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)

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.

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.

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.

Figure 3

Overexpression of Vps35 in DA neurons rescues the shortened lifespan of LRRK2(I2020T) flies. N = 8-11 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 Ddc/+;LRRK2(I2020T)/+is denoted as # for P < 0.05.

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.

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.

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.