Small interfering RNA targeting the PINK1 induces apoptosis in dopaminergic cells SH-SY5Y

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Abstract

PTEN-induced kinase 1 (PINK1) is a recently identified gene, mutations of which cause levodopa-responsive parkinsonism. An over-expression of wild-type PINK1 protects neurons from stress-induced mitochondrial dysfunction and apoptosis. We studied the effects of PINK1 suppression using small interfering RNA (siRNA), which can inhibit PINK1 mRNA expression up to 87%, and decrease PINK1 protein up to 80% in human dopaminergic cell line SH-SY5Y. Incubation with PINK1 siRNA decreased SH-SY5Y cell viability and significantly increased MPP+ or rotenone-induced cytotoxicity. Our results indicate that reduction in PINK1 expression can trigger apoptotic process that can be exacerbated by the presence of MPP+ or rotenone. These findings support the hypothesis that PINK1 participates in the protection of dopaminergic neurons.

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Materials and methods

Cell culture, siRNA transfection, and RNA extraction. Human dopaminergic cell line SH-SY5Y was maintained in DMEM, supplemented with 10% fetal calf serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). Cells from passages 10 to 20 were used and they were seeded at 40% confluency into 30 mm six-well dish. The required amount of PINK1 siRNA (Ambion) and 5 μl lipofectamine 2000 (Invitrogen) were diluted into a final volume of 100 μl in Opti-MEM I (Gibco), respectively, and gently mixed and

Results

Real-time RT-PCR analysis showed a dose-dependent reduction of PINK1 mRNA amplification following transfection with 5–160 nM PINK1 siRNA. At 80–160 nM PINK1 siRNA significantly reduced PINK1 mRNA as much as 87% (Fig. 1A). The inhibitory effects lasted up to 4 days following a single dose of siRNA treatment and there was no significant difference between 80 and 160 nM siRNA treatment for 24 h (13.3 ± 5.2 and 13.4 ± 4.3 of control #3 siRNA treated group, respectively, p = 0.974). The protein levels of PINK1

Discussion

Parkinson disease (PD) is a progressive neurodegenerative disorder, associated with the selective loss of dopaminergic neurons in the substantia nigra pars compacta. Current hypothesis suggests that neurodegeneration in PD is associated with a cascade of events that involve genetic factors, oxidative stress, mitochondrial abnormalities, excitatory amino acids, and a rise in intracytoplasmic free calcium. At least five genes have been identified as causal genes for PD: α-synuclein (PARK1) [9],

Acknowledgments

This work was supported by Grants NS 043567 and NS 40370 from the National Institute of Neurological Disorders and Stroke. The authors thank Dr. Junjiang Fu (Department of Molecular and Cellular Biology), Dr. Jonathan Lu and Dr Max Huang (Departments of Medicine, Baylor College of Medicine) for their technical assistance in this study.

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