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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 Vps35 in DA neurons rescues locomotor deficits of other PD mutants. (A) Overexpression of Vps35 in LRRK2(Y1699C) mutants.(B) Overexpression of Vps35 in LRRK2(I1122V) mutants. All flies were reared at 29°C. N = 3-7 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 < 0.001, or *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T)/+is denoted as ### for P < 0.0001.
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Figure 4: Overexpression of Vps35 in DA neurons rescues locomotor deficits of other PD mutants. (A) Overexpression of Vps35 in LRRK2(Y1699C) mutants.(B) Overexpression of Vps35 in LRRK2(I1122V) mutants. All flies were reared at 29°C. N = 3-7 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 < 0.001, or *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T)/+is denoted as ### for P < 0.0001.

Mentions: Similar to LRRK2(I2020T), expressing either one of the two mutant forms of LRRK2 in DA neurons caused a significant impairment locomotor activity on day 5 (24.71% +/- 3.44% of LRRK2(Y1699C) flies were able to reach the line within 5 seconds, compared to 63.21% +/- 5.30% of control, P < 0.0001; F (3, 177) = 50.60; and 39.93% +/- 2.73% of LRRK2(I1122V) flies were able to reach the line within 5 seconds, compared to 72.75% +/- 4.27% of control flies, P < 0.0001; F (3, 126) = 23.82, respectively) (Figure 4A and B), which is consistent with our previous results [13]. Importantly, overexpressing Vps 35 in either one of the two LRRK2 mutant lines resulted in a complete rescue of the locomotor impairment (74.40% +/-3.42% and 65.01% +/- 3.55%, respectively, of the double transgenic flies were able to reach the top within 5 seconds on day 5; P < 0.0001) (Figure 4A and B). The results were very similar on Day 10 (Figure 4A and B). These results demonstrate that the functional interaction between LRRK2 and Vps35 is not exclusive to the kinase domain mutant, and provide further evidence that LRRK2 may play a role in the retromer-dependent 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)

Overexpression of Vps35 in DA neurons rescues locomotor deficits of other PD mutants. (A) Overexpression of Vps35 in LRRK2(Y1699C) mutants.(B) Overexpression of Vps35 in LRRK2(I1122V) mutants. All flies were reared at 29°C. N = 3-7 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 < 0.001, or *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T)/+is denoted as ### for P < 0.0001.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Overexpression of Vps35 in DA neurons rescues locomotor deficits of other PD mutants. (A) Overexpression of Vps35 in LRRK2(Y1699C) mutants.(B) Overexpression of Vps35 in LRRK2(I1122V) mutants. All flies were reared at 29°C. N = 3-7 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 < 0.001, or *** for P < 0.0001. Statistically significant difference compared to Ddc/+;LRRK2(I2020T)/+is denoted as ### for P < 0.0001.
Mentions: Similar to LRRK2(I2020T), expressing either one of the two mutant forms of LRRK2 in DA neurons caused a significant impairment locomotor activity on day 5 (24.71% +/- 3.44% of LRRK2(Y1699C) flies were able to reach the line within 5 seconds, compared to 63.21% +/- 5.30% of control, P < 0.0001; F (3, 177) = 50.60; and 39.93% +/- 2.73% of LRRK2(I1122V) flies were able to reach the line within 5 seconds, compared to 72.75% +/- 4.27% of control flies, P < 0.0001; F (3, 126) = 23.82, respectively) (Figure 4A and B), which is consistent with our previous results [13]. Importantly, overexpressing Vps 35 in either one of the two LRRK2 mutant lines resulted in a complete rescue of the locomotor impairment (74.40% +/-3.42% and 65.01% +/- 3.55%, respectively, of the double transgenic flies were able to reach the top within 5 seconds on day 5; P < 0.0001) (Figure 4A and B). The results were very similar on Day 10 (Figure 4A and B). These results demonstrate that the functional interaction between LRRK2 and Vps35 is not exclusive to the kinase domain mutant, and provide further evidence that LRRK2 may play a role in the retromer-dependent 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