Limits...
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

Dopamine receptors act antagonistically to modulate the sensitivity of tsp-17 (gt1681) mutants to 6-OHDA.Worms of the indicated genotypes were intoxicated with A. 50 mM, B. 10 mM 6-OHDA and C. 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). D. 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. E. 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.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4256090&req=5

pgen-1004767-g007: Dopamine receptors act antagonistically to modulate the sensitivity of tsp-17 (gt1681) mutants to 6-OHDA.Worms of the indicated genotypes were intoxicated with A. 50 mM, B. 10 mM 6-OHDA and C. 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). D. 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. E. 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.

Mentions: Our combined genetic, pharmacological and biochemical analysis suggests that TSP-17 modulates DAT-1 activity. Previous studies using tissue culture-based assays demonstrated that dopamine receptor activation might promote DAT-1 activity [25], [50], [51]. Consistent with these results, we found dop-2 and dop-3 mutant worms to be partially resistant to high doses of 6-OHDA compared to wild-type (Figure 7A). We therefore investigated whether tsp-17 genetically interacts with dopamine receptors to modify DAT-1 activity and confer differential 6-OHDA sensitivity. This was done by assessing the sensitivity of tsp-17 mutants in the absence of the C. elegans DOP-1 D1-like receptor and/or in the absence of the DOP-2 and/or DOP-3 D2-like receptors. C. elegans DOP-1 is expressed in a variety of cells, including cholinergic neurons, mechanosensory neurons, head muscles and neuronal support cells. DOP-3 is expressed postsynaptically and its antagonism of DOP-1 in cholinergic neurons is required for the regulation of locomotion [33]. The DOP-2 receptor is expressed both postsynaptically and presynaptically. When expressed presynaptically, it acts as an autoreceptor on the plasma membrane of dopaminergic neurons. We found that dop-1; tsp-17 (gt1681) was as sensitive to 6-OHDA as the respective tsp-17 single mutant. In contrast, 6-OHDA hypersensitivity was reduced in dop-2; tsp-17 (gt1681) and dop-2; tsp-17 (tm4994) and in dop-3; tsp-17 (gt1681) and dop-3; tsp-17 (tm4994) double mutant worms (Figure 7B, C and Figure S9) Our genetic data thus argue that TSP-17 might inhibit DOP-2 and DOP-3 function, which in turn might be required for full DAT-1 transporter activity (Figure 7A, E). Given that deletion of dop-2 and dop-3 only partially rescues 6-OHDA hypersensitivity in tsp-17 mutants, we speculate that TSP-17 also inhibits DAT-1 activity independently of DOP-2 and DOP-3.


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)

Dopamine receptors act antagonistically to modulate the sensitivity of tsp-17 (gt1681) mutants to 6-OHDA.Worms of the indicated genotypes were intoxicated with A. 50 mM, B. 10 mM 6-OHDA and C. 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). D. 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. E. 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.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004767-g007: Dopamine receptors act antagonistically to modulate the sensitivity of tsp-17 (gt1681) mutants to 6-OHDA.Worms of the indicated genotypes were intoxicated with A. 50 mM, B. 10 mM 6-OHDA and C. 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). D. 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. E. 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.
Mentions: Our combined genetic, pharmacological and biochemical analysis suggests that TSP-17 modulates DAT-1 activity. Previous studies using tissue culture-based assays demonstrated that dopamine receptor activation might promote DAT-1 activity [25], [50], [51]. Consistent with these results, we found dop-2 and dop-3 mutant worms to be partially resistant to high doses of 6-OHDA compared to wild-type (Figure 7A). We therefore investigated whether tsp-17 genetically interacts with dopamine receptors to modify DAT-1 activity and confer differential 6-OHDA sensitivity. This was done by assessing the sensitivity of tsp-17 mutants in the absence of the C. elegans DOP-1 D1-like receptor and/or in the absence of the DOP-2 and/or DOP-3 D2-like receptors. C. elegans DOP-1 is expressed in a variety of cells, including cholinergic neurons, mechanosensory neurons, head muscles and neuronal support cells. DOP-3 is expressed postsynaptically and its antagonism of DOP-1 in cholinergic neurons is required for the regulation of locomotion [33]. The DOP-2 receptor is expressed both postsynaptically and presynaptically. When expressed presynaptically, it acts as an autoreceptor on the plasma membrane of dopaminergic neurons. We found that dop-1; tsp-17 (gt1681) was as sensitive to 6-OHDA as the respective tsp-17 single mutant. In contrast, 6-OHDA hypersensitivity was reduced in dop-2; tsp-17 (gt1681) and dop-2; tsp-17 (tm4994) and in dop-3; tsp-17 (gt1681) and dop-3; tsp-17 (tm4994) double mutant worms (Figure 7B, C and Figure S9) Our genetic data thus argue that TSP-17 might inhibit DOP-2 and DOP-3 function, which in turn might be required for full DAT-1 transporter activity (Figure 7A, E). Given that deletion of dop-2 and dop-3 only partially rescues 6-OHDA hypersensitivity in tsp-17 mutants, we speculate that TSP-17 also inhibits DAT-1 activity independently of DOP-2 and DOP-3.

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