Figure 7. TNC-scTNFR2-mediated neuroregeneration is depen- dent on PI3K-PKB/Akt signaling. (A) LUHMES cells were stimulated with LY294002 (25 mM) for either 24 hours or for 1 hour. If stimulated for 1 hour, medium was exchanged to differentiation medium and cells were cultivated for further 23 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values 6 SEM). (B,C) LUHMES cells were stimulated with hydrogen peroxide (H2O2; 100 mM) with or without LY294002 (25 mM). After one hour the cells were washed with medium und regenerated in medium with or without TNC-scTNFR2 (100 ng/ml). (B) Cells were incubated for 24 hours and cell viability was measured using the MTT assay (n = 3, shown are the mean values 6 SEM). (C) Cells were regenerated for one hour, fixed with 4% PFA and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). At least 5 different image sections containing a minimum of 250 cells were used to determine the number of total and TUNEL-positive cells (n = 3, shown are the mean values 6 SEM). **p values less than 0.001 were considered to be significant. doi:10.1371/journal.pone.0027621.g007 selectivity in order to avoid dose limiting severe TNFR1-related inflammatory responses. Fusion of the trimerization domain of tenascin C to a TNFR2-selective scTNF was shown in a previous study to exert memTNF-like activity [16]. We generated a fully human, optimized version of this fusion protein suitable to be further developed into a therapeutic. The HPLC-SEC and SDS-PAGE data indicate that for scTNFR2 the majority of the molecules were present in monomeric Neuroregeneration by a TNFR2 Agonist form, i.e. resembling a soluble trimeric TNF, whereas the TNC- scTNFR2 assembled into functional multimers, most likely trimers, thus resembling a nonameric TNF molecule. The introduced double mutation D143N/A145R leads to a complete loss of binding to TNFR1 [12]. Therefore, as demonstrated in an apoptosis assay with a cell line selectively expressing TNFR1 (MEF TNFR1-Fas), TNC-scTNFR2 did not activate TNFR1. In contrast, TNC-scTNFR2 efficiently activated TNFR2, indicating that the oligomeric TNF mutein is able to mimic memTNF. As a consequence, TNC-scTNFR2 efficiently induced nuclear translo- cation of NFkB p65 in Kym-1 cells within 30 minutes, whereas stimulation with scTNFR2 only resulted in a marginal transloca- tion after 30 minutes and a partial and delayed response at 60 minutes in Kym-1 cells. In these cells, this weak and late signal seemed to be strong enough to induce endogenous expression and secretion of TNF, which in an autocrine manner activated TNFR1 and subsequently the apoptotic signaling-cascade leading to Kym- 1 cell death (Fig. 2C), as shown previously [22,29]. Receptors of the TNF family are activated by ligand-mediated oligomerization [30] and efficient signal initiation requires the formation of larger ligand/receptor complexes [21,31]. In previous studies, the initiation of cluster formation of TNFR2 was found to be dependent on exogenous cross-linking of the ligand-receptor complexes and correlated with the particular signaling strength [21]. Using the recently introduced huTNFR2- expressing cell line R2 MEF [23], the superior bioactivity of the multimeric TNC-scTNFR2 was further evident from rapid formation of TNFR2 signaling complexes, which can be readily visualized by fluorescence microscopy of TRAF2 recruitment. Summarizing, the soluble TNC-scTNFR2 ligand mimics memTNF-like activation of TNFR2 and can be used to selectively induce TNFR2 signaling in vitro and in vivo. Parkinson’s disease is a neurological disorder, which is characterized by the loss of dopamine-producing neurons in the substantia nigra causing reduced dopamine levels in the striatum [32]. This disease [33], like a variety of other neurodegenerative diseases, including Alzheimer’s disease [34], amyotrophic lateral sclerosis [33] and conditions such as ischemia and excitotoxicity [35], has been associated with mitochondrial dysfunction and oxidative damage mediated by excessive production and exposure of cells to reactive oxygen species (ROS) resulting in neuronal death [36]. Oxidative stress can be simulated in vitro by brief exposure of cultured neurons to H2O2, resulting in the induction of cell death via the mitochondrial apoptosis pathway [37,38]. In addition, neurotoxins, such as 1-methyl-4-phenyl-1,2,3,6-tetrahy- dropyridine (MPTP) or 6-OHDA mimic many of the hallmark characteristics of PD and are widely used as an oxidative stress model for PD [39,40]. Preconditioning cells via TNFR2 signaling protected primary cortical neurons from excitotoxic cell death in an NFkB- dependent manner [7,8]. In contrast, we activated TNFR2 by TNC-scTNFR2 subsequent to the toxic stimulus. Interestingly, under these conditions, simulating a therapeutic intervention, a strongly increased survival was noted, too, indicating that TNFR2 signaling can interfere with an already activated cell death program and promote cell survival. Several pathways activated by TNFR2 mediate protective or regenerative responses. In particular, PKB/Akt-dependent activa- tion of NFkB induces expression of anti-apoptotic and/or neurotrophic factors [7,41]. In addition, PKB/Akt signaling directly exerts anti-apoptotic effects [24] and is involved in mediating cell protection in neurodegenerative diseases [25,26,27]. Since TNFR2 activation by TNC-scTNFR2 subsequent to the toxic exposure to H2O2 resulted in a fast protective response in a PLoS ONE | www.plosone.org 7 November 2011 | Volume 6 | Issue 11 | e27621