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1,25-dyhydroxyvitamin D3 Attenuates l-DOPA-Induced Neurotoxicity in Neural Stem Cells

Abstract

The neurotoxicity of levodopa (l-DOPA) on neural stem cells (NSCs) and treatment strategies to protect NSCs from this neurotoxicity remain to be elucidated. Recently, an active form of vitamin D3 has been reported to display neuroprotective properties. Therefore, we investigated the protective effect of 1,25-dyhydroxyvitamin D3 (calcitriol) on l-DOPA-induced NSC injury. We measured cell viability via the cell counting kit-8 (CCK-8) and lactate dehydrogenase (LDH) assays and Annexin V/PI staining followed by flow cytometry, cell proliferation using the BrdU and colony-forming unit (CFU) assays, cell differentiation via immunocytochemistry, the levels of free radicals via 2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA) staining, apoptosis via DAPI and TUNEL staining, and intracellular signaling protein expression via Western blot. Antibody microarrays were also employed to detect changes in the expression of prosurvival- and death-related proteins. Treatment of NSCs with l-DOPA reduced their viability and proliferation. This treatment also increased the levels of free radicals and decreased the expression levels of intracellular signaling proteins that are associated with cell survival. However, simultaneous exposure to calcitriol significantly reduced these effects. The calcitriol-mediated protection against l-DOPA toxicity was blocked by the phosphoinositide 3-kinase (PI3K) inhibitor LY294004. l-DOPA also inhibited the expression of Nestin and Ki-67, and co-treatment with calcitriol alleviated these effects. The expression levels of GFAP, DCX, and Tuj1 were not significantly affected by treatment with l-DOPA or calcitriol. Calcitriol protects against l-DOPA-induced NSC injury by promoting prosurvival signaling, including activation of the PI3K pathway, and reducing oxidative stress.

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Acknowledgments

This work was supported by the NanoBio R&D Program of the Korea Science and Engineering Foundation, the Ministry of Education, Science, and Technology (2007–04717), and the Korea Healthcare Technology R&D Project, Ministry for Health, Welfare & Family Affairs, Republic of Korea (A101712).

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The authors declare that they have no conflicts of interest.

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Correspondence to Hee-Tae Kim or Seong-Ho Koh.

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Wooyoung Jang and Hyun-Hee Park contributed equally to this work.

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Supplementary Fig. 1

The effect of calcitriol on the proliferation of NSCs. The BrdU assays revealed that treatment with high-dose calcitriol (100 μM) reduces NSC proliferation. *p < 0.05. (PDF 85 kb)

Supplementary Fig. 2

Immunostaining of NSC proliferation and the expression of neuronal markers in NSCs exposed to various concentrations of calcitriol. Treatment with calcitriol at concentrations greater than 10 μM decreased the expression of Nestin and Ki-67 but displayed no effect on that of DAPI or Tuj-1. *p < 0.05 and **p < 0.01 (compared to the control group). (PDF 359 kb)

Supplementary Fig. 3

The levels of TH and AADC activity in NSCs after exposure to 200 μM l-DOPA for 48 hr and different concentrations of calcitriol. There was no significant difference in the TH and AADC activity levels between the control, l-DOPA alone, and l-DOPA and various concentrations of calcitriol groups. (PDF 191 kb)

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Jang, W., Park, HH., Lee, KY. et al. 1,25-dyhydroxyvitamin D3 Attenuates l-DOPA-Induced Neurotoxicity in Neural Stem Cells. Mol Neurobiol 51, 558–570 (2015). https://doi.org/10.1007/s12035-014-8835-1

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  • DOI: https://doi.org/10.1007/s12035-014-8835-1

Keywords

  • l-DOPA
  • Neural stem cells
  • 1,25-dyhydroxyvitamin D3
  • Neuroprotection