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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

gt1681 mutants are hypersensitive to 6-OHDA.A. The extent of dopaminergic degeneration is indicated for wild-type and gt1681 mutant worms after intoxication with 10 mM 6-OHDA. Neurodegeneration of L1 worms was scored after 24, 48 and 72 h as described in Materials and Methods, 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. Representative images showing progressive stages of dopaminergic neurodegeneration. Absence of degeneration in wild-type (upper left panel) and complete degeneration in gt1681 mutant worms 72 h post 6-OHDA intoxication (upper right panel, complete degeneration); lower panel and inset are examples of partial degeneration in gt1881. Arrows indicate ‘blebs in degenerating neurons. C. Extent of neurodegeneration at various developmental stages in wild-type and gt1681 mutant worms 72 h post 6-OHDA intoxication. D. In wild-type and gt1681 worms, development is equally retarded following treatment with 6-OHDA. Progression to various developmental stages was scored once 95% of untreated worms reached adulthood.
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pgen-1004767-g001: gt1681 mutants are hypersensitive to 6-OHDA.A. The extent of dopaminergic degeneration is indicated for wild-type and gt1681 mutant worms after intoxication with 10 mM 6-OHDA. Neurodegeneration of L1 worms was scored after 24, 48 and 72 h as described in Materials and Methods, 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. Representative images showing progressive stages of dopaminergic neurodegeneration. Absence of degeneration in wild-type (upper left panel) and complete degeneration in gt1681 mutant worms 72 h post 6-OHDA intoxication (upper right panel, complete degeneration); lower panel and inset are examples of partial degeneration in gt1881. Arrows indicate ‘blebs in degenerating neurons. C. Extent of neurodegeneration at various developmental stages in wild-type and gt1681 mutant worms 72 h post 6-OHDA intoxication. D. In wild-type and gt1681 worms, development is equally retarded following treatment with 6-OHDA. Progression to various developmental stages was scored once 95% of untreated worms reached adulthood.

Mentions: In order to find genes that protect dopaminergic neurons, we performed a genetic screen for mutants conferring hypersensitivity to 6-OHDA. By adapting procedures initially established by Nass et al. [3] and using the same pdat-1::GFP reporter that highlights dopaminergic neurons, we screened ∼2500 F2 ethyl methanesulfonate (EMS)-mutagenized worms at the L1 developmental stage by incubating with 10 mM 6-OHDA for 1 h. This procedure, which is based on reduced, altered, or absent pdat-1::GFP expression, does not lead to neurodegeneration in >95% of wild-type worms, thus allowing the identification of mutants conferring hypersensitivity to 6-OHDA. Of the initial five mutant candidates, only gt1681 maintained a strong hypersensitive phenotype upon backcrossing (Figure 1A, Figure S1). 6-OHDA-induced degeneration of both wild-type and gt1681 neurons exhibits the same morphological features and pattern of degeneration initially described by Nass et al. [3]. Axonal blebbing becomes apparent (Figure 1B, inset, arrows) a feature also consistent with morphological changes previously observed by electron microscopy. Worms were scored 24, 48 and 72 h after intoxication. Neurons were lost in less than 10% of wild-type worms after 72 h. In contrast, all dopaminergic neurons were lost in ∼40% of gt1681 worms and partial dopaminergic loss was observed in an additional ∼30% of mutant worms after only 24 h (Figure 1A). The extent of neurodegeneration was further increased 72 h after intoxication, with ∼90% of worms displaying total dopaminergic loss at the adult stage (Figure 1A). Enhanced neurodegeneration in the gt1681 background, albeit to a lesser extent, also occurred in L2, L3 and L4 larvae treated with 6-OHDA; no such enhancement was seen in adults (Figure 1C). To exclude the possibility that neurodegeneration might be caused by increased net 6-OHDA uptake at the organismal level, we took advantage of the partial growth retardation conferred by 6-OHDA treatment. By scoring for progression to ensuing developmental stages, we found the growth of wild-type and gt1681 worms to be similarly retarded upon toxin treatment, suggesting that gt1681 specifically affects dopaminergic neurons (Figure 1D).


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)

gt1681 mutants are hypersensitive to 6-OHDA.A. The extent of dopaminergic degeneration is indicated for wild-type and gt1681 mutant worms after intoxication with 10 mM 6-OHDA. Neurodegeneration of L1 worms was scored after 24, 48 and 72 h as described in Materials and Methods, 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. Representative images showing progressive stages of dopaminergic neurodegeneration. Absence of degeneration in wild-type (upper left panel) and complete degeneration in gt1681 mutant worms 72 h post 6-OHDA intoxication (upper right panel, complete degeneration); lower panel and inset are examples of partial degeneration in gt1881. Arrows indicate ‘blebs in degenerating neurons. C. Extent of neurodegeneration at various developmental stages in wild-type and gt1681 mutant worms 72 h post 6-OHDA intoxication. D. In wild-type and gt1681 worms, development is equally retarded following treatment with 6-OHDA. Progression to various developmental stages was scored once 95% of untreated worms reached adulthood.
© Copyright Policy
Related In: Results  -  Collection

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

pgen-1004767-g001: gt1681 mutants are hypersensitive to 6-OHDA.A. The extent of dopaminergic degeneration is indicated for wild-type and gt1681 mutant worms after intoxication with 10 mM 6-OHDA. Neurodegeneration of L1 worms was scored after 24, 48 and 72 h as described in Materials and Methods, 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. Representative images showing progressive stages of dopaminergic neurodegeneration. Absence of degeneration in wild-type (upper left panel) and complete degeneration in gt1681 mutant worms 72 h post 6-OHDA intoxication (upper right panel, complete degeneration); lower panel and inset are examples of partial degeneration in gt1881. Arrows indicate ‘blebs in degenerating neurons. C. Extent of neurodegeneration at various developmental stages in wild-type and gt1681 mutant worms 72 h post 6-OHDA intoxication. D. In wild-type and gt1681 worms, development is equally retarded following treatment with 6-OHDA. Progression to various developmental stages was scored once 95% of untreated worms reached adulthood.
Mentions: In order to find genes that protect dopaminergic neurons, we performed a genetic screen for mutants conferring hypersensitivity to 6-OHDA. By adapting procedures initially established by Nass et al. [3] and using the same pdat-1::GFP reporter that highlights dopaminergic neurons, we screened ∼2500 F2 ethyl methanesulfonate (EMS)-mutagenized worms at the L1 developmental stage by incubating with 10 mM 6-OHDA for 1 h. This procedure, which is based on reduced, altered, or absent pdat-1::GFP expression, does not lead to neurodegeneration in >95% of wild-type worms, thus allowing the identification of mutants conferring hypersensitivity to 6-OHDA. Of the initial five mutant candidates, only gt1681 maintained a strong hypersensitive phenotype upon backcrossing (Figure 1A, Figure S1). 6-OHDA-induced degeneration of both wild-type and gt1681 neurons exhibits the same morphological features and pattern of degeneration initially described by Nass et al. [3]. Axonal blebbing becomes apparent (Figure 1B, inset, arrows) a feature also consistent with morphological changes previously observed by electron microscopy. Worms were scored 24, 48 and 72 h after intoxication. Neurons were lost in less than 10% of wild-type worms after 72 h. In contrast, all dopaminergic neurons were lost in ∼40% of gt1681 worms and partial dopaminergic loss was observed in an additional ∼30% of mutant worms after only 24 h (Figure 1A). The extent of neurodegeneration was further increased 72 h after intoxication, with ∼90% of worms displaying total dopaminergic loss at the adult stage (Figure 1A). Enhanced neurodegeneration in the gt1681 background, albeit to a lesser extent, also occurred in L2, L3 and L4 larvae treated with 6-OHDA; no such enhancement was seen in adults (Figure 1C). To exclude the possibility that neurodegeneration might be caused by increased net 6-OHDA uptake at the organismal level, we took advantage of the partial growth retardation conferred by 6-OHDA treatment. By scoring for progression to ensuing developmental stages, we found the growth of wild-type and gt1681 worms to be similarly retarded upon toxin treatment, suggesting that gt1681 specifically affects dopaminergic neurons (Figure 1D).

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