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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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Knocking down components of the cargo-recognition subunit of the retromer complex causes an eye phenotype and a locomotor impairment. The phenotypes are not additive with LRRK2(I2020T) phenotypes. A) Representative stereomicroscope images and B) Quantification of black lesions in the eyes Vps26 knock-down flies. Total of 444 eyes from 3-9 independent crosses/genotype (males and females) was analyzed. Statistically significant difference compared to control (GMR alone) is denoted as * for P < 0.05 and ** for P < 0.001. Data were statistically analyzed by One-Way ANOVA, Bonferroni’s post-test. C) Effect of Vps35 or Vps29 knock-down on locomotor activity. N = 3-13 cohorts of ten. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05, ** for P < 0.001, or *** for P < 0.0001. All flies were reared at 29°C.
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Figure 5: Knocking down components of the cargo-recognition subunit of the retromer complex causes an eye phenotype and a locomotor impairment. The phenotypes are not additive with LRRK2(I2020T) phenotypes. A) Representative stereomicroscope images and B) Quantification of black lesions in the eyes Vps26 knock-down flies. Total of 444 eyes from 3-9 independent crosses/genotype (males and females) was analyzed. Statistically significant difference compared to control (GMR alone) is denoted as * for P < 0.05 and ** for P < 0.001. Data were statistically analyzed by One-Way ANOVA, Bonferroni’s post-test. C) Effect of Vps35 or Vps29 knock-down on locomotor activity. N = 3-13 cohorts of ten. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05, ** for P < 0.001, or *** for P < 0.0001. All flies were reared at 29°C.

Mentions: First, we analyzed the effect in the eye. Knocking down expression of Vps26 in the eye caused a significant eye phenotype (35.84% +/- 5.43% of Vps26 knock-down eyes had a black lesion, compared to 2.78% +/- 3.4% of control eyes; P < 0.05; F (3, 15) = 7.01) (Figure 5A and B). This phenotype was similar to the eye phenotype of the LRRK2(I2020T) mutant (44.71 +/- 6.49% of LRRK2(I2020T) eyes had black lesions) (Figure 5A and B). Our next goal was to assess whether these two phenotypes are additive. An additive effect would indicate that the two genes likely act on two independent cellular pathways. We observed that the eye phenotypes of LRRK2(I2020T) expression and Vps26 knock-down were not additive (36.21% +/- 3.02% of double transgenic eyes had a black lesion) (Figure 5A and B), suggesting that the two genes act on the same pathway.


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)

Knocking down components of the cargo-recognition subunit of the retromer complex causes an eye phenotype and a locomotor impairment. The phenotypes are not additive with LRRK2(I2020T) phenotypes. A) Representative stereomicroscope images and B) Quantification of black lesions in the eyes Vps26 knock-down flies. Total of 444 eyes from 3-9 independent crosses/genotype (males and females) was analyzed. Statistically significant difference compared to control (GMR alone) is denoted as * for P < 0.05 and ** for P < 0.001. Data were statistically analyzed by One-Way ANOVA, Bonferroni’s post-test. C) Effect of Vps35 or Vps29 knock-down on locomotor activity. N = 3-13 cohorts of ten. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05, ** for P < 0.001, or *** for P < 0.0001. All flies were reared at 29°C.
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4126812&req=5

Figure 5: Knocking down components of the cargo-recognition subunit of the retromer complex causes an eye phenotype and a locomotor impairment. The phenotypes are not additive with LRRK2(I2020T) phenotypes. A) Representative stereomicroscope images and B) Quantification of black lesions in the eyes Vps26 knock-down flies. Total of 444 eyes from 3-9 independent crosses/genotype (males and females) was analyzed. Statistically significant difference compared to control (GMR alone) is denoted as * for P < 0.05 and ** for P < 0.001. Data were statistically analyzed by One-Way ANOVA, Bonferroni’s post-test. C) Effect of Vps35 or Vps29 knock-down on locomotor activity. N = 3-13 cohorts of ten. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05, ** for P < 0.001, or *** for P < 0.0001. All flies were reared at 29°C.
Mentions: First, we analyzed the effect in the eye. Knocking down expression of Vps26 in the eye caused a significant eye phenotype (35.84% +/- 5.43% of Vps26 knock-down eyes had a black lesion, compared to 2.78% +/- 3.4% of control eyes; P < 0.05; F (3, 15) = 7.01) (Figure 5A and B). This phenotype was similar to the eye phenotype of the LRRK2(I2020T) mutant (44.71 +/- 6.49% of LRRK2(I2020T) eyes had black lesions) (Figure 5A and B). Our next goal was to assess whether these two phenotypes are additive. An additive effect would indicate that the two genes likely act on two independent cellular pathways. We observed that the eye phenotypes of LRRK2(I2020T) expression and Vps26 knock-down were not additive (36.21% +/- 3.02% of double transgenic eyes had a black lesion) (Figure 5A and B), suggesting that the two genes act on the same pathway.

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