Pathogenic mutation in VPS35 impairs its protection against MPP(+) cytotoxicity

Abstract

Parkinson's disease primarily results from progressive degeneration of dopaminergic neurons in the substantia nigra. Both neuronal toxicants and genetic factors are suggested to be involved in the disease pathogenesis. The mitochondrial toxicant 1-methyl-4-phenylpyridinium (MPP(+)) shows a highly selective toxicity to dopaminergic neurons. Recent studies indicate that mutation in the vacuolar protein sorting 35 (vps35) gene segregates with Parkinson's disease in some families, but how mutation in the vps35 gene causes dopaminergic cell death is not known. Here, we report that enhanced VPS35 expression protected dopaminergic cells against MPP(+) toxicity and that this neuroprotection was compromised by pathogenic mutation in the gene. A loss of neuroprotective functions contributes to the pathogenesis of VPS35 mutation in Parkinson's disease.

Keywords: 1-methyl-4-phenylpyridinium; MPP; Parkinson's disease; VPS35; vacuolar protein sorting 35.

Figure 1

Mutant VPS35 overexpression produces no effect on cell growth in dopaminergic cell culture. (A) A diagram showing the construction of adenoviral vector that expresses wild-type or mutant (D620N substitution) human VPS35 gene. The open reading frame (ORF) of human VPS35 is driven by CMV promoter and the ORF of green fluorescence protein (GFP) gene is also integrated in the same transcript and is translated from the internal ribosome entry site (IRES). The expression cassette is recombined into adenoviral vector for producing viral particles in AD293 cells. (B-G) Fluorescence microscopy reveals that GFP was detectable in AD-VPS35 infected N27 cells (E-G) and not in uninfected cells (B-D). (H-J) Double-labeling fluorescence staining reveals that GFP colocalized with the immunostaining for VPS35 in AD-VPS35 infected cells. Scale bars: 100 µm (B-G) and 25 µm (H-J). (K) Western blotting reveals that VPS35 was overexpressed in the cells infected with adenovirus expressing wild-type (WT) or mutant (D620N) human VPS35. GAPDH immunoreactivity serves as an internal control for equal loading. (L) MTS assay reveals no effect of VPS35 overexpression on cell viability in N27 cell culture. N27 cells were infected with adenovirus expressing WT or D620N human VPS35 for 72 hours and cell viability was assayed with MTS kit. Data are means ± SEM (n = 5).

Figure 2

Pathogenic mutation in VPS35 impairs its protection against MPP⁺ toxicity to N27 cells. (A) MTS assay reveals that MPP⁺ dose-dependently induced cell death in N27 cell culture and that VPS35 overexpression protected the cells against MPP⁺ cytotoxicity. (B) Pathogenic mutation in VPS35 impaired its protection against MPP⁺ toxicity. Cells were infected with adenovirus expressing wild-type or mutant (D620N substitution) human VPS35 for 24 hours and then were treated with MPP⁺ at indicated concentrations for 48 hours. Cell viability was assayed with MTS kit afterwards. Data are means ± SEM (n = 5). * p < 0.05. (C) Immunoblotting reveals no effects of MPP⁺ (MPP) on endogenous VPS35 expression in N27 cells. GAPDH immunoreactivity serves as an internal control for equal loading. N27 cells were treated with MPP⁺ (200µM) for 48 hours and were then harvested for analysis of VPS35 induction.

Figure 3

Pathogenic mutation in VPS35 impairs its protection against MPP⁺ toxicity to primary ventral mesencephalic neurons. (A-F) Representative photos show live (green) and dead (red) cells in rat mesencephalic culture. Ventral mesencephalon was dissected from rat embryos (E-15) and mesencephalic neurons were cultured for 4 days before viral transduction. Primary neurons were transduced with adenoviral vectors and were further treated with MPP⁺ at 24 hours after viral transduction. Cell viability was assessed at 48 hours after MPP⁺ treatment. (G) Rat ventral mesencephalic culture was immunostained with an antibody against rat tyrosine hydroxylase (TH) to label dopaminergic neurons. Scale bars: 40 µm (A-G). (H) Cell viability in rat mesencephalic culture was quantitated. Data are means ± SEM (n = 7). * p < 0.05.

Figure 4

VPS35 expression knockdown produces no effect on VPS35-mediated protection against MPP⁺ toxicity. (A) A diagram showing the construction of adenoviral vector for expressing short-hairpin RNA (shRNA) against VPS35. VPS35 shRNA was inserted in the intron for co-expression with GFP. (B) Quantitative PCR reveals that the VPS35 mRNA level was reduced by expressing VPS35 shRNA. Data are means + SEM (n = 3). (C) Immunoblotting reveals that VPS35 expression was knocked down at protein levels by VPS35-shRNA. (D) Knocking down VPS35 expression produced no effect on cell viability in N27 cell culture. Data are means ± SEM (n =5). Cells were infected with adenovirus expressing VSP35 shRNA (shRNA-886, -887, or -888) for 72 hours and VPS35 knockdown and cell viability was assayed afterwards (B-D). (E) N27 cells were infected with shRNA viral vector for 24 hours and then were treated with MPP⁺ for 48 hours. After MPP⁺ treatment, cell viability was determined with MTS kit. Data are means ± SEM (n = 5). * p < 0.05.