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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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Effect of Vps35 overexpression and Vps35 knockdown on rotenone toxicity. A)Vps35 overexpression in DA neurons improves survival rates following exposure to rotenone, and LRRK2(I2020T) and Vps35 act synergistically to protect from rotenone. B)Vps35 overexpression in DA neurons protects from locomotor deficits seen in LRRK2(I2020T) or control flies exposed to rotenone. Knocking down Vps35 has no significant effect on survival (A), or locomotor activity (C). N = 20-40 flies/genotype. Statistical analysis by Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) and control (Ddc alone) was denoted as * *for P < 0.001; statistically significant difference compared to control and compared to Ddc/Vps35;LRRK2(I2020T)/+was denoted as § for P < 0.05.
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Figure 6: Effect of Vps35 overexpression and Vps35 knockdown on rotenone toxicity. A)Vps35 overexpression in DA neurons improves survival rates following exposure to rotenone, and LRRK2(I2020T) and Vps35 act synergistically to protect from rotenone. B)Vps35 overexpression in DA neurons protects from locomotor deficits seen in LRRK2(I2020T) or control flies exposed to rotenone. Knocking down Vps35 has no significant effect on survival (A), or locomotor activity (C). N = 20-40 flies/genotype. Statistical analysis by Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) and control (Ddc alone) was denoted as * *for P < 0.001; statistically significant difference compared to control and compared to Ddc/Vps35;LRRK2(I2020T)/+was denoted as § for P < 0.05.

Mentions: Treating control flies with rotenone has a profound effect on their survival. Although we previously showed that pan-neuronal expression of LRRK2(I2020T) sensitized flies to low doses of rotenone [13], overexpression of LRRK2(I2020T) in DA neurons did not seem to significantly affect survival of flies treated with 1 mM rotenone (Figure 6A). Importantly, overexpressing Vps35 alone offered a mild but significant protection against rotenone, compared to control flies (Figure 6A). To our surprise however, co-overexpressing Vps35 and LRRK2 in DA neurons resulted in a substantial synergistic protective effect against rotenone, demonstrated as a significantly better survival of these flies compared to all other groups (P < 0.0001; F (74, 190) = 3.45) (Figure 6A).


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)

Effect of Vps35 overexpression and Vps35 knockdown on rotenone toxicity. A)Vps35 overexpression in DA neurons improves survival rates following exposure to rotenone, and LRRK2(I2020T) and Vps35 act synergistically to protect from rotenone. B)Vps35 overexpression in DA neurons protects from locomotor deficits seen in LRRK2(I2020T) or control flies exposed to rotenone. Knocking down Vps35 has no significant effect on survival (A), or locomotor activity (C). N = 20-40 flies/genotype. Statistical analysis by Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) and control (Ddc alone) was denoted as * *for P < 0.001; statistically significant difference compared to control and compared to Ddc/Vps35;LRRK2(I2020T)/+was denoted as § for P < 0.05.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 6: Effect of Vps35 overexpression and Vps35 knockdown on rotenone toxicity. A)Vps35 overexpression in DA neurons improves survival rates following exposure to rotenone, and LRRK2(I2020T) and Vps35 act synergistically to protect from rotenone. B)Vps35 overexpression in DA neurons protects from locomotor deficits seen in LRRK2(I2020T) or control flies exposed to rotenone. Knocking down Vps35 has no significant effect on survival (A), or locomotor activity (C). N = 20-40 flies/genotype. Statistical analysis by Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) and control (Ddc alone) was denoted as * *for P < 0.001; statistically significant difference compared to control and compared to Ddc/Vps35;LRRK2(I2020T)/+was denoted as § for P < 0.05.
Mentions: Treating control flies with rotenone has a profound effect on their survival. Although we previously showed that pan-neuronal expression of LRRK2(I2020T) sensitized flies to low doses of rotenone [13], overexpression of LRRK2(I2020T) in DA neurons did not seem to significantly affect survival of flies treated with 1 mM rotenone (Figure 6A). Importantly, overexpressing Vps35 alone offered a mild but significant protection against rotenone, compared to control flies (Figure 6A). To our surprise however, co-overexpressing Vps35 and LRRK2 in DA neurons resulted in a substantial synergistic protective effect against rotenone, demonstrated as a significantly better survival of these flies compared to all other groups (P < 0.0001; F (74, 190) = 3.45) (Figure 6A).

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