the Caenorhabditis elegans egl-21 gene encodes a protein that is very similar to carboxypeptidase E (CPE) and is broadly expressed in the nervous system. Mutants lacking either egl-21 CPE or egl-3, which encodes the C. elegans ortholog of PC type 2 (PC2), were defective for processing endogenously expressed FMRFamide (Phe-Met-Arg-Phe-NH2)-related peptides (FaRPs). Mutants lacking the unc-104 kinesin motor protein were defective for anterograde movement of dense-core vesicle components, including egl-3 PC2, egl-21 CPE, and FaRPs. We provide evidence that egl-3 PC2 and egl-21 CPE mutants have diminished acetylcholine release at neuromuscular junctions (NMJs). Taken together, these results suggest that egl-21 CPE and egl-3 PC2 process endogenous neuropeptides that facilitate acetylcholine release at C. elegans NMJs.

Acetylcholine↗

PubMed2003

Mutations in the ligand-binding and pore domains control exit of glutamate receptors from the endoplasmic reticulum in C. elegans.

The abundance of ion channels and neurotransmitter receptors in the plasma membrane is limited by the efficiency of protein folding and subunit assembly in the endoplasmic reticulum (ER). The ER has a quality-control system for monitoring nascent proteins, which prevents incompletely folded and assembled proteins from being transported from the ER. Chaperone proteins identify unfolded and misassembled proteins in the ER via retention motifs that are normally buried at intersubunit contacts or via carbohydrate residues that are attached to misfolded domains. Here, we examined the trafficking of a C. elegans non-NMDA glutamate receptor (GLR-1). We show that mutations in the pore domain (predicted to block ion permeation) and mutations in the ligand-binding domain (predicted to block glutamate binding) both caused a dramatic reduction in the synaptic abundance of GLR-1 and increased retention of GLR-1 in the ER. These results suggest that the structural integrity of the ligand-binding site and the pore domain of GLR-1 are monitored in the ER during the process of quality control.

Animals↗

PubMed2002

Ubiquitin and AP180 regulate the abundance of GLR-1 glutamate receptors at postsynaptic elements in C. elegans.

Regulated delivery and removal of alpha-amino-3-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) glutamate receptors (GluRs) from postsynaptic elements has been proposed as a mechanism for regulating synaptic strength. Here we test the role of ubiquitin in regulating synapses that contain a C. elegans GluR, GLR-1. GLR-1 receptors were ubiquitinated in vivo. Mutations that decreased ubiquitination of GLR-1 increased the abundance of GLR-1 at synapses and altered locomotion behavior in a manner that is consistent with increased synaptic strength. By contrast, overexpression of ubiquitin decreased the abundance of GLR-1 at synapses and decreased the density of GLR-1-containing synapses, and these effects were prevented by mutations in the unc-11 gene, which encodes a clathrin adaptin protein (AP180). These results suggest that ubiquitination of GLR-1 receptors regulates synaptic strength and the formation or stability of GLR-1-containing synapses.

Adaptor Proteins, Vesicular Transport↗