Genetic risk for Parkinson's disease correlates with alterations in neuronal manganese sensitivity between two human subjects - PubMed (original) (raw)

Genetic risk for Parkinson's disease correlates with alterations in neuronal manganese sensitivity between two human subjects

Asad A Aboud et al. Neurotoxicology. 2012 Dec.

Abstract

Manganese (Mn) is an environmental risk factor for Parkinson's disease (PD). Recessive inheritance of PARK2 mutations is strongly associated with early onset PD (EOPD). It is widely assumed that the influence of PD environmental risk factors may be enhanced by the presence of PD genetic risk factors in the genetic background of individuals. However, such interactions may be difficult to predict owing to the complexities of genetic and environmental interactions. Here we examine the potential of human induced pluripotent stem (iPS) cell-derived early neural progenitor cells (NPCs) to model differences in Mn neurotoxicity between a control subject (CA) with no known PD genetic risk factors and a subject (SM) with biallelic loss-of-function mutations in PARK2 and family history of PD but no evidence of PD by neurological exam. Human iPS cells were generated from primary dermal fibroblasts of both subjects. We assessed several outcome measures associated with Mn toxicity and PD. No difference in sensitivity to Mn cytotoxicity or mitochondrial fragmentation was observed between SM and CA NPCs. However, we found that Mn exposure was associated with significantly higher reactive oxygen species (ROS) generation in SM compared to CA NPCs despite significantly less intracellular Mn accumulation. Thus, this report offers the first example of human subject-specific differences in PD-relevant environmental health related phenotypes that are consistent with pathogenic interactions between known genetic and environmental risk factors for PD.

Copyright © 2012 Elsevier Inc. All rights reserved.

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Figures

Fig. 1

Fig. 1. Experimental Design

iPS cells were plated for each experiment on Matrigel-covered 12 well or 96 well plates at a density of 6×104 cell/ml. On the fourth day after plating they reach a confluency of approximately 90 per cent and that is when neuralization starts (day 1) with the dual-SMAD inhibition protocol. On day 7 of neuralization 80-90% of the cell population in culture are NPCs (PAX6+). We exposed the cells in all of our experiments on day 7 for 24 hours followed by assessment of Mn-related outcome measures.

Fig. 2

Fig. 2. Mn cytotoxicity in NPCs

CA and SM NPCs were treated with Mn and cell viability was assessed using MTT assay. Two-way ANOVA revealed significant exposure effect (p < 0.001) without a significant genotype effect (p = 0.345). CA lines used in this experiment include CA6 (n=2) and CA11 (n=2), and SM lines include SM3 (n=1), SM5 (n=1), and SM14 (n=1). Data are collected from 3 independent replicates. Error bars represent SEM.

Fig. 3

Fig. 3. ROS generation in NPCs

NPCs are treated with two concentrations of manganese and the level of ROS generation was assessed using DCF dye. Two-way ANOVA on the log-transformed values returned genotype difference (P=0.001) but no genotype × exposure interaction. CA lines used in these experiments include CA6 (n=3) and CA11 (n=3); and SM lines include SM3 (n=2) and SM14 (n=2). Data are collected from four independent 500 μM and six independent 1000 μM MnCl2 experiments. Error bars represent SEM.

Fig. 4

Fig. 4. Quantification of mitochondrial fragmentation in NPCs using MitoTracker Red

CA and SM NPCs are treated with 500μM MnCl2 for 24 hours followed by mitochondrial staining using MitoTracker Red dye (100 nM). 50 PAX6+ cells were counted for each condition and scored from 1 to 4 increasing with severity of mitochondrial fragmentation. The average score of cells assessed for three independent experiments after exposure to 0 or 500 μM Mn. Two-way ANOVA on the log-transformed data returned high significance for the effect of Mn exposure, but no genotype or genotype × exposure interaction effects. Paired two-tailed t-test was used for post hoc analysis of the three independent paired experiments: CA6 with SM4 (n=1), and CA6 with SM5 (n=2). Error bars represent SEM.

Fig. 5

Fig. 5. Quantification of mitochondrial fragmentation in NPCs using CellLight Mitochondria-GFP

Representative images are shown for CA and SM vehicle- and Mn-treated NPCs after staining with CellLight Mitochondria-GFP and TO-PRO-3 nuclear counterstain. PAX6+ NPCs were analyzed for mitochondrial fragmentation as described in section 2.5. (CA6: SM3 n=2, CA11:SM14 n=1). Scale bar = 10 μm.

Fig. 6

Fig. 6. Measurement of total Mn accumulation in SM and CA NPCs using (GFAAS)

Intracellular Mn accumulation was assessed in CA and SM NPCs after exposure to vehicle and 500 μM Mn. Outputs were normalized to the protein concentration of each sample. Two-way ANOVA revealed significant genotype difference in total Mn level after Mn exposure (P = 009). CA6 and SM5 lines were used in these experiments. Data are collected from 3 independent vehicle exposed samples and 4 independent Mn treated samples. Error bars represent SEM.

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