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Vacuolar protein sorting 35 (Vps35) rescues locomotor deficits and shortened lifespan in Drosophila expressing a Parkinson's disease mutant of Leucine-Rich Repeat Kinase 2 (LRRK2).

Linhart R, Wong SA, Cao J, Tran M, Huynh A, Ardrey C, Park JM, Hsu C, Taha S, Peterson R, Shea S, Kurian J, Venderova K - Mol Neurodegener (2014)

Bottom Line: Strikingly, overexpressing Vps35 alone protects from toxicity of rotenone, a neurotoxin commonly used to model parkinsonism, both in terms of lifespan and locomotor activity of the flies, and this protection is sustained and even augmented in the presence of mutant LRRK2.Finally, we demonstrate that knocking down expression of Vps35 in dopaminergic neurons causes a significant locomotor impairment.From these results we conclude that LRRK2 plays a role in the retromer pathway and that this pathway is involved in PD pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Pharmacology, Thomas J, Long School of Pharmacy and Health Sciences, University of the Pacific, 751 Brookside Rd, Stockton, CA 95211, USA. kvenderova@pacific.edu.

ABSTRACT

Background: Parkinson's disease (PD) is the most common movement neurodegenerative movement disorder. An incomplete understanding of the molecular pathways involved in its pathogenesis impedes the development of effective disease-modifying treatments. To address this gap, we have previously generated a Drosophila model of PD that overexpresses PD pathogenic mutant form of the second most common causative gene of PD, Leucine-Rich Repeat Kinase 2 (LRRK2).

Findings: We employed this model in a genetic modifier screen and identified a gene that encodes for a core subunit of retromer - a complex essential for the sorting and recycling of specific cargo proteins from endosomes to the trans-Golgi network and cell surface. We present evidence that overexpression of the Vps35 or Vps26 component of the cargo-recognition subunit of the retromer complex ameliorates the pathogenic mutant LRRK2 eye phenotype. Furthermore, overexpression of Vps35 or Vps26 significantly protects from the locomotor deficits observed in mutant LRRK2 flies, as assessed by the negative geotaxis assay, and rescues their shortened lifespan. Strikingly, overexpressing Vps35 alone protects from toxicity of rotenone, a neurotoxin commonly used to model parkinsonism, both in terms of lifespan and locomotor activity of the flies, and this protection is sustained and even augmented in the presence of mutant LRRK2. Finally, we demonstrate that knocking down expression of Vps35 in dopaminergic neurons causes a significant locomotor impairment.

Conclusions: From these results we conclude that LRRK2 plays a role in the retromer pathway and that this pathway is involved in PD pathogenesis.

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Eye-specific overexpression of Vps26 or Vps35 rescues the black lesion phenotype caused by expression of LRRK2(I2020T). A) Representative stereomicroscope images of eyes overexpressing Vps26 (upper panel) and Vps35 (lower panel). B) Quantification of black lesions in flies overexpressing Vps26. Total of 314 eyes from 2-6 independent crosses/genotype (males and females) was analyzed. C) Quantification of black lesions in flies overexpressing Vps35. Total of 312 eyes from 3 independent crosses/genotype (males and females) was analyzed. All flies were reared at 29°C. Statistical analysis by One-Way ANOVA, Bonferroni’s post-test (P‹0.0001). Statistically significant difference compared to control (GMR alone) is denoted as ** for P < 0.001, or *** for P < 0.0001. Statistically significant difference compared to GMR/+;LRRK2(I2020T)/+is denoted as # for P < 0.05, or ### for P < 0.0001.
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Figure 1: Eye-specific overexpression of Vps26 or Vps35 rescues the black lesion phenotype caused by expression of LRRK2(I2020T). A) Representative stereomicroscope images of eyes overexpressing Vps26 (upper panel) and Vps35 (lower panel). B) Quantification of black lesions in flies overexpressing Vps26. Total of 314 eyes from 2-6 independent crosses/genotype (males and females) was analyzed. C) Quantification of black lesions in flies overexpressing Vps35. Total of 312 eyes from 3 independent crosses/genotype (males and females) was analyzed. All flies were reared at 29°C. Statistical analysis by One-Way ANOVA, Bonferroni’s post-test (P‹0.0001). Statistically significant difference compared to control (GMR alone) is denoted as ** for P < 0.001, or *** for P < 0.0001. Statistically significant difference compared to GMR/+;LRRK2(I2020T)/+is denoted as # for P < 0.05, or ### for P < 0.0001.

Mentions: In our studies, we employed the commonly used UAS-Gal4 system for a cell/tissue-specific expression [19]. As we have previously shown [13], expression of one of the PD-causing mutants of LRRK2, LRRK2(I2020T), under an eye-specific (GMR) promoter at 29°C causes a rough eye phenotype with pigmentation deficits. Notably, 50.03%+/-6.58% of LRRK2(I2020T) eyes have black lesions (Figure 1). Similar lesions were reported in other fly models of neurodegeneration [20-24] and seem to be indicative of neuronal (photoreceptor) death occurring later in the eye development, after a full differentiation [21]. Such black lesions are very rare in control (GMR alone) flies (3.03%+/-3.03% of eyes) (Figure 1). Employing this LRRK2(I2020T) eye phenotype as a read-out in a genetic modifier screen, we identified Vacuolar protein sorting 26 (Vps26) (GenBank: NM_130596.2) as a new LRRK2 interacting gene. Specifically, overexpressing endogenous Drosophila Vps26 in the eye caused a mild eye phenotype, including an occasional presence of black lesions (11.21% +/- 2.12% of flies) (Figure 1A and B). Strikingly, overexpressing Vps26 in the LRRK2(I2020T) flies rescued the black lesion eye phenotype of the LRRK2 mutants (10.10% +/- 2.12%; P < 0.05; F(3, 9) = 13.30) (Figure 1A and B).


Vacuolar protein sorting 35 (Vps35) rescues locomotor deficits and shortened lifespan in Drosophila expressing a Parkinson's disease mutant of Leucine-Rich Repeat Kinase 2 (LRRK2).

Linhart R, Wong SA, Cao J, Tran M, Huynh A, Ardrey C, Park JM, Hsu C, Taha S, Peterson R, Shea S, Kurian J, Venderova K - Mol Neurodegener (2014)

Eye-specific overexpression of Vps26 or Vps35 rescues the black lesion phenotype caused by expression of LRRK2(I2020T). A) Representative stereomicroscope images of eyes overexpressing Vps26 (upper panel) and Vps35 (lower panel). B) Quantification of black lesions in flies overexpressing Vps26. Total of 314 eyes from 2-6 independent crosses/genotype (males and females) was analyzed. C) Quantification of black lesions in flies overexpressing Vps35. Total of 312 eyes from 3 independent crosses/genotype (males and females) was analyzed. All flies were reared at 29°C. Statistical analysis by One-Way ANOVA, Bonferroni’s post-test (P‹0.0001). Statistically significant difference compared to control (GMR alone) is denoted as ** for P < 0.001, or *** for P < 0.0001. Statistically significant difference compared to GMR/+;LRRK2(I2020T)/+is denoted as # for P < 0.05, or ### for P < 0.0001.
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Related In: Results  -  Collection

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Figure 1: Eye-specific overexpression of Vps26 or Vps35 rescues the black lesion phenotype caused by expression of LRRK2(I2020T). A) Representative stereomicroscope images of eyes overexpressing Vps26 (upper panel) and Vps35 (lower panel). B) Quantification of black lesions in flies overexpressing Vps26. Total of 314 eyes from 2-6 independent crosses/genotype (males and females) was analyzed. C) Quantification of black lesions in flies overexpressing Vps35. Total of 312 eyes from 3 independent crosses/genotype (males and females) was analyzed. All flies were reared at 29°C. Statistical analysis by One-Way ANOVA, Bonferroni’s post-test (P‹0.0001). Statistically significant difference compared to control (GMR alone) is denoted as ** for P < 0.001, or *** for P < 0.0001. Statistically significant difference compared to GMR/+;LRRK2(I2020T)/+is denoted as # for P < 0.05, or ### for P < 0.0001.
Mentions: In our studies, we employed the commonly used UAS-Gal4 system for a cell/tissue-specific expression [19]. As we have previously shown [13], expression of one of the PD-causing mutants of LRRK2, LRRK2(I2020T), under an eye-specific (GMR) promoter at 29°C causes a rough eye phenotype with pigmentation deficits. Notably, 50.03%+/-6.58% of LRRK2(I2020T) eyes have black lesions (Figure 1). Similar lesions were reported in other fly models of neurodegeneration [20-24] and seem to be indicative of neuronal (photoreceptor) death occurring later in the eye development, after a full differentiation [21]. Such black lesions are very rare in control (GMR alone) flies (3.03%+/-3.03% of eyes) (Figure 1). Employing this LRRK2(I2020T) eye phenotype as a read-out in a genetic modifier screen, we identified Vacuolar protein sorting 26 (Vps26) (GenBank: NM_130596.2) as a new LRRK2 interacting gene. Specifically, overexpressing endogenous Drosophila Vps26 in the eye caused a mild eye phenotype, including an occasional presence of black lesions (11.21% +/- 2.12% of flies) (Figure 1A and B). Strikingly, overexpressing Vps26 in the LRRK2(I2020T) flies rescued the black lesion eye phenotype of the LRRK2 mutants (10.10% +/- 2.12%; P < 0.05; F(3, 9) = 13.30) (Figure 1A and B).

Bottom Line: Strikingly, overexpressing Vps35 alone protects from toxicity of rotenone, a neurotoxin commonly used to model parkinsonism, both in terms of lifespan and locomotor activity of the flies, and this protection is sustained and even augmented in the presence of mutant LRRK2.Finally, we demonstrate that knocking down expression of Vps35 in dopaminergic neurons causes a significant locomotor impairment.From these results we conclude that LRRK2 plays a role in the retromer pathway and that this pathway is involved in PD pathogenesis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Department of Physiology and Pharmacology, Thomas J, Long School of Pharmacy and Health Sciences, University of the Pacific, 751 Brookside Rd, Stockton, CA 95211, USA. kvenderova@pacific.edu.

ABSTRACT

Background: Parkinson's disease (PD) is the most common movement neurodegenerative movement disorder. An incomplete understanding of the molecular pathways involved in its pathogenesis impedes the development of effective disease-modifying treatments. To address this gap, we have previously generated a Drosophila model of PD that overexpresses PD pathogenic mutant form of the second most common causative gene of PD, Leucine-Rich Repeat Kinase 2 (LRRK2).

Findings: We employed this model in a genetic modifier screen and identified a gene that encodes for a core subunit of retromer - a complex essential for the sorting and recycling of specific cargo proteins from endosomes to the trans-Golgi network and cell surface. We present evidence that overexpression of the Vps35 or Vps26 component of the cargo-recognition subunit of the retromer complex ameliorates the pathogenic mutant LRRK2 eye phenotype. Furthermore, overexpression of Vps35 or Vps26 significantly protects from the locomotor deficits observed in mutant LRRK2 flies, as assessed by the negative geotaxis assay, and rescues their shortened lifespan. Strikingly, overexpressing Vps35 alone protects from toxicity of rotenone, a neurotoxin commonly used to model parkinsonism, both in terms of lifespan and locomotor activity of the flies, and this protection is sustained and even augmented in the presence of mutant LRRK2. Finally, we demonstrate that knocking down expression of Vps35 in dopaminergic neurons causes a significant locomotor impairment.

Conclusions: From these results we conclude that LRRK2 plays a role in the retromer pathway and that this pathway is involved in PD pathogenesis.

Show MeSH
Related in: MedlinePlus