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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

TSP-17::GFP expression.Analysis of the TG2439 strain containing dopaminergic neurons labeled by mCherry and tsp-17 C-terminally fused to GFP and driven by its own promoter. A, D, G, J. Dopaminergic neurons expressing the mCherry marker. Neurons are indicated. White arrows highlight dendrites and axons. B, E. H, K. Expression of TSP-17::GFP. C, F, I, L. Merged images. White arrows highlight dendrites and axons. K, L, N. The arrow-heads indicate TSP-17::GFP signal enrichment around the nucleus. Expression in the vulva (M), the spermatheca (N), a NSM neuron (O), and in body wall muscle cells (P).
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pgen-1004767-g003: TSP-17::GFP expression.Analysis of the TG2439 strain containing dopaminergic neurons labeled by mCherry and tsp-17 C-terminally fused to GFP and driven by its own promoter. A, D, G, J. Dopaminergic neurons expressing the mCherry marker. Neurons are indicated. White arrows highlight dendrites and axons. B, E. H, K. Expression of TSP-17::GFP. C, F, I, L. Merged images. White arrows highlight dendrites and axons. K, L, N. The arrow-heads indicate TSP-17::GFP signal enrichment around the nucleus. Expression in the vulva (M), the spermatheca (N), a NSM neuron (O), and in body wall muscle cells (P).

Mentions: To assess the TSP-17 expression pattern, we used biolistic bombardment to generate transgenic worms (TG2439) expressing a tsp-17::GFP gene fusion (NM001) under the control of its own promoter and 3′UTR. A dat-1 (promoter)::mCherry fusion (PBI001) was co-bombarded to mark dopaminergic neurons. The tsp-17::GFP gene fusion largely suppressed the hypersensitivity phenotype conferred by tsp-17, thus confirming its functionality (Figure 2C, bar 3). Importantly, fusion protein expression was observed in all dopaminergic neurons: it was uniform along axons and dendrites of both dorsal and ventral pairs of CEP neurons, as well as in ADE neurons (Figure 3A–I, arrows indicate axons and dendrites) and in the posterior PDE neurons. Within the cell body, the TSP-17::GFP fusion seems to be excluded from the nucleus, a pattern that is more evident in a “close-up” image of a PDE neuron, where the signal appears to form a ring-like structure around the nucleus (Figure 3J–L arrowheads). mCherry aggregates (which are not linked to neurodegeneration) form dot-like structures in dendrites and axons (arrows), and the surrounding TSP-17 fluorescent signal suggests plasma membrane expression (arrow, Figure 3K). TSP-17 enrichment at the plasma membrane can be observed most prominently in the large cells of the vulva and the sheath cells enclosing the spermatheca (Figure 3M, N). In the spermatheca, TSP-17::GFP expression is also clearly enriched around the nucleus (Figure 3N, arrowheads), possibly localizing to the nuclear membrane or endoplasmic reticulum (Figure 3N, arrowhead). Analysis of subcellular localization in the vulva and spermatheca revealed that the TSP-17::GFP (gt1681) mutant protein is uniformly expressed in the cytoplasm, with a loss of enrichment at the plasma membrane and around the nucleus (Figure S3A). Thus, the gt1681 mutation, which leads to an amino acid change in the fourth transmembrane domain, might compromise the membrane localization of TSP-17 and therefore block its function. TSP-17::GFP is also expressed in multiple neurons throughout worm development. For instance, the NSM serotonergic neuron, which is characterized by extensive axon sprouting, shows TSP-17::GFP expression along its entire length (Figure 3O). Prominent expression was also observed in the muscles of early stage larvae (Figure 3P). Finally expression also appears to be apparent in muscles of the adult head (Figure 3B, C, H, I). In summary, the TSP-17::GFP expression indicates that TSP-17 is expressed in dopaminergic neurons. Transgene expression in dopaminergic neurons was also confirmed by analyzing a TSP-17::GFP expressing transgenic strain crossed to a DAT-1 reporter strain (Figure S3B). We cannot rule out expression of TSP-17 not uncovered by the transgene, due to missing regulatory sequences. We next wanted to investigate whether TSP-17 expression in dopaminergic neurons protects them from 6-OHDA-mediated neurodegeneration. By direct injection of transgenes into the gonad, we generated transgenic worms overexpressing TSP-17 under the control of the dat-1 promoter. Consistent with TSP-17 expression in dopaminergic neurons, we found partial rescue of the hypersensitivity conferred by gt1681 (Figure 2C, compare bars 1, 2 and 4). Interestingly, overexpression of TSP-17 and TSP-17 (gt1681) under the dat-1 promoter led to spontaneous neurodegeneration (Figure S4A, B, respectively). This phenotype tended to be more severe following TSP-17 (gt1681) overexpression. Taken together, these data indicate that TSP-17 indeed functions in dopaminergic neurons, and that excessive TSP-17, especially the mutant form, leads to spontaneous neurodegeneration.


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)

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

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getmorefigures.php?uid=PMC4256090&req=5

pgen-1004767-g003: TSP-17::GFP expression.Analysis of the TG2439 strain containing dopaminergic neurons labeled by mCherry and tsp-17 C-terminally fused to GFP and driven by its own promoter. A, D, G, J. Dopaminergic neurons expressing the mCherry marker. Neurons are indicated. White arrows highlight dendrites and axons. B, E. H, K. Expression of TSP-17::GFP. C, F, I, L. Merged images. White arrows highlight dendrites and axons. K, L, N. The arrow-heads indicate TSP-17::GFP signal enrichment around the nucleus. Expression in the vulva (M), the spermatheca (N), a NSM neuron (O), and in body wall muscle cells (P).
Mentions: To assess the TSP-17 expression pattern, we used biolistic bombardment to generate transgenic worms (TG2439) expressing a tsp-17::GFP gene fusion (NM001) under the control of its own promoter and 3′UTR. A dat-1 (promoter)::mCherry fusion (PBI001) was co-bombarded to mark dopaminergic neurons. The tsp-17::GFP gene fusion largely suppressed the hypersensitivity phenotype conferred by tsp-17, thus confirming its functionality (Figure 2C, bar 3). Importantly, fusion protein expression was observed in all dopaminergic neurons: it was uniform along axons and dendrites of both dorsal and ventral pairs of CEP neurons, as well as in ADE neurons (Figure 3A–I, arrows indicate axons and dendrites) and in the posterior PDE neurons. Within the cell body, the TSP-17::GFP fusion seems to be excluded from the nucleus, a pattern that is more evident in a “close-up” image of a PDE neuron, where the signal appears to form a ring-like structure around the nucleus (Figure 3J–L arrowheads). mCherry aggregates (which are not linked to neurodegeneration) form dot-like structures in dendrites and axons (arrows), and the surrounding TSP-17 fluorescent signal suggests plasma membrane expression (arrow, Figure 3K). TSP-17 enrichment at the plasma membrane can be observed most prominently in the large cells of the vulva and the sheath cells enclosing the spermatheca (Figure 3M, N). In the spermatheca, TSP-17::GFP expression is also clearly enriched around the nucleus (Figure 3N, arrowheads), possibly localizing to the nuclear membrane or endoplasmic reticulum (Figure 3N, arrowhead). Analysis of subcellular localization in the vulva and spermatheca revealed that the TSP-17::GFP (gt1681) mutant protein is uniformly expressed in the cytoplasm, with a loss of enrichment at the plasma membrane and around the nucleus (Figure S3A). Thus, the gt1681 mutation, which leads to an amino acid change in the fourth transmembrane domain, might compromise the membrane localization of TSP-17 and therefore block its function. TSP-17::GFP is also expressed in multiple neurons throughout worm development. For instance, the NSM serotonergic neuron, which is characterized by extensive axon sprouting, shows TSP-17::GFP expression along its entire length (Figure 3O). Prominent expression was also observed in the muscles of early stage larvae (Figure 3P). Finally expression also appears to be apparent in muscles of the adult head (Figure 3B, C, H, I). In summary, the TSP-17::GFP expression indicates that TSP-17 is expressed in dopaminergic neurons. Transgene expression in dopaminergic neurons was also confirmed by analyzing a TSP-17::GFP expressing transgenic strain crossed to a DAT-1 reporter strain (Figure S3B). We cannot rule out expression of TSP-17 not uncovered by the transgene, due to missing regulatory sequences. We next wanted to investigate whether TSP-17 expression in dopaminergic neurons protects them from 6-OHDA-mediated neurodegeneration. By direct injection of transgenes into the gonad, we generated transgenic worms overexpressing TSP-17 under the control of the dat-1 promoter. Consistent with TSP-17 expression in dopaminergic neurons, we found partial rescue of the hypersensitivity conferred by gt1681 (Figure 2C, compare bars 1, 2 and 4). Interestingly, overexpression of TSP-17 and TSP-17 (gt1681) under the dat-1 promoter led to spontaneous neurodegeneration (Figure S4A, B, respectively). This phenotype tended to be more severe following TSP-17 (gt1681) overexpression. Taken together, these data indicate that TSP-17 indeed functions in dopaminergic neurons, and that excessive TSP-17, especially the mutant form, leads to spontaneous neurodegeneration.

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