The effects of pdr1, djr1.1 and pink1 loss in manganese-induced toxicity and the role of α-synuclein in C. elegans

Abstract

Parkinson's disease (PD) is a neurodegenerative brain disorder characterized by selective dopaminergic (DAergic) cell loss that results in overt motor and cognitive deficits. Current treatment options exist to combat PD symptomatology, but are unable to directly target its pathogenesis due to a lack of knowledge concerning its etiology. Several genes have been linked to PD, including three genes associated with an early-onset familial form: parkin, pink1 and dj1. All three genes are implicated in regulating oxidative stress pathways. Another hallmark of PD pathophysiology is Lewy body deposition, associated with the gain-of-function genetic risk factor α-synuclein. The function of α-synuclein is poorly understood, as it shows both neurotoxic and neuroprotective activities in PD. Using the genetically tractable invertebrate Caenorhabditis elegans (C. elegans) model system, the neurotoxic or neuroprotective role of α-synuclein upon acute Mn exposure in the background of mutated pdr1, pink1 or djr1.1 was examined. The pdr1 and djr1.1 mutants showed enhanced Mn accumulation and oxidative stress that was reduced by α-synuclein. Moreover, DAergic neurodegeneration, while unchanged with Mn exposure, returned to wild-type (WT) levels for pdr1, but not djr1.1 mutants expressing α-synuclein. Taken together, this study uncovers a novel, neuroprotective role for WT human α-synuclein in attenuating Mn-induced toxicity in the background of PD-associated genes, and further supports the role of extracellular dopamine in exacerbating Mn neurotoxicity.

Fig. 1. pdr1 mutants are hypersensitive to an acute Mn exposure

(A,B) Doseresponse survival curves following acute Mn exposure and the respective LD50 doses. All values were compared to non-treated worms set to 100% survival and plotted against the logarithmic scale of the used Mn concentrations. (A) N2 (wildtype, WT) and pdr1, pink1 and djr1.1 deletion mutants were treated for 30 min at L1 (larval) stage with increasing concentrations of MnCl2. (B) UA44 (WT; human α-Syn), UA88 (pdr1 KO; human α-Syn), UA86 (pink1 KO; human α-Syn) and UA84 (djr1.1 KO; human α-Syn) were treated for 30 min at L1 stage with increasing concentrations of MnCl2. (A,B) Data are expressed as means ± SEM from at least four independent experiments. Statistical analysis of the LD50: **p < 0.01 versus respective wildtype worms. (C) Statistical comparison of respective non-α-Syn and α-Syn containing worms: *p < 0.05. KO = deletion mutants.

Fig. 2. Enhanced Mn accumulation in pdr1 and djr1.1 mutants is reversed by WT α-Syn expression

(A-D) Intraworm Mn content after acute treatment with MnCl2 was quantified by ICP-MS/MS. All values were normalized to non-treated wildtype (WT) worms. (A) N2 (WT) and pdr1, pink1 and djr1.1 deletion mutants were treated at L1 stage for 30 min with increasing concentrations of MnCl2. (B) UA44 (WT; human α-Syn), UA88 (pdr1 KO; human α-Syn), UA86 (pink1 KO; human α-Syn) and UA84 (djr1.1 KO; human α-Syn) were treated at L1 stage for 30 min with increasing concentrations of MnCl2. (C) Comparing intraworm Mn content of pdr1 mutants and UA88 (pdr1 KO; human α-Syn) after 30 min treatment with MnCl2 (data from A,B). (D) Comparing intraworm Mn content in djr1.1 mutants and UA84 (djr1.1 KO; human α-Syn) after 30 min treatment with MnCl2 (data from A,B). (A-D) Data are expressed as means + SEM from at least six independent experiments. Statistical analysis by two-way ANOVA: (A) interaction p < 0.001, genotype p < 0.001, concentration p < 0.001; (B) interaction ns, genotype p < 0.001, concentration p < 0.001; (C) interaction ns, genotype ns, concentration p < 0.001; (D) interaction p < 0.001, genotype p < 0.001, concentration p < 0.001. ***p < 0.001, **p < 0.01, *p < 0.05. (E) 2D images and respective microscope images of WT worms (I, nontreated; II, 10 mM MnCl2); djr1.1 deletion mutants (III) and UA84 (djr1.1 KO; human α-Syn) (IV) incubated with 10 mM MnCl2 for 30 min. KO = deletion mutants; ns = not significant.

Fig. 3. Absence of Mn-induced DAergic neurodegeneration

(A) Representative confocal images used in the scoring system: normal worms (I), worms showing puncta (II), shrunken soma (III) or loss of dendrites and soma (IV). (B) The CEP architecture of 15 worms per group of WT, pdr1 and djr1.1 mutants and the respective α-Syncontaining strains (UA44, UA86, UA88) were scored 72 hours after an acute, 30 min treatment with MnCl2. Shown are mean values + SEM of at least four experiments each. *p < 0.05 versus respective non-treated worms without α-Syn. KO = deletion mutants; ns = not significant.

Fig. 4. Mn-induced oxidative stress is exacerbated in pdr1 and djr1.1 mutants, but rescued by α-Syn expression

(A) (a) Effect of MnCl2 on the RONS induction in N2 (WT) and pdr1, pink1 and djr1.1 deletion mutants after 1 h dye loading and subsequent MnCl2 post-treatment with their respective LD25 doses. (b) Effect of MnCl2 on the RONS induction of UA44 (WT; human α-Syn), UA88 (pdr1 KO; human α-Syn), UA86 (pink1 KO; human α-Syn) and UA84 (djr1.1 KO; human α-Syn) after 1 h dye loading and subsequent MnCl2 post-treatment with their respective LD25 doses. (c) Comparing Mn-induced RONS induction of pdr1 mutants and UA88 (pdr1 KO; human α-Syn) (see B,a and B,b). (d) Comparing Mn-induced RONS induction of djr1.1 mutants and UA84 (djr1.1 KO; human α-Syn) (see B,a and B,b). (A) Shown are mean values (+ SEM) of at least four measurements, which were normalized to the respective dye-loaded worms at the respective time-point. Statistical analysis by two-way ANOVA: (a,b,c) interaction p < 0.001, genotype p < 0.001, time p < 0.001; (d) interaction ns, genotype p < 0.001, time p < 0.001. (B) Total glutathione level of N2 (WT) and pdr1, pink1 and djr1.1 deletion mutants following 30 min exposure with increasing concentrations of MnCl2. Data are expressed as means + SEM from at least five independent experiments. Statistical analysis by twoway ANOVA: interaction ns, genotype p < 0.001, concentration p < 0.01. (A-C) p < 0.001. ***p < 0.001, **p < 0.01, *p < 0.05. KO = deletion mutants; ns = not significant.

Fig. 5. Increased skn-1 mRNA expression in djr1.1 and pink1 mutants

SKN-1 expression after acute treatment with MnCl₂. Relative gene expression was determined by qRT-PCR. (A) N2 (WT) and pdr1, pink1 and djr1.1 deletion mutants were treated at L1 stage for 30 min with MnCl₂ at the respective LD₂₅ and LD₅₀ doses. (B) UA44 (WT; human α-Syn), UA88 (pdr1 KO; human α-Syn), UA86 (pink1 KO; human α-Syn) and UA84 (djr1.1 KO; human α-Syn) were treated at L1 stage for 30 min with MnCl₂ at the respective LD₂₅ and LD₅₀ doses. (A, B) Shown are mean values ± SEM of four independent experiments in duplicates normalized to the untreated wildtype and relative to afd-1/β-actin mRNA. Statistical analysis by two-way ANOVA: (A) interaction ns, genotype p < 0.01, concentration; (B) interaction ns, genotype ns, concentration ns. **p < 0.01, *p < 0.05 versus respective wildtype worms. KO = deletion mutants; ns = not significant.

Fig. 6. Decreased dat-1 expression in djr1.1 deletion mutants - dat-1

mRNA expression after acute treatment with MnCl₂. Relative gene expression was determined by qRT-PCR. UA44 (WT; human α-Syn), UA88 (pdr1 KO; human α-Syn)) and UA84 (djr1.1 KO; human α-Syn) were treated at L1 stage for 30 min with MnCl₂ at the respective LD₅₀. Data are expressed as means + SEM from at least four independent experiments in duplicates normalized to the untreated wildtype and relative to afd-1/β-actin mRNA. Statistical analysis by two-way ANOVA: interaction ns, genotype p < 0.001, concentration ns. ***p < 0.001, *p < 0.05 versus respective wildtype worms. KO = deletion mutants; ns = significant.