Neuroregeneration by a TNFR2 Agonist Figure 5. TNC-scTNFR2 induces formation of TNFR2 signaling complexes. (A,B) R2 MEF were transfected with pTRAF2-eGFP. After 24 hours, cells were incubated with or without 80M2 (1 mg/ml) for 5 minutes on ice and subsequently incubated with scTNFR2 or TNC-scTNFR2 (10 ng/ml) for 10 minutes at 37uC. Then cells were fixed with 4% PFA, permeabilized with 0.1% Triton X-100 and localization of huTNFR2 (A) or huTNF (B) was detected with specific antibodies and Alexa-Fluor546-labeled secondary antibodies. Cell nuclei were visualized using DAPI. Pictures are optical sections obtained by confocal fluorescence microscopy (bar = 10 mm). White arrows indicate areas of colocalization. doi:10.1371/journal.pone.0027621.g005 decrease in the number of cells was noted, which was further enhanced after four hours. In contrast, no significant loss of cells was observed at two and four hours in the samples treated with TNC-scTNFR2 one hour after H2O2 exposure. The rapid H2O2- induced cell death is at least partially due to apoptosis induction, as revealed from TUNEL-staining of exposed cells (Fig. 6E). A reduced amount of TUNEL-positive cells after regeneration in presence of TNC-scTNFR2 further indicates that TNFR2 signaling rapidly interferes with the H2O2-induced apoptotic signal pathways. TNC-scTNFR2-mediated protection from superoxide induced cell death is dependent on PI3K-PKB/Akt signaling PKB/Akt exerts anti-apoptotic effects [24] and mediates cell protection in neurodegenerative diseases [25,26,27]. Furthermore, preconditioning via TNFR2-signaling spared primary neurons from glutamate-induced excitotoxicity in vitro in a PI3K-PKB/Akt- dependent manner [7,8]. In accordance with these data, we could show that the protective effect mediated by TNC-scTNFR2 is dependent on PKB/Akt activity. First, whereas transient blocking of PI3K for one hour with the specific inhibitor LY294002 did not influence neuronal viability, persistent blocking of PI3K activity resulted in a dramatic decrease in cell viability after 24 hours of otherwise untreated neuronal cells (Fig. 7A), verifying the general importance of PI3K-PKB/Akt signaling for neuronal cell survival [18]. Second, upon oxidative stress already a transient inhibition of PI3K drastically affected protection by TNFR2 stimulation, as evident from reduced cell viability after 24 hours (Fig. 7B) and the elevated number of TUNEL-positive cells one hour post TNC-scTNFR2 treatment (Fig. 7C). This further demonstrates that activation of the PI3K-PKB/Akt pathway via TNFR2 results in anti-apoptotic signaling, thereby preventing the cell death induced by H2O2. TNC-scTNFR2 rescues neurons from catecholaminergic cell death LUHMES cells differentiate into neurons with a dopaminergic phenotype [18]. The neurotoxin 6-hydroxydopamine (6-OHDA) PLoS ONE | www.plosone.org 5 November 2011 | Volume 6 | Issue 11 | e27621