Neuroregeneration by a TNFR2 Agonist Figure 4. TNC-scTNFR2 induces TNFR2 signaling in R2 MEF. (A–C) R2 MEF were stimulated with TNC-scTNFR2 (20 ng/ml) for the indicated times. (A) TNFR2 was immunoprecipitated using MR2-1 antibodies and protein G agarose. The precipitates were analyzed by immunoblot analysis using anti-huTNFR2 (HP9003) and anti-TRAF2 antibodies (NB = non-bound; B = bound). (B) Localization of NFkB p65 was visualized via immunofluorescence microscopy as shown in the upper panel and the number of nuclei showing NFkB translocation was quantified. At least 200 cells per experiment were analyzed (n = 3; shown are the mean values 6 SEM of percent NFkB positive nuclei; bar = 20 mm). (C) Phospho-Akt (Ser473) levels in cell lysates were analyzed using immunoblot analysis. Akt was used as a loading control. Representative blot and bar graph show the quantification of the phospho-Akt (Ser473) band. *p values less than 0.05 versus untreated cells were considered to be significant (n = 3; shown are the mean values 6 SEM). doi:10.1371/journal.pone.0027621.g004 R2 MEF expressing TRAF2-eGFP showed a homogenous cytosolic distribution of the reporter. As expected [23], cross- linking of TNFR2 by 80M2 resulted in a spotted appearance of the TNFR2 signal at the plasma membrane, yet did not induce colocalization with the TRAF2-eGFP signal (Fig. 5A). Similar, stimulation with scTNFR2 alone did not result in a detectable colocalization of the TRAF2-eGFP signal with either huTNFR2 (Fig. 5A) or scTNFR2 itself, stained with antibodies against human TNF (huTNF; Fig. 5B) during the observation period (10 min). In contrast, scTNFR2 in combination with 80M2 induced TNFR2 clustering and TRAF2 recruitment, apparent as a spotted appearance of huTNFR2 (Fig. 5A) or huTNF (Fig. 5B) signal colocalizing with TRAF2-eGFP. This finding indicates TNFR2 signaling complex formation [23]. In contrast to scTNFR2, TNC- scTNFR2 alone was sufficient to rapidly induce TNFR2 clustering and TRAF2 recruitment and thus efficient formation of TNFR2 signaling complexes (Fig. 5A and B). Summarizing, TNC- scTNFR2 showed superior bioactivity compared to scTNFR2 and was able to efficiently activate TNFR2 in distinct cell lines. TNC-scTNFR2 preserves neurons from superoxide-induced cell death TNFR2 signaling has been associated with protection of neurons against cytotoxic insults [7,8,9]. Potential neuroprotective properties of TNC-scTNFR2 were investigated using the human cell line LUHMES, which has been shown to display typical biochemical and morphological features of human dopaminergic neurons [17,18,19,20]. Differentiated LUHMES cells developed long dendrites, formed a neural-like network and expressed typical neuronal markers such as b-III-tubulin, a-synapsin, microtubule- associated protein 2 and neurofilament [18,19] (data not shown). Short hydrogen peroxide (H2O2) exposure was used to induce cell death in vitro, simulating oxidative stress, a process that contributes to the damage in neurodegenerative diseases in vivo. To investigate the sensitivity of LUHMES cells to H2O2-induced cell death, the cells were incubated for one hour with the toxic chemical, then medium was exchanged to remove H2O2 and cells were cultivated for additional 24 hours. LUHMES cells started to die at H2O2 concentrations above 20 mM, with a half maximal effective concentration (EC50) of approximately 75 mM (Fig. 6A). Addition of TNC-scTNFR2 one hour after exposure to a toxic concentration of H2O2, counteracted the H2O2-triggered cell death pathway such that nearly 80% of the cells survived the treatment, compared to 30% survival without TNC-scTNFR2 (Fig. 6B). To investigate the protective effect of TNC-scTNFR2 in detail, LUHMES cells were fixed two or four hours after starting the H2O2-treatment and the b-III-tubulin content (Fig. 6C) as well as cell number (Fig. 6D) were determined by immunofluorescence analyses. Already two hours after H2O2 exposure, a significant PLoS ONE | www.plosone.org 4 November 2011 | Volume 6 | Issue 11 | e27621