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Tetraspanin (TSP-17) protects dopaminergic neurons against 6-OHDA-induced neurodegeneration in C. elegans.

Masoudi N, Ibanez-Cruceyra P, Offenburger SL, Holmes A, Gartner A - PLoS Genet. (2014)

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee, United Kingdom.

ABSTRACT
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 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.

No MeSH data available.


Related in: MedlinePlus

Evidence for DAT-1 hyperactivation in tsp-17 worms.A. dat-1::yfp 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 tsp-17 worms (****p<0.00001) B. More imipramine than in wild-type worms is needed to prevent neurodegeneration in tsp-17 mutants and in dat-1::yfp 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) C., D. [3H]-dopamine (DA) uptake in wild-type and tsp-17 worms. Uptake assays were performed using 50 nM (C) and 250 nM [3H]-DA (D).
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pgen-1004767-g006: Evidence for DAT-1 hyperactivation in tsp-17 worms.A. dat-1::yfp 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 tsp-17 worms (****p<0.00001) B. More imipramine than in wild-type worms is needed to prevent neurodegeneration in tsp-17 mutants and in dat-1::yfp 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) C., D. [3H]-dopamine (DA) uptake in wild-type and tsp-17 worms. Uptake assays were performed using 50 nM (C) and 250 nM [3H]-DA (D).

Mentions: To systematically test whether TSP-17 protects dopaminergic neurons by modulating dopamine metabolism, catabolism, reuptake or signaling, we performed a genetic epistasis analysis. As expected, tsp-17 dat-1 double mutants were completely resistant to 6-OHDA-induced neurodegeneration, consistent with the notion that TSP-17 does not bypass 6-OHDA uptake by the DAT-1 dopamine transporter (Figure 6A). We observed no alterations in 6-OHDA sensitivity in cat-2 (tyrosine hydroxylase), bas-1 (aromatic amino acid decarboxylase/AAADC) and cat-1 (VMAT ortholog required for dopamine packaging) tsp-17 double mutants, indicating that TSP-17 is unlikely to affect levels of dopamine synthesis or packaging (Figure S6).


Tetraspanin (TSP-17) protects dopaminergic neurons against 6-OHDA-induced neurodegeneration in C. elegans.

Masoudi N, Ibanez-Cruceyra P, Offenburger SL, Holmes A, Gartner A - PLoS Genet. (2014)

Evidence for DAT-1 hyperactivation in tsp-17 worms.A. dat-1::yfp 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 tsp-17 worms (****p<0.00001) B. More imipramine than in wild-type worms is needed to prevent neurodegeneration in tsp-17 mutants and in dat-1::yfp 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) C., D. [3H]-dopamine (DA) uptake in wild-type and tsp-17 worms. Uptake assays were performed using 50 nM (C) and 250 nM [3H]-DA (D).
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Related In: Results  -  Collection

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pgen-1004767-g006: Evidence for DAT-1 hyperactivation in tsp-17 worms.A. dat-1::yfp 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 tsp-17 worms (****p<0.00001) B. More imipramine than in wild-type worms is needed to prevent neurodegeneration in tsp-17 mutants and in dat-1::yfp 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) C., D. [3H]-dopamine (DA) uptake in wild-type and tsp-17 worms. Uptake assays were performed using 50 nM (C) and 250 nM [3H]-DA (D).
Mentions: To systematically test whether TSP-17 protects dopaminergic neurons by modulating dopamine metabolism, catabolism, reuptake or signaling, we performed a genetic epistasis analysis. As expected, tsp-17 dat-1 double mutants were completely resistant to 6-OHDA-induced neurodegeneration, consistent with the notion that TSP-17 does not bypass 6-OHDA uptake by the DAT-1 dopamine transporter (Figure 6A). We observed no alterations in 6-OHDA sensitivity in cat-2 (tyrosine hydroxylase), bas-1 (aromatic amino acid decarboxylase/AAADC) and cat-1 (VMAT ortholog required for dopamine packaging) tsp-17 double mutants, indicating that TSP-17 is unlikely to affect levels of dopamine synthesis or packaging (Figure S6).

Bottom Line: 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 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.

View Article: PubMed Central - PubMed

Affiliation: Centre for Gene Regulation and Expression, University of Dundee, Dow Street, Dundee, United Kingdom.

ABSTRACT
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 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.

No MeSH data available.


Related in: MedlinePlus