Loer, Curtis et al. (2009) International Worm Meeting "Functions of biopterin synthesis and recycling genes in C. elegans."

Werner, Ernst, Calvo, Ana, Loer, Curtis, Werner-Felmayer, Gabriele, Martinez, Aurora

[International Worm Meeting, 2009]

In mammals, the pterin cofactor tetrahydrobiopterin (BH4) is required for the function of aromatic amino acid hydroxylases (AAHs), nitric oxide synthases (NOSs), and glycerol ether monooxygenase (GEM). BH4 is synthesized de novo from GTP via 4 steps catalyzed by 3 enzymes: GTP cyclohydrolase I (GCH), pyruvoyl tetrahydropterin synthase (PTPS), and sepiapterin reductase (SR). The last enzyme, SR, carries out two final reducing steps. When used as a cofactor in the AAH reaction, BH4 is oxidized in the catalytic cycle. Reduced BH4 can be regenerated by 2 enzymes: pterin-4-a-carbinolamine dehydratase (PCBD) and quinonoid dihydropteridine reductase (QDPR). The AAHs requiring BH4 include the rate-limiting enzymes in the synthesis of serotonin and catecholamines (TPH and TH, respectively), and in phenylalanine catabolism (PAH, phenylalanine hydroxylase). C. elegans has clear orthologs of most biopterin synthesis and recycling genes. Some BH4-using enzymes are clearly present: all the AAHs exist (tph-1, cat-2 and pah-1 genes), but no clear NOS homologs are found in C. elegans. Mutants in the cat-4/GCH gene are serotonin- and dopamine-deficient, and are hypersensitive, perhaps due to a ''leaky'' cuticle; mutants in the ptps-1 gene appear to have an identical phenotype. The hypersensitivity phenotype may be caused by dysfunction of an unrecognized C. elegans enzyme that requires BH4. C. elegans lacks a clear SR ortholog; its function may be mediated by other aldo-keto-type reductases/short chain dehydrogenases. There are many candidate genes for this function in the C. elegans genome; none we have examined to date likely function as SR. We are also examining putative pcbd-1 and qdpr-1 mutants. Preliminary results with cat-4 and ptps-1 mutants show the predicted reduction or loss of GCH and PTPS activity, respectively, and accompanying reductions in pterin content. We are also characterizing the expression pattern of cat-4::GFP reporters (kindly provided by A. Chisholm); the reporter is expressed in 5HT and DA neurons, hypodermis and intestine.

Loer, Curtis M. et al. (2013) International Worm Meeting "Regulation of serotonergic neuron patterning in C. elegans by Wnt signaling genes."

Williams, Erin, Loer, Curtis M.

[International Worm Meeting, 2013]

In C. elegans, the homeotic complex (HOM-C) gene lin-39 is necessary for proper specification of many cells in the central body, including neurons in the ventral nerve cord (VNC). In the male, six central body VNC neurons (CP1 - CP6) normally express the neurotransmitter serotonin. In lin-39 mutant males, these cells fail to make serotonin or die. We have found that in lin-39; ced-3 double mutants - in which programmed cell death is blocked - all surviving CPs fail to make serotonin. Furthermore, the loss of serotonergic fate in the CPs in lin-39 mutants is complete: four different genes required for making and using serotonin (tph-1, bas-1, cat-1 and cat-4) all fail to be expressed normally in lin-39 mutants, as assessed by GFP reporters. Other serotonergic neurons express serotonin and these genes normally in lin-39 mutants. To find other genes that function similarly to lin-39 in specifying serotonergic fate in the CP neurons, we are knocking down candidate genes by RNAi. Since lin-39 is known to be regulated in vulval development by Wnt signaling, RTK signaling, and HOM-C protein regulators, we have screened these and other genes for effects of knockdown on serotonergic marker expression in CP neurons. We have found that knockdown of hmp-2, sys-1, and sem-4 all reduce serotonergic marker gene expression. Both hmp-2 and sys-1 function in Wnt/MAPK signaling, encoding b-catenin-like proteins. The sem-4 gene encodes a zinc-finger protein previously shown to interact with lin-39 in vulval development in the central body. At the same time, we are testing which 'neuronal RNAi enhancing' mutants or transgenics - such as uIs69 (with unc-119::sid-1 - neuronal expression of dsRNA transporter SID-1) with or without a sid-1 mutation - work best to increase RNA interference in these neurons (and/or their precursors).

Loer, Curtis et al. (2021) International Worm Meeting "Characterizing the nervous system of the nematode Pristionchus pacificus - similarities and differences with C. elegans"

Hobert, Oliver, Cook, Steve, Witte, Hanh, Loer, Curtis, Sommer, Ralf

[International Worm Meeting, 2021]

The nervous systems of many nematodes are remarkably similar despite considerable divergence of their genomes. We are examining the evolution of nematode nervous systems by characterizing the nervous system of the nematode Pristionchus pacificus (Ppa) to compare with that of the highly described C. elegans (Cel), which boasts a complete cell lineage, and a complete set of neurons and their connectivities in both sexes (Sulston & Horvitz, 1977; Sulston et al., 1983; White et al., 1986; Cook et al., 2019, Nature 571: 63-71). Within a few years, we and others seek to approach a similar level of characterization for Ppa. For example, the connectivity and likely homologies for head sensory neurons in Ppa have been determined, demonstrating considerable similarities plus a few striking differences, such as the absence of amphid 'winged' cilia found in Cel (Hong et al., 2019, eLife 2019;8:e47155). Considerable progress has been made in transgenics, traditional reporters and CRISPR generation of mutants and short knock-ins. To study neuronal specification in Ppa, we have epitope-tagged transcription factor genes to examine their expression patterns, including Ppa orthologs of unc-3, unc-86 and unc-42. We have also found that an antiserum to C. elegans unc-86 works in Ppa, showing a pattern identical to that of the epitope-tagged locus. The pattern of Ppa-unc-86 expression is remarkably similar to that of Cel; one difference is in the absence of clear HSN homologs in hermaphrodites. This is consistent with the absence of HSNs in Ppa seen with anti-serotonin. Other differences in serotonergic neurons suggest possible differences in the role of Ppa-unc-86 in Ppa vs. Cel. In Cel, the 'terminal selector' unc-86 plays a role in specifying serotonergic fate (Zhang et al., 2014, Development 141: 422-435). For example, in Cel, the AIM neurons express both unc-86 and serotonin; in Ppa presumptive AIM neurons lack serotonin but still express unc-86. We will present these and other results of our analysis of nervous system similarities and difference in C. elegans and P. pacificus.

Curtis Loer et al. (2007) International Worm Meeting "Evolution of Neuronal Patterning in Rhabditid Nematodes: Serotonergic Head Neurons."

Curtis Loer, Laura Rivard

[International Worm Meeting, 2007]

We have examined the serotonin-immunoreactive (serotonin-IR) neurons found in 17 species of free-living nematodes and compared them to the identified serotonin-IR neurons of C. elegans. We have previously described the sex-specific neurons found in the bodies of these worms (Loer & Rivard, 2007, J. Comp. Neurol., in press); here we describe neurons of the head which are typically the same in both sexes. Among the brightest, most reliably serotonin-IR neurons in C. elegans are the two NSMs (neurosecretory motoneurons) which are part of the pharyngeal nervous system. All nematode species we examined had a pair of neurons in the pharynx that, based on location and morphology, were clearly homologous to C. elegans NSMs. All the putative NSM homologs had the same basic neurite structure: a bifurcation near the soma sending two processes posteriorly, one in the dorsal pharyngeal isthmus and one in the ventral isthmus. In many species, we found amphid sensory neurons that may be homologous ADF in C. elegans. We found many other head neurons among the species examined - up to an additional 14 cells, mostly bilaterally paired. For many of these, homology to known C. elegans neurons is unclear.C. elegans.

Curtis M Loer et al. (2002) West Coast Worm Meeting "The cat-4/GTP cyclohydrolase I /F32G8.6 gene of C. elegans"

Scott DePaul, Curtis M Loer

[West Coast Worm Meeting, 2002]

We are characterizing genes used by serotonergic neurons in C. elegans to learn how they are regulated; among these is the cat-4 gene. Worms with mutations in the cat-4 gene lack serotonin and dopamine, and are hypersensitive to a variety of agents. The cat-4 gene appears to correspond to the predicted gene F32G8.6, which encodes GTP Cyclohydrolase I (GCH), an enzyme necessary for synthesis of biopterin. Biopterin is a cofactor required by (among others) all aromatic amino acid hydroxylases (AAAHs); these include the enzymes catalyzing synthesis of serotonin, dopamine and tyrosine. We have found that the original cat-4 mutant allele (e1141) has a missense mutation in the GCH coding sequence altering a highly conserved amino acid. A new mutant (kindly provided by the C.e. Gene Knockout Consortium) that deletes the F32G8.6/GCH gene is also serotonin-deficient, hypersensitive, and fails to complement cat-4(e1141). We hypothesize that hypersensitivity of cat-4 mutants is caused by tyrosine deficiency during cuticle synthesis, leading to a reduction in tyrosine cross-linking in cuticle proteins, and a 'leaky' cuticle. We are continuing characterization of the new cat-4 deletion allele, including comparing the presence of dityrosine cross-links in the cuticles of mutant vs. wild type worms.

Curtis M Loer (2004) West Coast Worm Meeting "EVOLUTION OF SEROTONIN SYNTHETIC ENZYME GENES AND THE PATTERNING OF SEROTONERGIC NEURONS IN CAENORHABDITIS AND OTHER NEMATODES"

Curtis M Loer

[West Coast Worm Meeting, 2004]

The biogenic amine neurotransmitters serotonin and dopamine are synthesized via two enzymatic steps by an aromatic amino acid hydroxylase (AAH) and aromatic amino acid decarboxylase (AADC). The AADC enzyme, which has a broad substrate specificity, is shared by both synthetic pathways in all animals, including the vertebrates, insects and nematodes. AADC belongs to a larger family of pyridoxal phosphate-dependent decarboxylases (PLP-DC) that includes histidine DC (histamine sythesis), tyrosine DC (octopamine synthesis in animals, alkaloid synthesis in plants) and the more distantly related glutamate decarboxylase (GABA synthesis). We have previously identified the serotonin- and dopamine synthetic AADC in the nematode C. elegans as encoded by the bas-1 gene. The C. elegans genome encodes five additional PLP-DC genes, some of which have no obvious homolog in other animals. In addition, two close relatives of bas-1 found in C. elegans (C05D2.3, F12A10.3) are missing from the C. briggsae genome. Phylogenetic analysis reveals that the bas-1 -like genes (or a common ancestor) were originally present in the C. briggsae lineage, but were lost. In C. elegans , the bas-1 -like gene F12A10.3 appears to be an expressed pseudogene. C05D2.3, which is just downstream of bas-1 (aka C05D2.4), encodes a protein lacking amino acids critical to the function of an AADC. Comparing ratios of non-synonymous to synonymous substitutions (K A /K S ) suggests that the bas-1 -like genes are under relaxed selection, unlike the bas-1 and other AADC genes, which are under strong purifying selection. Ongoing sequencing of other Caenorhabditis genomes should provide additional insight into questions about the evolutionary fates of duplicate genes such as these. We have also recently begun an examination of the patterning and function of serotonergic neurons in free-living nematode relatives of C. elegans . We have found considerable variability in the numbers and patterns of serotonergic neurons, especially those regulating reproductive behaviors. Supported by NIH R15-GM60203.

Latzer, Joachim et al. (2015) International Worm Meeting "Completing the de novo synthesis pathway for the coenzyme tetrahydrobiopterin (BH4) in C. elegans by structural modeling and mutant screening."

Latzer, Joachim, Knapp, Alec, Vitomirov, Aleks, Loer, Curtis

[International Worm Meeting, 2015]

The small molecule coenzyme tetrahydrobiopterin (BH4) is required in animals for the function of three enzymes that metabolize aromatic amino acids and another that breaks down ether lipids. BH4-deficient worms lack the neurotransmitters dopamine and serotonin, derived from Trp and Tyr, and have leaky, fragile cuticles caused by aberrant lipid metabolism in the hypodermis. The defective cuticle in BH4-deficient worms also alters a worm's susceptibility to bacterial pathogens (Loer et al., 2015, Genetics 200: in press). The first two steps of BH4 synthesis are performed by GTP Cyclohydrolase I (GTPCH1, gene cat-4) and 6-Pyruvoyl Tetrahydrobiopterin Synthetase (PTPS, gene ptps-1). The final two synthetic steps are typically carried out in animals by Sepiapterin Reductase (SR), although other reductases can perform the function in some cases. C. elegans lacks an SR ortholog, but has over 100 reductase/dehydrogenase genes with weak homology to known SRs. We are using computational methods to predict which reductase enzymes in C. elegans might be able to bind BH4-related molecules, generating structural homology models with I-TASSER, followed by docking a biopterin substrate with Chimera Autodock Vina. Using AMBER, binding free energies of BH4 bound to candidate protein structures are compared with those of a reference BH4-bound structure. At the same time, we are screening all available mutants in candidate reductase genes for dopamine and serotonin synthesis and cuticle strength to try to identify the genes(s) required for the final synthesis steps of BH4 in C. elegans..

Bassett KD et al. (1996) East Coast Worm Meeting "Aromatic amino acid hydroxylase genes from C. elegans"

Bassett KD, Loer CM

[East Coast Worm Meeting, 1996]

We are seeking to identify genes that regulate the expression of the neurotransmitter phenotype of serotonergic neurons of C. elegans. We are using two approaches: 1) isolation and characterization of serotonin deficient mutants, some of which should identify regulatory genes, and 2) identification of marker genes used by serotonergic neurons, the genes controlled by the regulators of neurotransmitter type. As a part of this second approach, we have attempted to identify genes encoding serotonin synthetic enzymes. The first step in synthesis of the neurotransmitter serotonin (5- hydroxytryptamine) is catalyzed by an aromatic amino acid hydroxylase (AAAH) that converts tryptophan to 5-hydroxytryptophan. In the vertebrates, this enzyme is called tryptophan hydroxylase (TrpH); it is one of three structurally related AAAH's, including phenylalanine hydroxylase (PheH), which synthesizes tyrosine from phenylalanine, and tyrosine hydroxylase (TH), which catalyzes the first step in dopamine synthesis. We previously cloned an AAAH gene from C. elegans using PCR with degenerate oligonucleotides similar to a Phe/Trp hydroxylase (C. Loer and C. Kenyon, unpublished results). Reporter lacZ fusions suggest that the gene is expressed in the hypodermic and not in serotonergic neurons. We subsequently expressed the protein in bacteria, and tested the substrate specificity of the resulting enzyme. Although the enzyme is capable of hydroxylating both Phe and Trp, it is more effective as a PheH (B. Davidson and C. Loer, unpublished results). The C. elegans Genome Sequencing Consortium has identified a second AAAH gene on LG II, located on cosmic ZK1290. This sequence is most similar to other TrpH genes, and thus may encode the serotonin synthetic AAAH of C. elegans. We are making reporter constructs to test this hypothesis.

Nicholas J D Gower et al. (2003) International Worm Meeting "Multiple roles for IP3 signalling in male fertility."

Howard A Baylis, Nicholas J D Gower

[International Worm Meeting, 2003]

Males have complex anatomical and behavioural peculiarities that reflect the process of mating. Although the roles of IP3 signalling in hermaphrodites have been studied in detail the roles of this important signalling pathway in males have not been determined. Reduction-of-function of the IP3 receptor, itr-1, in temperature sensitive itr-1(sa73) mutants or by RNAi leads to complete infertility of males as assayed in matings with fer-1 animals. More detailed observation and analysis of male mating behaviour using the system of Loer and Kenyon (1993) reveals that itr-1 reduction-of-function causes defects in turning and in spicule insertion. However the level of these defects is not sufficient to explain the sterility. Fluorescent assays reveal that there is no transfer of sperm in these animals suggesting a fundamental defect in this process. Sperm from these animals appear normal and activate normally. Expression of itr-1::GFP reporter constructs from the 3 promoters of itr-1 reveals that each drives expression in male specific structures including male specific muscles and the vas deferens. Thus the expression pattern and functional data may well correlate. Thus IP3 signalling is required for male fertility and is involved in several steps during mating. We are now extending this study to identify the roles of the upstream phospholipase C genes in this process. We have determined the expression pattern of all C. elegans phospholipase C subtypes in males and have assayed the effect of RNAi knock-down on male fertility. Detailed analysis of the relevant phenotypes is now underway in order to delimit the signalling pathways involved in IP3 mediated male fertility. Loer, C. M., and Kenyon, C. J. (1993). J Neurosci 13, 5407-17.

Loer, C.M. et al. (2019) International Worm Meeting "Feedback regulation of tetrahydrobiopterin (BH4) synthesis by GFRP in C. elegans."

Loer, C.M., Moreno, T.

[International Worm Meeting, 2019]

Synthesis of neurotransmitters serotonin and dopamine, conversion of Phe to Tyr, and breakdown of ether lipids in all animals involve enzymes that require the cofactor 5,6,7,8-tetrahydrobiopterin (BH4) to function. Worms that can't synthesize BH4 are serotonin- and dopamine-deficient, and have leaky, fragile cuticles caused by aberrant lipid metabolism in the hypodermis. The defective cuticle in BH4-deficient worms also alters their susceptibility to bacterial pathogens (Loer et al., 2015, Genetics 200: 237). The first and rate-limiting step in BH4 synthesis is performed by GTP Cyclohydrolase I (GTPCH1, encoded in C. elegans by cat-4). This first enzyme is regulated by 'end product feedback inhibition' whereby BH4 inhibits GTPCH1 activity. BH4 does not bind to GTPCH1 alone, but requires an additional small protein called 'GTPCH1 feedback regulatory protein' (GFRP) to inhibit GTPCH1. Crystal structures of rat GTPCH1 and GFRP with the BH4 analog BH2 show that the biopterin binds at the interface between the proteins in a large complex (Maita et al., 2004, J Biol Chem 279: 51534). Phe, which stimulates GTPCH1, also binds at this interface, mainly interacting with GFRP. C. elegans encodes an ortholog of GFRP (gene gfrp-1); sequence analysis and structural predictions suggest worm GFRP will function like the mammalian protein, binding both BH4 and Phe (D. Sykora & C. Loer, unpublished). In mammals, GTPCH1 and GFRP can also bind the selective inhibitor 2,4-Diamino-6-hydroxypyrimidine (DAHP), which is structurally similar to both GTP (substrate) and BH4 (pathway end product). At low concentrations, DAHP apparently inhibits GTPCH1 like BH4 by binding with GFRP; this inhibition is GFRP-dependent. At higher concentrations, DAHP acts as a competitive inhibitor, binding at the GTPCH1 active site like GTP; this inhibition is GFRP-independent (Xie et al., 1998, J Biol Chem 273: 21091). Although DAHP has no obvious effect on wildtype C. elegans, serotonin levels (a proxy for BH4 levels) are reduced by DAHP in worms with a reduction-of-function mutation [cat-4(e3015)] that already have less BH4. Preliminary evidence using a gfrp-1 missense mutant suggests this effect is GFRP-dependent. We are currently testing GFRP function using a CRISPR-generated deletion allele of gfrp-1.