Neuroregeneration by a TNFR2 Agonist Figure 6. TNC-scTNFR2 induces neuroregeneration after H2O2-induced oxidative stress. (A) Differentiated LUHMES cells were incubated with different concentrations of hydrogen peroxide (H2O2) for one hour. Then cells were washed with medium und cultivated for additional 24 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations 6 SEM). (B–E) LUHMES cells were stimulated with H2O2 (100 mM). After one hour the cells were washed with medium und regenerated for the indicated time intervals in medium with or without TNC-scTNFR2 (100 ng/ml). (B) LUHMES cells were regenerated 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 or three hours, fixed with 4%PFA, permeabilized with 0.1% Triton-X100 and b- III-tubulin was detected with specific antibodies. Cell nuclei were visualized using DAPI. Pictures are projections of eight optical sections (0.4 mm; bar = 50 mm). (D) Number of cells was determined by counting the nuclei (DAPI staining). (E) Cells were regenerated for one hour and apoptotic cells were identified by terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-FITC nick end labeling (TUNEL). (D,E) At least 10 different image sections containing a minimum of 500 cells were used to determine the number of total and TUNEL-positive cells. *p values less than 0.05 (** p-value less than 0.001) were considered to be significant (n = 2, shown are the mean values 6 SD). doi:10.1371/journal.pone.0027621.g006 is widely used as an oxidative stress in vitro model, which has relevance for PD. 6-OHDA generates free radicals and selectively induces cell death in catecholamine-containing neurons [28]. LUHMES cells were sensitive towards 6-OHDA-induced cell death with an EC50 of approximately 5 mM (Fig. 8A). Treatment of LUHMES cells with TNC-scTNFR2 one or two hours after exposure to a toxic concentration of 6-OHDA, counteracted the cell death pathway such that more than 50% of the cells survived the treatment, compared to 20% survival without TNC-scTNFR2 (Fig. 8B), further demonstrating the protective role of TNFR2 activation in oxidative stress-induced cell death. Discussion Signaling via TNFR2 has been implicated in protection and regeneration of tissues, such as pancreas, liver, heart and brain [6]. Accordingly, TNF variants selectively activating TNFR2 could be a useful therapeutic regimen in a variety of diseases, including heart failure, autoimmune and neurodegenerative diseases. Towards this aim, here we describe a human TNFR2-selective TNF ligand with memTNF-mimetic activity (TNC-scTNFR2) that rescues differentiated neurons from cell death post insultem, i.e. under conditions that reflect the time span of a potential therapeutic intervention, for example to limit ischemic lesions after stroke. Soluble TNF is a strong mediator of inflammation, predomi- nantly through TNFR1, which is efficiently activated by the membrane-bound as well as the soluble form of TNF in the picomolar range [15]. Whereas TNFR2 binds soluble TNF as well as memTNF, it is only fully activated by the latter [15]. Therefore, potential TNFR2-specific therapeutics have to comply with two basic requirements: mimicry of memTNF function and receptor PLoS ONE | www.plosone.org 6 November 2011 | Volume 6 | Issue 11 | e27621