1,25-dyhydroxyvitamin D 3 Attenuates L-DOPA-Induced Neurotoxicity in Neural Stem Cells Wooyoung Jang & Hyun-Hee Park & Kyu-Yong Lee & Young Joo Lee & Hee-Tae Kim & Seong-Ho Koh Received: 24 February 2014 /Accepted: 25 July 2014 # Springer Science+Business Media New York 2014 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 D 3 has been reported to display neu- roprotective properties. Therefore, we investigated the protec- tive effect of 1,25-dyhydroxyvitamin D 3 (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 immuno- cytochemistry, 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 mi- croarrays were also employed to detect changes in the expres- sion 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 pro- teins that are associated with cell survival. However, simulta- neous exposure to calcitriol significantly reduced these ef- fects. The calcitriol-mediated protection against L-DOPA tox- icity 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 activa- tion of the PI3K pathway, and reducing oxidative stress. Keywords L-DOPA . Neural stem cells . 1,25-dyhydroxyvitamin D 3 . Neuroprotection Introduction Parkinson’ s disease (PD), a movement disorder characterized by resting tremors, rigidity, bradykinesia, and a loss of pos- tural reflex, is a common cause of disability among the elderly [1]. Since L-3,4-dihydrophenylalanine (L-DOPA) was first introduced in clinical trials in the 1960s, it remains the gold standard for the treatment of PD [1–3]. Although L-DOPA exerts a clear beneficial effect on PD symptoms, it can be toxic to dopaminergic neurons and induce or accelerate the degeneration of nigral neurons [4–7]. Several studies have suggested that L-DOPA exerts a cytotoxic effect Wooyoung Jang and Hyun-Hee Park contributed equally to this work. Electronic supplementary material The online version of this article (doi:10.1007/s12035-014-8835-1) contains supplementary material, which is available to authorized users. W. Jang : H.<H. Park : K.<Y. Lee : Y. J. Lee : H.<T. Kim : S.<H. Koh Department of Neurology, Hanyang University College of Medicine, Seoul, South Korea W. Jang Department of Neurology, Gangneung Asan Hospital, College of Medicine, University of Ulsan, Gangneung, South Korea S.<H. Koh Department of Translational Medicine, Hanyang University Graduate School of Biomedical Science and Engineering, Seoul, Republic of Korea S.<H. Koh (*) Department of Neurology, Hanyang University College of Medicine, 249-1 Hanyang University Guri Hospital Gyomun-dong, Guri-si, Gyeonggi-do 471-701, South Korea e-mail: ksh213@hanyang.ac.kr H.<T. Kim (*) Department of Neurology, Hanyang University College of Medicine, 17 Haengdang-dong, Seongdong-gu, Seoul 133-792, South Korea e-mail: kimht@hanyang.ac.kr Mol Neurobiol DOI 10.1007/s12035-014-8835-1