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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

The TSP-17 tetraspanin family member protects dopaminergic neurons from 6-OHDA.A. Extent of neurodegeneration in heterozygous and trans-heterozygous worms 72 h post 6-OHDA intoxication. B. Schematic gene model of the two isoforms of tsp-17. Alleles used in this study are indicated. C. Complementation of tsp-17 expressed under its own promoter (3rd column strain TG2439) and under the dat-1 promoter (4th column, strain TG2440). Data presented is from scoring the extent of neurodegeneration 72 h post 6-OHDA intoxication. Asterisks represent statistical significance of differences between tsp-17 and the rescuing lines (****p<0.0001). D. 6-OHDA hypersensitivity conferred by various tsp-17 alleles. E. Alignment of nematode TSP-17 to the most closely related human tetraspanins. Blue bars indicate transmembrane domains and brown bars designate extracellular loops (EC1 and EC2). The arrow indicates amino acid G109, which is mutated in the C. elegans gt1681 and vc2026 mutants. The red box indicates the CCG motif in the EC2, which is highly conserved throughout the tetraspanin protein family. Hs, Homo sapiens; Ce, Caenorhabditis elegans; Cbn, C. brenneri; Cre, C. remanei; Cbr, C. briggsae.
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pgen-1004767-g002: The TSP-17 tetraspanin family member protects dopaminergic neurons from 6-OHDA.A. Extent of neurodegeneration in heterozygous and trans-heterozygous worms 72 h post 6-OHDA intoxication. B. Schematic gene model of the two isoforms of tsp-17. Alleles used in this study are indicated. C. Complementation of tsp-17 expressed under its own promoter (3rd column strain TG2439) and under the dat-1 promoter (4th column, strain TG2440). Data presented is from scoring the extent of neurodegeneration 72 h post 6-OHDA intoxication. Asterisks represent statistical significance of differences between tsp-17 and the rescuing lines (****p<0.0001). D. 6-OHDA hypersensitivity conferred by various tsp-17 alleles. E. Alignment of nematode TSP-17 to the most closely related human tetraspanins. Blue bars indicate transmembrane domains and brown bars designate extracellular loops (EC1 and EC2). The arrow indicates amino acid G109, which is mutated in the C. elegans gt1681 and vc2026 mutants. The red box indicates the CCG motif in the EC2, which is highly conserved throughout the tetraspanin protein family. Hs, Homo sapiens; Ce, Caenorhabditis elegans; Cbn, C. brenneri; Cre, C. remanei; Cbr, C. briggsae.

Mentions: The gt1681 mutant is recessive in hermaphrodites (Figure 2A). Genetic linkage was established by single nucleotide polymorphism (SNP) mapping, which placed gt1681 on the left arm of the X chromosome. Using unc-20 and lon-2 genetic markers to perform three-factor mapping, the locus was further refined to ∼10 map units. A cross between an unc-20 gt1681 lon-2 triple mutant and the CB4856 “Hawaii” mapping strain enabled us to assess the position of single recombination events relative to gt1681. This analysis localized gt1681 to an interval between nucleotides 3,659,480 and 3,737,466 on the physical map. In parallel, next generation sequencing revealed a single exonic mutation within this interval, leading to a guanine to adenine substitution in the C02F12.1 open reading frame and resulting in a glycine to glutamic acid change at position 109 of the encoded protein (Figure 2B). C02F12.1 encodes a tetraspanin family, integral membrane protein called TSP-17 (see below). Rescue of the phenotype by a fosmid (WRM0626aC02) encompassing tsp-17 and by a tsp-17-encoding transgene (Figure 2C) provides further evidence that gt1681 confers 6-OHDA hypersensitivity. Hypersensitivity is also conferred by the vc2026 allele, a substitution obtained via the Million Mutation Project [38] that results in a glycine to arginine change at position 109 (Figure 2B, 2D). Finally, two deletion alleles, generously provided by the Japanese Knockout Consortium, affecting the first exons of tsp-17 also confer hypersensitivity to 6-OHDA-mediated neurotoxicity (Figure 2B, 2D) as does the trans-heterozygous gt1681/tm4995 mutant combination (Figure 2A).


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)

The TSP-17 tetraspanin family member protects dopaminergic neurons from 6-OHDA.A. Extent of neurodegeneration in heterozygous and trans-heterozygous worms 72 h post 6-OHDA intoxication. B. Schematic gene model of the two isoforms of tsp-17. Alleles used in this study are indicated. C. Complementation of tsp-17 expressed under its own promoter (3rd column strain TG2439) and under the dat-1 promoter (4th column, strain TG2440). Data presented is from scoring the extent of neurodegeneration 72 h post 6-OHDA intoxication. Asterisks represent statistical significance of differences between tsp-17 and the rescuing lines (****p<0.0001). D. 6-OHDA hypersensitivity conferred by various tsp-17 alleles. E. Alignment of nematode TSP-17 to the most closely related human tetraspanins. Blue bars indicate transmembrane domains and brown bars designate extracellular loops (EC1 and EC2). The arrow indicates amino acid G109, which is mutated in the C. elegans gt1681 and vc2026 mutants. The red box indicates the CCG motif in the EC2, which is highly conserved throughout the tetraspanin protein family. Hs, Homo sapiens; Ce, Caenorhabditis elegans; Cbn, C. brenneri; Cre, C. remanei; Cbr, C. briggsae.
© Copyright Policy
Related In: Results  -  Collection

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getmorefigures.php?uid=PMC4256090&req=5

pgen-1004767-g002: The TSP-17 tetraspanin family member protects dopaminergic neurons from 6-OHDA.A. Extent of neurodegeneration in heterozygous and trans-heterozygous worms 72 h post 6-OHDA intoxication. B. Schematic gene model of the two isoforms of tsp-17. Alleles used in this study are indicated. C. Complementation of tsp-17 expressed under its own promoter (3rd column strain TG2439) and under the dat-1 promoter (4th column, strain TG2440). Data presented is from scoring the extent of neurodegeneration 72 h post 6-OHDA intoxication. Asterisks represent statistical significance of differences between tsp-17 and the rescuing lines (****p<0.0001). D. 6-OHDA hypersensitivity conferred by various tsp-17 alleles. E. Alignment of nematode TSP-17 to the most closely related human tetraspanins. Blue bars indicate transmembrane domains and brown bars designate extracellular loops (EC1 and EC2). The arrow indicates amino acid G109, which is mutated in the C. elegans gt1681 and vc2026 mutants. The red box indicates the CCG motif in the EC2, which is highly conserved throughout the tetraspanin protein family. Hs, Homo sapiens; Ce, Caenorhabditis elegans; Cbn, C. brenneri; Cre, C. remanei; Cbr, C. briggsae.
Mentions: The gt1681 mutant is recessive in hermaphrodites (Figure 2A). Genetic linkage was established by single nucleotide polymorphism (SNP) mapping, which placed gt1681 on the left arm of the X chromosome. Using unc-20 and lon-2 genetic markers to perform three-factor mapping, the locus was further refined to ∼10 map units. A cross between an unc-20 gt1681 lon-2 triple mutant and the CB4856 “Hawaii” mapping strain enabled us to assess the position of single recombination events relative to gt1681. This analysis localized gt1681 to an interval between nucleotides 3,659,480 and 3,737,466 on the physical map. In parallel, next generation sequencing revealed a single exonic mutation within this interval, leading to a guanine to adenine substitution in the C02F12.1 open reading frame and resulting in a glycine to glutamic acid change at position 109 of the encoded protein (Figure 2B). C02F12.1 encodes a tetraspanin family, integral membrane protein called TSP-17 (see below). Rescue of the phenotype by a fosmid (WRM0626aC02) encompassing tsp-17 and by a tsp-17-encoding transgene (Figure 2C) provides further evidence that gt1681 confers 6-OHDA hypersensitivity. Hypersensitivity is also conferred by the vc2026 allele, a substitution obtained via the Million Mutation Project [38] that results in a glycine to arginine change at position 109 (Figure 2B, 2D). Finally, two deletion alleles, generously provided by the Japanese Knockout Consortium, affecting the first exons of tsp-17 also confer hypersensitivity to 6-OHDA-mediated neurotoxicity (Figure 2B, 2D) as does the trans-heterozygous gt1681/tm4995 mutant combination (Figure 2A).

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