Neuroscience Letters 496 (2011) 100–105 Contents lists available at ScienceDirect Neuroscience Letters journal homepage: www.elsevier.com/locate/neulet Cystatin C induces apoptosis and tyrosine hydroxylase gene expression through JNK-dependent pathway in neuronal cells XueYun Liang a,b , Atsushi Nagai a,∗ , Masaharu Terashima c , Abdullah Md. Sheikh a , Yuri Shiota a , Shingo Mitaki d , Seung U. Kim e,f , Shuhei Yamaguchi d a Department of Laboratory Medicine, Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan b Department of Laboratory Medicine, Affiliated Hospital of Ningxia Medical University, Yinchuan, PR China c Department of Registered Dietitian and Nutritional Science, Tokaigakuen University Faculty of Human Wellness, Nagoya, Japan d Department of Internal Medicine III, Shimane University Faculty of Medicine, Izumo, Japan e Department of Neurology, UBC Hospital, University of British Columbia, Vancouver, Canada f Medical Research Institute, Chung-Ang University College of Medicine, Seoul, Republic of Korea article info Article history: Received 20 December 2010 Received in revised form 28 March 2011 Accepted 29 March 2011 Keywords: Cystatin C Neuron Apoptosis Differentiation JNK abstract Cystatin C (CysC), an endogenous cysteine protease inhibitor, has been implicated in the apoptosis and differentiation processes of neuronal cells. In this study, we have investigated the pathway involved in the process. A human neuronal hybridoma cell line (A1 cell) was treated with CysC in both undifferentiated and retinoic acid (RA)-induced differentiated conditions, which decreased overall process length in both conditions. Also, CysC increased apoptotic cell number time-dependently, as revealed by TUNEL assay. Western blot analysis demonstrated that in differentiated A1 cells, CysC treatment decreased Bcl-2 and increased active caspase-9 protein level time-dependently. Immunocytochemistry results revealed that, CysC treatment significantly increased active form of Bax expressing cell number, which co-localized with mitochondria. Mitogen activated protein (MAP) kinase inhibition experiments showed that Bax mRNA induction and Bcl-2 mRNA inhibition by CysC treatment were c-Jun N-terminal kinase (JNK)-dependent. After RA-induced differentiation, choline acetyltransferase (ChAT) and neurofilament (NF) mRNA lev- els were increased in A1 cells. CysC treatment inhibited NF mRNA level in both undifferentiated and RA-differentiated, and increased TH mRNA in differentiated A1 neurons. Analysis of signal transduction pathway demonstrated that TH gene induction was also JNK-dependent. Thus, our results demonstrated the significance of JNK-dependent pathways on CysC-induced apoptosis and TH gene expression in neu- ronal cells, which might be an important target in the management of CysC dependent neurodegenerative processes. © 2011 Elsevier Ireland Ltd. All rights reserved. Cystatin C (CysC) is an endogenous cysteine protease inhibitor, which is ubiquitously expressed in the nucleated cells and secreted in the body fluids. By inhibiting cysteine proteases such as cathep- sins, it plays important roles in the regulation of diverse biological functions including inflammation, tumor invasion and neuronal cell differentiation. In the central nervous system (CNS), where CysC concentration is 5.5 times higher in cerebrospinal fluid (CSF) compared to serum [1], it might play an important role in CNS pathophysiology by balancing the protease activities. Indeed, Abbreviations: CysC, cystatin C; RA, retinoic acid; ChAT, choline acetyltrans- ferase; NF, neurofilament; TH, tyrosin hydroxylase; JNK, c-Jun N-terminal kinase; CNS, central nervous system; CSF, cerebrospinal fluid; ER, endoplasmic reticulum; DMEM, Dulbecco’s modified eagle’s medium; TUNEL, terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling; RT, room temperature; MAPK, mitogen- activated protein kinase. ∗ Corresponding author. Tel.: +81 853 20 2409; fax: +81 853 20 2409. E-mail address: anagai@med.shimane-u.ac.jp (A. Nagai). its concentration is reported to be decreased in neuroinflam- matory disease conditions [14], leptomeningeal metastasis [13] and cerebral amyloid angiopathy [19]. Conversely, enhanced CysC expression is observed in cell-stress conditions such as facial nerve axotomy [10], hypophysectomy [6], transient forebrain ischemia [17] and 6-OHDA-induced niagrostraiatal neuronal degeneration in vivo [22]. It has been shown that CysC possesses functions other than lysosomal cysteine protease inhibition. Such as, it could influ- ence the fibrillation process of Alzheimer’s amyloid peptide [11], or deposited intracellularly in endoplasmic reticulum (ER) [9]. Moreover, there are reports that have shown that CysC provides neuroprotection in serum deprived neuroblastoma cells [21], and increases nestin-positive neuronal progenitor cells and neuro- sphere [7]. On the other hand, we and others have found that CysC treatment increases the apoptosis of neuronal cells [2,11]. Inter- estingly, using CysC knockout mice, a report showed that CysC modulates both neurodegeneration and neurogenesis in a status 0304-3940/$ – see front matter © 2011 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.neulet.2011.03.091