Novel Mutations in Synaptic Transmission Genes Suppress Neuronal Hyperexcitation inCaenorhabditis 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-α 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.

Sphingosine Kinase Activates the Mitochondrial Unfolded Protein Response and Is Targeted to Mitochondria by Stress

“…The sphk-1(ju831) mutant encodes a P177S substitution located adjacent to the nucleotide binding site in the SPHK-1 kinase domain at a residue that is conserved between C. elegans and mammals but is not conserved in budding yeast ( Pitson et al, 2002 ). Thus, we speculate that residual kinase activity in this strain may account for the weaker UPR mt defects seen in sphk-1(ju831) mutants compared to sphk-1 null mutants ( McCulloch et al, 2017 ). Nonetheless, we observed full rescue of sphk-1(ok1097) with two independent SPHK-1 transgenes (Figures 1C and S1F ), providing strong support for the conclusion that SPHK-1 activates the UPR mt .…”

“…The defects in UPR mt activation in sphk-1 mutants were similar to those seen when atfs-1 was knocked down by RNAi ( Figure 1A ; Haynes et al, 2010 ). Knockdown of sphk-1 by RNAi or by the independently isolated sphk-1(ju831) missense mutation ( McCulloch et al, 2017 ) attenuated paraquat-induced Phsp-6::GFP expression, albeit not as strongly as sphk-1(ok1097) mutations ( Figures S1A and S1B ). sphk-1 mutations did not attenuate the activation of the SKN-1/Nrf2-mediated antioxidant response (P gst-4::GFP reporter induction; Figure S1C ) ( Inoue et al, 2005 ) or the endoplasmic reticulum UPR (UPR er , P hsp-4::GFP reporter induction; Figure S1D ) ( Glover-Cutter et al, 2013 ).…”

Expanded genetic screening in Caenorhabditis elegans identifies new regulators and an inhibitory role for NAD+ in axon regeneration

“…MFSD family proteins have 10–12 transmembrane regions ( Yan, 2015 ); some mediate nutrient transport across the blood-brain barrier ( Ceder et al, 2017 ; Perland et al, 2017 ), but most are of unknown function. C. elegans MFSD-6 was previously identified as a regulator of motor circuit activity and mfsd-6(0) mutants are resistant to inhibitors of cholinesterase ( McCulloch et al, 2017 ). mfsd-6 is expressed in most neurons, including mechanosensory neurons ( Ogurusu et al, 2015 ).…”

Genetic Suppression of Basement Membrane Defects inCaenorhabditis elegansby Gain of Function in Extracellular Matrix and Cell-Matrix Attachment Genes

“…Worm genomic DNA was prepared for sequencing following the Gentra Puregene Tissue Kit DNA purification protocol. Whole genome sequencing (WGS) was performed by BGI, and analyzed as described ( McCulloch et al 2017 ). Strains were sequenced in batches, using the most outcrossed version of each suppressor available at the time.…”