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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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Overexpression of Vps26 or Vps35 in DA neurons rescues LRRK2(I2020T) locomotor deficits. (A) Overexpression of Vps26 in DA neurons. (B) Overexpression of Vps35 in DA neurons. N = 3-12 cohorts of ten per genotype. All flies were reared at 29°C. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05 and *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) is denoted as ## for P < 0.001 and ### for P < 0.0001.
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Figure 2: Overexpression of Vps26 or Vps35 in DA neurons rescues LRRK2(I2020T) locomotor deficits. (A) Overexpression of Vps26 in DA neurons. (B) Overexpression of Vps35 in DA neurons. N = 3-12 cohorts of ten per genotype. All flies were reared at 29°C. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05 and *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) is denoted as ## for P < 0.001 and ### for P < 0.0001.

Mentions: To quantify locomotor activity, we employed a well-established negative geotaxis climbing assay. As we have shown previously, overexpressing LRRK2(I2020T) in DA neurons causes significant locomotor deficits [13]. Compared to control, the climbing ability of LRRK2(I2020T) flies was reduced by 61.45% +/- 7.49% on day 5 (specifically, 28.04% +/- 5.45% of LRRK2 flies were able to reach the line within 5 seconds, compared to 72.75% +/- 7.89% of control) (Figure 2). Similar to the eye, this LRRK2 phenotype can be fully rescued either by overexpressing Vps35 in DA neurons (70.00% +/- 6.36% of flies overexpressing Vps35 and mutant LRRK2 were able to cross the line within 5 seconds) (P < 0.0001; F (4, 77) = 8.20) (Figure 2B), or by overexpressing Vps26 (64.12% +/- 3.28% of flies overexpressing Vps26 and mutant LRRK2 in DA neurons crossed the line within 5 seconds) (P < 0.0001; F (3, 225) = 11.31) (Figure 2A). Similar to day 5, Vps35 or Vps26 overexpression rescued the LRRK2(I2020T) phenotype on day 10 (Figure 2A and B). By day 20 however, the locomotor activity of all flies, including control, was severely impaired due to age (Figure 2A and B). Please note that the locomotor activity of flies was always assessed at three different time intervals (5, 10 and 30 seconds), with similar results (data not shown). In addition to the locomotor activity, we also assessed survival of these flies. Compared to the control (Ddc alone), survival of flies expressing LRRK2(I2020T) in DA neurons was significantly shorter. This phenotype was completely rescued by overexpression of Vps35 (Figure 3). Altogether, these data validate that mutant LRRK2(I2020T) functionally interacts with Vps35 and Vps26 in DA neurons and that this interaction is important for the locomotor activity and basal survival of the flies.


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)

Overexpression of Vps26 or Vps35 in DA neurons rescues LRRK2(I2020T) locomotor deficits. (A) Overexpression of Vps26 in DA neurons. (B) Overexpression of Vps35 in DA neurons. N = 3-12 cohorts of ten per genotype. All flies were reared at 29°C. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05 and *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) is denoted as ## for P < 0.001 and ### for P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Overexpression of Vps26 or Vps35 in DA neurons rescues LRRK2(I2020T) locomotor deficits. (A) Overexpression of Vps26 in DA neurons. (B) Overexpression of Vps35 in DA neurons. N = 3-12 cohorts of ten per genotype. All flies were reared at 29°C. Statistical analysis: Two-Way ANOVA, Tukey’s post-test. Statistically significant difference compared to control (Ddc alone) is denoted as * for P < 0.05 and *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T) is denoted as ## for P < 0.001 and ### for P < 0.0001.
Mentions: To quantify locomotor activity, we employed a well-established negative geotaxis climbing assay. As we have shown previously, overexpressing LRRK2(I2020T) in DA neurons causes significant locomotor deficits [13]. Compared to control, the climbing ability of LRRK2(I2020T) flies was reduced by 61.45% +/- 7.49% on day 5 (specifically, 28.04% +/- 5.45% of LRRK2 flies were able to reach the line within 5 seconds, compared to 72.75% +/- 7.89% of control) (Figure 2). Similar to the eye, this LRRK2 phenotype can be fully rescued either by overexpressing Vps35 in DA neurons (70.00% +/- 6.36% of flies overexpressing Vps35 and mutant LRRK2 were able to cross the line within 5 seconds) (P < 0.0001; F (4, 77) = 8.20) (Figure 2B), or by overexpressing Vps26 (64.12% +/- 3.28% of flies overexpressing Vps26 and mutant LRRK2 in DA neurons crossed the line within 5 seconds) (P < 0.0001; F (3, 225) = 11.31) (Figure 2A). Similar to day 5, Vps35 or Vps26 overexpression rescued the LRRK2(I2020T) phenotype on day 10 (Figure 2A and B). By day 20 however, the locomotor activity of all flies, including control, was severely impaired due to age (Figure 2A and B). Please note that the locomotor activity of flies was always assessed at three different time intervals (5, 10 and 30 seconds), with similar results (data not shown). In addition to the locomotor activity, we also assessed survival of these flies. Compared to the control (Ddc alone), survival of flies expressing LRRK2(I2020T) in DA neurons was significantly shorter. This phenotype was completely rescued by overexpression of Vps35 (Figure 3). Altogether, these data validate that mutant LRRK2(I2020T) functionally interacts with Vps35 and Vps26 in DA neurons and that this interaction is important for the locomotor activity and basal survival of the flies.

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