A TNF Receptor 2 Selective Agonist Rescues Human Neurons from Oxidative Stress-Induced Cell Death Roman Fischer1*, Olaf Maier1, Martin Siegemund1, Harald Wajant2, Peter Scheurich1, Klaus Pfizenmaier1 1 Institute of Cell Biology and Immunology, University Stuttgart, Stuttgart, Germany, 2 Division of Molecular Internal Medicine, Department of Internal Medicine II, University Hospital Wu¨ rzburg, W u¨rzburg, Germany Abstract Tumor necrosis factor (TNF) plays a dual role in neurodegenerative diseases. Whereas TNF receptor (TNFR) 1 is predominantly associated with neurodegeneration, TNFR2 is involved in tissue regeneration and neuroprotection. Accordingly, the availability of TNFR2-selective agonists could allow the development of new therapeutic treatments of neurodegenerative diseases. We constructed a soluble, human TNFR2 agonist (TNC-scTNFR2) by genetic fusion of the trimerization domain of tenascin C to a TNFR2-selective single-chain TNF molecule, which is comprised of three TNF domains connected by short peptide linkers. TNC-scTNFR2 specifically activated TNFR2 and possessed membrane-TNF mimetic activity, resulting in TNFR2 signaling complex formation and activation of downstream signaling pathways. Protection from neurodegeneration was assessed using the human dopaminergic neuronal cell line LUHMES. First we show that TNC-scTNFR2 interfered with cell death pathways subsequent to H2O2 exposure. Protection from cell death was dependent on TNFR2 activation of the PI3K-PKB/Akt pathway, evident from restoration of H2O2 sensitivity in the presence of PI3K inhibitor LY294002. Second, in an in vitro model of Parkinson disease, TNC-scTNFR2 rescues neurons after induction of cell death by 6-OHDA. Since TNFR2 is not only promoting anti-apoptotic responses but also plays an important role in tissue regeneration, activation of TNFR2 signaling by TNC-scTNFR2 appears a promising strategy to ameliorate neurodegenerative processes. Citation: Fischer R, Maier O, Siegemund M, Wajant H, Scheurich P, et al. (2011) A TNF Receptor 2 Selective Agonist Rescues Human Neurons from Oxidative Stress-Induced Cell Death. PLoS ONE 6(11): e27621. doi:10.1371/journal.pone.0027621 Editor: Malu´ G. Tansey, Emory University, United States of America Received August 3, 2011; Accepted October 20, 2011; Published November 14, 2011 Copyright: 2011 Fischer et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Funding: This work was supported by a grant from European Community Sixth Framework Programme, Project NeuroproMiSe, contract # LSHM-CT-2005- 018637. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing Interests: The authors have declared that no competing interests exist. * E-mail: roman.fischer@izi.uni-stuttgart.de Introduction Tumor necrosis factor (TNF), plays a dominant role in the initiation and perpetuation of chronic inflammation [1], a condition that can lead to a variety of diseases. Blocking of TNF signaling has been evaluated in various inflammatory diseases and is successfully used for treatment of autoimmune diseases such as rheumatoid arthritis, Crohn’s disease and psoriasis [2,3]. Chronic inflammation is a common feature of neurodegenerative diseases of the central nervous system, such as Alzheimer’s disease, Parkinson’s disease (PD) and multiple sclerosis (MS), too [4], and TNF signaling has been implicated as an important factor for the onset of demyelinating diseases. However, despite promising results in mouse models of MS, clinical trials with TNF neutralizing reagents in MS patients failed to ameliorate the disease and in some cases even led to disease exacerbation [5]. Since then, the dual role of TNF in the CNS has been investigated in various mouse models, revealing significant beneficial effects predominantly mediated by TNF receptor (TNFR) 2, whereas TNFR1, directly and indirectly promotes neurotoxicity [2,6]. In particular, it has been demonstrated that TNFR2 can protect neurons against excitotoxic insults in vitro [7,8] and promotes neuronal survival as well as oligodendrocyte regeneration after ischemic and neurotoxic insults, respectively [9,10]. In contrast, TNFR1 exacerbated axonal and neuronal damage through its potent pro-inflammatory effects, which became particularly obvious under chronic inflammatory situa- tions [11]. Therefore, TNF remains a primary therapeutic target for the treatment of neuroimmune diseases, but therapeutic interference should be strictly receptor selective. Using genetic engineering we have designed soluble human TNFR2-selective agonists. TNFR2 selectivity has been ensured by introducing known TNFR discriminating mutations in the TNF molecule (D143N/A145R; [12]). We used the TNFR2-selective mutant in the single-chain TNF format (scTNFR2), consisting of three TNF monomers connected by short peptide linkers [13,14]. Since TNFR2 is only fully activated by membrane-bound TNF (memTNF) but not by soluble TNF trimers [15], the trimerization domain of tenascin C (TNC) was fused to the N-terminus of the TNFR2-selective scTNF. This molecule (TNC-scTNFR2) exists in a trimeric assembly of the single stranded fusion protein thus resembling a nonameric TNF molecule, which by its increased avidity mimics memTNF activity. In pilot studies with a prototype of TNC-scTNFR2 it has been verified that this molecule format allows activation of human TNFR2 without the requirement of secondary cross-linking of TNFR2 [16]. In the current work, we describe a fully human TNC-scTNFR2 in which peptide linker sequences were reduced and overall nucleic acid sequence was optimized for improved expression and function. After verifying selective human TNFR2 activation we evaluated the neuroprotective effect of TNC-scTNFR2 on LUHMES cells, an established neuronal precursor cell line that PLoS ONE | www.plosone.org 1 November 2011 | Volume 6 | Issue 11 | e27621