Neuroligin-Deficient Mutants of C. Elegans Have Sensory Processing Deficits and Are Hypersensitive to Oxidative Stress and Mercury Toxicity

Neuroligin-deficient mutants of C. elegans have sensory processing deficits and are hypersensitive to oxidative stress and mercury toxicity

Jerrod W. Hunter1,2,*, Gregory P. Mullen1,*, John R. McManus1, Jessica M. Heatherly1,3, Angie Duke1 and James B. Rand1,2,3,‡

SUMMARY Neuroligins are postsynaptic cell adhesion proteins that bind specifically to presynaptic membrane proteins called neurexins. Mutations in human neuroligin genes are associated with autism spectrum disorders in some families. The nematode Caenorhabditis elegans has a single neuroligin gene (nlg-1), and approximately a sixth of C. elegans neurons, including some sensory neurons, interneurons and a subset of cholinergic motor neurons, express a neuroligin transcriptional reporter. Neuroligin-deficient mutants of C. elegans are viable, and they do not appear deficient in any major motor functions. However, neuroligin mutants are defective in a subset of sensory behaviors and sensory processing, and are hypersensitive to oxidative stress and mercury compounds; the behavioral deficits are strikingly similar to traits frequently associated with autism spectrum disorders. Our results suggest a possible link between genetic defects in synapse formation or function, and sensitivity to environmental factors in the development of autism spectrum disorders. DMM INTRODUCTION Moessner et al., 2007; The Autism Genome Project Consortium, Neuroligins are a family of postsynaptic cell adhesion proteins that 2007; Kim et al., 2008). It is now generally accepted that mutations were originally isolated on the basis of their binding to presynaptic affecting structural components of synapses provide a significant proteins called neurexins (Ichtchenko et al., 1995; Ichtchenko et risk factor for the development of ASDs. al., 1996; Boucard et al., 2005; Chih et al., 2006). Although early In the present study, we have examined the expression, studies demonstrated that, under certain conditions, the interaction localization and biological function of the synaptic protein between neuroligin and neurexin was capable of inducing neuroligin in a simple model organism, the nematode synaptogenesis (Scheiffele et al., 2000; Dean et al., 2003; Graf et al., Caenorhabditis elegans. Historically, studies of C. elegans synaptic 2004), recent studies suggest that neuroligins function primarily proteins and mutants have been instrumental in elaborating the in the maturation, stability and/or maintenance of synapses, rather roles of synaptic proteins in neuronal function and development than synaptogenesis per se (Varoqueaux et al., 2006; Südhof, 2008). (Piechotta et al., 2006; Jin and Garner, 2008). We now report that There are four neuroligin genes in mammals, and several neuroligin-deficient mutants of C. elegans are defective in a subset important studies have shown that mutations in the human genes of sensory behaviors and sensory processing, and are hypersensitive encoding neuroligin 3 and neuroligin 4 are associated with autism to oxidative stress and heavy metal toxicity; the behavioral deficits spectrum disorders (ASDs) (Jamain et al., 2003; Laumonnier et al., are strikingly similar to traits frequently associated with ASDs. Our Disease Models & Mechanisms 2004; Yan et al., 2005). Although subsequent studies have shown data thus provide clear connections between the nematode that neuroligin mutations are only associated with ASDs in a limited equivalent of an autism-associated synaptic mutation, altered number of family pedigrees (Vincent et al., 2004; Gauthier et al., sensory behaviors and hypersensitivity to environmental toxins. We 2004; Ylisaukko-oja et al., 2005), these data provide a crucial believe that these data support an important model for how both connection between defects in a specific type of synaptic molecule genetic and environmental contributions to a neurological disorder and a pervasive developmental disorder of the nervous system. can have a single underlying basis. Based on the association between neuroligin mutations and autism, a model emerged that emphasized the importance of RESULTS genetic perturbations of synaptic structure and/or synaptic The nlg-1 gene encodes the C. elegans homolog of vertebrate transmission in the etiology of autism (Zoghbi, 2003; Garber, 2007). neuroligins Support for this model increased with reports that mutations in The C. elegans genome contains a single gene (cosmid designation the genes encoding shank 3 (a postsynaptic scaffolding protein) C40C9.5) encoding a homolog of vertebrate neuroligins. This gene, and neurexin 1 are also associated with autism (Durand et al., 2007; now designated nlg-1, spans approximately 5.7 kb of genomic sequence (Fig. 1). The reading frame corresponding to the longest possible transcript (see below) encodes an 847-amino acid protein 1Genetic Models of Disease Research Program, Oklahoma Medical Research (including a 17-amino acid N-terminal signal sequence). The Foundation, Oklahoma City, OK 73104, USA structure of the predicted NLG-1 protein is similar to those of the 2 3 Department of Cell Biology and Oklahoma Center for Neuroscience, University of mammalian neuroligins: a single-pass type I membrane protein Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA *These authors contributed equally to this work with a large extracellular cholinesterase-like domain, and a small ‡Author for correspondence ([email protected]) intracellular domain terminating in a PDZ-binding motif (Fig. 2).

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Fig. 1. nlg-1 transcripts, deletion mutations and reporter constructs. The diagram shows the exon structure corresponding to the yk497a9 cDNA (GenBank accession FJ825295), augmented by the 5Ј-terminal 20 nucleotides and the 22-nucleotide SL1 sequence, which are from cDNA clone yk1657a10 (GenBank accession BJ767300). Blue regions are coding sequence; dark grey regions are untranslated regions. SL1 represents the trans-spliced leader sequence found at the 5Ј-end of many C. elegans mRNAs (Krause and Hirsh, 1987). The vertical arrowheads above the exon diagram correspond to sites of alternative splicing; the white arrowheads indicate exons 13 and 14, which are variably present in some transcripts and may be (independently) skipped, and the black arrowheads indicate the documented tandem alternative splice acceptor sites at the 5Ј-ends of exons 4 and 16, and the putative tandem alternative splice donor sites at the 3Ј-end of exon 14. Also shown are the extents of the tm474 and ok259 deletions, and the reporter constructs FRM77 and FRM253 (details in Methods).

NLG-1 is 26-28% identical (45-47% similar) to the four human the head, and the two PVD mechanosensory and two HSN motor neuroligins and is most similar overall (28% identical, 47% similar) neurons in the body, as nlg-1-expressing cells. Of these cells, the to human neuroligin 4 (supplementary material Fig. S1). AIY interneurons are cholinergic (Altun-Gultekin et al., 2001), the Through a combination of cDNA sequencing and reverse PVD neurons are glutamatergic (Lee et al., 1999), and the HSN transcription PCR (RT-PCR) analysis of transcripts, we neurons release both serotonin and acetylcholine (ACh) (Desai et documented several types of nlg-1 alternative splicing (Figs 1, 2). al., 1988; Duerr et al., 2001). Neurotransmitter assignments have Exons 13 and 14 are variably present in nlg-1 transcripts; the not been reported for the remaining nlg-1-expressing neurons;

DMM skipping of these two exons occurs independently, and we have however, they do not express GABAergic, dopaminergic, detected transcripts containing only exon 13, only exon 14, both serotonergic or glutamatergic reporters (see Methods). Finally, we exons, and neither exon. In addition, we have identified tandem also observed faint Pnlg-1::YFP expression in body wall muscles alternative splice acceptor sites at the 5Ј-ends of exons 4 and 16, (Fig. 3G). and tandem alternative splice donor sites at the 3Ј-end of exon 14 (Figs 1, 2). If these splicing events are independent, there could be The NLG-1 protein is localized to synapses as many as 24 distinct NLG-1 isoforms. To examine the subcellular localization of the NLG-1 protein, we generated a transgenic NLG-1::YFP fusion protein under the nlg-1 is expressed in a subset of neurons and muscle cells control of the nlg-1 promoter (FRM253, Fig. 1). This NLG-1::YFP We used a transgenic transcriptional reporter, with the nlg-1 fusion protein rescues all nlg-1 mutant behaviors (see below). promoter driving YFP expression (FRM77, Fig. 1), to examine the Confocal microscopy revealed that NLG-1::YFP is present at or near cellular expression of nlg-1. We found that nlg-1 is expressed in a synapses (Fig. 4A-C); localization in the synapse-rich nerve ring subset of neurons in C. elegans adults, including ~20 cells in the and ventral nerve cord is observed in embryos, and persists ventral nerve cord and ~20 cells in the head (Fig. 3). We identified throughout development. NLG-1::YFP was also present in some the nlg-1-expressing cells in the ventral nerve cord as the cholinergic neuronal cell bodies (such cells also express the FRM77 Pnlg-1::YFP VA and DA motor neurons (Fig. 3). We also identified the two AIY transcriptional reporter); this may reflect modest overexpression Disease Models & Mechanisms and two URB interneurons and the four URA motor neurons in of the NLG-1::YFP transgene.

Fig. 2. Structure of the C. elegans neuroligin protein (NLG-1). The diagram indicates the positions corresponding to the ok259 and tm474 deletions, sites of alternative splicing, and the placement of yellow fluorescent protein (YFP) in functional fusion transgenes. The expanded sequence (below) shows the C- terminal region of the protein (utilizing the single-letter amino acid code) from the transmembrane domain (TMD) to the terminal PDZ-binding motif. Pro-rich, proline-rich region; N-glyco, putative N-linked glycosylation sites; O-glyco, region rich in putative O-linked glycosylation sites.

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Fig. 4. A functional neuroligin-YFP fusion protein is localized to synaptic regions. Transgenic animals (expressing FRM253) were stained with anti- green fluorescent protein (GFP) (green; A,B,D,E) and anti-UNC-10/RIM (red; Disease Models & Mechanisms B,C,E,F). (A-C)The head of a young adult hermaphrodite. The positions of the nerve ring (nr), dorsal nerve cord (dnc) and ventral nerve cord (vnc) are indicated. Anterior is to the left and ventral is down. (D-F)An enlarged section of a sublateral nerve cord. Bars, ~10m (A-C); ~2.5m (D-F).

Fig. 3. Neuroligin-expressing cells. Confocal images of young adult transgenic animals expressing a Pnlg-1::YFP reporter (FRM77, see Fig. 1). nerve cords. In adult animals, each of these nerve cords contains Anterior is to the left and ventral is down. The reporter is expressed in ~45 five axons: SIA, SIB, SMB and SMD axons projecting from cell neurons in the head and body (out of the adult complement of 302 neurons). An adult head view is shown in A-C. The nlg-1 reporter is shown in green and a bodies in the head, and ALN/PLN axons projecting from cell bodies ttx-3 reporter [specific for AIY neurons (Altun-Gultekin et al., 2001)] is shown in in the tail (White et al., 1986). These axons make periodic en passant red (nr, nerve ring; vnc, ventral nerve cord). A portion of the ventral nerve cord synapses (neuromuscular junctions) onto the adjacent body wall is shown in D-F. The neuroligin reporter is shown in green and a cholinergic muscles. None of the neurons with axons in the sublateral nerve reporter is shown in red. The neuroligin reporter is expressed in a subset of cords expresses nlg-1, but the gene is expressed in the body wall cholinergic motor neurons in the ventral cord. A region of an adult body is muscles (Fig. 3G). We observed NLG-1::YFP-containing puncta enlarged in G, showing that the neuroligin reporter is expressed in body wall along the sublateral nerve cords (Fig. 4D); these are of necessity muscles. Bars, ~10m. muscle-derived, and therefore postsynaptic. Furthermore, these NLG-1::YFP puncta were apposed to presynaptic active zones We compared the distribution of NLG-1::YFP with that of the (UNC-10/RIM-containing puncta) present in the axons (Fig. 4D- presynaptic protein UNC-10/RIM (an active zone protein originally F). We conclude that, at least in some cells, NLG-1::YFP is localized identified as a Rab3-interacting molecule) in the four sublateral to postsynaptic regions.

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Neuroligin-deficient mutants are viable and superficially wild type mutants are far more likely than wild-type animals to cross the We characterized the phenotypes associated with two independent barrier in response to the attractant. nlg-1 alleles: ok259 and tm474. The nlg-1(ok259) mutation removes approximately half of the nlg-1 coding sequence (2341 base pairs; Defects in thermal response Figs 1, 2) and is almost certainly a null mutation. The tm474 When well-fed wild-type nematodes are placed in a thermal mutation is associated with a smaller (583-base pair) deletion, which gradient, they preferentially accumulate at the temperature at which removes exon 7 and part of exon 8 (Figs 1, 2). Animals homozygous they were raised (Hedgecock and Russell, 1975). However, nlg-1 for either of these alleles are viable and superficially wild type in mutants do not accumulate at a specific temperature, but instead their appearance, development and behavior. In addition, the move independently of temperature (Fig. 5C). The mutants appear nervous system of nlg-1 mutants is grossly normal: expression to lack completely a thermal response; this atactic behavior was of neuronal reporters in live animals and indirect independent of the temperature at which the animals were grown immunofluorescence for several different synaptic antigens in or their feeding state (not shown). A normal thermal response was fixed specimens revealed no apparent difference between nlg- restored by transgenic expression of an NLG-1::YFP fusion protein 1(ok259) mutants and wild-type animals (data not shown). (Fig. 5C). We conclude that nlg-1 mutants are either unable to sense We used two quantitative assays to measure general synaptic temperature, or are indifferent to changes in ambient temperature. function in nlg-1 mutants. The response to the acetylcholinesterase inhibitor aldicarb is commonly used as a measure of cholinergic Spontaneous reversal frequency is decreased in nlg-1 mutants neurotransmission in C. elegans; resistance to aldicarb is typically For approximately 20 minutes after wild-type C. elegans are associated with decreased ACh release (Miller et al., 1996). removed from food, they display a stereotypic locomotory behavior Locomotory behavior in swimming assays provides a general (Hills et al., 2004; Gray et al., 2005): the animals move forward for measure of motor neuron function and neurotransmitter release. 30-40 seconds, and then spontaneously reverse direction and back We found that nlg-1 mutants did not differ from wild-type animals up for approximately three body lengths. They then start moving

DMM in their response to aldicarb in an acute response assay (not shown) forward again, usually in a different direction. We found that nlg- and were not appreciably deficient in swimming behavior (wild 1 mutants move forward for a much longer time than wild-type type157±4 body bends/minute; nlg-1(ok259)143±11 body animals before initiating backward movement (Fig. 5D). This bends/minute; details in Methods). We therefore conclude that decrease in spontaneous reversal rate, sometimes called a ‘hypo- elimination of nlg-1 function does not lead to dramatic deficits in reversal’ phenotype (Tsalik and Hobert, 2003), was rescued by synapse formation or function. However, as described below, we transgenic expression of the NLG-1::YFP fusion protein (Fig. 5D). have identified a number of significant behavioral and biochemical However, although the likelihood of a reversal event was greatly differences between nlg-1 mutants and wild-type animals. reduced in nlg-1 mutants, the duration of backing, once initiated, was approximately normal (wild type5.8±0.9 seconds; nlg- nlg-1 mutants have specific deficits in chemosensation and the 1(ok259)7.2±1.8 seconds). We also observed that the decrease in processing of chemosensory cues reversal likelihood was progressive: the decrease was significant in Wild-type C. elegans hermaphrodites respond to a wide variety of young larvae, but was far more pronounced in adults (Fig. 5D). We volatile and water-soluble chemical cues (Bargmann et al., 1993; note that other phenotypes of nlg-1 mutants described above (e.g. Bargmann, 2006), and nlg-1 mutants responded normally to most the lack of response to 1-octanol and insensitivity to temperature) attractants and repellants. However, we were able to identify some do not appear to be progressive. specific chemosensory deficits in nlg-1 mutants. For example, nlg- Disease Models & Mechanisms 1 mutants are not repelled by the normally aversive chemical 1- Sensitivity to oxidative stress and heavy metals octanol: the chemotaxis index for wild type–0.68±0.11; nlg- The progressive nature of the spontaneous reversal phenotype in 1–0.04±0.08; and transgenic rescue by FRM253–0.52±0.05 nlg-1 mutants suggested that the absence of neuroligin might trigger (negative values indicate repulsion; details in Methods). The some type of degenerative process. We therefore evaluated the difference between nlg-1 and wild type is statistically significant sensitivity of nlg-1 mutants to oxidative stress. Exposure to paraquat (P<0.0001). The octanol-sensing deficit can not be ascribed to a (N,NЈ-dimethyl-4,4Ј-bipyridinium dichloride) is commonly used as general insensitivity to volatile chemicals (nlg-1 mutants respond a paradigm for oxidative stress in C. elegans (Ishii et al., 1990). We normally to the volatile attractant diacetyl), or to an insensitivity found that nlg-1 mutants were significantly more sensitive to to repellants in general (the mutants have a normal response to paraquat than wild-type animals, and that this hypersensitivity was the repellant cupric acetate), or even to a general insensitivity to rescued by transgenic expression of a NLG-1::YFP fusion protein volatile repellants (the mutants have a normal response to the (Fig. 6A). In most organisms, including C. elegans, hypersensitivity volatile repellant nonanone). The behavior seems instead to be a to oxidative stress is associated with decreased life span (Ishii et specific lack of response to 1-octanol. al., 1998; Senoo-Matsuda et al., 2001; Kondo et al., 2005), and this In addition to defects in response to specific chemical cues, nlg- was also true for nlg-1 mutants (supplementary material Fig. S2). 1 mutants also exhibit defects in the processing of two conflicting We extended these studies by examining the toxic effects of chemosensory inputs. For example, wild-type animals and nlg-1 mercury compounds, and we found that nlg-1 mutants were mutants are comparably attracted to diacetyl and repelled by cupric significantly more sensitive than wild-type animals to both acetate (Fig. 5A,B). However, when animals are presented with these inorganic (HgCl2) and organic [thimerosal (sodium two compounds simultaneously (i.e. with a cupric acetate barrier ethylmercurithiosalicylate)] forms of mercury (Fig. 6B). The nlg-1 between the animals and the attractant; see Fig. 5A,B), nlg-1 mutants were also more sensitive than wild-type animals to the

Disease Models & Mechanisms 369 RESEARCH ARTICLE Neuroligin mutants and oxidative stress DMM

Fig. 5. nlg-1 mutant behaviors. (A,B)‘Approach/avoidance’ paradigm. The experimental setup is shown in A; the data are shown in B. Assays were performed with only the cupric acetate barrier (and a mock attractant; ‘Cu(+) Diacetyl(–)’), only the diacetyl attractant (and a mock barrier; ‘Cu(–) Diacetyl(+)’), and with both compounds present (‘Cu(+) Diacetyl(+)’). Values were calculated according to the formula in A. Each bar represents the mean of three assays (approximately 75 animals/assay) ± standard deviation. N2 is wild type; ‘Rescue’nlg-1 mutants expressing an integrated functional NLG-1::YFP transgene (FRM253). The asterisk (*) indicates a statistically significant difference (P0.0002) between nlg-1 and wild type. (C)Thermal response. Animals were grown at 20°C and placed on a thermal gradient as described in the Methods. Wild-type nematodes accumulated at their growth temperature. nlg-1 mutants did not accumulate at a specific temperature, but instead moved independently of temperature. Transgenic expression of a NLG-1::YFP fusion protein (FRM253) rescued the thermotaxis defect. Each data point represents the mean of six trials of 50 animals each ± standard deviation. The asterisk (*) for the 20°C temperature point indicates a statistically significant difference (P<0.0001) between nlg-1 and wild type. (D)Spontaneous reversal behavior. Animals were removed from food and monitored for spontaneous reversal of direction. Each animal was observed for 3 minutes; each data bar represents the mean of 25 animals ± standard deviation. The nlg-1 mutants moved forward for a much longer time before initiating backward movement. This phenotype is progressive: 4-day-old (adult) animals have a much stronger phenotype than 2-day-old (L3) animals. Transgenic expression of a NLG-1::YFP fusion protein (FRM253) rescued this mutant phenotype. An asterisk (*) indicates a statistically significant difference (P<0.0001) between nlg-1 and wild-type animals.

toxic effects of copper (cupric acetate) (supplementary material Fig. of the NLG-1::YFP fusion protein reduced protein carbonylation S3). However, there was no difference between the mutants and levels to those of the wild type (Fig. 6C), confirming the specificity Disease Models & Mechanisms wild-type animals in their survival on cadmium (CdCl2) of this biochemical phenotype. Levels of carbonylated proteins in (supplementary material Fig. S3); nlg-1 mutants are therefore not untreated and treated mev-1 mutants were comparable to those hypersensitive to all heavy metals. observed in similarly treated nlg-1 mutants. We conclude that, even in the absence of paraquat, nlg-1 mutants experience elevated An oxidative biomarker is elevated in nlg-1 mutants oxidative stress. To examine oxidative damage directly, we measured protein carbonylation (an irreversible oxidative modification) in wild-type, DISCUSSION nlg-1 and mev-1 strains. This approach has been used previously In this study we report that neuroligin is expressed in a subset of to assess oxidative damage in C. elegans (Adachi et al., 1998; Yasuda neurons and in muscles of C. elegans, and is localized to synaptic et al., 1999). The mev-1 gene encodes a subunit of succinate regions. Neuroligin-deficient mutants of C. elegans are defective dehydrogenase cytochrome b, part of complex II of the in specific sensory behaviors and sensory processing, and are mitochondrial electron transport chain (Ishii et al., 1998); it was hypersensitive to oxidative stress and heavy metal toxicity. Our data included in our analysis because mev-1 mutants are hypersensitive provide clear connections between the nematode equivalent of an to paraquat (Ishii et al., 1990) and exhibit elevated levels of autism-associated synaptic mutation, altered sensory behaviors, and carbonylated proteins (Adachi et al., 1998; Yasuda et al., 1999). We hypersensitivity to environmental toxins. found that nlg-1 mutants had a significantly higher level of protein carbonylation than wild-type animals (Fig. 6C). Exposure to The C. elegans neuroligin gene and protein structure paraquat significantly increased the protein carbonylation levels of The C. elegans genome contains a single neuroligin homolog all of the strains tested, although the levels remained much higher (C40C9.5), encoded by the nlg-1 gene. The nlg-1 transcripts in nlg-1 mutants than in wild-type animals. Transgenic expression undergo extensive alternative splicing, giving rise to a number of

370 dmm.biologists.org Neuroligin mutants and oxidative stress RESEARCH ARTICLE

Fig. 6. Neuroligin-deficient mutants exhibit oxidative stress. (A)Sensitivity to paraquat. Young adults were transferred to plates containing 1.8 mM of paraquat and monitored daily for survival. Each data point represents the mean of six trials of 10 animals each ± standard deviation. The difference in mean survival times between nlg-1 (3.7±0.6 days) and wild type (5.7±0.4 days) is statistically significant (P<0.0001). (B)Sensitivity to thimerosal. Young adults were transferred to plates containing 91 nM of thimerosal. Each data point represents the mean of three trials with at least 46 animals in each trial ± standard deviation. The difference in mean survival times between nlg-1 (1.3±0.03 days) and wild type (3.0±0.1 days) is statistically significant (P<0.0001). (C)nlg-1 mutants have elevated levels of oxidized proteins. Young adults were transferred to plates containing 1.8 mM of paraquat (or control) and grown for 2 days, then assayed by ELISA for carbonyl modification of proteins. Values were normalized to protein concentration and are presented relative to the value of wild type without paraquat (N22.43±0.94 ng carbonyl/g protein). Bars represent the means of four separate experiments ± standard deviation. ‘Rescue’nlg-1 mutants expressing an integrated functional NLG-1::YFP transgene (FRM253). mev-1 mutants were previously shown to be hypersensitive to paraquat and to have elevated levels of oxidized protein (Ishii et al., 1990; Adachi et al., 1998). ‘+Pq’ and ‘–Pq’ represent growth with or without paraquat, respectively. The asterisk (*)

DMM indicates a statistically significant difference (P<0.0001) between N2 with and without paraquat; the dagger (†) indicates a statistically significant difference (P0.0005) between nlg-1 without paraquat and wild type without paraquat; and the double dagger (‡) indicates a statistically significant difference (P0.0011) between mev-1 without paraquat and wild type without paraquat.

NLG-1 isoforms. However, in contrast to vertebrate neuroligins, present in both presynaptic and postsynaptic regions of C. elegans where alternative splicing primarily affects the extracellular domain neurons (Feinberg et al., 2008). In the DA9 motor neuron, for of the protein and modulates binding interactions with neurexins example, strong NLG-1::YFP fluorescence was present in the (Comoletti et al., 2006), alternative splicing of C. elegans nlg-1 ventral postsynaptic region and weaker fluorescence was present transcripts leads almost exclusively to diversity in the intracellular in the dorsal presynaptic region. The significance of this presynaptic domain of the protein (Fig. 2). It is possible that the isoform diversity localization is unclear, although we note that rodent neurexins are generated by alternative splicing of nematode neuroligin localized to both presynaptic and postsynaptic regions (Taniguchi corresponds to some of the diversity provided by multiple et al., 2007), and the same may be true for neuroligins. neuroligin genes in mammals. For example, the intracellular domain of mammalian neuroligin 2 has a proline-rich region that is not Integration of multiple sensory inputs found in the other mammalian neuroligins. Similarly, exon 14 of Although sensory problems are not part of the official diagnostic Disease Models & Mechanisms C. elegans nlg-1 encodes a proline-rich region; perhaps nematode criteria for ASDs, difficulties with the processing and/or integration isoforms containing this exon correspond to mammalian neuroligin of sensory inputs are often part of the presentation (American 2. However, we note that transgenic expression of a single isoform Psychiatric Association, 1994; Filipek et al., 1999; Filipek et al., containing both exons 13 and 14 (Fig. 1) provided complete rescue 2000). It is therefore particularly intriguing that nlg-1 mutants have of the thermosensory, chemosensory, spontaneous reversal, toxin deficits in the processing of conflicting sensory inputs, as measured sensitivity and protein oxidation phenotypes associated with nlg- in an approach-avoidance paradigm. nlg-1 mutants respond 1 mutants (Fig. 5B-D; Fig. 6A-C). normally to the volatile attractant diacetyl and the repellent cupric acetate; however, their response to the simultaneous presentation Neuroligin expression and localization of these two compounds is clearly not normal (Fig. 5A,B). We Our reporter studies indicate that neuroligin is expressed in believe that the mutant phenotype should not be interpreted as a approximately 45 of the 302 neurons present in adult failure to process the two conflicting signals; rather, the discrete hermaphrodites. Aside from the DA and VA motor neurons in the sensory inputs are processed, but the processing appears to use an ventral nerve cord, there is no clear pattern (e.g. neurotransmitter, alternative algorithm. This may reflect functional deficits in one circuitry, etc.) linking the neuroligin-expressing neurons. In or more of the processing interneurons. addition to neurons, we observed nlg-1 expression in the body wall muscles (Fig. 3); muscle expression has also been reported for Neuroligin and behavioral circuits human neuroligin 4 (Bolliger et al., 2001). The circuits mediating C. elegans thermotaxis and spontaneous We report that neuroligin is localized to synaptic regions in C. reversal behaviors have been described in considerable detail. elegans, and is present in postsynaptic puncta in at least a subset Thermotaxis involves input from the AFD sensory neurons, and of cells. Interestingly, a recent study found that neuroligin is processing by the AIY, AIZ and RIA interneurons (Mori and

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Ohshima, 1995); control of ‘spontaneous’ reversal involves input medium (Sun and Lambie, 1997), modified by the addition of from many sensory neurons (including the AFD thermosensory streptomycin and mycostatin to reduce contamination, and the use neurons), followed by processing through the AIY, AIZ, RIB and of the streptomycin-resistant bacterial strain OP50/1 (Johnson et RIM interneurons (Zheng et al., 1999; Tsalik and Hobert, 2003). al., 1988). The nlg-1(ok259) mutant was provided by the C. elegans Although the AFD sensory neurons and the AIY and AIZ Gene Knockout Consortium, and the nlg-1(tm474) mutant was interneurons participate in both circuits, only the AIY cells appear obtained from Shohei Mitani (Tokyo Women’s Medical College, to express neuroligin. However, laser ablation of the AIY neurons Tokyo, Japan). The nlg-1(ok259) allele was used for these studies, leads to cryophilic (cold-seeking) behavior (Mori and Ohshima, and observations were confirmed with tm474. The pha-1(e2123) 1995) and a ‘hyper-reversal’ phenotype (Tsalik and Hobert, 2003), mutant (see below) was obtained from the Caenorhabditis Genetics whereas nlg-1 mutants are athermotactic and have a ‘hypo-reversal’ Center (University of Minnesota, Minneapolis, MN). phenotype. The lack of neuroligin in the AIY neurons may alter the strength of specific synaptic connections rather than simply Sequencing of mutants and transcript analysis render these cells non-functional. nlg-1 deletion mutations were analyzed by amplification of specific nlg-1 genomic regions from mutant animals (Barstead and Oxidative stress and autism Waterston, 1989), followed by sequencing of the purified PCR We utilized an established model of oxidative stress in C. elegans product. The cDNA clone yk497a9 was obtained from Yuji Kohara (Yamamoto et al., 1996; Yanase et al., 2002) to demonstrate that (National Institute of Genetics, Mishima, Japan) and the insert was neuroligin-deficient mutants are hypersensitive to paraquat (Fig. 6A). fully sequenced (GenBank accession FJ825295); it includes 51 We believe that this sensitivity results from an increased basal level nucleotides of 5Ј-untranslated region (UTR) (out of a predicted 71 of oxidative stress, and we have shown that nlg-1 mutants exhibit a nucleotides), 2526 nucleotides of coding sequence (including both higher level of biochemical protein modifications characteristic of exons 13 and 14; see Fig. 1), 276 nucleotides of 3Ј-UTR, and a 19- oxidative stress (Fig. 6C). We do not yet understand how the lack of nucleotide polyA sequence. RNA was isolated from wild-type C.

DMM neuroligin in only a subset of muscle cells and neurons is able to elegans using standard procedures, and analyzed using RT-PCR sensitize the entire organism to toxins such as paraquat and mercury. with exon-specific primers, followed by DNA sequencing with There is a body of literature documenting the presence of internal primers. All DNA sequencing was performed at the OMRF biomarkers associated with oxidative stress in individuals with DNA Sequencing Core Facility, using oligonucleotide primers autism. These biomarkers include a significant decrease in the ratio obtained from IDT (Coralville, IA). of reduced to oxidized glutathione in plasma (James et al., 2006; Geier et al., 2009), an increase in 3-nitrotyrosine in cerebellar extracts Reporter constructs and transgenic methods (Sajdel-Sulkowska et al., 2008), increased urinary excretion of 8- Plasmids containing the cyan fluorescent protein (CFP), YFP or isoprostane-F2 (a non-enzymatic oxidation product of arachidonic GFP coding sequences were derived from the pPD95.67, pPD132.12 acid) (Ming et al., 2005), and increased plasma levels of or pPD133.48 plasmids (Miller, D. M. et al., 1999). A plasmid malonyldialdehyde (an end product of peroxidation of containing a modified mCherry gene (‘wormCherry’) (McNally et polyunsaturated fatty acids and related esters) (Chauhan et al., 2004). al., 2006; Green et al., 2008) was a generous gift from Anjon Audhya Although it is unclear how the absence of neuroligin leads to and Karen Oegema (Ludwig Institute for Cancer Research, La Jolla, oxidative stress in C. elegans, it is plausible that a comparable CA). Some reporter constructs were generated using a PCR fusion mechanism might link autism-associated mutations in humans to method (Horton et al., 1989; Hobert, 2002). The neuroligin the autism-associated oxidative phenotypes that have been reported. transcriptional fusion construct (FRM77) was generated by PCR Disease Models & Mechanisms amplification of the 5Ј-end of the nlg-1 gene (from 3563 base pairs Implications for ASDs upstream of the SL1 trans-splice site through the first 45 base pairs Human twin studies have shown that the concordance among of exon 3), and fusing this product in-frame to the YFP coding monozygotic twins for a strict diagnosis of autism is 60%, which is sequence followed by the unc-54 3Ј-UTR. The neuroligin functional up to 12-fold higher than the concordance among dizygotic twins fusion protein (FRM253) had the YFP inserted after amino acid (Bailey et al., 1995; Muhle et al., 2004). This clearly provides strong E661 (using the protein sequence derived from the yk497a9 cDNA; evidence for a hereditary basis for autism, but also highlights the GenBank accession ACO52513). FRM253 was generated by importance of non-hereditary (environmental) factors. Our data amplifying a PCR product containing the same upstream region on the sensitivity of neuroligin-deficient mutants to oxidative that was used to make FRM77, but extending downstream to exon stress (e.g. paraquat) and mercury compounds demonstrate a clear 11. This amplification product was fused in-frame to the YFP connection between the nematode equivalent of an autism- coding sequence, followed by nlg-1 exons 12-16 and the nlg-1 3Ј- associated synaptic mutation and hypersensitivity to environmental UTR, which were derived from the yk497a9 cDNA. The structures toxins. We believe that this provides an important model for of these reporters are shown in Fig. 1. understanding how both genetic and environmental contributions Transgenic nematodes were obtained by microinjection of DNA to a neurological disorder can have a single underlying basis. (plasmids and/or PCR products), essentially as described by Mello (Mello et al., 1991). Transformation markers included the pBX METHODS plasmid (Heinke and Ralf Schnabel, Max-Plank-Institute fur Strains and strain maintenance Biochemie), which rescues the temperature-sensitive lethality of Standard laboratory methods for C. elegans were described by pha-1(e2123) mutants (Granato et al., 1994). For experiments Brenner (Brenner, 1974). Worms were grown on NGM-Lite solid utilizing the pBX plasmid, we constructed appropriate recipient

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strains for transformation containing the pha-1(e2123) mutation. C.I.(A–B)/(A+B). A positive C.I. value (up to 1.00) indicates For some DNA injections, we used a modified unc-122 promoter attraction, a negative value (down to –1.00) indicates repulsion, to drive reporter expression only in coelomocytes (Loria et al., and a value near 0.00 indicates neutrality. 2004). Extrachromosomal arrays were integrated by gamma To test the integration of simultaneous attractive and repellant irradiation (Schade et al., 2005). cues, a repellant barrier (50 l of 0.5 mM cupric acetate) was placed in a line across the middle of a 100-mm plate (Ishihara et al., 2002). Cell identification After ~16 hours, a chemo-attractant (2 l of 0.1% diacetyl in nlg-1-expressing cells were identified on the basis of position and ethanol) was placed on one side of the barrier, and approximately morphology (Altun and Hall, 2008), and also through the use of 75 worms were placed on the opposite side. The worms were specific transgenic reporters for colocalization and/or to provide allowed to move freely on the plate for 30 minutes, and the fraction cellular landmarks. The cells identified as nlg-1 positive were found of worms that had crossed the barrier was then scored. In control to express the Pnlg-1::YFP reporter (FRM77) in 100% of animals experiments, when either cupric acetate or diacetyl was presented observed, with at least 10 animals examined for each genotype. without the other, we did not observe significant differences The promoters used for these studies included unc-25 for between wild-type animals and nlg-1 mutants in their responses GABAergic neurons (Eastman et al., 1999), unc-17 for cholinergic to varying concentrations of either compound (data not shown). neurons (Alfonso et al., 1993; Duerr et al., 2008), eat-4 for a subset of glutamatergic neurons (Lee et al., 1999), dat-1 for dopaminergic Life span measurements neurons (Sulston et al., 1975; Nass et al., 2002), tph-1 for Synchronous populations were initiated by transferring adults to serotonergic neurons (Sze et al., 2000), acr-5 for DB and VB motor a plate, letting them lay eggs at 20°C for ~2 hours, and then neurons (Winnier et al., 1999), ttx-3 for AIY interneurons (Altun- removing the adults. The eggs were permitted to hatch, and the Gultekin et al., 2001), gcy-8 for AFD sensory neurons (Yu et al., resulting populations of synchronized animals (~25 animals per 1997), glr-3 for RIA interneurons (Brockie et al., 2001a), odr-2(2b) plate in triplicate for each strain) were used for life span

DMM for AIB, AIZ, ASG, AVG, IL2, PVP, RIF, RIV and SIAV neurons measurements. Animals were grown at 20°C and transferred away (Chou et al., 2001), and nmr-1 for AVA, AVD, AVE, RIM, AVG and from their progeny to new plates as necessary (usually three times). PVC interneurons (Brockie et al., 2001b). Dye filling of ciliated Animals were assessed each day for viability; those that had ceased sensory neurons with DiI (Hedgecock et al., 1985) provided pharyngeal pumping and failed to move, even after repeated additional cellular landmarks in the head. prodding, were scored as no longer viable. Ages are given as days from hatching. Behavioral assays All behavioral measurements were performed on young adults at Immunofluorescence staining ~22°C on NGM-Lite plates unless stated otherwise; statistical Antibodies used in this study included a rabbit polyclonal -GFP significance was determined using the Student’s t-test. Swimming antibody (which also recognizes YFP; Invitrogen/Molecular Probes, rates were measured using hermaphrodites raised at 20°C, as Carlsbad, CA), a mouse monoclonal -UNC-17/VAChT antibody described previously (Miller, K. G. et al., 1999). Acute response to (mAb1403) (Lickteig et al., 2001; Duerr et al., 2008) and a aldicarb (2-methyl-2-[methylthio]proprionaldehyde-O-[methyl- chicken polyclonal -RIM antibody (pAb271) (Koushika et al., carbamoyl]oxime) (Chem Service, West Chester, PA) was assayed, 2001). Secondary antibodies were obtained from Jackson as described previously (Lackner et al., 1999), using 2 mM aldicarb. ImmunoResearch (West Grove, PA). Nematodes were stained To score reversal behavior, worms were grown at 20°C and using a modified freeze-fracture procedure, as described previously Disease Models & Mechanisms transferred to unseeded plates at room temperature. After 2 (Duerr et al., 1999; Mullen et al., 2006). minutes of equilibration, the animals were scored visually for changes in direction for 10 minutes, and the data were recorded Microscopy and imaging using the Etho 1.2.2 program (provided by Dr James H. Thomas, Confocal images were collected on a Leica TCS NT confocal Genome Sciences, University of Washington). Thermal response microscope. Low-resolution images were collected with a 40ϫ Plan assays were performed after establishing a thermal gradient on an Fluotar 1.0 NA oil immersion objective, at 512ϫ512 or 1024ϫ1024 unseeded 100-mm plate by placing a vial of frozen glacial acetic pixels, with 0.5 micron Z-steps. Higher-resolution images were acid (16.7°C) in the center of an inverted plate in a 25°C incubator collected with a 63ϫ Plan APO 1.4 NA oil immersion objective, at (Hedgecock and Russell, 1975). Approximately 50 worms were 512ϫ512 pixels, with a 4ϫ zoom and 0.2 micron Z-steps. Images transferred to the thermal gradient plate and allowed to move freely were cropped to size, assembled, and annotated using Adobe for 30 minutes; their positions were then scored on an overlay of Photoshop CS2. Digital manipulations were limited to rotating and concentric circles demarking eight equal areas. cropping (Photoshop Bicubic) of images, as well as minor level For chemotaxis assays, 2 l of a chemo-attractant or repellant adjustments. was placed on one side of a 100-mm plate (the ‘A’ side) and diluent was placed on the opposite side of the plate (the ‘B’ side). Toxicity studies Approximately 50 worms were placed on the middle of the plate Stock solutions of paraquat (Sigma-Aldrich), HgCl2, CdCl2, cupric and were allowed to move freely for 20 minutes, and then scored. acetate or thimerosal (Sigma-Aldrich) were added to molten growth Only worms within 2 cm of the test or control spots were scored, medium. Final concentrations in the medium were: paraquat, 1.8 and therefore worms in neutral areas were disregarded. A mM; HgCl2, 7.3 M; CdCl2, ranged from 4 to 28 mM; cupric acetate, chemotaxis index (C.I.) was calculated using the formula 0.9 mM; thimerosal, 91 nM. Toxicity was assessed by placing 20

Disease Models & Mechanisms 373 RESEARCH ARTICLE Neuroligin mutants and oxidative stress

young adults/plate on NGM-Lite plates containing the toxin (or TRANSLATIONAL IMPACT solvent), and scoring survival every 24 hours. Clinical issue Analysis of protein carbonylation by ELISA Autism spectrum disorders (ASDs) are common neurological disorders that include autistic disorder, Asperger disorder and PDD-NOS (pervasive To quantify protein carbonylation, we used an ELISA method developmental disorder-not otherwise specified). The Centers for Disease described by Alamdari et al. (Alamdari et al., 2005), with some Control and Prevention now estimate an overall ASD prevalence of 1 case per modifications. Approximately 50 L4 worms were transferred to 110 children. The three essential criteria for an ASD diagnosis are: (1) impaired NGM-Lite plates with or without paraquat, and allowed to grow verbal and nonverbal communication; (2) impaired social interaction; and (3) for 2 days prior to harvest, with the exception of mev-1 mutants, restricted, repetitive and stereotyped patterns of behavior, interests and which were harvested after 1 day. Nematode proteins were activities. Family studies show that ASDs have a strong hereditary basis, apparently involving a large number of genes. However, it is also clear that extracted in lysis buffer [20 mM 2-(N-morpholino)- environmental factors play a significant role in the etiology and severity of ethanesulfonate (MES) buffer, pH 5.5, containing 0.1% Triton X- these disorders. 100, complete protease inhibitor cocktail (one tablet/50 ml) Mutations affecting structural components of synapses are significant risk (Roche Diagnostics), and 100 M butylated hydroxytoluene factors for developing ASDs. The best-studied of these are the neuroligins: (BHT)] on ice, and sonicated on ice with a Sonic Dismembrator postsynaptic adhesion/signaling proteins that bind specifically to a set of model 100 (Fisher Scientific). The protein concentrations of the presynaptic membrane proteins called neurexins. There are four neuroligin- samples were measured by the Micro BCA spectro-photometric encoding (NLGN) genes in humans, and mutations disrupting NLGN3 and NLGN4 are associated with autism. However, it is not clear how disruption of a assay (Pierce), and initially adjusted to 10 g/ml with lysis buffer broadly expressed synaptic protein results in the relatively specific behavioral (some samples needed to be diluted further to stay within the deficits associated with ASDs. linear working range of the assay). The diluted standards (10 g protein in 1 ml PBS), samples (10 g protein in 1 ml PBS), and Results Here, the authors investigate the effects of neuroligin-disrupting mutations in PBS without protein (blank) were added in duplicate to wells of DMM Caenorhabditis elegans. C. elegans neuronal proteins are structural and an ELISA plate. DNPH (2,4-dinitrophenylhydrazine) (0.05 mM, functional homologs of mammalian proteins, making it a powerful model for pH 6.2, freshly prepared) solution was added, incubated for 45 analyzing synapse structure, function and development. Worms have a single minutes at room temperature in the dark, and then neutralized neuroligin gene (nlg-1), and C. elegans neuroligin is structurally similar to with 0.1 N NaOH. Samples were transferred to a new ELISA plate, mammalian homologs. Mutants lacking the neuroligin protein have and ELISAs were performed using rabbit anti-DNPH and goat superficially normal growth and behavior, and apparently normal nervous anti-rabbit HRP-conjugated IgG antibodies (Chemicon), and systems. However, the authors show that nlg-1 null mutants have several sensory deficits: they do not respond normally to some chemical cues, they standard methods. are insensitive to temperature changes and they have altered processing of ACKNOWLEDGEMENTS conflicting sensory inputs. We are grateful to Bob Barstead and the C. elegans Gene Knockout Consortium for nlg-1 mutants also have increased levels of oxidative stress, which results the nlg-1(ok259) allele; Shohei Mitani for the tm474 allele; Yuji Kohara for the from excessive free radicals and reactive oxygen species (ROS) that can yk497a9 cDNA; Erik Jorgensen for information about modifying the unc-122 promoter; Anjon Audhya and Karen Oegema for the wormCherry clone; Mike damage cellular components (e.g. proteins, lipids and DNA). nlg-1 mutants are Nonet for antibodies to UNC-10/RIM; Jim Thomas for the Etho software; Ellie hypersensitive to paraquat toxicity (an herbicide that produces excess free Mathews for assistance with swimming assays; Kiely Grundahl, Mary Wikswo and radicals and ROS) suggesting elevated levels of endogenous free radicals. Amber Sherrill for technical assistance; Jim Henthorn for assistance with confocal Oxidative damage to proteins in nlg-1 mutants is elevated compared with imaging; and Ken Hensley, Dario Ramirez and Ellie Mathews for helpful wild-type animals, and mutants are also hypersensitive to the toxic effects of discussions. These studies were supported by a research grant from Autism

Disease Models & Mechanisms copper- and mercury-containing compounds. Speaks. Some strains were obtained from the Caenorhabditis Genetics Center, which is supported by the National Center for Research Resources. Implications and future directions The relationship between ASDs and oxidative stress is unclear, although it has COMPETING INTERESTS The authors declare no competing financial interests. been proposed that oxidative stress, from environmental toxins, may contribute to the disorders. These studies show that loss of the synaptic AUTHOR CONTRIBUTIONS protein neuroligin causes oxidative stress. This raises the possibility that J.W.H., G.P.M. and J.B.R. designed the experiments. J.W.H., G.P.M., J.R.M., J.M.H. and specific types of neuronal disruption might be the cause, rather than the A.D. performed the experiments. J.W.H., G.P.M. and J.B.R. analyzed the data and result, of oxidative stress. In addition, these data demonstrate a clear wrote the manuscript. connection between an autism-associated synaptic mutation in C. elegans and SUPPLEMENTARY MATERIAL hypersensitivity to environmental toxins (e.g. paraquat, mercury compounds, Supplementary material for this article is available at etc.). This provides an important example of how both genetic and http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.003442/-/DC1 environmental contributions to a neurological disorder can have a single Received 8 April 2009; Accepted 14 September 2009. underlying basis. ASDs are often associated with changes in sensitivity to sensory inputs, as REFERENCES well as the ability to process and integrate these inputs. It is therefore Adachi, H., Fujiwara, Y. and Ishii, N. (1998). Effects of oxygen on protein carbonyl and aging in Caenorhabditis elegans mutants with long (age-1) and short (mev-1) life intriguing that nlg-1 mutants have specific sensory deficits, as well as deficits spans. J. Gerontol. A. Biol. Sci. Med. Sci. 53, B240-B244. in the processing of conflicting sensory inputs. This characterization of a C. Alamdari, D. H., Kostidou, E., Paletas, K., Sarigianni, M., Konstas, A. G. P., elegans ASD model indicates a role for neuroligin in processing sensory Karapiperidou, A. and Koliakos, G. (2005). High sensitivity enzyme-linked information and the importance of proper synaptic function in regulating immunosorbent assay (ELISA) method for measuring protein carbonyl in samples oxidative stress. with low amounts of protein. Free Radic. Biol. Med. 39, 1362-1367. Alfonso, A., Grundahl, K., Duerr, J. S., Han, H.-P. and Rand, J. B. (1993). The doi:10.1242/dmm.005173 Caenorhabditis elegans unc-17 gene: A putative vesicular acetylcholine transporter. Science 261, 617-619.

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Altun, Z. F. and Hall, D. H. (2008). Atlas of C. elegans anatomy. Filipek, P. A., Accardo, P. J., Ashwal, S., Baranek, G. T., Cook, E. H., Dawson, G., http://www.wormatlas.org. Gordon, B., Gravel, J. S., Johnson, C. P., Kallen, R. J. et al. (2000). Practice Altun-Gultekin, Z., Andachi, Y., Tsalik, E. L., Pilgrim, D., Kohara, Y. and Hobert, O. parameter: screening and diagnosis of autism: report of the quality standards (2001). A regulatory cascade of three homeobox genes, ceh-10 ttx-3 and ceh-23, subcommittee of the american academy of neurology and the child neurology controls cell fate specification of a defined interneuron class in C. elegans. society. Neurology 55, 468-479. Development 128, 1951-1969. Garber, K. (2007). Autism’s cause may reside in abnormalities at the synapse. Science American Psychiatric Association (1994). Diagnostic and statistical manual of mental 317, 190-191. disorders (4th edn). Washington, D.C.: American Psychiatric Association. Gauthier, J., Bonnel, A., St Onge, J., Karemera, L., Laurent, S., Mottron, L., Bailey, A., Le, C. A., Gottesman, I., Bolton, P., Simonoff, E., Yuzda, E. and Rutter, M. Fombonne, E., Joober, R. and Rouleau, G. A. (2004). NLGN3/NLGN4 gene (1995). Autism as a strongly genetic disorder: evidence from a British twin study. mutations are not responsible for autism in the Quebec population. Am. J. Med. Psychol. Med. 25, 63-77. Genet. B Neuropsychiatr. Genet. 132B, 74-75. Bargmann, C. I. (2006). Chemosensation in C. elegans. In WormBook: The Online Review Geier, D., Kern, J., Garver, C., Adams, J., Audhya, T. and Geier, M. (2009). A of C. elegans Biology (ed. The C. elegans Research Community): prospective study of transsulfuration biomarkers in autistic disorders. Neurochem. http://www.wormbook.org. Res. 34, 386-393. Bargmann, C. I., Hartweig, E. and Horvitz, H. R. (1993). Odorant-selective genes and Graf, E. R., Zhang, X., Jin, S. X., Linhoff, M. W. and Craig, A. M. (2004). Neurexins neurons mediate olfaction in C. elegans. Cell 74, 515-527. induce differentiation of GABA and glutamate postsynaptic specializations via Barstead, R. J. and Waterston, R. H. (1989). The basal component of the nematode neuroligins. Cell 119, 1013-1026. dense-body is vinculin. J. Biol. Chem. 264, 10177-10185. Granato, M., Schnabel, H. and Schnabel, R. (1994). Genesis of an organ: Molecular Bolliger, M. F., Frei, K., Winterhalter, K. H. and Gloor, S. M. (2001). Identification of a analysis of the pha-1 gene. Development 120, 3005-3017. novel neuroligin in humans which binds to PSD-95 and has a widespread Gray, J. M., Hill, J. J. and Bargmann, C. I. (2005). A circuit for navigation in expression. Biochem. J. 356, 581-588. Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 102, 3184-3191. Boucard, A. A., Chubykin, A. A., Comoletti, D., Taylor, P. and Südhof, T. C. (2005). A splice code for trans-synaptic cell adhesion mediated by binding of neuroligin 1 to Green, R. A., Audhya, A., Pozniakovsky, A., Dammermann, A., Pemble, H., Monen, J., Portier, N., Hyman, A., Desai, A. and Oegema, K. (2008). Expression and - and -neurexins. Neuron 48, 229-236. Brenner, S. (1974). The genetics of Caenorhabditis elegans. Genetics 77, 71-94. Imaging of Fluorescent Proteins in the C. elegans Gonad and Early Embryo. In Brockie, P. J., Madsen, D. M., Zheng, Y., Mellem, J. and Maricq, A. V. (2001a). Methods in Cell Biology: Fluorescent Proteins (ed. F. S. Kevin), pp. 179-218: Academic Differential expression of glutamate receptor subunits in the nervous system of Press. Caenorhabditis elegans and their regulation by the homeodomain protein UNC-42. J. Hedgecock, E. M. and Russell, R. L. (1975). Normal and mutant thermotaxis in the Neurosci. 21, 1510-1522. nematode Caenorhabditis elegans. Proc. Natl. Acad. Sci. USA 72, 4061-4065. DMM Brockie, P. J., Mellem, J. E., Hills, T., Madsen, D. M. and Maricq, A. V. (2001b). The C. Hedgecock, E. M., Culotti, J. G., Thomson, J. N. and Perkins, L. A. (1985). Axonal elegans glutamate receptor subunit NMR-1 is required for slow NMDA-activated guidance mutants of Caenorhabditis elegans identified by filling sensory neurons currents that regulate reversal frequency during locomotion. Neuron 31, 617-630. with fluorescein dyes. Dev. Biol. 111, 158-170. Chauhan, A., Chauhan, V., Brown, W. T. and Cohen, I. (2004). Oxidative stress in Hills, T., Brockie, P. J. and Maricq, A. V. (2004). Dopamine and glutamate control area- autism: Increased lipid peroxidation and reduced serum levels of ceruloplasmin and restricted search behavior in Caenorhabditis elegans. J. Neurosci. 24, 1217-1225. transferrin-the antioxidant proteins. Life Sci. 75, 2539-2549. Hobert, O. (2002). PCR fusion-based approach to create reporter gene constructs for Chih, B., Gollan, L. and Scheiffele, P. (2006). Alternative splicing controls selective expression analysis in transgenic C. elegans. Bio. Techniques 32, 728-730. trans-synaptic interactions of the neuroligin-neurexin complex. Neuron 51, 171-178. Horton, R. M., Hunt, H. D., Ho, S. N., Pullen, J. K. and Pease, L. R. (1989). Engineering Chou, J. H., Bargmann, C. I. and Sengupta, P. (2001). The Caenorhabditis elegans odr- hybrid genes without the use of restriction enzymes: gene splicing by overlap 2 gene encodes a novel Ly-6-related protein required for olfaction. Genetics 157, extension. Gene 77, 61-68. 211-224. Ichtchenko, K., Hata, Y., Nguyen, T., Ullrich, B., Missler, M., Moomaw, C. and Comoletti, D., Flynn, R. E., Boucard, A. A., Demeler, B., Schirf, V., Shi, J., Jennings, Südhof, T. C. (1995). Neuroligin 1, A splice site-specific ligand for -neurexins. Cell L. L., Newlin, H. R., Südhof, T. C. and Taylor, P. (2006). Gene selection, alternative 81, 435-443. splicing, and post-translational processing regulate neuroligin selectivity for - Ichtchenko, K., Nguyen, T. and Südhof, T. C. (1996). Structures, alternative splicing, Neurexins. Biochemistry 45, 12816-12827. and neurexin binding of multiple neuroligins. J. Biol. Chem. 271, 2676-2682. Dean, C., Scholl, F. G., Choih, J., DeMaria, S., Berger, J., Isacoff, E. and Scheiffele, P. Ishihara, T., Iino, Y., Mohri, A., Mori, I., Gengyo-Ando, K., Mitani, S. and Katsura, I. (2003). Neurexin mediates the assembly of presynaptic terminals. Nat. Neurosci. 6, (2002). HEN-1, a secretory protein with an LDL receptor motif, regulates sensory 708-716. integration and learning in Caenorhabditis elegans. Cell 109, 639-649. Desai, C., Garriga, G., McIntire, S. L. and Horvitz, H. R. (1988). A genetic pathway for Ishii, N., Takahashi, K., Tomita, S., Keino, T., Honda, S., Yoshino, K. and Suzuki, K. Disease Models & Mechanisms the development of the Caenorhabditis elegans HSN motor neurons. Nature 336, (1990). A methyl viologen-sensitive mutant of the nematode Caenorhabditis elegans. 638-646. Mutat. Res. 237, 165-171. Duerr, J. S., Frisby, D. L., Gaskin, J., Duke, A., Asermely, K., Huddleston, D., Eiden, Ishii, N., Fujii, M., Hartman, P. S., Tsuda, M., Yasuda, K., Senoo-Matsuda, N., L. E. and Rand, J. B. (1999). The cat-1 gene of C. elegans encodes a vesicular Yanase, S., Ayusawa, D. and Suzuki, K. (1998). A mutation in succinate monoamine transporter required for specific monoamine-dependent behaviors. J. dehydrogenase cytochrome b causes oxidative stress and ageing in nematodes. Neurosci. 19, 72-84. Nature 394, 694-697. Duerr, J. S., Gaskin, J. and Rand, J. B. (2001). Identified neurons in C. elegans co- Jamain, S., Quach, H., Betancur, C., Rastam, M., Colineaux, C., Gillberg, I. C., express vesicular transporters for acetylcholine and monoamines. Am. J. Physiol. Cell Soderstrom, H., Giros, B., Leboyer, M., Gillberg, C. et al. (2003). Mutations of the Physiol. 280, C1616-C1622. X-linked genes encoding neuroligins NLGN3 and NLGN4 are associated with autism. Duerr, J. S., Han, H.-P., Fields, S. D. and Rand, J. B. (2008). Identification of major Nat. Genet. 34, 27-29. classes of cholinergic neurons in the nematode Caenorhabditis elegans. J. Comp. Neurol. 506, 398-408. James, S. J., Melnyk, S., Jernigan, S., Cleves, M. A., Halsted, C. H., Wong, D. H., Durand, C. M., Betancur, C., Boeckers, T. M., Bockmann, J., Chaste, P., Fauchereau, Cutler, P., Bock, K., Boris, M., Bradstreet, J. J. et al. (2006). Metabolic F., Nygren, G., Rastam, M., Gillberg, I. C., Anckarsater, H. et al. (2007). Mutations endophenotype and related genotypes are associated with oxidative stress in in the gene encoding the synaptic scaffolding protein SHANK3 are associated with children with autism. Am. J. Med. Genet. B Neuropsychiatr. Genet. 141B, 947-956. autism spectrum disorders. Nat. Genet. 39, 25-27. Jin, Y. and Garner, C. C. (2008). Molecular mechanisms of presynaptic differentiation. Eastman, C., Horvitz, H. R. and Jin, Y. (1999). Coordinated transcriptional regulation Annu. Rev. Cell Dev. Biol. 24, 237. of the unc-25 glutamic acid decarboxylase and the unc-47 GABA vesicular Johnson, C. D., Rand, J. B., Herman, R. K., Stern, B. D. and Russell, R. L. (1988). The transporter by the Caenorhabditis elegans UNC-30 homeodomain protein. J. Neurosci. acetylcholinesterase genes of C. elegans: identification of a third gene (ace-3) and 19, 6225-6234. mosaic mapping of a synthetic lethal phenotype. Neuron 1, 165-173. Feinberg, E. H., VanHoven, M. K., Bendesky, A., Wang, G., Fetter, R. D., Shen, K. Kim, H. G., Kishikawa, S., Higgins, A. W., Seong, I.-S., Donovan, D. J., Shen, Y., Lally, and Bargmann, C. I. (2008). GFP Reconstitution Across Synaptic Partners (GRASP) E., Weiss, L. A., Najm, J., Kutsche, K. et al. (2008). Disruption of Neurexin 1 defines cell contacts and synapses in living nervous systems. Neuron 57, 353-363. associated with autism spectrum disorder. Am. J. Hum. Genet. 82, 199-207. Filipek, P. A., Accardo, P. J., Baranek, G. T., Cook, E. H., Dawson, G., Gordon, B., Kondo, M., Senoo-Matsuda, N., Yanase, S., Ishii, T., Hartman, P. S. and Ishii, N. Gravel, J. S., Johnson, C. P., Kallen, R. J., Levy, S. E. et al. (1999). The screening and (2005). Effect of oxidative stress on translocation of DAF-16 in oxygen-sensitive diagnosis of autistic spectrum disorders. J. Autism. Dev. Disord. 29, 439-484. mutants, mev-1 and gas-1 of Caenorhabditis elegans. Mech. Ageing Dev. 126, 637-641.

Disease Models & Mechanisms 375 RESEARCH ARTICLE Neuroligin mutants and oxidative stress

Koushika, S. P., Richmond, J. E., Hadwiger, G., Weimer, R. M., Jorgensen, E. M. and Gs pathway and define a third major branch of the synaptic signaling network. Nonet, M. L. (2001). A post-docking role for active zone protein Rim. Nat. Neurosci. 4, Genetics 169, 631-649. 997-1005. Scheiffele, P., Fan, J., Choih, J., Fetter, R. and Serafini, T. (2000). Neuroligin expressed Krause, M. and Hirsh, D. (1987). A trans-spliced leader sequence on actin mRNA in C. in nonneuronal cells triggers presynaptic development in contacting axons. Cell 101, elegans. Cell 49, 753-761. 657-669. Lackner, M. R., Nurrish, S. J. and Kaplan, J. M. (1999). Facilitation of synaptic Senoo-Matsuda, N., Yasuda, K., Tsuda, M., Ohkubo, T., Yoshimura, S., Nakazawa, transmission by EGL-30 Gq and EGL-8 PLC: DAG binding to UNC-13 is required to H., Hartman, P. S. and Ishii, N. (2001). A defect in the cytochrome b large subunit in stimulate acetylcholine release. Neuron 24, 335-346. complex II causes both superoxide anion overproduction and abnormal energy Laumonnier, F., Bonnet-Brilhault, F., Gomot, M., Blanc, R., David, A., Moizard, M. metabolism in Caenorhabditis elegans. J. Biol. Chem. 276, 41553-41558. P., Raynaud, M., Ronce, N., Lemonnier, E., Calvas, P. et al. (2004). X-linked mental Südhof, T. C. (2008). Neuroligins and neurexins link synaptic function to cognitive retardation and autism are associated with a mutation in the NLGN4 gene, a disease. Nature 455, 903-911. member of the neuroligin family. Am. J. Hum. Genet. 74, 552-557. Sulston, J., Dew, M. and Brenner, S. (1975). Dopaminergic neurons in the nematode Lee, R. Y. N., Sawin, E. R., Chalfie, M., Horvitz, H. R. and Avery, L. (1999). EAT-4, a Caenorhabditis elegans. J. Comp. Neurol. 163, 215-226. homolog of a mammalian sodium-dependent inorganic phosphate cotransporter, is Sun, A. Y. and Lambie, E. J. (1997). gon-2, a gene required for gonadogenesis in necessary for glutamatergic neurotransmission in Caenorhabditis elegans. J. Neurosci. Caenorhabditis elegans. Genetics 147, 1077-1089. 19, 159-167. Sze, J. Y., Victor, M., Loer, C., Shi, Y. and Ruvkun, G. (2000). Food and metabolic Lickteig, K. M., Duerr, J. S., Frisby, D. L., Hall, D. H., Rand, J. B. and Miller, D. M. signalling defects in a Caenorhabditis elegans serotonin-synthesis mutant. Nature (2001). Regulation of neurotransmitter vesicles by the homeodomain protein UNC-4 403, 560-564. and its transcriptional corepressor UNC-37/Groucho in Caenorhabditis elegans Taniguchi, H., Gollan, L., Scholl, F. G., Mahadomrongkul, V., Dobler, E., Limthong, cholinergic motor neurons. J. Neurosci. 21, 2001-2014. N., Peck, M., Aoki, C. and Scheiffele, P. (2007). Silencing of neuroligin function by Loria, P. M., Hodgkin, J. and Hobert, O. (2004). A conserved postsynaptic postsynaptic neurexins. J. Neurosci. 27, 2815-2824. transmembrane protein affecting neuromuscular signaling in Caenorhabditis elegans. The Autism Genome Project Consortium (2007). Mapping autism risk loci using J. Neurosci. 24, 2191-2201. genetic linkage and chromosomal rearrangements. Nat. Genet. 39, 319-328. McNally, K., Audhya, A., Oegema, K. and McNally, F. J. (2006). Katanin controls Tsalik, E. L. and Hobert, O. (2003). Functional mapping of neurons that control mitotic and meiotic spindle length. J. Cell Biol. 175, 881-891. locomotory behavior in Caenorhabditis elegans. J. Neurobiol. 56, 178-197. Mello, C. C., Kramer, J. M., Stinchcomb, D. and Ambros, V. (1991). Efficient gene Varoqueaux, F., Aramuni, G., Rawson, R. L., Mohrmann, R., Missler, M., Gottmann, transfer in C. elegans: extrachromosomal maintenance and integration of K., Zhang, W., Südhof, T. C. and Brose, N. (2006). Neuroligins determine synapse transforming sequences. EMBO J. 10, 3959-3970. maturation and function. Neuron 51, 741-754. Miller, D. M., Desai, N. S., Hardin, D. C., Piston, D. W., Patterson, G. H., Fleenor, J., Vincent, J. B., Kolozsvari, D., Roberts, W. S., Bolton, P. F., Gurling, H. M. and DMM Xu, S. and Fire, A. (1999). Two-color GFP expression system for C. elegans. Scherer, S. W. (2004). Mutation screening of X-chromosomal neuroligin genes: no BioTechniques 26, 914-921. mutations in 196 autism probands. Am. J. Med. Genet. B Neuropsychiatr. Genet. 129B, Miller, K. G., Alfonso, A., Nguyen, M., Crowell, J. A., Johnson, C. D. and Rand, J. B. 82-84. (1996). A genetic selection for Caenorhabditis elegans synaptic transmission mutants. White, J. G., Southgate, E., Thomson, J. N. and Brenner, S. (1986). The structure of Proc. Natl. Acad. Sci. USA 93, 12593-12598. the nervous system of the nematode Caenorhabditis elegans. Phil. Trans. R. Soc. Lond. Miller, K. G., Emerson, M. D. and Rand, J. B. (1999). Go and diacylglycerol kinase B 314, 1-340. negatively regulate the Gq pathway in C. elegans. Neuron 24, 323-333. Winnier, A. R., Meir, J. Y. J., Ross, J. M., Tavernarakis, N., Driscoll, M., Ishihara, T., Ming, X., Stein, T. P., Brimacombe, M., Johnson, W. G., Lambert, G. H. and Wagner, Katsura, I. and Miller, D. M. (1999). UNC-4/UNC-37-dependent repression of motor G. C. (2005). Increased excretion of a lipid peroxidation biomarker in autism. neuron-specific genes controls synaptic choice in Caenorhabditis elegans. Genes Dev. Prostaglandins Leukot Essent Fatty Acids 73, 379-384. 13, 2774-2786. Moessner, R., Marshall, C. R., Sutcliffe, J. S., Skaug, J., Pinto, D., Vincent, J., Yamamoto, K., Honda, S. and Ishii, N. (1996). Properties of an oxygen-sensitive Zwaigenbaum, L., Fernandez, B., Roberts, W., Szatmari, P. et al. (2007). mutant mev-3 of the nematode Caenorhabditis elegans. Mutation Research 358, 1-6. Contribution of SHANK3 mutations to autism spectrum disorder. Am. J. Hum. Genet. Yan, J., Oliveira, G., Coutinho, A., Yang, C., Feng, J., Katz, C., Sram, J., Bockholt, A., 81, 1289-1297. Jones, I. R., Craddock, N. et al. (2005). Analysis of the neuroligin 3 and 4 genes in Mori, I. and Ohshima, Y. (1995). Neural regulation of thermotaxis in Caenorhabditis autism and other neuropsychiatric patients. Mol. Psychiatry 10, 329-332. elegans. Nature 376, 344-348. Yanase, S., Yasuda, K. and Ishii, N. (2002). Adaptive responses to oxidative damage in Muhle, R., Trentacoste, S. V. and Rapin, I. (2004). The genetics of autism. Pediatrics three mutants of Caenorhabditis elegans (age-1, mev-1 and daf-16) that affect life 113, E472-E486. span. Mech. Ageing Dev. 123, 1579-1587. Mullen, G. P., Mathews, E. A., Saxena, P., Fields, S. D., McManus, J. R., Moulder, G., Yasuda, K., Adachi, H., Fujiwara, Y. and Ishii, N. (1999). Protein carbonyl Barstead, R. J., Quick, M. W. and Rand, J. B. (2006). The Caenorhabditis elegans snf- accumulation in aging Dauer formation-defective (daf) mutants of Caenorhabditis Disease Models & Mechanisms 11 gene encodes a sodium-dependent GABA transporter required for clearance of elegans. J. Gerontol. A. Biol. Sci. Med. Sci. 54, B47-B51. synaptic GABA. Mol. Biol. Cell 17, 3021-3030. Ylisaukko-oja, T., Rehnstrom, K., Auranen, M., Vanhala, R., Alen, R., Kempas, E., Nass, R., Hall, D. H., Miller, D. M. and Blakely, R. D. (2002). Neurotoxin-induced Ellonen, P., Turunen, J. A., Makkonen, I., Riikonen, R. et al. (2005). Analysis of four degeneration of dopamine neurons in Caenorhabditis elegans. Proc. Natl. Acad. Sci. neuroligin genes as candidates for autism. Eur. J. Hum. Genet. 13, 1285-1292. USA 99, 3264-3269. Yu, S., Avery, L., Baude, E. and Garbers, D. L. (1997). Guanylyl cyclase expression in Piechotta, K., Dudanova, I. and Missler, M. (2006). The resilient synapse: insights from specific sensory neurons: A new family of chemosensory receptors. Proc. Natl. Acad. genetic interference of synaptic cell adhesion molecules. Cell Tissue Res. 326, 617-642. Sci. USA 94, 3384-3387. Sajdel-Sulkowska, E. M., Lipinski, B., Windom, H., Audhya, T. and McGinnis, W. Zheng, Y., Brockie, P. J., Mellem, J. E., Madsen, D. M. and Maricq, A. V. (1999). (2008). Oxidative Stress in Autism: Elevated Cerebellar 3-nitrotyrosine Levels. Am. J. Neuronal control of locomotion in C. elegans is modified by a dominant mutation in Biochem. Biotechnol. 4, 73-84. the GLR-1 ionotropic glutamate receptor. Neuron 24, 347-361. Schade, M. A., Reynolds, N. K., Dollins, C. M. and Miller, K. G. (2005). Mutations that Zoghbi, H. Y. (2003). Postnatal neurodevelopmental disorders: Meeting at the rescue the paralysis of Caenorhabditis elegans ric-8 (synembryn) mutants activate the synapse? Science 302, 826-830.

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