Tetraspanin (TSP-17) Protects Dopaminergic Neurons against 6-OHDA-Induced Neurodegeneration in <i>C. elegans</i>

Parkinson's disease (PD), the second most prevalent neurodegenerative disease after Alzheimer's disease, is linked to the gradual loss of dopaminergic neurons in the substantia nigra. Disease loci causing hereditary forms of PD are known, but most cases are attributable to a combination of genetic and environmental risk factors. Increased incidence of PD is associated with rural living and pesticide exposure, and dopaminergic neurodegeneration can be triggered by neurotoxins such as 6-hydroxydopamine (6-OHDA). In C. elegans, this drug is taken up by the presynaptic dopamine reuptake transporter (DAT-1) and causes selective death of the eight dopaminergic neurons of the adult hermaphrodite. Using a forward genetic approach to find genes that protect against 6-OHDA-mediated neurodegeneration, we identified tsp-17, which encodes a member of the tetraspanin family of membrane proteins. We show that TSP-17 is expressed in dopaminergic neurons and provide genetic, pharmacological and biochemical evidence that it inhibits DAT-1, thus leading to increased 6-OHDA uptake in tsp-17 loss-of-function mutants. TSP-17 also protects against toxicity conferred by excessive intracellular dopamine. We provide genetic and biochemical evidence that TSP-17 acts partly via the DOP-2 dopamine receptor to negatively regulate DAT-1. tsp-17 mutants also have subtle behavioral phenotypes, some of which are conferred by aberrant dopamine signaling. Incubating mutant worms in liquid medium leads to swimming-induced paralysis. In the L1 larval stage, this phenotype is linked to lethality and cannot be rescued by a dop-3 null mutant. In contrast, mild paralysis occurring in the L4 larval stage is suppressed by dop-3, suggesting defects in dopaminergic signaling. In summary, we show that TSP-17 protects against neurodegeneration and has a role in modulating behaviors linked to dopamine signaling

<i>gt1681</i> mutants are hypersensitive to 6-OHDA.

<p><b>A</b>. The extent of dopaminergic degeneration is indicated for wild-type and <i>gt1681</i> mutant worms after intoxication with 10 mM 6-OHDA. Neurodegeneration of L1 worms was scored after 24, 48 and 72 h as described in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1004767#s4" target="_blank">Materials and Methods</a>, and categorized as “complete loss,” “partial loss” or “no loss” phenotypes (labeled black, white and gray, respectively). Asterisks represent statistical significance of differences from wild-type (****p<0.00001). <b>B</b>. Representative images showing progressive stages of dopaminergic neurodegeneration. Absence of degeneration in wild-type (upper left panel) and complete degeneration in <i>gt1681</i> mutant worms 72 h post 6-OHDA intoxication (upper right panel, complete degeneration); lower panel and inset are examples of partial degeneration <i>in gt1881</i>. Arrows indicate ‘blebs in degenerating neurons. <b>C</b>. Extent of neurodegeneration at various developmental stages in wild-type and <i>gt1681</i> mutant worms 72 h post 6-OHDA intoxication. <b>D</b>. In wild-type and <i>gt1681</i> worms, development is equally retarded following treatment with 6-OHDA. Progression to various developmental stages was scored once 95% of untreated worms reached adulthood.</p

Dopamine receptors act antagonistically to modulate the sensitivity of <i>tsp-17 (gt1681)</i> mutants to 6-OHDA.

<p>Worms of the indicated genotypes were intoxicated with <b>A</b>. 50 mM, <b>B</b>. 10 mM 6-OHDA and <b>C</b>. 5 mM 6-OHDA and scored for neurodegeneration 72 h post intoxication. Experiments were done in triplicate and the average data is presented. N, total number of animals examined for each strain. Error bars represent the standard error of the mean. Asterisks represent statistically significant differences (***p<0.0001, ****p<0.00001). <b>D</b>. Evidence for a direct interaction between DOP-2 and TSP-17. Growth on -Leu, -Trp, -His (left panel) and -Leu, -Trp, -His, -Ade (middle panel) plates is shown. The right panel depicts a β-galactosidase assay. <b>E</b>. Working model as to how TSP-17 might interact with DAT-1 and DOP-2 to modulate level of DAT-1 activity. Arrows indicate activation. T-bars indicate repression. The question mark indicates that we do not know the mechanism of DAT-1 inhibition by TSP-17.</p

Evidence for DAT-1 hyperactivation in <i>tsp-17</i> worms.

<p><b>A</b>. <i>dat-1::yfp</i> transgenic worms (TG2470) do not exhibit hypersensitivity to 10 mM 6-OHDA. The extent of neurodegeneration was scored 72 h post 6-OHDA intoxication. Asterisks represent statistically significant differences compared to <i>tsp-17</i> worms (****p<0.00001) <b>B</b>. More imipramine than in wild-type worms is needed to prevent neurodegeneration in <i>tsp-17</i> mutants and in <i>dat-1::yfp</i> overexpression worms co-treated with 50 mM 6-OHDA. Data presented is from scoring the extent of neurodegeneration 72 h post 6-OHDA intoxication. The imipramine concentration is indicated on the x axis. N, total number of worms from each strain examined for every treatment. Error bars represent the standard error of the mean. Asterisks (below top bar) represent statistically significant differences compared to wild-type; worms treated with 0.125 mM imipramine are compared (****p<0.00001). Lower bars indicate difference within individual strains (no imipramine compared to 0.125 mM imipramine; *p<0.05, *p<0.005, ****p<0.00001) <b>C., D</b>. [³H]-dopamine (DA) uptake in wild-type and <i>tsp-17</i> worms. Uptake assays were performed using 50 nM (<b>C</b>) and 250 nM [³H]-DA (<b>D</b>).</p

<i>tsp-17 (gt1681)</i> enhances the neurodegeneration phenotype of <i>cat-2</i>-overexpressing lines, and <i>cat-2</i> overexpression protects against 6-OHDA toxicity.

<p><b>A</b>. <i>cat-2</i> induced neurodegeneration. Analysis of <i>cat-2</i>-overexpressing stains UA57 <i>baIn4 [pdat-1::gfp pdat-1::cat-2]</i> and TG2402 <i>baIn4[pdat-1::gfp pdat-1::cat-2]; tsp-17(gt1681)</i>. Error bars represent standard deviation. Asterisks represent statistical difference between <i>cat-2::gfp</i> adults on days 5 and 7 (**p<0.005). <b>B</b>. <i>cat-2</i> overexpression suppresses 6-OHDA-induced neurotoxicity. Experiments were done in triplicate and the average is shown. Data presented is from scoring the extent of neurodegeneration 72 h post 6-OHDA intoxication.</p

Behavioral phenotypes associated with <i>tsp-17</i> mutants.

<p><b>A</b>. Quantitative analysis of SWIP behavior at L4-stage, over 30 minutes. <b>B</b>. The SWIP phenotype of <i>tsp-17(tm4995)</i> in L4-stage worms is rescued by <i>dop-3</i> deletion. <b>C</b>. Quantitative analysis of SWIP behavior in L1-stage worms over 20 min. <b>D</b>. The SWIP phenotype of <i>tsp-17(tm4995)</i> at L1 stage is not rescued by <i>dop-3</i> deletion. Assays were done in triplicate for the total number of worms indicated by N values. Error bars represent the standard error of the mean. Asterisks represent statistically significant differences from the wild-type (***p<0.01). To facilitate comparison, strains are indicated by the same color code.</p

The TSP-17 tetraspanin family member protects dopaminergic neurons from 6-OHDA.

<p><b>A</b>. Extent of neurodegeneration in heterozygous and trans-heterozygous worms 72 h post 6-OHDA intoxication. <b>B</b>. Schematic gene model of the two isoforms of <i>tsp-17</i>. Alleles used in this study are indicated. <b>C</b>. Complementation of <i>tsp-17</i> expressed under its own promoter (3^rd column strain TG2439) and under the <i>dat-1</i> promoter (4^th column, strain TG2440). Data presented is from scoring the extent of neurodegeneration 72 h post 6-OHDA intoxication. Asterisks represent statistical significance of differences between <i>tsp-17</i> and the rescuing lines (****p<0.0001). <b>D</b>. 6-OHDA hypersensitivity conferred by various <i>tsp-17</i> alleles. <b>E</b>. Alignment of nematode TSP-17 to the most closely related human tetraspanins. Blue bars indicate transmembrane domains and brown bars designate extracellular loops (EC1 and EC2). The arrow indicates amino acid G109, which is mutated in the <i>C. elegans gt1681</i> and <i>vc2026</i> mutants. The red box indicates the CCG motif in the EC2, which is highly conserved throughout the tetraspanin protein family. Hs, <i>Homo sapiens</i>; Ce, <i>Caenorhabditis elegans</i>; Cbn, <i>C. brenneri</i>; Cre, <i>C. remanei</i>; Cbr, <i>C. briggsae</i>.</p

TSP-17::GFP expression.

<p>Analysis of the TG2439 strain containing dopaminergic neurons labeled by mCherry and <i>tsp-17</i> C-terminally fused to GFP and driven by its own promoter. <b>A, D, G, J</b>. Dopaminergic neurons expressing the mCherry marker. Neurons are indicated. White arrows highlight dendrites and axons. <b>B, E. H, K</b>. Expression of TSP-17::GFP. <b>C, F, I, L</b>. Merged images. White arrows highlight dendrites and axons. <b>K, L, N</b>. The arrow-heads indicate TSP-17::GFP signal enrichment around the nucleus. Expression in the vulva (<b>M</b>), the spermatheca (<b>N</b>), a NSM neuron (<b>O</b>), and in body wall muscle cells (<b>P</b>).</p