tat-derived cpps  (GenScript corporation)

Journal: EMBO Molecular Medicine

Article Title: Prevention of excitotoxicity‐induced processing of BDNF receptor TrkB‐FL leads to stroke neuroprotection

doi: 10.15252/emmm.201809950

Figure Lengend Snippet: A Effect of TFL 457 on MEF2‐promoter activity. Cultures transfected with pMEF2 (two minimal wild‐type MEF2 elements) or pMEF2mut (mutant) were preincubated with CPPs (25 μM, 30 min) and treated with NMDA (100 μM, 2 h). Means ± SEM of luciferase activities obtained in excitotoxicity, relative to values found in cells treated with same peptide and no NMDA, are presented. Significance was analyzed by Student's t ‐test (* P = 0.039; n .s. = non‐significant, P = 0.79; n = 8). B Effect on CRE‐promoter activity. pCRE contains two minimal CREs. Peptide preincubation was as above with or without KG‐501 (10 μM). Mean ± SEM luciferase activities is given relative to values in cells treated with the same peptide but no NMDA or KG‐501. Differences found in excitotoxicity were analyzed by ANOVA test followed by post hoc Tukey's HSD test (* P = 0.023; ** P = 0.003; n.s. = non‐significant, P = 0.20; n = 6). C Effect on NMDAR‐subunit promoters. Cells transfected with pGluN1 or pGluN2A were treated and analyzed as in panel (A). Significance was analyzed by Student's t ‐test (pGluN1, * P = 0.016; pGluN2A, * P = 0.029; n = 14). D Effects on BDNF promoter III or TrkB promoter. Cells transfected with pIIIBDNF ( n = 9), pTrkB ( n = 7), or pCRE ( n = 5), as a control, were processed as above but using 20 μM NMDA for 4 h. Data are presented and analyzed as in panel (A). Significance was analyzed by Student's t ‐test (pCRE, ** P = 0.0098; pIIIBDNF, * P = 0.029; pTrkB, * P = 0.033). E Effects of TFL 457 on mRNA levels of CREB/MEF2‐regulated genes. Total RNA was extracted from cultures preincubated with CPPs (25 μM, 30 min) and treated or not with NMDA (100 μM, 4 h). Levels of mRNA were normalized to NSE (genes expressed in neurons, left panel) or GAPDH (neuronal and glial expression, right panel). Means ± SEM of levels obtained in excitotoxicity relative to values found in cells treated with same peptide and no NMDA are presented. Differences found in excitotoxicity were analyzed by ANOVA test followed by post hoc Tukey's HSD test (GluN1, *** P = 0.00096; GluN2A, P = 0.98; TrkB‐FL, *** P = 0.00037; BDNF, * P = 0.045; n = 8).

Article Snippet: When indicated, primary cultures were preincubated for 30 min with Tat‐derived CPPs (> 95% purity; GenScript; 25 μM, unless otherwise indicated) or BDNF (100 ng/ml) before NMDA treatment.

Techniques: Activity Assay, Transfection, Mutagenesis, Luciferase, Control, Expressing

Journal: EMBO Molecular Medicine

Article Title: Prevention of excitotoxicity‐induced processing of BDNF receptor TrkB‐FL leads to stroke neuroprotection

doi: 10.15252/emmm.201809950

Figure Lengend Snippet: A TFL 457 interference of RIP and calpain processing in excitotoxicity. Cultures were preincubated with peptides (25 μM, 30 min) or left untreated before NMDA addition (2 h). Different exposures are shown to facilitate visualization of FL, f42/39, or f32. B Quantitation of normalized f32 and f42/39 levels. Means ± SEM are shown relative to cultures treated with NMDA and no peptide. Analysis was performed by unpaired Student's t ‐test (* P = 0.014, ** P = 0.00198; n = 6). C Effect of TFL 457 on neuronal viability after calpain inhibition. After 30 min preincubation with calpeptin (Calp, 10 μM) and calpain inhibitor III (CiIII, 10 μM), cultures were treated with CPPs and NMDA (4 h) as before. Means ± SEM relative to those obtained in untreated cultures are given. We performed ANOVA test followed by post hoc Tukey's HSD test (* P = 0.035, ** P = 0.0058, *** P = 0.00009; n = 12). D Effect of TFL 457 on neuronal viability after metalloproteinase inhibition. Cultures preincubated with GM6001 (10 μM, 30 min) were treated with CPPs and NMDA as before. Analysis was performed as above (without GM6001, *** P = 0.0007; with GM6001, *** P = 0.0006; n = 4). E Effect of NMDA on total and cell‐surface TrkB‐FL levels. Cultures were incubated with NMDA (100 μM) or BDNF (100 ng/ml) for 1 h. Membrane proteins were labeled and purified and compared to corresponding total lysates. TrkB‐FL levels are expressed relative to untreated cells. Mean ± SEM is presented and analyzed by Student's t ‐test (* P = 0.027; n.s. = non‐significant, P = 0.1; n = 7). F Effect of TFL 457 on the decrease in TrkB‐FL surface levels induced by NMDA. Analysis was performed as before in cells incubated with CPPs (25 μM, 30 min) and NMDA‐treated (1 h). Results obtained in excitotoxic cultures are expressed relative to cells treated with the same peptide but no NMDA. Data are analyzed as in panel (E). Mean ± SEM is presented and analyzed by Student's t ‐test (* P = 0.019; n.s. = non‐significant, P = 0.44; n = 4). G Experimental design to study the effect of endocytosis inhibition on TrkB‐FL downregulation in ischemia. Mice were i.p. injected with vehicle or dynasore followed by Rose Bengal. Vessel occlusion and brain damage were induced by cold‐light irradiation. Animals were sacrificed early after damage initiation. H Effect of endocytosis inhibition on TrkB‐FL downregulation. The infarcted area of the ipsilateral hemisphere (I) was compared to the corresponding region of the contralateral area (C). Results from representative mice injected with dynasore or vehicle are shown. Different exposures are presented to facilitate visualization of dynasore effects on TrkB‐FL and f32. I Quantitation of TrkB‐FL and TrkB‐T1 in the infarcted area. Normalized protein levels are expressed relative to those of the corresponding contralateral region. Mean ± SEM is presented and analyzed by Student's t ‐test (* P = 0.04; n.s. = non‐significant, P = 0.14; n = 9). Source data are available online for this figure.

Article Snippet: When indicated, primary cultures were preincubated for 30 min with Tat‐derived CPPs (> 95% purity; GenScript; 25 μM, unless otherwise indicated) or BDNF (100 ng/ml) before NMDA treatment.

Techniques: Quantitation Assay, Inhibition, Incubation, Membrane, Labeling, Purification, Injection, Irradiation