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Neuronal Growth and Survival Mediated by Eif5a, a Polyamine-Modified Translation Initiation Factor

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Neuronal Growth and Survival Mediated by Eif5a, a Polyamine-Modified Translation Initiation Factor Neuronal growth and survival mediated by eIF5A, a polyamine-modified translation initiation factor Yunfei Huang*, Daniel S. Higginson*, Lynda Hester*, Myung Hee Park†, and Solomon H. Snyder*‡§¶ *The Solomon H. Snyder Department of Neuroscience, ‡Department of Pharmacology and Molecular Sciences, and §Department of Psychiatry and Behavioral Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; and †Oral and Pharyngeal Cancer Branch, National Institute of Dental and Craniofacial Research, National Institutes of Health, Bethesda, MD 20892-4340 Contributed by Solomon H. Snyder, December 27, 2006 (sent for review December 7, 2006) Lys 50 Eukaryotic translation initiation factor 5A (eIF5A), the only known HN protein containing the polyamine-derived amino acid hypusine, Arginine eIF5A -(CH2 )4 -NH2 H2 N NH2 modulates protein synthesis. We show that neurotrophic and Arginase Precursor Spermidine neuroprotective actions of nerve growth factor (NGF) are mediated by hypusinated eIF5A, which can account for the known roles of Deoxyhypusine Ornithine Synthase Ornithine polyamines in cell growth and survival. NGF treatment of PC12 cells (DHS) Decarboxylase Lys 50 stimulates eIF5A formation. Moreover, prevention of hypusine H formation by a selective inhibitor of deoxyhypusine synthase and Putrescine eIF5A -(CH2 )4 -N NH2 Deoxyhypusinated by its depletion with RNA interference blocks the NGF-elicited Spermidine augmentation of neurite outgrowth and cell survival of PC12 cells. Synthase Deoxyhypusine In brain cultures, inhibition of hypusine formation also inhibits Spermidine Hydroxylase neuronal process extension. (DOHH) Spermine Lys 50 Synthase H OH ͉ ͉ ͉ ͉ hypusine spermidine nerve growth factor arginase eIF5A -(CH2 )4 -N NH2 Spermine deoxyhypusine synthase Hypusinated Fig. 1. Pathway of polyamine biosynthesis and hypusine modification in eIF5A. n all eukaryotic organisms, protein translation is regulated by a Arginase catalyzes the production of ornithine, which is subsequently converted Ivariety of translation initiation factors, which in the nervous into putrescine, spermidine, and spermine. Hypusine modification involves two system are responsible for neuronal survival and neurite extension sequential steps. DHS transfers the 4-aminobutyl moiety from spermidine to the (1, 2). One of these, eukaryotic translation initiation factor 5A ␧-amino group of one specific lysine residue (Lys-50 in the human protein) in (eIF5A), is the only known cellular protein to contain the unique eIF5A generating eIF5A intermediate, which is then hydroxylated by deoxyhy- polyamine-derived amino acid hypusine (3) (Fig. 1). The name pusine hydroxylase to form the mature hypusinated eIF5A. hypusine reflects the composition of this amino acid, a combination of hydroxyputrescine and lysine (3). Hypusine is formed from spermidine by the sequential action of two enzymes. Deoxyhy- Results pusine synthase (DHS) transfers the 4-aminobutyl group of sper- Arginase-I is induced by neurotrophic stimuli in dorsal root ganglia midine to the epsilon-amino group of a specific lysine in eIF5A with neurons (27) and superior cervical ganglion (28). In PC12 cells the resultant deoxyhypusine intermediate then hydroxylated by stimulated with nerve growth factor (NGF) after serum starvation, deoxyhypusine hydroxylase (DOHH) (3, 4). eIF5A, a 17-kDa acidic we observe a 2-fold increase in arginase-I protein (Fig. 2 A and B). protein highly conserved throughout eukaryotes, associates with The increased protein levels of arginase-I are associated with a translation machinery (5, 6) and enhances methionyl-puromycin substantial augmentation of arginine transformation to its products synthesis in a model assay for translation initiation (7), and its with a 3-fold increase in urea generation (Fig. 2C). Additionally, the 14 deletion in yeast is lethal (8). eIF5A is implicated in the regulation generation of CO2 from carboxyl-labeled [ C]arginine is increased of p53 expression and thereby p53-dependent apoptosis through 2- to 3-fold after NGF treatment (Fig. 2D). This increase is interactions with syntenin, independent of its influences on protein abolished after treatment with the arginase inhibitor S-(2- translation (9). Other proposed functions of eIF5A include serving boronoethyl)-L-cysteine (BEC) or the ODC inhibitor difluoro- as a cofactor of HIV-1 REV (10, 11) to regulate nuclear export (12) methylornithine (DMFO) (Fig. 2D), consistent with findings that and RNA turnover (13–15) as well as maintaining cell wall integrity NGF induces expression of ODC in superior cervical ganglion (29) and actin polarity (16, 17). and PC12 cells (30). Arginine, a dietary precursor of polyamines, is converted by To monitor hypusinated eIF5A, we used ion exchange arginase to ornithine, which is subsequently transformed to pu- chromatography to analyze the formation of 3H-labeled hy- trescine, spermidine, and spermine (18–20) (Fig. 1). Polyamine pusine (31). Treatment of PC12 cells with NGF elicits a 2- to biosynthesis and ornithine decarboxylase (ODC) are dramatically 3-fold augmentation in hypusine (Fig. 3A) and 3H-labeled augmented in numerous forms of rapid tissue growth and many eIF5A (Fig. 3B), consistent with the increase in polyamine tumors (21, 22). ODC inhibitors and other agents interfering with formation. Gel Coomassie staining does not display any major polyamine synthesis prevent rapid tissue growth (20, 22). Arginine is also the direct precursor of nitric oxide (NO), as NO synthase Author contributions: Y.H., M.H.P., and S.H.S. designed research; Y.H. and M.H.P. per- (NOS) abstracts NO from the guanidino group of arginine giving formed research; Y.H., D.S.H., L.H., and M.H.P. contributed new reagents/analytic tools; rise to citrulline as a by-product (23). Both NO and polyamines have Y.H. and M.H.P. analyzed data; and Y.H. and S.H.S. wrote the paper. been implicated in neuronal growth and survival (24, 25), and The authors declare no conflict of interest. differential movement of arginine into the polyamine and NO Abbreviations: eIF5A, eukaryotic initiation factor 5A; DHS, deoxyhypusine synthase; NGF, pathways influences neuronal survival (26). In the present study, we nerve growth factor; GC-7, N-guanyl-1,7-diaminoheptane; BEC, S-(2-boronoethyl)- provide evidence that hypusinated eIF5A physiologically regulates L-cysteine; DMFO, difluoromethylornithine; ODC, ornithine decarboxylase. nerve process extension and neuronal survival both in PC12 cells ¶To whom correspondence should be addressed. E-mail: [email protected]. and brain neurons. © 2007 by The National Academy of Sciences of the USA 4194–4199 ͉ PNAS ͉ March 6, 2007 ͉ vol. 104 ͉ no. 10 www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611609104 Downloaded by guest on September 23, 2021 8000 A Cont NGF C * A Control NGF 6000 Arginase I 4000 (DPM) 2000 Arginase activity Arginase GAPDH NGF + BEC NGF + DMFO 0 Cont NGF NGF BEC B D 500 2.5 * * 400 2.0 B 50 2 No or Short 300 1.5 40 * Long 200 30 * 1.0 * (Fold increase) Arginase I protein Arginase * 20 100 * 0.5 Production of CO (DPM) Number of Cells 10 0 0 Cont NGF NGF NGF Cont NGF 0 BEC DMFO Cont NGF NGF NGF Fig. 2. NGF stimulates arginine metabolism in primed PC12 cells. (A) Induction BEC DMFO of arginase I expression in primed PC12 cells after NGF treatment. Western blot NEUROSCIENCE of lysates from PC12 cells treated with NGF (50 ng/ml) for 14 h is shown. (B) Fig. 4. Inhibition of arginase or ODC attenuates NGF-induced neurite out- Quantification of arginase I expression (control, 1.15 Ϯ 0.02; NGF, 1.92 Ϯ 0.08). growth in primed PC12 cells. (A) Primed PC12 cells, which express GFP, were Data are presented as mean Ϯ SEM from three experiments. *, P Ͻ 0.01. (C) plated in serum-free medium or in medium with NGF (50 ng/ml), NGF plus 25 Arginase activity. Primed PC12 cells were treated with NGF (50 ng/ml) for 14 h ␮M BEC, or NGF plus 2 mM DMFO as indicated. Cells were fixed 14 h after followed by addition of arginine labeled with 14C at the guanido group and incubation and visualized with fluorescent microscopy. (B) Quantification of incubated for 1 h. Radiolabeled urea was separated by TLC and quantified by neurite outgrowth. Cells were divided into two groups with long vs. short scintillation counting (control, 2,061 Ϯ112; NGF, 6,976 Ϯ226; NGF plus BEC, 257 Ϯ processes as described in the experimental procedure. Approximately 40 cells 29 in dpm). Results are presented as mean Ϯ SEM from three independent were counted in each experiment (the number of cells with long processes in experiments. *, P Ͻ 0.01. (D) ODC. Primed PC12 cells were treated with NGF (50 control, 4 Ϯ 1.5; NGF, 37 Ϯ 1; NGF plus BEC, 26.3 Ϯ 1.7; NGF plus DMFO, 23.5 Ϯ ng/ml) for 14 h followed by addition of arginine labeled with 14C at the carboxyl 1.6 vs. the number of cells with short processes in control, 36 Ϯ 1.5; NGF, 3.8 Ϯ group and incubated for 1 h. CO2 was captured by 1 M KOH and measured by 0.4; NGF plus BEC, 12.3 Ϯ 1.3; and NGF plus DMFO, 17.3 Ϯ 1.6). Data are scintillation counting (control, 164 Ϯ 7.5; NGF, 397 Ϯ 33; NGF plus BEC, 110 Ϯ 9.4; presented as mean Ϯ SEM from four experiments. *, P Ͻ 0.01. NGF Ϯ DMFO, 94 Ϯ 10 in dpm). Results are presented as mean Ϯ SEM from three independent experiments. *, P Ͻ 0.01. We wondered whether the increase in hypusine synthesis and change in overall protein level, indicating that increased hypusinated eIF5A formation mediates the neurotrophic and pro- hypusine generation reflects augmented formation of hypusi- survival actions of NGF. First, we examined whether neurite nated eIF5A (Fig. 3B). outgrowth is influenced by inhibition of polyamine formation (Fig. A 1400 B Cont NGF Cont NGF 1200 1000 800 Control 600 NGF 400 3 [ H] Hypusine(DPM) 3 200 [ H] eIF5A 0 12345678910111213 Fractions Coomassie Fluorogram Fig.
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