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

Abstract

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.

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 5′-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 5′-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 5′-ends of exons 4 and 16, and the putative tandem alternative splice donor sites at the 3′-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).

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.

Fig. 3.

Neuroligin-expressing cells. Confocal images of young adult transgenic animals expressing a Pnlg-1::YFP reporter (FRM77, see Fig. 1). Anterior is to the left and ventral is down. The reporter is expressed in ∼45 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 ttx-3 reporter [specific for AIY neurons (Altun-Gultekin et al., 2001)] is shown in red (nr, nerve ring; vnc, ventral nerve cord). A portion of the ventral nerve cord is shown in D–F. The neuroligin reporter is shown in green and a cholinergic reporter is shown in red. The neuroligin reporter is expressed in a subset of cholinergic motor neurons in the ventral cord. A region of an adult body is enlarged in G, showing that the neuroligin reporter is expressed in body wall muscles. Bars, ∼10 μm.

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; 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 μm (A–C); ∼2.5 μm (D–F).

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.

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/μg 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 (*) 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.