PMC

Extrasynaptic glutamate model for swip-10-dependent hyperdopaminergia. Schematic rendition of the hypothesis that, whereas Swip of dat-1 animals arises from an inability to clear DA at normal rates of Glu-dependent DA neuron excitation, paralysis of swip-10 animals arises from an increase in extrasynaptic Glu that results in changes in DA neuron excitability and excess DA release, overwhelming DA clearance mechanisms.

DA- and dop-3-dependent Swip mutants vt29 and vt33 harbor mutations in F53B1.6. A, Genomic localization of mutations in vt29 and vt33 and gene diagram of F53B1.6. Lines vt29 and vt33 harbor, respectively, nonsense and a nonconservative missense mutation in exon 10 of FB3B1.6. The tm5915 allele deletes 339 bp spanning F53B1.6 exons 2, 3, and 4 along with a 2 bp insertion. B, Manual Swip analyses of vt29, vt33, and tm5915 demonstrated that these lines exhibit DA synthesis (cat-2)- and DA receptor (dop-3)-dependent Swip. Assays were performed as described in the Materials and Methods. Data were analyzed using one-way ANOVA with multiple Bonferroni posttests where ****p < 0.0001 and error bars represent SEM. Asterisks indicate a comparison of double mutants back to the single mutant vt29, vt33, and tm5915, respectively. Loss of cat-2 and dop-3 alone does not result in a significant change in swimming.

Expression pattern of swip-10. A, Summary diagram of constructs used for swip-10 promoter GFP fusion experiments. Data derive from L4 animals. PCR products were generated via overlap PCR as described in the Materials and Methods. B, GFP expression under the control of the swip-10^a promoter in hypodermal cells. C–E, GFP expression driven by swip-10^b is visible along processes (arrowheads) that run parallel to DA neuron dendrites as revealed by coexpression with dat-1: mCherry. F–H, swip-10^b:GFP is expressed in a number of cells located in the head that do not overlap with DA neurons. Arrowheads in F denote processes similar to C–E. I–K, swip-10^c:GFP is expressed in multiple head cells, include low expression in DA neurons (arrowheads). L–N, swip-10^b:GFP expression (L) is colocalized in multiple cells with reporter driven by the pan-glial promoter ptr-10 (M). Scale bars, 10 μm in all images. Dotted lines denote the outline of the worm head as revealed by DIC imaging.

swip-10 paralysis is dependent on vesicular Glu release and GluRs. A, Automated thrashing analysis reveals that swip-10 paralysis suppressed by loss of the vesicular GLT eat-4. Curves represent the average thrashing frequency of at least 25 independent animals recorded over the course of several sessions. eat-4;swip-10(vt29) is significantly suppressed from swip-10(vt29) progeny during minutes 1–3 and eat-4;swip-10(vt33) is significantly suppressed from swip-10(vt33) from minutes 2–10. We did not observe any significant difference between eat-4 and N2 (data not shown). B, Loss of glr-4, glr-6, and mgl-1 additively suppress swip-10 paralysis. Individual mutations of these GluRs have no impact on swimming behavior. C, Loss of glr-4 significantly suppresses swip-10(vt29). glr-4;swip-10 is significantly elevated from swip-10(vt29) starting in the first minute. D, Loss of glr-6 significantly suppresses swip-10(tm5915) in minutes 4–10. E, Loss of mgl-1 significantly suppresses swip-10(tm5915) in minutes 3–10. F, Loss of mgl-1 significantly restores dat-1 Swip, whereas loss of glr-4 or glr-6 does not affect dat-1 paralysis. For all panels, double and triple mutant strains were constructed and assays were performed as described in the Materials and Methods. For B and F, data were analyzed by one-way ANOVA with multiple Bonferroni posttests. ***p < 0.001, ****p < 0.0001. For A, B, D, and E, single worm recordings were performed as described in the Materials and Methods and analyzed with SwimR. Data were analyzed by two-way ANOVA with multiple Bonferroni posttests at each time point.

Loss of GLT genes produces DA-dependent Swip. A, Manual Swip assays of glutamate transporter alleles. Loss of glt-1, glt-3, glt-4, or glt-6 produces a significant Swip phenotype relative to N2 when assessed at a single 10 min time point. B, Automated thrashing analysis reveals that loss of glt-1 produces a significant Swip phenotype relative to N2 (significant reduction at all time points). Multiple comparisons of swip-10 versus swip-10 glt-1 mutants show that swip-10 glt-1 mutants are significantly reduced from swip-10 mutants from time points 0–48 s. C, Automated thrashing analysis reveals that loss of glt-3 produces a significant Swip phenotype relative to N2 (significant reduction at scattered time points during minutes 6–9 from 9:13–10:00). Multiple comparisons of swip-10 versus glt-3;swip-10 mutants show that glt-3;swip-10 mutants are significantly reduced from swip-10 mutants from time points 1:12–1:18, 1:29–1:32, and 1:34–1:39. D, Automated thrashing analysis reveals that loss of glt-4 produces a significant Swip phenotype relative to N2 (significant reduction at scattered time points during minutes 2–6). Multiple comparisons of swip-10 versus swip-10 glt-4 mutants show that swip-10 glt-4 mutants are significantly reduced from swip-10 mutants from time points 0:55–1:31, 1:37–2:01, and 2:07–2:19. E, Heat maps of the first minute of automated recordings for N2, swip-10, glt-1, and swip-10 glt-1 strains. Colors represent normalized thrashing values within that strain where red is the highest thrashing value and green is no movement. F, Swip in glt-1, glt-3, and glt-4 is DA dependent because loss of cat-2 restores swimming behavior in glt-1, glt-3, and glt-4 to wild-type swimming levels. For A and F, data were analyzed using one-way ANOVA with multiple Bonferroni posttests where ****p < 0.0001 and error bars represent SEM. For B–D, single worm recordings were performed as described in the Materials and Methods and analyzed with SwimR. Data were analyzed by two-way ANOVA with multiple Bonferroni posttests at each time point. n ≥ 33 for each strain.

Expression of F53B1.6 in glial cells is sufficient to rescue Swip. Expression of a swip-10 genomic fragment significantly restores swimming behavior in vt29 (second bar) and vt33 (seventh bar) mutants. Similarly, expression of swip-10 cDNA under the control of a pan glial promoter ptr-10, and not in DA neurons, significantly rescues vt29, vt33, and tm5915. Mutation of histidine residues within the conserved canonical β-lactamase motif significantly reduces the ability of the p_ptr-10: swip-10 cDNA to rescue vt29. For all experiments, transgenic lines were generated and tested as described in the Materials and Methods. The promoter used to drive expression of the swip-10 gene, and swip-10 coding sequences (CDS; either genomic or cDNA) are noted beneath the bars. The basal Swip values from nontransgenic animals shown for swip-10(vt29), swip-10(vt33), and swip-10(tm5915) are representative averages obtained during these experiments and consistent with values presented in . Values from at least three independent transgenic lines were pooled to generate the averages shown. Data were analyzed using an unpaired Student's t test comparing nontransgenic and transgenic progeny assayed in parallel. **p < 0.01, ***p < 0.001, ****p < 0.0001, ####p < 0.0001 from a t test between averages of transgenic progeny.

Loss of swip-10 results in elevated DA neuron excitability that is restored by expression of swip-10 in glia. A, Left, Schematic of recording apparatus and assay conditions. Top right, Illustration of background transgenic strain used for CARIBN experiments. Bottom right, Representative trace of wild-type background strain before entering a lawn of food (white bar). Scale bar, ΔR/R of 20% and 20 s. B, Food triggers an increase in DA neuron activity that is greater in worms lacking swip-10. Expression of swip-10 under a ptr-10 promoter restores the magnitude of the GCaMP/dsRed ratio to wild-type levels. Data represent average traces of WT, swip-10, and rescued strains after encountering food (red arrow). n = at least 17 worms per group. The WT strain is XuIs14(p_dat-1: GCaMP, p_dat-1:dsRed); lite-1(Xu7). C, Area under the curve quantification for B. swip-10(tm5915) displays a significantly elevated AUC from WT levels that is significantly restored by expression of swip-10 expressed with a ptr-10 promoter. Data were analyzed by one-way ANOVA with multiple Bonferroni posttests where *, **, and *** indicate a *p < 0.05, **p < 0.01, ***p < 0.001, respectively. No significant differences were found between WT and strains containing ex227 and ex228.

Loss of swip-10 results in elevated vesicular release in DA terminals and DA-dependent reductions in crawling speed. A, Schematic illustration of the experimental transgenes used in B. The pH-sensitive GFP fluorophore pHlourin fused in frame to the C terminus of the synaptic vesicle protein SNB-1 was expressed solely in DA neurons via the asic-1 promoter as in . Vesicular fusion rates at DA neuron synapses was then monitored using FRAP. Images illustrate a wild-type PDE neuron synapse before bleaching and during different points of recovery. B, Loss of swip-10 results in a significantly elevated rate of fluorescence recovery, indicative of a more rapid vesicular fusion rate. Data were fit to a one phase exponential using nonlinear regression. K (rate constant) = 0.032 ± 0.002 s⁻¹ for N2 vs 0.063 ± 0.01 s⁻¹. p < 0.05, Student's two-tailed t test. Red arrow denotes the time point immediately after photobleaching. Confocal images and FRAP were performed as described in the Materials and Methods. C, Loss of swip-10 results in a DA-dependent decrease in crawling on solid substrate. Crawling videos and tracking were performed using WormLab as described in the Materials and Methods. Data were analyzed using one-way ANOVA with multiple Tukey posttests. Unlike dat-1, swip-10(vt29) demonstrates a significantly reduced speed relative to N2 that is fully restored by loss of cat-2. ***p < 0.001.

Phylogenetic conservation of swip-10 and CNS expression of the swip-10 mouse homolog Mblac1. A, The SWIP-10 protein exhibits significant conservation with its nematode homolog CBG14084 across phylogeny, concentrated at the protein's C-terminal MBD. The vertebrate homologs have much shorter protein sequences and only begin to align at 260 aa into the SWIP-10 protein sequence. Shaded residues indicate amino acids that match the SWIP-10 sequence. Red bar indicates the span of the MBD that begins with a canonical “ILVDTG” motif. Blue boxes indicate histidine residues that are predicted to be critical for metal binding, including a canonical HxHxDH motif that is typical of the entire metallo β-lactamase superfamily. Yellow boxes indicate amino acids that are altered in the strains isolated from the Swip-based mutagenesis screen. swip-10(vt29) harbors a SNP that results in the conversion of a conserved tryptophan at position 377 to a stop codon, resulting in a truncated MBD. The mutation in swip-10(vt33) converts a conserved glycine at position 362 to glutamic acid. Sequences were aligned using ClustalW (DNAStar). B, Dendrogram generated from multiple sequence alignment of swip-10 with Mblac1, MBLAC1, and another MBD containing protein in mouse and human named Mblac2 and MBLAC2, respectively. swip-10 exhibits greater identity with Mblac1 and MBLAC1. C, RT-PCR of Mblac1, Mblac2, and Gapdh from different areas of the mouse brain, heart, lung, and liver. We observed the presence of both RNAs in tissue harvested from all tissues assayed, but not in whole-brain lysates lacking reverse transcriptase.