Neuroregeneration by a TNFR2 Agonist Figure 8. TNC-scTNFR2 protects neurons against catecholaminergic cell death. (A) Differentiated LUHMES cells were incubated with different concentrations of 6-hydroxydopamine (6-OHDA) for 20 hours. Cell viability was measured using the MTT assay (n = 3; shown are the mean values of triplicate determinations 6 SEM). (B) LUHMES cells were stimulated with 6-OHDA (32 mM). Cells were stimulated with TNC-scTNFR2 (100 ng/ ml) one, two or four hours after 6-OHDA addition and incubated for a total time period of 20 hours. Cell viability was measured using the MTT assay (n = 3, shown are the mean values 6 SEM). *p values less than 0.05 (** p-value less than 0.001) were considered to be significant. doi:10.1371/journal.pone.0027621.g008 PKB/Akt-dependent manner, we assume that the protective effect is not dependent on de novo gene expression. Indeed, PKB/Akt has been shown to interfere with early apoptotic signals [42] and thus could mediate protection by directly interacting with components involved in the apoptotic signal pathway. The molecular mechanisms of PKB/Akt-mediated survival from H2O2-induced toxicity in the neuronal cells studied here are not known yet. However, several components of the apoptotic machinery share the PKB/Akt consensus phosphorylation se- quence (RXRXXS/T), e.g. pro-death components such as Bad, caspase-9 and caspase-8 or pro-survival factors like Bcl-2, cIAP and xIAP [42]. PKB/Akt therefore could regulate apoptosis by either inhibiting cell death or promoting cell survival. In particular, phosphorylation of Bad by PKB/Akt induces the dissociation of Bad from Bcl-XL, which then can promote cell survival. In addition, PKB/Akt was reported to directly block cell death after mitochondrial cytochrome C release, e.g. by phosphorylating caspase-9 at serine 196 [43] thereby inactivating the caspase. PKB/Akt therefore could potentially also interfere with the apoptotic pathway even at this rather late stage. The mechanisms of the TNFR2-mediated protection from 6- OHDA-induced cell death are not characterized yet. However, Jorda´n et al. [44] present evidence that Bcl-XL seems to prevent mitochondrial multiple conductance channel opening, cytochrome c release and caspase-3 like activity following 6-OHDA treatment. Therefore similar mechanisms could contribute to the protection of TNC-scTNFR2 against 6-OHDA-induced cell death as described for protection against H2O2-induced cell death, e.g activation of Bcl-XL through phosphorylation of Bad. In conclusion, we constructed a human TNFR2-selective TNF ligand possessing memTNF-mimetic activity and show that this molecule can rescue differentiated neurons from cell death subsequent to a toxic exposure, i.e. under conditions that reflect the time span of a potential therapeutic intervention. Our data, together with clear evidence that TNFR2 signaling promotes tissue regeneration in several organs [6,10], indicate that selective activation of TNFR2 signaling may be a promising approach to treat various diseases, including autoimmune and neurodegener- ative diseases. Being the first fully human TNFR2 agonist, TNC- scTNFR2 is suitable not only for further studies on the protective and regenerative mechanisms of TNFR2 signaling in different disease models but represents a prototype of a TNFR2-selective therapeutic which is in principle applicable in man as a novel therapeutic approach to promote tissue homeostasis and regener- ation. Materials and Methods Antibodies and chemicals The TNFR2 specific antibody 80M2 has been described [15]. LY294002 and the antibodies against NFkB p65, TRAF2, Akt and phospho Akt (Ser473) were from Cell Signaling Technology (Boston, MA). The antibody anti-6-his was from Biovision (San Francisco, CA), the antibodies against huTNFR2 (HP9003; MR2- 1) were from Hbt (Uden, The Netherlands), the antibody against huTNF (AF-210-NA) was from R&D Systems (Wiesbaden, Germany) and the antibody against b-III-tubulin was purchased from Acris Antibodies (Hiddenhausen, Germany). Secondary antibodies coupled to horseradish peroxidase (HRP) and Alexa- Fluor488 or Alexa-Fluor546 were from Jackson ImmunoResearch (Suffolk, UK) and Invitrogen (Karlsruhe, Germany), respectively. DAPI, MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyl tetrazoli- um bromide), 6-hydroxydopamine (6-OHDA) and H2O2 (hydro- gen peroxide) were from Sigma-Aldrich (Steinheim, Germany). All other chemicals were of analytical grade. Cloning of scTNFR2 and TNC-scTNFR2 The TNFR2-selective scTNFR2 (D143N/A145R) and the human tenascin C (TNC) domain (aa 110–139) were synthesized with optimized eukaryotic codon usage (Geneart, Regensburg, Germany). The sequence of scTNFR2 was subcloned in frame into the expression vector pSecTag A (Invitrogen) containing an IgG secretion signal. Then TNC-scTNFR2 was generated by cloning of the TNC domain sequence to the N-terminal end of the scTNFR2 construct. All vector sequences were verified by sequencing. Cell culture Immortalized mouse fibroblasts generated from TNFR12/2/ TNFR22/2 mice and stably transfected with human TNFR1-Fas (MEF TNFR1-Fas) or TNFR2-Fas (MEF TNFR2-Fas) [21] or with human TNFR2 (R2 MEF) [23] have been described elsewhere. HEK293T cells and mouse fibroblasts were grown in RPMI1640 medium (Invitrogen) supplemented with 5% (v/v) heat-inactivated FCS (PAN Biotech, Aidenbach, Germany) and 100units/ml penicillin and 100 mg/ml streptomycin (Invitrogen). Kym-1 cells were cultivated in RPMI1640 medium supplemented PLoS ONE | www.plosone.org 8 November 2011 | Volume 6 | Issue 11 | e27621