Endocrinol Metab.  2014 Mar;29(1):62-69. 10.3803/EnM.2014.29.1.62.

Functional Role of Parkin against Oxidative Stress in Neural Cells

• Hwang M
• Lee JM
• Kim Y
• Geum D

• KMID: 2169353
• DOI: http://doi.org/10.3803/EnM.2014.29.1.62

Abstract

BACKGROUND
Parkinson disease (PD) is caused by selective cell death of dopaminergic neurons in the substantia nigra. An early onset form of PD, autosomal recessive juvenile parkinsonism has been associated with a mutation in the parkin gene. The function of parkin is known to remove misfolding proteins and protect cell death. We aimed to investigate the role of parkin against oxidative stress in neuronal cells.
METHODS
Parkin knockout embryonic stem cells (PKO ES cells) were differentiated into neurons by adherent monolayer culture method. Oxidative stress was induced by the treatment of 1-methyl-4-phenylpyridinium (MPP+) in neurons derived from wild type and PKO ES cells, and cell viability was examined by MTT assay. After exposure to MPP+, Tuj1-positive cell population was compared between PKO and wild type cells by fluorescence activated cell sorter (FACS) analysis. The activated caspase3 protein level was also measured by Western blot analysis, FACS and immunocytochemistry.
RESULTS
There was no difference in the efficiency of neuronal differentiation between wild type and PKO ES cells. After exposure to MPP+, no significant differences were found in cell viability and Tuj1-positive cell population between the two groups determined by MTT assay and FACS analysis, respectively. The activated caspase3 protein levels examined by Western blot analysis, FACS and immunocytochemistry were not changed in PKO cells compared with those of wild type cells after MPP+ treatment.
CONCLUSION
These results suggest that PKO neuronal cells including dopaminergic neurons are not sensitive to caspase3-dependent cell death pathway during the response against MPP+-induced oxidative stress.

Keyword

Parkin; Dopaminergic neurons; Oxidative stress; Cell death; Embryonic stem cells

MeSH Terms

1-Methyl-4-phenylpyridinium
Blotting, Western
Cell Death
Cell Survival
Dopaminergic Neurons
Embryonic Stem Cells
Fluorescence
Immunohistochemistry
Neurons
Oxidative Stress*
Parkinson Disease
Parkinsonian Disorders
Substantia Nigra
1-Methyl-4-phenylpyridinium

Figure

• 

  Fig. 1 Induction of dopaminergic neurons from wild-type (WT) and parkin knockout embryonic stem (PKO ES) cells by the adherent monolayer culture method. (A) Induction of neural cells from WT and PKO ES cells by the adherent monolayer method. Representative images were taken 0, 5, 10, and 15 days after differentiation. Immunocytochemistry shows MAP2-positive mature neural cells, and 6-diamidino-2-phenylindole (DAPI) was used for nuclear staining. (B) Expression of tyrosine hydroxylase (TH) 15 days after differentiation. TH+ neurons were analyzed by immunocytochemistry with an anti-TH antibody 15 days after differentiation. The 10% to 12% of cells were identified as TH-positive cells. (C) Reverse-transcriptional polymerase chain reaction analysis of dopaminergic neuron markers, such as Nurr1, TH, Pitx3, aromatic L-amino acid decarboxylase (AADC), and dopamine receptor 2 (D2R) at 15 days after differentiation. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as an internal control. Data are expressed as mean±SEM (n=3 per group). Statistical analysis was performed by Student t test. No significant differences in dopaminergic neuron marker expression were observed between WT and PKO cells.

• 

  Fig. 2 Cell viability analysis after treatment with 1-methyl-4-phenylpyridinium (MPP+) by MTT assay. MPP+ was treated at various concentrations (0, 100, 500, 750, 1,000, 1,250, and 1,500 µM) for 24 hours, at 15 days after differentiation. MTT assay was performed for viability of differentiated cells. Data are expressed as mean±SEM (n=6 per group). Statistical analysis was performed by two-way analysis of variance. No significant differences in the MPP+ susceptibility were observed between wild-type (WT) and parkin knockout (PKO) cells.

• 

  Fig. 3 Activity of caspase 3 in 1-methyl-4-phenylpyridinium (MPP+) treated wild-type (WT) and parkin knockout (PKO) neuronal cells. (A) Fluorescence-activated cell sorting analysis of WT and PKO cells treated with 1 mM MPP+ for 24 hours with anti-Tuj1 and antiactivated caspase 3 antibodies. Double+ cells represent colabeling with Tuj1 and activated caspase 3. The right panel shows the percentage of double+ cells per Tuj1 positive cells. (B) Western blot analysis of caspase 3 activity in WT and PKO cells. Cells were harvested 24 hours after 1 mM MPP+ treatment and Western blot analysis was performed with an activated caspase 3 antibody. β-Actin was used as an internal control. (C) Immunocytochemistry of WT and PKO cells treated with 1 mM MPP+ for 24 hours with anti-Tuj1 (green) and activated caspase 3 (red) antibody (scale bar, 20 µm). While MPP+ treatment causes a significant increase in caspase 3 activity, no significant differences in caspase 3 activity are observed between WT and PKO neural cells. Data are expressed as the mean±SEM (n=3 per group). Statistical analysis was performed by Student t test. CTL, control without MPP+ treatment; DAPI, 4, 6-diamidino-2-phenylindole.

• 

  Fig. 4 Activity of caspase 3 in 1-methyl-4-phenylpyridinium (MPP+) treated wild-type (WT) and parkin knockout (PKO) dopaminergic neurons. (A) Fluorescence-activated cell sorting analysis of WT and PKO cells treated with 1 mM MPP+ for 24 hours with tyrosine hydroxylase (TH) and activated caspase 3 antibodies. Double+ cells represent colabeling with TH and activated caspase 3 antibodies. Graphs show the percentage of double positive cells per TH-positive cells. (B) Immunocytochemistry of WT and PKO cells treated with 1 mM MPP+ for 24 hours with TH (red) and activated caspase 3 (green) antibodies (scale bar, 20 µm). No significant differences in caspase 3 activity were observed between WT and PKO dopaminergic neurons. Data are expressed as mean±SEM (n=3 per group). Statistical analysis was performed by Student t test. CTL, control without MPP+ treatment; DAPI, 4, 6-diamidino-2-phenylindole.

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