(A) BDNF induces the nuclear translocation of RelA. DIV 7 cortical neurons were transiently transfected with a plasmid encoding RelA-GFP. Fourty hours later, neurons were treated with BDNF (100 ng/ml) alone, or together with K252a (50 nM) for 1 hour, and examined under a fluorescent microscope for mobilization of RelA-GFP from the cytoplasm to the nucleus. (B) BDNF induces NF-κB activity in a TrkB-dependent manner. DIV 7 cortical neurons were transfected with an NF-κB luciferase reporter plasmid and were treated with BDNF (100ng/ml), or BDNF plus K252a (50 nM), for 8 hours. The results shown are mean (of fold induction ± SEM) of three independent observations. # equals p<0.05 as compared to BDNF-treated cells.

(A) Expression of TrkB and ARMS expression is developmentally regulated. Whole cell lysates of primary cortical neurons were prepared from cultures incubated for various days in vitro (DIV) and immunoblot analyses were performed as indicated. (B) The extent of NF-κB activation induced by BDNF correlates with the expression of TrkB and ARMS. TransAM NF-κB DNA binding ELISAs were performed using nuclear extracts of primary cortical neurons treated with BDNF on different DIV. The data (expressed as relative light units) is the mean of three independent observations (bars indicate SEM). ### equals p<0.001. (C) Specificity of NF-κB ELISA. Nuclear extracts derived from neuronal cultures (DIV 9) treated with BDNF (100 ng/ml) were used for TransAM ELISA in the presence of 20× excess of oligonucleotides containing either WT or Mutant NF-κB consensus sequences. These assays demonstrate that the measured activity is indeed specific to NF-κB. ### equals p<0.001 as compared to ‘None’.

(A) Overexpression of ARMS augments BDNF-induced activation of NF-κB. DIV 7 cortical neurons were co-transfected with NF-κB luciferase reporter plasmid and either empty vector or increasing amounts of full-length ARMS plasmid. The recipient cells were then treated with BDNF (100ng/mL) for 8 hours and luciferase activity was measured. Data is presented as the mean (fold induction) of 3 independent observations, the bars represents SEM. # denotes p<0.05 as compared to untreated cells (NT). (B) Similar luciferase assays were performed to measure Oct-1 activity in cortical neurons. The results (Mean of fold induction ± SEM of 3 experiments) show that the over-expression of ARMS has no effect on Oct-1 activity. (C) Overexpression of ARMS does not influence TNFα mediated activation of NF-κB. NF-κB-dependent luciferase assays were performed in which HEK293 cells were treated with TNFα (20ng/mL) for 8 hours. Results revealed similar levels of NF-κB activity in untreated as well as TNFα-treated cells. (D) Expression of TrkB and ARMS is sufficient for BDNF to induce NF-κB activity. HEK293 cells were co-transfected with NF-κB luciferase reporter plasmid and TrkB alone, or TrkB plus full-length ARMS. After this, the recipient cells were treated as indicated. # denotes p<0.05 derived from three sets of experiments, shown as Mean of fold induction ± SEM. (E) Interaction between TrkB and ARMS is required for BDNF to induce NF-κB. Neuronal cells were co-transfected with an NF-κB luciferase reporter and indicated plasmids. ARMS-TM4 encodes transmembrane region (amino acids 2041 to 2673) of ARMS. Luciferase activity is presented as a mean of fold induction ± SEM. ## equals p<0.01.

(A) Generation of ARMS shRNA-GFP expressing lentivirus. The ARMS shRNA cassette was subcloned into a plasmid (pBluescript) downstream of the human U6 RNA polymerase III promoter. The plasmid region containing the cassette and the promoter was then subcloned into the FG12 lentiviral vector, which also encodes for GFP under the control of Ubiquitin C (UbiC) promoter. (B) Analysis of the efficiency of lentiviral transduction of primary cortical neurons. Primary cortical neurons were transduced with ARMS shRNA-GFP lentivirus (MOI 10) as outlined in Materials and Methods. Panel on the left-hand indicates GFP-positive cells, whereas the right-hand panel shows Hoechst 33342 staining of the same microscopic field (C) Analysis of ARMS expression following lentivirus infection. Cell lysates were prepared from infected cortical neurons and immunoblot analyses were performed using antibodies specific to ARMS and α-tubulin. ARMS expression was reduced by approximately 80%. (D) Functional validation of ARMS shRNA-GFP expressing lentivirus. Primary cortical neurons were infected with indicated lentiviruses for 7 days. On DIV9, 10μM of FM1-43 was added to the medium for 15 minutes. After washing, the cells were resuspended in HBSS plus 100mM KCl for depolarization. The release of FM1-43 was monitored and recorded over 60 minutes at 1 minute intervals. As previously reported (Cortes et al., 2007), synaptic activity was increased in the absence of ARMS.

(A) Depletion of ARMS reduces basal as well as BDNF-stimulated transcriptional activity of NF-κB. Primary cortical neurons were transfected with an NF-κB luciferase reporter plasmid plus either empty vector (vector) or plasmid expressing ARMS shRNA (ARMS shRNA). 40 hours post-transfection, the cells were treated with BDNF (100ng/mL) for 8 hours. Data shows that the loss of ARMS results in a reduction of NF-κB driven gene expression. (B) Analogous experiments were performed in HEK293 cells that were treated with TNFα (20ng/mL). Expression of ARMS shRNA had no effect on NF-κB elicited by TNFα. (C) Primary cortical neurons were either mock infected of infected with ARMS shRNA-GFP lentivirus. After 7 days, cells were exposed to either nothing, BDNF (100ng/mL), or TNFα (20ng/mL) for 2 hours. EMSA was then performed to analyze DNA binding activity of NF-κB and Oct-1(control transcription factor). Both BDNF-, and TNFα-treatment led to increased binding of NF-κB complexes (RelA:RelA and RelA:p50 are indicated) in mock infected cells. Cells expressing ARMS shRNA-GFP exhibited increased activity of NF-κB only in response to TNFα, but not BDNF. In all the cases, activity of Oct-1 remained unaltered. (D) Quantitative analysis of the DNA binding activity of NF-κB after expression of ARMS shRNA. A TransAM NF-κB DNA binding ELISA was performed using nuclear extracts of GFP and ARMS shRNA-GFP infected primary cortical neurons. BDNF was unable to stimulate DNA binding activity in cells expressing the ARMS shRNA. (E) The loss of ARMS does not effect DNA binding activity of NF-YA in a TransAM NF-YA DNA binding ELISA. The results presented here are derived from two (C), three (B and D), and four (A) sets of experiments. Data represents Mean ± SEM of fold induction as a function of the luciferase activity in untreated (and vector-transfected) cells; ### shows P<0.01 (A) and P<0.05 (D), as compared to BDNF-treated control groups. Vehicle-treated cells are shown as ‘NT’.

(A) ARMS regulates MAPK activation. Following lentiviral infection, neurons were treated with BDNF (100 ng/ml) and whole cell lysates were prepared at indicated times (NT denotes non-treated cells). These lysates were then analyzed for the phosphorylation status of ERK1/2 and AKT by using specific antibodies. Increased immunoreactivity to phospho ERK1/2-specific antibodies, but not to that of AKT, was observed in control cells (GFP-expressing cells) that were treated with BDNF. BDNF failed to elicit such an effect in ARMS-depleted cells (ARMS shRNA-GFP expressing cells). (B) MAPK is involved in the activation of NF-κB by BDNF. NF-κB luciferase assays were performed in which neurons were treated with BDNF (100ng/mL), and MAPK blockers, U0126 (10μM) or PD98059 (25μM), for 8 hours. Results (Mean ± SEM) are representative of three independent observations. ### equals p<0.001 as compared to BDNF-treated groups. (C) ARMS interferes with IKK activation. ARMS-depleted, and control neurons were treated with BDNF (100ng/mL) for 30 minutes and IKK complexes were purified using IKKα-specific antibodies. The immune-complexes were then incubated with GST-IκBα and γ [³²P] ATP for 30 min. at room temperature. The incorporation of ³²P in GST-IκBα was then measured by SDS-PAGE and autoradiography. Strong phosphorylation of GST-IκBα in response to BDNF was observed in mock-, and GFP-infected samples, but not in those infected with ARMS shRNA-GFP lentivirus. (D) Role of IKK in BDNF-mediated activation of NF-κB. Luciferase assays were performed in DIV 7 neurons following transfection with NF-κB luciferase reporter and IKKα expressing plasmid (WT, wild type; KM, dominant negative mutant). BDNF-induced luciferase transcription was completely blocked in IKKα KM-expressing cells, indicating a key role for IKK in BDNF-elicited NF-κB signaling. The data (Mean ± SEM) is derived from three parallel experiments. ## denotes (p<0.01) statistically significant inhibition of NF-κB activity by IKKα KM. (E) ARMS mediates phosphorylation of RelA at IKK-sensitive site. Immunoblot analysis was carried out by using lysates of infected neurons and phospho (Ser536)-specific antibodies to RelA. As shown, both BDNF (100ng/mL) and TNFα (20ng/mL) exerted enhanced phosphorylation of RelA at serine 536 in mock, as well as GFP infected cells. This effect was observed only in TNFα-exposed ARMS-depleted cells.

(A) Neuroprotective effect of BDNF is mediated by ARMS. Primary cortical neurons infected with ARMS shRNA-GFP or GFP lentivirus were treated with BDNF (100 ng/ml) for 18 hours, followed by incubation of the cells with gp120 (5nM) for an additional 72h. Staining of the cultures with Hoechst 33342 dye revealed a higher percentage of apoptotic nuclei in gp120-exposed cells (middle panels). Whereas, co-administration of BDNF inhibited gp120-induced apoptosis in control cells (cells expressing GFP alone, upper right panel) but not in ARMS shRNA-GFP expressing cells (lower right panel). Apoptotic cells are marked with arrows, whereas arrow-heads show healthy cells. (B) Quantitative analysis of data from three independent experiments that examined survival of neurons as outlined above. The data are presented as Mean ± SEM of percent apoptotic neurons. ## indicates P<0.01 as compared to gp120-treated control cells (GFP, filled bar). (C) Loss of ARMS does not affect BDNF-induced internalization of TrkB. Surface proteins of cells infected with ARMS shRNA-GFP or GFP expressing lentivirus were labeled by EZ Link Sulfo NHS-SS-biotin. TrkB internalization was then initiated by incubation with BDNF (100 ng/ml) for 30 minutes at 37°C. Glutathione cleavage buffer removed the remaining biotin on the cell surface. The biotinylated, internalized TrkB was precipitated by streptavidin followed by immunoblot using a TrkB antibody. Both full-length (145 kD) and truncated (95 kD) TrkB receptors are shown. Total surface biotinylated TrkB (Total biotin) was measured in cells held on ice; the efficiency of biotin cleavage is demonstrated in (Cleaved); and spontaneous TrkB internalization induced by BDNF is shown (4C). Cells expressing either GFP lentivirus or ARMS shRNA-GFP expressing lentivirus exhibit similar levels of basal and BDNF-induced TrkB internalization. (D) Receptor internalization is required for BDNF-induced NF-κB transcriptional activity. DIV7 primary cortical neurons were transfected with NF-κB luciferase plasmid for 40 hours. Cells were then stimulated with BDNF (100ng/mL) alone, BDNF plus the internalization inhibitor, MDC (25μM), or vehicle alone for 8 hours. ‘NT’ represents non-treated cells. Analysis of luciferase gene expression (presented as a fold induction) shows that inhibition of receptor internalization blocks NF-κB activity induced by BDNF. The bars represents SEMs and the data is representative of three separate experiments. # indicates statistical significance p<0.05 as compared to BDNF-treated cells.