Neuronal growth and survival mediated by eIF5A, a polyamine-modified translation initiation factor

Abstract

Eukaryotic translation initiation factor 5A (eIF5A), the only known protein containing the polyamine-derived amino acid hypusine, modulates protein synthesis. We show that neurotrophic and neuroprotective actions of nerve growth factor (NGF) are mediated by hypusinated eIF5A, which can account for the known roles of polyamines in cell growth and survival. NGF treatment of PC12 cells stimulates eIF5A formation. Moreover, prevention of hypusine formation by a selective inhibitor of deoxyhypusine synthase and by its depletion with RNA interference blocks the NGF-elicited augmentation of neurite outgrowth and cell survival of PC12 cells. In brain cultures, inhibition of hypusine formation also inhibits neuronal process extension.

Fig. 1.

Pathway of polyamine biosynthesis and hypusine modification in eIF5A. Arginase catalyzes the production of ornithine, which is subsequently converted into putrescine, spermidine, and spermine. Hypusine modification involves two sequential steps. DHS transfers the 4-aminobutyl moiety from spermidine to the ε-amino group of one specific lysine residue (Lys-50 in the human protein) in eIF5A generating eIF5A intermediate, which is then hydroxylated by deoxyhypusine hydroxylase to form the mature hypusinated eIF5A.

Fig. 2.

NGF stimulates arginine metabolism in primed PC12 cells. (A) Induction of arginase I expression in primed PC12 cells after NGF treatment. Western blot of lysates from PC12 cells treated with NGF (50 ng/ml) for 14 h is shown. (B) Quantification of arginase I expression (control, 1.15 ± 0.02; NGF, 1.92 ± 0.08). Data are presented as mean ± SEM from three experiments. ∗, P < 0.01. (C) Arginase activity. Primed PC12 cells were treated with NGF (50 ng/ml) for 14 h followed by addition of arginine labeled with ¹⁴C at the guanido group and incubated for 1 h. Radiolabeled urea was separated by TLC and quantified by scintillation counting (control, 2,061 ± 112; NGF, 6,976 ± 226; NGF plus BEC, 257 ± 29 in dpm). Results are presented as mean ± SEM from three independent experiments. ∗, P < 0.01. (D) ODC. Primed PC12 cells were treated with NGF (50 ng/ml) for 14 h followed by addition of arginine labeled with ¹⁴C at the carboxyl group and incubated for 1 h. CO₂ was captured by 1 M KOH and measured by scintillation counting (control, 164 ± 7.5; NGF, 397 ± 33; NGF plus BEC, 110 ± 9.4; NGF ± DMFO, 94 ± 10 in dpm). Results are presented as mean ± SEM from three independent experiments. ∗, P < 0.01.

Fig. 3.

NGF enhances hypusine modification of eIF5A. Primed PC12 cells were treated with NGF and labeled with 1,8-[³H]spermidine·HCl. (A) Formation of ³H-labeled hypusine was monitored by ion-exchange chromatography. (B) ³H-labeled hypusinated eIF5A was detected by a fluorogram (Right), and total proteins were revealed by Coomassie blue staining of an SDS/PAGE (Left).

Fig. 4.

Inhibition of arginase or ODC attenuates NGF-induced neurite outgrowth in primed PC12 cells. (A) Primed PC12 cells, which express GFP, were plated in serum-free medium or in medium with NGF (50 ng/ml), NGF plus 25 μM BEC, or NGF plus 2 mM DMFO as indicated. Cells were fixed 14 h after incubation and visualized with fluorescent microscopy. (B) Quantification of neurite outgrowth. Cells were divided into two groups with long vs. short processes as described in the experimental procedure. Approximately 40 cells were counted in each experiment (the number of cells with long processes in control, 4 ± 1.5; NGF, 37 ± 1; NGF plus BEC, 26.3 ± 1.7; NGF plus DMFO, 23.5 ± 1.6 vs. the number of cells with short processes in control, 36 ± 1.5; NGF, 3.8 ± 0.4; NGF plus BEC, 12.3 ± 1.3; and NGF plus DMFO, 17.3 ± 1.6). Data are presented as mean ± SEM from four experiments. ∗, P < 0.01.

Fig. 5.

Inhibition of hypusine modification attenuates NGF-induced neurite outgrowth. (A) Primed PC12 cells were treated with GC-7 at concentrations of 0.1, 1, or 10 μM, respectively and labeled with 1,8-[³H]spermidine·HCl. Formation of [³H]hypusine was monitored by ion exchange chromatography. (B) Fluorogram for detecting [³H]hypusinated eIF5A (Right) and Coomassie blue staining for total protein (Left). (C) PC12 cells transfected with a control or a DHS RNAi plasmid were labeled with 1,8-[³H]spermidine·HCl. [³H]hypusine was monitored by ion-exchange chromatography. (D) Fluorogram of ³H-hypusinated eIF5A (Right) and Coomassie blue staining for total proteins (Left). (E) Primed PC12 cells were replated in serum-free medium or in medium with NGF (50 ng/ml), NGF plus 1 μM GC-7, or NGF plus DHS RNAi for 24 h, followed by fixation and imaging. (F) Quantification of neurite outgrowth (the number of cells with long processes in control, 5.3 ± 0.7; NGF, 40.8 ± 2.4; NGF plus GC-7, 27.3 ± 2.7; NGF plus DHS RNAi, 15.3 ± 1.1 vs. the number of cells with short processes in control, 34.5 ± 2.1; NGF, 4.3 ± 0.6; NGF plus GC-7, 13.5 ± 1.3; NGF plus DHS RNAi, 24.8 ± 2.5). Data are presented as mean ± SEM from four experiments. ∗, P < 0.01.

Fig. 6.

Hypusine modification is required for NGF-dependent survival of primed PC12 cells. (A) Primed PC12 cells were cultured as described above for 24 h, followed by fixation and DAPI staining. Arrows indicate the condensed nuclei in dying cells. (B) Quantification of cells 24 h after plating (control, 12 ± 1.3; NGF, 59.5 ± 4.7; NGF plus BEC, 25.5 ± 2.4; NGF plus DMFO, 26 ± 2.6; NGF plus GC-7, 20.8 ± 1.3; NGF plus DHS RNAi, 33.3 ± 2.8). Data are presented as mean ± SEM from four experiments. ∗, P < 0.01. (C) Trypan blue staining for dying cells. Percentages of Trypan blue-positive cells are 50 ± 4.3 for control, 4.5 ± 0.3 for NGF, 27.3 ± 2.6 for NGF plus BEC, 28.3 ± 2.2 for NGF plus DMFO, 30.8 ± 3.8 for NGF plus GC-7, and 22.3 ± 4.1 for NGF plus DHS RNAi. Data are presented as mean ± SEM from four experiments. ∗, P < 0.01.

Fig. 7.

Inhibition of DHS attenuates neurite outgrowth of primary hippocampal neurons. (A) Primary hippocampal cultures at in vitro day 3 were treated with 1 μM GC-7, fixed 2 days after treatment, and visualized by F-actin staining. (B) Quantification of neurite outgrowth (the number of cells with long processes in control, 33.5 + 2.4 and GC-7, 13.5 + 1.5 vs. with short processes in control, 9.5 ± 1.7 and GC-7, 29 ± 2.8). Data are presented as mean ± SEM from four experiments. ∗, P < 0.01.