PHYSIOLOGY Development of a serum-free supplement for primary neuron culture reveals the interplay of selenium and vitamin E in neuronal survival Stephan Roth, SiJie Zhang, Jazmin Chiu, Eva K Wirth, Ulrich Schweizer n Neurobiology of Selenium, Institute for Experimental Endocrinology, Charite´-Universit¨ atsmedizin Berlin, Augustenburger Platz 1, 13353 Berlin, Germany article info Article history: Received 3 November 2009 Accepted 14 January 2010 Keywords: Selenium Vitamin E Peroxide Oxidative stress abstract Serum-free media require a number of supplements in order to support long-term neuronal survival. Commercially available B27 TM , in combination with Neurobasal TM medium, supports neuronal survival and suppresses glial proliferation. However, B27 contains many biological antioxidants as well as catalase and superoxide dismutase, eventually demanding the application of unphysiologically high peroxide concentrations in survival assays. Moreover, optimal amounts of selenium (Se) are included in ‘‘B27 supplement minus antioxidants’’, a commercially available supplement used for the study of the role of antioxidants. Hence, Se-dependent enzymes like glutathione peroxidase are maximally expressed when this supplement is used and Se-depletion studies are not possible without changing the medium composition. We have therefore developed a modified serum-free media supplement which allows for free variation of all constituents. Our supplement was comparable to B27 with regard to cell survival and expression of neurochemical markers. Reduction of Se content in the supplement reduced selenoprotein expression and made cortical neurons more sensitive towards challenges with peroxides. Withdrawal from the medium supplement of vitamin E alone did not alter the survival of neurons in response to peroxides, while simultaneous reduction of Se and vitamin E rendered neurons hypersensitive towards peroxide challenge. This finding implied that adequate Se supply of neurons is required to minimize lipid peroxidation. Our medium supplement is easily prepared, inexpensive, and should be applicable to the analysis of survival mechanisms beyond peroxide challenge. & 2010 Elsevier GmbH. All rights reserved. Introduction The concept of oxidative stress has been exceptionally influential to biochemical aspects of the pathophysiology of ageing and degenerative diseases [1]. Recently, it has been refined and now also encompasses disturbances in redox signal transduc- tion [2]. Markers of oxidative stress are elevated in neurodegen- erative disorders, including Alzheimer’s and Parkinson’s disease, among others [3–5]. The mechanisms leading to the formation of and protection from reactive oxygen or nitrogen species (ROS and RNS, respectively) in response to toxic insults or mutated proteins are a matter of intense studies. Cell culture models of primary neurons are important approaches for studying development and maintenance of neurons, as well as their reaction to challenges. In vitro cultures allow for a maximum of manipulations, both chemically and genetically. Thus, serum-free cell culture models have been developed for central neurons from rodents. A particularly successful and widely applied medium supplement allowing for the long-term survival of cultivated neurons is B27 TM in combination with Neurobasal TM medium [6,7]. However, since the exact formulation is proprietary, researchers are not able to vary specific ingredients. Moreover, B27 contains several enzymatic and non-enzymatic antioxidants at concentra- tions optimized for long-term survival of neurons. Therefore, additions to the medium are possible, while concentrations below the optimum are not achievable. Hence, cellular glutathione peroxidase activity is maximal under selenium (Se)-replete conditions as in both normal B27 and ‘‘B27 supplement minus antioxidants’’ (B27-AO TM ). One approach to experimentally reduce the concentration of antioxidants and Se, at least in part, was the dilution of B27/Neurobasal with Eagle’s minimal essential medium [8]. Since we are interested in the roles of Se-dependent proteins in neuronal survival and function, the question of Se status was particularly pertinent to our research. The trace element Se is essential for mammals. It is cotranslationally incorporated into the growing peptide-chain in the form of the rare amino acid selenocysteine (SeCys) [9]. The most well-known selenoprotein is cellular glutathione peroxidase (GPx1), a hydrogen peroxide- degrading enzyme [10], inactivation of which is known to exacerbate neuronal damage in cerebral ischemia or upon application of neurotoxins [11]. However, not all Se effects are ARTICLE IN PRESS Contents lists available at ScienceDirect journal homepage: www.elsevier.de/jtemb Journal of Trace Elements in Medicine and Biology 0946-672X/$ - see front matter & 2010 Elsevier GmbH. All rights reserved. doi:10.1016/j.jtemb.2010.01.007 n Corresponding author. Tel.: + 49 30 450524080; Fax.: + 49 30 450524922 E-mail address: ulrich.schweizer@charite.de (U. Schweizer). Journal of Trace Elements in Medicine and Biology 24 (2010) 130–137