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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

Behavioral phenotypes associated with tsp-17 mutants.A. Quantitative analysis of SWIP behavior at L4-stage, over 30 minutes. B. The SWIP phenotype of tsp-17(tm4995) in L4-stage worms is rescued by dop-3 deletion. C. Quantitative analysis of SWIP behavior in L1-stage worms over 20 min. D. The SWIP phenotype of tsp-17(tm4995) at L1 stage is not rescued by dop-3 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.
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pgen-1004767-g005: Behavioral phenotypes associated with tsp-17 mutants.A. Quantitative analysis of SWIP behavior at L4-stage, over 30 minutes. B. The SWIP phenotype of tsp-17(tm4995) in L4-stage worms is rescued by dop-3 deletion. C. Quantitative analysis of SWIP behavior in L1-stage worms over 20 min. D. The SWIP phenotype of tsp-17(tm4995) at L1 stage is not rescued by dop-3 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.

Mentions: Since these genetic interactions suggest that dopamine levels could be altered in tsp-17 mutants, we next investigated behavioral phenotypes associated with dopamine. Dopamine synthesis and release are required for the basal slowing response, in which worms reduce their speed when encountering a bacterial lawn [36]. We did not observe a defect in this response, indicating that both dopamine synthesis and extracellular dopamine sensing by receptors are intact in tsp-17 mutants (Figure S5A). One of the most accessible phenotypes thought to be associated with excessive extracellular dopamine is the SWIP (Swimming Induced Paralysis) phenotype [37]. While wild-type worms placed into a drop of water maintain their thrashing frequency dat-1 mutants become progressively paralyzed. The SWIP phenotype is ascribed to excessive extracellular dopamine as a consequence of the reuptake defect in the dat-1 mutant. Excessive extracellular dopamine triggers paralysis by hyperactivating the DOP-3 receptor expressed on cholinergic neurons and hence blocking acetylcholine release [33]. To perform this experiment, we placed L4 worms into drops of water and scored their ability to swim over a period of 30 minutes. As expected, we found that wild-type but not dat-1 mutant worms can swim for 30 minutes with no change in the speed or pattern of swimming. All four tsp-17 mutants showed a partial SWIP phenotype (Figure 5A). This phenotype is probably caused by dopaminergic signaling because it can be rescued by deletion of the dop-3 dopamine receptor and by deletion of the cat-2 tyrosine hydroxylase (Figure 5A and Figure 5B). It was surprising to find a SWIP phenotype in tsp-17 mutants as we argue that tsp-17 inhibits dat-1 function (see below). While elucidating the exact mechanism of how TSP-17 affects behavioral phenotypes will require further investigation we speculate that hyper-activation of DAT-1 in tsp-17 strains could trigger a feedback loop that transiently enhance extracellular dopamine levels inducing the weak SWIP phenotype we observe.


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)

Behavioral phenotypes associated with tsp-17 mutants.A. Quantitative analysis of SWIP behavior at L4-stage, over 30 minutes. B. The SWIP phenotype of tsp-17(tm4995) in L4-stage worms is rescued by dop-3 deletion. C. Quantitative analysis of SWIP behavior in L1-stage worms over 20 min. D. The SWIP phenotype of tsp-17(tm4995) at L1 stage is not rescued by dop-3 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.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4256090&req=5

pgen-1004767-g005: Behavioral phenotypes associated with tsp-17 mutants.A. Quantitative analysis of SWIP behavior at L4-stage, over 30 minutes. B. The SWIP phenotype of tsp-17(tm4995) in L4-stage worms is rescued by dop-3 deletion. C. Quantitative analysis of SWIP behavior in L1-stage worms over 20 min. D. The SWIP phenotype of tsp-17(tm4995) at L1 stage is not rescued by dop-3 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.
Mentions: Since these genetic interactions suggest that dopamine levels could be altered in tsp-17 mutants, we next investigated behavioral phenotypes associated with dopamine. Dopamine synthesis and release are required for the basal slowing response, in which worms reduce their speed when encountering a bacterial lawn [36]. We did not observe a defect in this response, indicating that both dopamine synthesis and extracellular dopamine sensing by receptors are intact in tsp-17 mutants (Figure S5A). One of the most accessible phenotypes thought to be associated with excessive extracellular dopamine is the SWIP (Swimming Induced Paralysis) phenotype [37]. While wild-type worms placed into a drop of water maintain their thrashing frequency dat-1 mutants become progressively paralyzed. The SWIP phenotype is ascribed to excessive extracellular dopamine as a consequence of the reuptake defect in the dat-1 mutant. Excessive extracellular dopamine triggers paralysis by hyperactivating the DOP-3 receptor expressed on cholinergic neurons and hence blocking acetylcholine release [33]. To perform this experiment, we placed L4 worms into drops of water and scored their ability to swim over a period of 30 minutes. As expected, we found that wild-type but not dat-1 mutant worms can swim for 30 minutes with no change in the speed or pattern of swimming. All four tsp-17 mutants showed a partial SWIP phenotype (Figure 5A). This phenotype is probably caused by dopaminergic signaling because it can be rescued by deletion of the dop-3 dopamine receptor and by deletion of the cat-2 tyrosine hydroxylase (Figure 5A and Figure 5B). It was surprising to find a SWIP phenotype in tsp-17 mutants as we argue that tsp-17 inhibits dat-1 function (see below). While elucidating the exact mechanism of how TSP-17 affects behavioral phenotypes will require further investigation we speculate that hyper-activation of DAT-1 in tsp-17 strains could trigger a feedback loop that transiently enhance extracellular dopamine levels inducing the weak SWIP phenotype we observe.

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