Dataset Information

Novel Mutations in Synaptic Transmission Genes Suppress Neuronal Hyperexcitation in Caenorhabditis elegans.

ABSTRACT: Acetylcholine (ACh) receptors (AChR) regulate neural circuit activity in multiple contexts. In humans, mutations in ionotropic acetylcholine receptor (iAChR) genes can cause neurological disorders, including myasthenia gravis and epilepsy. In Caenorhabditis elegans, iAChRs play multiple roles in the locomotor circuit. The cholinergic motor neurons express an ACR-2-containing pentameric AChR (ACR-2R) comprised of ACR-2, ACR-3, ACR-12, UNC-38, and UNC-63 subunits. A gain-of-function mutation in the non-? subunit gene acr-2 [acr-2(gf)] causes defective locomotion as well as spontaneous convulsions. Previous studies of genetic suppressors of acr-2(gf) have provided insights into ACR-2R composition and assembly. Here, to further understand how the ACR-2R regulates neuronal activity, we expanded the suppressor screen for acr-2(gf)-induced convulsions. The majority of these suppressor mutations affect genes that play critical roles in synaptic transmission, including two novel mutations in the vesicular ACh transporter unc-17 In addition, we identified a role for a conserved major facilitator superfamily domain (MFSD) protein, mfsd-6, in regulating neural circuit activity. We further defined a role for the sphingosine (SPH) kinase (Sphk) sphk-1 in cholinergic neuron activity, independent of previously known signaling pathways. Overall, the genes identified in our study suggest that optimal modulation of synaptic activity is balanced by the differential activities of multiple pathways, and the novel alleles provide valuable reagents to further dissect neuronal mechanisms regulating the locomotor circuit.

SUBMITTER: McCulloch KA

PROVIDER: S-EPMC5499116 | biostudies-other | 2017 Jul

REPOSITORIES: biostudies-other

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Publications

Novel Mutations in Synaptic Transmission Genes Suppress Neuronal Hyperexcitation in <i>Caenorhabditis elegans</i>.

McCulloch Katherine A KA Qi Yingchuan B YB Takayanagi-Kiya Seika S Jin Yishi Y Cherra Salvatore J SJ

G3 (Bethesda, Md.) 20170705 7

Acetylcholine (ACh) receptors (AChR) regulate neural circuit activity in multiple contexts. In humans, mutations in ionotropic acetylcholine receptor (iAChR) genes can cause neurological disorders, including myasthenia gravis and epilepsy. In <i>Caenorhabditis elegans</i>, iAChRs play multiple roles in the locomotor circuit. The cholinergic motor neurons express an ACR-2-containing pentameric AChR (ACR-2R) comprised of ACR-2, ACR-3, ACR-12, UNC-38, and UNC-63 subunits. A gain-of-function mutatio ...[more]

PMID: 28468816

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Project description:Synaptic vesicles (SVs) undergo a cycle of biogenesis and membrane fusion to release transmitter, followed by recycling. How exocytosis and endocytosis are coupled is intensively investigated. We describe an all-optical method for identification of neurotransmission genes that can directly distinguish SV recycling factors in C. elegans, by motoneuron photostimulation and muscular RCaMP Ca2+ imaging. We verified our approach on mutants affecting synaptic transmission. Mutation of genes affecting SV recycling (unc-26 synaptojanin, unc-41 stonin, unc-57 endophilin, itsn-1 intersectin, snt-1 synaptotagmin) showed a distinct 'signature' of muscle Ca2+ dynamics, induced by cholinergic motoneuron photostimulation, i.e. faster rise, and earlier decrease of the signal, reflecting increased synaptic fatigue during ongoing photostimulation. To facilitate high throughput, we measured (3-5 times) ~1000 nematodes for each gene. We explored if this method enables RNAi screening for SV recycling genes. Previous screens for synaptic function genes, based on behavioral or pharmacological assays, allowed no distinction of the stage of the SV cycle in which a protein might act. We generated a strain enabling RNAi specifically only in cholinergic neurons, thus resulting in healthier animals and avoiding lethal phenotypes resulting from knockdown elsewhere. RNAi of control genes resulted in Ca2+ measurements that were consistent with results obtained in the respective genomic mutants, albeit to a weaker extent in most cases, and could further be confirmed by opto-electrophysiological measurements for mutants of some of the genes, including synaptojanin. We screened 95 genes that were previously implicated in cholinergic transmission, and several controls. We identified genes that clustered together with known SV recycling genes, exhibiting a similar signature of their Ca2+ dynamics. Five of these genes (C27B7.7, erp-1, inx-8, inx-10, spp-10) were further assessed in respective genomic mutants; however, while all showed electrophysiological phenotypes indicative of reduced cholinergic transmission, no obvious SV recycling phenotypes could be uncovered for these genes.

Dataset Information

Novel Mutations in Synaptic Transmission Genes Suppress Neuronal Hyperexcitation in Caenorhabditis elegans.

Publications

Novel Mutations in Synaptic Transmission Genes Suppress Neuronal Hyperexcitation in <i>Caenorhabditis elegans</i>.

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