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.
Keywords: acetylcholine receptor, sphingosine kinase/sphk-1, major facilitator superfamily domain (MFSD) proteins, acetylcholine transporter, unc-17, lipid, seizure, epilepsy, locomotion
Cholinergic transmission underlies a variety of processes including learning, memory, and movement. iAChRs are evolutionarily conserved pentameric channels that regulate neuronal activity in the central nervous system and at the neuromuscular junction (Albuquerque et al. 2009). Multiple mutations in the human iAChR subunits encoded by CHRNA2 (α2), CHRNA4 (α4), and CHRNB2 (β4), have been linked to autosomal dominant forms of epilepsy (Boillot and Baulac 2016). Most disease-associated mutations in α2, α4, or β4 cluster in the second or third transmembrane (TM) domain and generally elicit gain-of-function phenotypes (Bertrand et al. 2002, 2005; Leniger et al. 2003; Hoda et al. 2008).
The Caenorhabditis elegans genome encodes over 30 AChR subunits (Hobert 2013). Decades of study have revealed the subunit composition of heteromeric and homomeric channels that act in different tissues or cells and that display differences in channel physiology and pharmacology. We previously characterized the ACR-2R pentameric ion channel that is expressed in cholinergic motorneurons (Jospin et al. 2009). A V309M gain-of-function mutation in the second TM domain of the ACR-2 subunit causes elevated cholinergic activity. Additionally, acr-2(gf) results in a cell nonautonomous decrease in the activity of inhibitory GABAergic neurons (Jospin et al. 2009; Stawicki et al. 2011). The concurrent increase in cholinergic excitation and decrease in GABA inhibition results in overexcitation of the motor circuit. This activity imbalance causes defective locomotion accompanied by spontaneous contractions of the body wall muscles, referred to as convulsions. Previous studies of genetic mutations that restored wild-type locomotion to acr-2(gf) animals identified UNC-38, UNC-63, and ACR-12 as the other subunits that form functional receptors with ACR-2 (Jospin et al. 2009). Additional suppressors of the acr-2(gf) convulsion phenotype defined a divalent cation transient receptor potential channel subfamily M (TRPM) that modulates locomotor circuit via ion homeostasis, and a novel mutation in unc-13 that affects synaptic transmission through positional docking of synaptic vesicles (Stawicki et al. 2011; Zhou et al. 2013).
To further characterize the molecular pathways that mediate the effects of the overactive ACR-2R(gf), we expanded the genetic suppressor screen of acr-2(gf). Here, we report the identification of novel mutations in multiple genes that regulate synaptic transmission. Many mutations are partial loss-of-function alleles in genes required for synaptic function. We identified multiple mutations affecting a conserved MFSD protein, mfsd-6. In addition, our analysis of sphk-1, the C. elegans homolog of human Sphk, suggests a neuronal subtype specific role for this kinase in promoting cholinergic activity in acr-2(gf) animals. This screen expands our understanding of the function of AChRs and provides a useful resource to dissect how synaptic transmission is modulated in the context of an in vivo neural circuit.
Materials and Methods
C. elegans genetics and mutagenesis screen
Strains were maintained at room temperature or 20° as described (Brenner 1974). Genetic crosses were performed using standard methods. The genotypes of strains are listed in Supplemental Material, Table S1. The previous suppressor mutations of acr-2(gf) were selected based on faster movement than acr-2(gf) (Jospin et al. 2009). Here, we performed a semiclonal screen, focusing on mutations that primarily reduced the convulsion frequency. Briefly, acr-2(n2420gf) L4 animals (CZ10402) were subjected to 50 mM ethyl methanesulfonate following standard protocols (Kutscher and Shaham 2014). Forty P0 animals were placed on individual plates to allow egg-laying for 24 hr, averaging 30–50 F1 per P0. The P0 animals were transferred to fresh plates every day for 3 d, giving rise to ∼20,000 mutagenized haploid genomes. The F2 progeny were screened for a reduction in convulsion frequency. Only one line derived from an individual P0 was kept for subsequent analysis, resulting in a total of 31 lines. Since levamisole-resistant mutations were overrepresented in the previous screen (Jospin et al. 2009), we first tested the suppressor mutations for levamisole sensitivity. Eight lines were found to be resistant to 1 mM levamisole and were not pursued in further analysis.
Mutation identification by outcrossing and whole-genome sequencing
Twenty-three suppressor lines that showed normal sensitivity to levamisole were outcrossed to N2. Three suppressor mutations were linked to the X-chromosome and were identified to be one intragenic loss-of-function mutation of acr-2(gf) and two loss-of-function mutations in acr-12. These suppressor lines were not subjected to further analysis. We obtained whole-genome sequencing data on 20 outcrossed suppressor strains. The Galaxy platform (Afgan et al. 2016) was used to analyze raw sequence files with a custom-designed workflow. We used custom-designed software to identify SNPs affecting restriction enzyme sites in the mutagenized suppressor strains compared to the reference N2 sequences. These SNPs were then used to follow chromosome linkage after further outcrossing of the suppressors to N2 (Table S2). For example, for mapping sphk-1(ju831), all of the outcrossed strains contained a SNP on chromosome II but not SNPs on chromosome III or IV. For mapping the unc-63(ju815) allele, CZ24017 (Table S2) was outcrossed to N2, and linkage of the ju815 mutation to chromosome I was determined based on cosegregation of the suppression effect with the unc-63 SNP and a SNP in anc-1(I), ∼1.36 map units from the unc-63 (I) locus (Table S1). Multiple outcrossings of unc-63(ju815) were then conducted to eliminate other SNPs nearby to generate CZ25251.
Among the remaining 20 independent lines, only unc-63(ju815) behaved as a completely dominant suppressor of acr-2(gf). To outcross the unc-63(ju815) mutation, wild-type males heterozygous for an integrated fluorescent transgene, either (juIs76) or (juIs14), were crossed into the ju815; acr-2(gf) strain. The fluorescent transgenes were used to verify isolation of suppressed cross progeny. Nonconvulsing F2s were isolated from heterozygous F1s carrying either juIs14 or juIs76 transgenes and verified as homozygous for the acr-2(gf) mutation by Sanger sequencing. Using the combination of whole-genome sequencing and SNP mapping analyses, we identified the causative mutations in all but two of the 20 levamisole-sensitive suppressor lines. Both ju807 and ju863 showed linkage to chromosome II; however, the causative mutations have not been determined (Table S2).
Convulsion behavioral observation and pharmacological analysis
All behavioral observations were made on mutations that were outcrossed with N2 at least four times. Convulsions were defined as simultaneous contraction of the body wall muscles producing a concerted shortening in body length. The convulsion frequency for d1 adult animals was calculated during a 90 sec period of visual observation.
For levamisole sensitivity, 10 d1 adult animals were transferred to fresh plates containing 1 mM levamisole and were monitored every 15 min for paralysis. For aldicarb sensitivity, 0.5 mM aldicarb was used for strains containing acr-2(gf) or mfsd-6 alleles, and 1.5 mM aldicarb was used on all other strains. Aldicarb sensitivity was assessed by transferring 10 d1 adults to fresh aldicarb plates, and by monitoring worms for paralysis every 30 min by gently touching the animal with a platinum wire. Aldicarb sensitivity was quantified for at least three independent experiments.
Fluorescent microscopy and image analysis
SPHK-1::GFP (nuIs197) was analyzed by confocal microscopy (LSM710, Zeiss) in wild-type and acr-2(gf) animals. The dorsal cord of L4 animals was imaged under identical settings for all samples, as previously described (Cherra and Jin 2016). Fluorescence intensity and area of each punctum was measured from a 0.5 μm Z-plane using the Analyze Particles function in NIH ImageJ.
Data availability
All reagents including strains and the diagnostic SNP analysis program are available upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.
Results and Discussion
To identify additional genes contributing to the acr-2(gf) convulsion phenotype, we performed a semiclonal genetic suppressor screen for worms that showed a reduction in convulsion frequency. Following pharmacological tests using levamisole, an agonist of muscle iAChRs, and aldicarb, an acetylcholinesterase inhibitor, as well as outcrossing and genetic mapping, we identified a total of 20 independent suppressor lines that showed normal sensitivity to levamisole. Two loss-of-function mutations affected the TRPM channel gtl-2 (Stawicki et al. 2011; Takayanagi-Kiya et al. 2016). One mutation, ju825, was characterized as a gain-of-function mutation in the ACC family of ligand-gated channel lgc-46 (Takayanagi-Kiya et al. 2016). Two suppressors were loss-of-function mutations in the neuronal calcium sensor protein ncs-2 (Zhou et al. 2017). Two suppressors were mapped to chromosome II, but the causative mutations have yet to be identified (Table 1). The 13 suppressors described here in detail can be organized into two major categories: novel mutations in ACR-2R subunits, and mutations that affect synaptic vesicle loading, exocytosis, or recycling (Table 1).
Table 1. acr-2(gf) suppressors.
Gene | Allele | Nucleotide Changea | Amino Acid Changeb |
---|---|---|---|
AChR subunits | |||
unc-38 | ju852 | cCc/cAc | P494H |
unc-38 | ju857 | Gga/Aga | G321R |
unc-63 | ju860 | atG/atA | M150I |
unc-63 | ju815 | tGt/tAt | C294Y |
Synaptic genes | |||
unc-31 | ju818 | Cga/Tga | R1180* |
sphk-1 | ju831 | Cca/Tca | P177S |
unc-17 | ju840 | tCc/tTc | S398F |
unc-17 | ju854 | Ccc/Tcc | P415S |
unc-41 | ju873 | ctG/ctA | Q91* |
unc-13 | ju874 | atttcaGcttccttg/atttcaActtccttg | Splice site: intron 26/exon 27 |
mfsd-6 | ju833 | Gga/Aga | G524R |
mfsd-6 | ju866 | gGa/gAa | G421E |
mfsd-6 | ju870 | ttG/ttA | Q76* |
Unidentified | |||
ju807(II) | |||
ju863(II) |
AChR, acetylcholine receptor.
Capital letters indicate mutated nucleotide. The left is the reference sequence and on the right, is the mutated sequence. For the ju874 mutation, underlined sequence is intronic sequence prior to splice site.
Amino acid position is based on that for protein isoform UNC-31B, UNC-41A, UNC-13A, respectively.*indicates stop codon.
Novel mutations in iAChR subunits suppress acr-2(gf)
UNC-63 and UNC-38 are both ACh-binding α subunits of the ACR-2R. We have previously identified multiple recessive alleles of unc-63 and unc-38 that suppress the acr-2(gf) convulsion frequency and also show strong resistance to levamisole (Jospin et al. 2009). Here, we found several levamisole-sensitive alleles of unc-63 and unc-38 that behaved as recessive suppressors of acr-2(gf) (Table 1). The M150I mutation in UNC-63 (ju860) and the G321R or P494H mutation in UNC-38 (ju852, ju857) showed disparate distribution throughout the receptors (Figure 1, A and B). These mutations may either alter the binding of ACh but not levamisole, or may alter the function of neuronal iAChRs but only mildly affect the muscle iAChRs. Of particular note, for UNC-63, animals harboring the C151Y mutation are resistant to levamisole (Lewis et al. 1980) but the M150I mutants remain sensitive to levamisole, highlighting the importance of obtaining a deeper understanding of the structure–function relationship of iAChR subunits.
Figure 1.
Levamisole-sensitive unc-63 and unc-38 mutants suppress acr-2(gf). (A) Protein diagrams illustrating known and new alleles of unc-63. New alleles isolated in the acr-2(gf) suppressor screen are indicated with a ju allele number. G121R, K130I, and P159S were previously isolated through an acr-2(gf) screen (Jospin et al. 2009), and C151Y was isolated through a levamisole resistance screen (Lewis et al. 1980). Levamisole-sensitive alleles are labeled in black and levamisole-resistant alleles are labeled in gray. unc-63(ju815) affects the TM2 domain at a residue conserved from C. elegans to mammals. Protein alignments labeled as: C. e. = C. elegans UNC-63, D. m. = Drosophila melanogaster ACH4, D. r. = Danio rerio ACHA2 and ACHB4, and H. s. = Homo sapiens ACHA2 and ACHA6. (B) Protein diagrams illustrating known and new alleles of unc-38. P159L was previously isolated in a levamisole resistance screen (Garcia et al. 2001), and P111L, P267L, and G477E were previously isolated in an acr-2(gf) suppressor screen (Jospin et al. 2009). Black and gray alleles indicate levamisole sensitivity and resistance, respectively. Protein alignments labeled as C. e. = C. elegans UNC-38, D. m. = Drosophila melanogaster ACH4, D. r. = Danio rerio ACHA2 and ACHB2, and H. s. = Homo sapiens ACHA2 and ACHA3. (C) unc-63(ju815) completely suppresses acr-2(gf) convulsions. N ≥ 17. (D) unc-63(ju815) does not cause strong resistance to 1 mM levamisole. unc-63(x37) results in a premature stop and is a null allele. N = 10 animals each trial, average of three trials in shown. *** P < 0.001, two-way ANOVA followed by Bonferroni’s post hoc test. aa, amino acid; TM, transmembrane.
While all other mutations in unc-63 or unc-38 were recessive for suppression of acr-2(gf), the ju815 mutation in unc-63, which affects TM2, acted in a dominant manner to suppress acr-2(gf) convulsion frequency (Figure 1, A–C). We verified the dominant suppression of ju815 following extensive outcrossing and reisolation (Figure 1C, Table S1, and Table S2). unc-63(ju815) completely suppressed acr-2(gf) convulsions but does not cause a noticeable defect in locomotion as compared to unc-63 null alleles (Figure 1C, File S1, File S2, and File S7). To more quantitatively compare unc-63(ju815) to an unc-63 null mutation, unc-63(x37) (Lewis et al. 1980), we assayed these mutants for sensitivity to levamisole. unc-63(ju815) mutants were not as resistant to levamisole as unc-63(x37) animals, but unc-63(ju815) animals showed mild resistant to levamisole as compared to wild-type (Figure 1D).
The highly conserved TM2 domain lines the receptor pore and is critical for regulating the activity of iAChRs (Unwin 2005). The heteromeric nature of iAChRs presents a difficulty for understanding the functional interactions of the subunits without disrupting the entire receptor complex. The observation that unc-63(ju815) dominantly reduces acr-2(gf)-induced behavior yet does not show resistance to levamisole suggests that the requirement for the UNC-63 α subunit in neuronal ACR-2R and muscle Lev-R may differ significantly to provide different gating properties or ion flux. Together with the missense mutations that were reported previously to be resistant to levamisole (Jospin et al. 2009), these alleles provide useful information to further tease apart how heteromeric iAChRs with similar subunit compositions can have separate functions to regulate neural circuit and muscle activity.
Novel mutations in presynaptic proteins suppress acr-2(gf)
Several suppressors affected a set of genes that are required to maintain the efficient transmission of neurotransmitters or neuropeptides. We found ju874 to be an allele of unc-13, which is a phorbol ester/diacylglycerol-binding protein with multiple C2 domains (Maruyama and Brenner 1991). UNC-13 and its Munc13 homologs are presynaptic active zone proteins required for synaptic vesicle priming, and loss of unc-13 greatly reduces synaptic transmission (Aravamudan et al. 1999; Augustin et al. 1999; Richmond et al. 1999). We previously reported that both strong loss-of-function mutations of unc-13 and a unique mutation in the C2A domain of UNC-13 suppress convulsions of acr-2(gf) (Zhou et al. 2013). The ju874 allele disrupts the splice site between intron 26 and exon 27 and would be predicted to affect the extreme C-terminus of the protein (Table 1). unc-13(ju874) behaved as partial loss-of-function, as the animals show normal locomotion.
The ju873 mutation affects unc-41, the C. elegans Stoned B homolog, which is generally agreed to function in synaptic vesicle recycling (Walther et al. 2004; Diril et al. 2006; Mullen et al. 2012). The unc-41 gene produces two isoforms: the A isoform is broadly expressed in the nervous system, while the B isoform is exclusively expressed in GABA motor neurons (Mullen et al. 2012). The unc-41(ju873) allele results in a premature stop codon in the first exon of the A isoform (Figure 2A and Table 1). While unc-41 null mutations are not lethal, they cause multiple defects in locomotion and egg-laying (Mullen et al. 2012). We confirmed that ju873 is a new allele of unc-41 through complementation tests with a null allele, unc-41(e268). Consistent with unc-41(ju873) being partial loss-of-function, the locomotion defects of unc-41(ju873) are less severe than for unc-41(e268) (File S3 and File S4). Interestingly, unc-41(e268); acr-2(gf) and unc-41(ju873); acr-2(gf) animals displayed strongly reduced convulsion frequencies (Figure 2B), suggesting that the function of the UNC-41A isoform is rate-limiting for synaptic transmission in acr-2(gf).
Figure 2.
Novel mutations that alter synaptic vesicle function suppress acr-2(gf). (A) Gene structures of unc-41a and unc-41b isoforms are depicted as described in (Mullen et al. 2012). unc-41(ju873) causes a premature stop in the first exon of unc-41a, which is broadly expressed in the nervous system. unc-41(e268) is a premature stop and is a null for both unc-41 isoforms, producing no functional protein (Mullen et al. 2012). (B) unc-41 loss-of-function suppresses acr-2(gf) convulsions. N ≥ 19 each genotype. *** P < 0.001, one-way ANOVA on ranks followed by Dunn’s post hoc test. n.s., not significant. (C) Diagram of UNC-17 protein with previously studied aa changes in the UNC-17 protein labeled in gray (Zhu et al. 2001). The mutations isolated in our screen, both of which alter evolutionarily conserved residues, are labeled in black. Protein alignments labeled as: C. e. = C. elegans UNC-17, D. m. = Drosophila melanogaster VAChT, D. r. = Danio rerio VACh-B, and H. s. = Homo sapiens VAChT. (D) unc-17(lf) mutations suppress acr-2(gf) convulsion frequency. N ≥ 10 *** P < 0.001, One-way ANOVA on ranks followed by Dunn’s post hoc test. aa, amino acid; VAChT, vesicular acetylcholine transporter.
Consistent with our previous studies that revealed neuropeptide modulation of acr-2(gf) (Stawicki et al. 2013; Zhou et al. 2013), we identified ju818 to be a new allele of unc-31, the calcium-dependent secretion activator (Table 1). unc-31(ju818) causes a premature termination in all but one predicted isoform, and behaves as a null allele of unc-31 based on the suppression of acr-2(gf).
In addition to genes that directly regulate synaptic vesicle release and recycling, we have also identified two mutations affecting unc-17 (Figure 2, C and D and Table 1), the C. elegans vesicular ACh transporter (VAChT). Null alleles of unc-17 are lethal, and most strong loss-of-function mutants are extremely defective in locomotion and growth. The unc-17 mutants isolated from our screen behave as hypomorphs and show essentially normal locomotion (File S5 and File S6). The new alleles of unc-17 map to the region around TM11, which contains one previously reported partial loss-of-function mutation, Y400N, isolated in aldicarb resistance screens (Figure 2C) (Zhu et al. 2001). A previous study of unc-17 mutations affecting TM domains has used pharyngeal pumping or animal thrashing assays to rank mutation severity: TM9 mutations > TM6 mutations > TM5 mutations ≥ TM10 mutations (Zhu et al. 2001). The new alleles, ju840 and ju854, reside in TM11, and are similar to or slightly weaker than mutations in TM5 or TM10 based on pharyngeal pumping (data not shown), thus placing these alleles at the lower end of the severity spectrum. Previous transport activity analysis of unc-17 mutations has led to the identification of the binding site for vesamicol, a VAChT antagonist (Zhu et al. 2001). The alleles isolated here will provide additional coverage to further understand how VAChT functions to transport ACh and how this transporter is modulated. Overall, reduction-of-function mutations in genes involved in either SV loading, release, or endocytosis are strong suppressors of acr-2(gf) phenotypes, likely through reducing the efficiency of neurotransmission.
Loss-of-function in a novel conserved MFSD gene, mfsd-6, suppresses acr-2(gf)
The MFSD proteins are generally characterized by 10–12 TMs and play broad roles as transporters in vesicular or plasma membranes (Yan 2013). Members of this family include GluT, VAChT, and VGAT. We mapped three acr-2(gf) suppressor mutations to the R13A5.9 open reading frame (Figure 3A). R13A5.9 displays > 23% identity to the vertebrate protein known as MFSD6; therefore, this gene is renamed mfsd-6. An independent mutation, tm3356, deletes 227 bases in exon six, removing TM4, TM5, and part of TM6, and is therefore likely a null mutation (Figure 3A). The tm3356 allele suppressed acr-2(gf) convulsion frequency to a similar degree as the point mutations isolated in mfsd-6, which suggests that the suppression is due to loss-of-function in mfsd-6 (Figure 3B). Animals harboring null mutations in mfsd-6 were homozygous viable with no obvious locomotion defects; however, these mutants were resistant to aldicarb, an acetylcholinesterase inhibitor that causes eventual paralysis in wild-type animals (Figure 3C). Similar results have been observed for a different deletion allele of mfsd-6 (Ogurusu et al. 2015). Mutations in mfsd-6 were not resistant to levamisole, suggesting that mfsd-6 mutants do not display defects in muscle response to ACh. Since mutations in mfsd-6 prevent the paralysis caused by aldicarb, which causes a build-up of ACh, leading to prolonged muscle contraction, we hypothesize that mutations in mfsd-6 suppress the acr-2(gf) convulsions by disrupting presynaptic release of ACh. MFSD-6 localizes to presynaptic terminals in or near synaptic vesicles (Ogurusu et al. 2015). Therefore, we speculate that MFSD-6 may regulate synaptic vesicle trafficking or exocytosis to enable efficient synaptic transmission.
Figure 3.
Loss-of-function in a novel conserved major facilitator superfamily domain (MFSD) gene, mfsd-6, suppresses acr-2(gf). (A) Diagram of MFSD-6 protein with transmembrane (TM) domains labeled in gray. Two of the mutations identified in this screen alter evolutionarily conserved residues. Protein alignments labeled as: C. e. = C. elegans MFSD-6, D. m. = Drosophila melanogaster uncharacterized encoded by jef, D. r. = Danio rerio MFSD6-A, and H. s. = Homo sapiens MFSD6. (B) Loss-of-function in mfsd-6 suppresses acr-2(gf) convulsions. N ≥ 8 worms per genotype *** P < 0.001, ** P < 0.01, one-way ANOVA on ranks followed by Dunn’s post hoc test. (C) mfsd-6 mutations cause aldicarb resistance. Percent of animals not paralyzed over time on plates with 0.5 mM drug are shown. N = 10 animals in each trial, average of four trials shown, *** P < 0.001, ** P < 0.01, two-way ANOVA followed by Bonferroni’s post hoc test.
The TMs of MFSD family transporters have been grouped into three functional classes: substrate coordination, TM1, 4, 7, and 10; interdomain interactions, TM2, 5, 8, and 11; and structural integrity, TM3, 6, 9, and 12 (Yan 2013). As found with UNC-17, the most severe unc-17 mutants fall in the structural TMs TM9 or TM6. Interestingly, our current screen has identified mutations in both unc-17 and mfsd-6 that affect the interdomain interactions modulated by TM8 or TM11. There are currently multiple hypotheses regarding how the MFSD family may transport solutes (Quistgaard et al. 2016), and these novel mutations may provide further insight into how these proteins function, for example by illuminating the underlying structural changes that occur during solute transport. Mammalian MFSD6 shows expression in many areas of the brain, including the cortex, hippocampus, and midbrain [Allen Mouse Brain Atlas, Lein et al. (2007)], and its in vivo function remains unknown. Overall, the mutations in mfsd-6 provide a valuable entry point to investigate the function of this conserved protein family.
Loss-of-function mutations in sphk-1 suppress acr-2(gf) hyperactivity
We identified the ju831 mutation as affecting sphk-1, the sole C. elegans homolog of the conserved Sphk, which phosphorylates the lipid SPH to generate SPH-1-phosphate (S1P) (Spiegel and Milstien 2003). Previous studies have shown that SPHK-1 is localized near presynaptic terminals and that sphk-1 loss-of-function mutants exhibit a reduced evoked release from excitatory motor neurons, possibly by modulating synaptic vesicle recycling (Chan et al. 2012; Chan and Sieburth 2012; Shen et al. 2014). sphk-1(ju831) causes a conserved P177S mutation in the kinase domain, close to the ATP-binding site (Figure 4A). sphk-1(ju831) strongly suppresses acr-2(gf) convulsion frequency, which was rescued by expressing an SPHK-1 cDNA transgene driven by the endogenous sphk-1 promoter (Figure 4B). The null allele, sphk-1(ok1097), suppressed acr-2(gf) locomotion defects, but reduced convulsion frequency to a lesser degree as compared to ju831 (Figure 4B), suggesting that sphk-1(ju831) might act as a dominant-negative mutation.
Figure 4.
Loss of sphk-1 function suppresses acr-2(gf) phenotypes. (A) ju831 affects the kinase domain of sphk-1. Shown is the sphk-1 genomic locus and protein alignment of part of the kinase domain. sphk-1(ju831) is G/A transition in the fourth exon of the gene, causing a P177S mutation. ok1097 is a large deletion and null allele. Protein alignments labeled as: C. e. = C. elegans SPHK-1, D. m. = Drosophila melanogaster Sphk1, D. r. = Danio rerio Sphk1, and H. s. = Homo sapiens Sphk1. (B) sphk-1(ju831) and sphk-1(ok1097) both suppress acr-2(gf) convulsions. Transgenic expression of SPHK-1 under its endogenous promoter or the unc-129 promoter rescues sphk-1(ju831) suppression of acr-2(gf). N ≥ 18 each genotype *** P < 0.001, n.s., not significant. One-way ANOVA on ranks followed by Dunn’s post hoc test. (C) sphk-1 mutations cause aldicarb resistance. Shown are percentages of animals not paralyzed after 3 hr on 1.5 mM aldicarb. (D) sphk-1(ju831) suppresses acr-2(gf) aldicarb hypersensitivity. Shown are the percentages of animals that were not paralyzed after 1 hr on 0.5 mM aldicarb. Pharmacological data are averaged from at least three trials, N ≥ 9 animals per strain per trial. *** P < 0.001, ** P < 0.01, n.s., not significant, one-way ANOVA followed by Bonferroni’s post hoc test. SPHK, sphingosine kinase.
Previous studies have found that sphk-1(ok1097) mutants are resistant to 1.5 mM aldicarb as compared to wild-type (Chan et al. 2012). sphk-1(ju831) animals also showed resistance to paralysis after 3 hr incubation on 1.5 mM aldicarb, relative to wild-type animals (Figure 4C and Table S3). Consistent with being a dominant-negative mutation, sphk-1(ju831) mutants are more resistant to aldicarb than sphk-1(ok1097). Interestingly, we observed that overexpression of sphk-1 also induced aldicarb resistance to a similar level as sphk-1(ok1097), suggesting that in these conditions, excessive levels of sphk-1 also inhibit neurotransmission. Overexpression of wild-type sphk-1 reduced the aldicarb resistance of sphk-1(ju831) animals to that of the overexpression line alone (Figure 4C). In contrast to sphk-1 mutants, acr-2(gf) animals are hypersensitive to aldicarb (Jospin et al. 2009), becoming paralyzed after just 1 hr on a lower concentration of drug, 0.5 mM (Figure 4D and Table S4). sphk-1(ju831) suppressed the aldicarb hypersensitivity of acr-2(gf) animals back to wild-type levels (Figure 4D). Transgenic overexpression of wild-type sphk-1 restored aldicarb hypersensitivity to sphk-1(ju831); acr-2(gf) double mutants (Figure 4D), indicating that sphk-1 is critical for regulating cholinergic synaptic activity.
We have previously shown that the acr-2(gf) mutation is capable of driving the convulsion phenotype when expressed under the unc-129 promoter in the cholinergic motorneurons that form synapses in the dorsal cord (Qi et al. 2013). Interestingly, we found that expression of sphk-1 was also required only in this subset of cholinergic motorneurons to rescue the suppression effect of sphk-1(ju831) on convulsion frequency of acr-2(gf) (Figure 4B). These data suggest that SPHK-1 acts directly in the cholinergic neurons to mediate the convulsion behavior of acr-2(gf) animals.
sphk-1 contributes to acr-2(gf) convulsions independently of its regulatory Gqα pathway
In C. elegans motor neurons, activation of Gqα signaling, through either treatment with arecoline, a muscarinic agonist, or by a gain-of-function mutation in egl-30/Gqα, caused increased punctal expression of SPHK-1::GFP in axons and increased sensitivity to aldicarb (Chan et al. 2012, 2013; Chan and Sieburth 2012). The activity-induced SPHK-1::GFP expression required a G-protein signaling pathway involving the muscarinic receptor gene gar-3, the guanine exchanger factor Trio unc-73, and a calmodulin-like calcium binding protein calm-1 (Chan et al. 2012, 2013; Chan and Sieburth 2012). We wanted to address whether the increased cholinergic activity caused by acr-2(gf) might promote sphk-1 expression through the GAR-3 G-protein signaling pathway. Therefore, we used genetic analyses to test whether this pathway was required for acr-2(gf) convulsions, similar to sphk-1.
Double mutant combinations were made between gar-3 or calm-1 null alleles, or unc-73(ce362), a partial loss-of-function allele, and acr-2(gf). None of these double mutants showed detectable suppression of convulsions (Figure 5A), although calm-1(0); acr-2(gf) animals showed a slight, but not statistically significant, increase in convulsion rate. We next investigated whether functional redundancy from similar receptors could mask a role for GAR-3 in the cholinergic neuron response to acr-2(gf). To test this, we examined gar-2, which is also expressed in the cholinergic motorneurons and is thought to inhibit circuit activity through Goα signaling (Dittman and Kaplan 2008). We found that both gar-2(0); acr-2(gf) and gar-2(0); gar-3(0); acr-2(gf) mutants were not significantly different from acr-2(gf) alone (Figure 5A). Altogether, these genetic data suggest that, while the activity of calm-1 or the G-protein signaling pathway regulates SPHK-1 localization and function in a wild-type background, they are not necessary for sphk-1 function when cholinergic motor neurons are hyperactivated in acr-2(gf) mutants.
Figure 5.
SPHK-1 regulates acr-2(gf) convulsions independent of known regulatory mechanisms. (A) Loss-of-function mutations in gar-2, gar-3, unc-73, and calm-1 genes do not suppress acr-2(gf) convulsion rate. N ≥ 19, n.s., not significant, one-way ANOVA on ranks followed by Dunn’s post hoc test. (B) Expression of Punc-129::SPHK-1::GFP in the dorsal cord of wild-type and acr-2(gf) animals. Bar, 10 µm. (C) Quantification of SPHK-1::GFP punctal density. (D) Quantification of SPHK-1::GFP intensity. N ≥ 19 each genotype. n.s., not significant (P > 0.05), *** P < 0.001, Student’s t-test. SPHK, sphingosine kinase.
Although the G-protein signaling pathway that regulates SPHK-1 localization did not affect acr-2(gf) convulsions, it was possible that hyperactive ACR-2R caused by the acr-2(gf) mutation alters SPHK-1 localization through a parallel pathway to cause convulsions. Therefore, we investigated the localization and fluorescence intensity of SPHK-1::GFP in cholinergic motor neurons. Compared to wild-type, acr-2(gf) caused a small but statistically significant decrease in SPHK-1::GFP fluorescence intensity without altering synapse number (Figure 5, A, B and C). Therefore, unlike activation of Gqα signaling, which increased the expression of SPHK-1 in cholinergic motor neurons, hyperactivation of the ACR-2R does not cause increased synaptic SPHK-1 levels.
Loss-of-function in SPH metabolism genes does not affect acr-2(gf) convulsions
SPH can be converted to ceramide by ceramide synthase enzymes or to S1P through the activity of Sphk (Figure 6A) (Spiegel and Milstien 2003; Maceyka et al. 2012). Furthermore, S1P is irreversibly degraded by the enzyme S1P lyase. sphk-1 is the sole Sphk homolog in C. elegans, and sphk-1(lf) mutants should lack S1P. We next tested whether other enzymes in the SPH metabolism pathway both upstream and downstream of SPHK-1, were involved in mediating acr-2(gf) behaviors.
Figure 6.
Mutations in sphingolipid metabolism genes do not modulate acr-2(gf) convulsions. (A) Diagram of the sphingosine metabolism pathway with C. elegans homologs noted. (B) Loss-of-function mutations in the sphingosine metabolism pathway do not suppress acr-2(gf) convulsion rate. The hyl-1 and lagr-1 mutations are small deletions that disrupt the catalytic domains of these proteins (Deng et al. 2008), while the tag-38 mutation is a large deletion resulting in a molecular null. N ≥ 19, n.s., not significant, one-way ANOVA on ranks followed by Dunn’s post hoc test. CDase, ceramidase.
The C. elegans homologs of ceramide synthase include the genes hyl-1 and lagr-1, while tag-38 encodes the worm S1P lyase homolog. Lipid profiling has found that both hyl-1(lf) and sphk-1(ok1097) mutants accumulate SPH, while hyl-1(lf) was also shown to cause decreased levels of long-chain ceramides (Menuz et al. 2009). Both tag-38 and hyl-1 are expressed in the C. elegans cholinergic motor neurons, and TAG-38 strongly colocalizes with SPHK-1 in axons (Chan and Sieburth 2012). Null or strong loss-of-function mutants of tag-38 and hyl-1 are superficially wild-type, with normal locomotion, although hyl-1(lf) animals are hypersensitive to aldicarb, possibly due to increased SPH and S1P levels (Chan and Sieburth 2012). Given the increased aldicarb sensitivity of hyl-1(lf) mutants, one prediction would be that increased SPH and/or S1P levels resulting from hyl-1(lf) or tag-38(0) might enhance acr-2(gf) phenotypes. However, we found that neither tag-38(0) nor hyl-1(lf) had any effect on acr-2(gf) convulsion rate (Figure 6B). In addition to hyl-1, the C. elegans genome contains another putative ceramide synthase gene, lagr-1. Although no locomotion phenotype has been reported for lagr-1(lf), this mutation conferred resistance to radiation-induced apoptosis in the germline (Deng et al. 2008). In contrast, sphk-1(ok1097) results in increased radiation-induced cell death in the germline. lagr-1(lf) was epistatic to sphk-1(ok1097) in germline apoptosis due to lack of a ceramide, a proapoptotic lipid. However, we found that neither lagr-1(lf) nor lagr-1(lf); hyl-1(lf) had any significant effect on acr-2(gf) convulsions (Figure 6B). Therefore, increased SPH and S1P levels do not seem to affect acr-2(gf) convulsion rate. These results are consistent with the observation that overexpression of SPHK-1 also does not enhance convulsions (Figure 4B). Taken together, these genetic data highlight a novel, specific function of sphk-1 in the motor circuit in the context of acr-2(gf) that may be independent of the SPH metabolism pathway.
S1P has been shown to function primarily as a signaling molecule that regulates multiple processes, particularly apoptosis, and has been well-studied for its role in cancer (Maceyka et al. 2012). Some reports from mouse models and cell culture studies also support a conserved function for Sphk1 in promoting excitatory neurotransmission. Work in murine models found a role for Sphk1 in excitatory transmission in the hippocampus to promote learning and memory (Kanno et al. 2010). In cell culture experiments, Sphk1 localized to sites of endocytosis, and knockdown of Sphk1 resulted in decreased rates of endocytic uptake (Shen et al. 2014). These studies suggest that Sphk1 may function to mediate activity-dependent effects on endocytic recycling in the nervous system. Coincidentally, our screen has revealed a selective role of unc-41/stoned and sphk-1, both implicated in promoting endocytosis, in modulating the effects of a hyperactive neuronal AChR in the locomotion circuit. Future studies will explore possible links between these pathways.
Conclusions
Neurological disorders such as epilepsy often result from hyperactivity of cholinergic receptors, which can lead to disruptions in a diverse set of genes and pathways. We have used C. elegans to understand how the activity of neural circuits can be modulated in the context of circuit hyperactivity. The primary effect of acr-2(gf) is increased cholinergic release, and disruption of the ACR-2R itself (Jospin et al. 2009) or components of presynaptic release machinery are key points to modulate acr-2(gf) phenotypes (Zhou et al. 2013). This is further supported by the identification of novel hypomorphic alleles of genes, known to function in presynaptic release in the cholinergic system, that strongly suppress acr-2(gf). Our recent studies of another suppressor mutation lgc-46(ju825) also revealed an ACC family of ligand-gated anion channels that localize to the presynaptic terminal and may provide rapid feedback inhibition on synaptic vesicle release (Takayanagi-Kiya et al. 2016). Altogether, our findings demonstrate the power of genetic pathway dissection using the suppression of acr-2(gf) as a functional readout.
Supplementary Material
Supplemental material is available online at www.g3journal.org/lookup/suppl/doi:10.1534/g3.117.042598/-/DC1.
Acknowledgments
We thank Bhavika Anandpura for assistance in mapping the ju815 allele. We thank Derek Sieburth for OJ802 and KP4010 strains and Shohei Mitani at National BioResource Project in Japan for deletion alleles. Some strains were provided by the Caenorhabditis Genetics Center, which is funded by the National Institutes of Health (NIH) Office of Research Infrastructure Programs (P40 OD010440). This work was supported in part by NIH institutional training grants (T32 NS007220 to K.A.M. and S.J.C. and T32 AG000216 to K.A.M.), and grants to S.J.C. (F32 NS081945 and K99 NS097638) and Y.J. (R01 NS035546). Y.J. is an investigator of the Howard Hughes Medical Institute.
Footnotes
Communicating editor: D. S. Fay
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Data Availability Statement
All reagents including strains and the diagnostic SNP analysis program are available upon request. The authors state that all data necessary for confirming the conclusions presented in the article are represented fully within the article.