Journal: Cell Death & Disease
Article Title: DAP5 increases axonal outgrowth of hippocampal neurons by enhancing the cap-independent translation of DSCR1.4 mRNA
Figure Lengend Snippet: BDNF makes cap-independent translation of DSCR1.4 mRNA more actively by increasing DAP5 expression. a , b BDNF treatment on DIV 3 hippocampal neuron increases protein levels of DAP5 and DSCR1.4 but not DSCR1.4 mRNA level. Vehicle (DDW) or 30 μM BDNF were treated for 1 h. a The protein levels were confirmed by Western blot. GAPDH and phosphorylation of ERK were used as a loading control and marker of BDNF activity, respectively. b Endogenous DSCR1.4 mRNA levels were analyzed by qRT-PCR and were normalized to β-actin. The bars represent the mean ± SEM ( n = 3). c Cap-independent translation is essential for DSCR1.4 protein accumulation by BDNF. DIV 3 hippocampal neurons were treated with vehicle (DMSO), 100 μM RAD001(RAD) or 50 mg/ml cycloheximide (CHX) for 3 h followed by BDNF treatment for 1 h. The levels of each protein were confirmed by Western blot. The numbers at the bottom indicate the fold relative to a vehicle. The amount of DSCR1.4 was normalized to GAPDH. d , e BDNF raises cap-independent translation activity of DSCR1.4 mRNA. At 24 h after d pRF hDSCR1.4 5′UTR or e pRF mDSCR1.4 5′UTR vectors were transfected into DIV 2 hippocampal neurons, Vehicle (DDW) and BDNF were treated to the neurons for 1 h. The bars represent the mean ± SEM ( e ; n = 5, F; n = 3). f BDNF increases the interaction between DAP5 and DSCR1.4 5′UTR. In vitro transcribed biotin-DSCR1.4 5′UTR was incubated with extracts of the vehicle (DDW) or 30 μM BDNF-treated DIV 3 mouse hippocampal neurons. DAP5 binding was measured by Western blot. Phospho-ERK was used to confirm the activity of BDNF. GADPH was used as a loading control and negative control. g , h BDNF increases the cap-independent local translation of DSCR1.4 mRNA in axon as well as soma. EGFP and pRF mDSCR1.4 5′ 3′ UTR vectors were co-transfected into DIV 2 mouse hippocampal neurons. At 24 h later, 100 μM anisomycin was treated for 3 h and then 30 μM BDNF was treated for 1 h, followed by 5 μM puromycin treatment for 40 min. To detect newly synthesized FLUC proteins, Puro-PLA assay was conducted. g Representative image obtained from confocal microscopy. h The graph shows relative fluorescence intensity measured by Image J. The bars represent the mean ± SEM (Vehicle; n = 11, Anisomycin; n = 12, BDNF; n = 11, BDNF + Anisomycin; n = 11). Scale bar, 30 μm. Data information: In d , e , h , * P
Article Snippet: Plasmids and RNA interference Bicistronic pRF DSCR1.4 5′UTR, ΔCMV RF DSCR1.4 5′UTR, and hp pRF DSCR1.4 5′UTR vectors for reporter assay were made by inserting the human DSCR1.4 (Accession no. NM_203418.1) or mouse DSCR1.4 (Accession no. NC_000082.6) 5′UTR. mDSCR1.4 5′UTR and hDSCR1.4 5′UTR were PCR-amplified by cDNA derived from N2A and SHSY5Y cells using primers as follows: hDSCR1.4 5′UTR forward primer 5′-AAGTCGACTGTCTGCCTGCAAGCATGC-3′, reverse primer 5′-GGGCTTGCTTTC TTACAGTGAAAG-3′, mDSCR1.4 5′UTR forward primers 5′- AAGTCGACCGTCTGCCCGAGGGCAT GC-3′, reverse primer 5′-GGGTCTGCTTTTTCACGGGGC-3′ (Macrogen, Seoul, Republic of Korea).
Techniques: Expressing, Western Blot, Marker, Activity Assay, Quantitative RT-PCR, Transfection, In Vitro, Incubation, Binding Assay, Negative Control, Synthesized, Proximity Ligation Assay, Confocal Microscopy, Fluorescence