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

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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.

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Fig. 3. NGF enhances hypusine modiﬁcation of eIF5A. Primed PC12 cells were treated with NGF and labeled with 1,8-[3H]spermidine⅐HCl. (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).

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Fig. 5. Inhibition of hypusine modification attenuates NGF-induced neurite % of outgrowth. (A) Primed PC12 cells were treated with GC-7 at concentrations of * ␮ 3 ⅐ 0 0 0.1, 1, or 10 M, respectively and labeled with 1,8-[ H]spermidine HCl. For- Cont NGF NGF NGF NGF NGF Cont NGF NGF NGF NGF NGF mation of [3H]hypusine was monitored by ion exchange chromatography. (B) BEC DMFO GC-7 DHS RNAi BEC DMFO GC-7 DHS RNAi Fluorogram for detecting [3H]hypusinated eIF5A (Right) and Coomassie blue Fig. 6. Hypusine modification is required for NGF-dependent survival of staining for total protein (Left). (C) PC12 cells transfected with a control or a primed PC12 cells. (A) Primed PC12 cells were cultured as described above for DHS RNAi plasmid were labeled with 1,8-[3H]spermidine⅐HCl. [3H]hypusine 24 h, followed by fixation and DAPI staining. Arrows indicate the condensed was monitored by ion-exchange chromatography. (D) Fluorogram of 3H- nuclei in dying cells. (B) Quantification of cells 24 h after plating (control, 12 Ϯ hypusinated eIF5A (Right) and Coomassie blue staining for total proteins 1.3; NGF, 59.5 Ϯ 4.7; NGF plus BEC, 25.5 Ϯ 2.4; NGF plus DMFO, 26 Ϯ 2.6; NGF (Left). (E) Primed PC12 cells were replated in serum-free medium or in medium plus GC-7, 20.8 Ϯ 1.3; NGF plus DHS RNAi, 33.3 Ϯ 2.8). Data are presented as with NGF (50 ng/ml), NGF plus 1 ␮M GC-7, or NGF plus DHS RNAi for 24 h, mean Ϯ SEM from four experiments. , P Ͻ 0.01. (C) Trypan blue staining for followed by fixation and imaging. (F) Quantification of neurite outgrowth * dying cells. Percentages of Trypan blue-positive cells are 50 Ϯ 4.3 for control, (the number of cells with long processes in control, 5.3 Ϯ 0.7; NGF, 40.8 Ϯ 2.4; 4.5 Ϯ 0.3 for NGF, 27.3 Ϯ 2.6 for NGF plus BEC, 28.3 Ϯ 2.2 for NGF plus DMFO, NGF plus GC-7, 27.3 Ϯ 2.7; NGF plus DHS RNAi, 15.3 Ϯ 1.1 vs. the number of cells 30.8 Ϯ 3.8 for NGF plus GC-7, and 22.3 Ϯ 4.1 for NGF plus DHS RNAi. Data are with short processes in control, 34.5 Ϯ 2.1; NGF, 4.3 Ϯ 0.6; NGF plus GC-7, presented as mean Ϯ SEM from four experiments. , P Ͻ 0.01. 13.5 Ϯ 1.3; NGF plus DHS RNAi, 24.8 Ϯ 2.5). Data are presented as mean Ϯ SEM * from four experiments. *, P Ͻ 0.01. RNA interference (RNAi) (Fig. 5 C and D), which reduces the 4). In PC12 cells treated with NGF, the arginase inhibitor BEC number of cells with long processes by half and more than quin- significantly reduces the number of neurites with long processes and tuples the number of cells with short processes (Fig. 5 E and F). triples cells with short processes. Similarly, the ODC inhibitor We explored whether hypusinated eIF5A mediates the ability of DMFO decreases the number of long processes and quintuples the NGF to enhance neuronal survival by monitoring the influence of BEC, DMFO, GC-7, and DHS depletion by RNAi (Fig. 6). All four number of cells with short processes. treatments reduce total cell number by 50% (Fig. 6B) and increase To determine whether hypusinated eIF5A mediates the influ- the number of dying, Trypan blue-positive cells by 5- to 6-fold ences of polyamines on neurite outgrowth, we prevented eIF5A (Fig. 6C). formation in two ways. First, we treated PC12 cells with the potent We wondered whether the increased number of dying cells DHS inhibitor N-guanyl-1,7-diaminoheptane (GC-7) (IC50,17nM; ␮ elicited by BEC, DMFO, and GC-7 reflects nonspecific toxicity. All refs. 32 and 33). GC-7 (1 M) inhibits hypusine synthesis in PC12 of the above experiments used primed PC12 cells. Accordingly, we cells (Fig. 5 A and B). The inhibition of hypusine synthesis by GC-7 examined the effects of these agents on PC12 cells that were not is cell-density dependent so that with lower cell densities, GC-7 primed but had also been treated with NGF with similar augmen- ␮ Ϸ more potently inhibits hypusine formation (1 M, 60% inhibition; tation of neurite extension and cell survival as in the primed cells. 10 ␮M, 90% inhibition) (data not shown). In NGF-treated PC12 These preparations are much less sensitive to effects of the drugs cells, GC7 substantially reduces the number of cells with long on number of dying cells than the primed cells, indicating that drug processes and triples the number with short processes (Fig. 5 E and actions in primed cells do not reflect nonspecific toxicity (data not F). These effects do not reflect any general effect on polyamine shown). The inhibition of neurite extension and increase of cell synthesis, because GC-7 does not alter polyamine pools in Chinese death caused by depletion of DHS with RNAi also cannot be hamster ovary cells (32) or in PC12 cells (data not shown). We also attributed to nonspecific toxicity, because a scrambled RNAi depleted hypusine synthesis and hypusinated eIF5A with DHS construct fails to elicit these effects.

4196 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611609104 Huang et al. Downloaded by guest on September 23, 2021 A Control B 40 No or Short Long

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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 ␮M 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 ϩ 2.4 and GC-7, 13.5 ϩ 1.5 vs. with short processes in control, 9.5 Ϯ 1.7 and GC-7, 29 Ϯ 2.8). Data are presented as mean Ϯ SEM from four experiments. *, P Ͻ 0.01.

We assessed the importance of hypusinated eIF5A for neuronal axotomized sympathetic neurons (43). Zigmond and associates (44) disposition in the brain by treating primary hippocampal neuronal showed that spermidine enhances regrowth of neurites in these NEUROSCIENCE cultures with GC-7 (1 ␮M) for 48 h and monitoring neurite length preparations. Treatment with polyamines also accelerates regen- (Fig. 7 A and B). GC-7 treatment reduces the number of neurons eration of damaged sympathetic nerves in rat superior cervical with the longest neurites by Ͼ50% and augments 2- to 3-fold those ganglia (45). In our experiments, inhibitors of polyamine biosyn- with the shortest neurites 2- to 3-fold (Fig. 7C). The cultures include thesis and hypusine formation were substantially more effective in pyramidal cells, granule cells, and interneurons, all of which are preventing influences of NGF on neurite outgrowth and survival in affected similarly by GC-7. primed than in unprimed PC12 cells. Thus, polyamines and hypusinated eIF5A may be particularly important for tissue restoration Discussion after damage. Such actions may involve a wide range of tissues. For The principal findings of our study are that NGF-dependent instance, Filbin and associates (27) recently showed that BDNF neurite outgrowth and survival of PC12 cells as well as mam- stimulates axonal growth of dorsal root ganglion (DRG) neurons malian brain neurons are critically dependent on hypusinated through induction of arginase I with biosynthesis of polyamines and eIF5A. Inhibition of polyamine formation by agents that block conceivably hypusinated eIF5A mediating these efforts. arginase and ODC prevents neurite outgrowth and reduces cell How might eIF5A impact tissue growth and survival? eIF5A viability. More importantly, highly selective inhibition of hy- associates with translational machinery components (5, 6) and pusine formation by the DHS inhibitor GC-7 and depletion of regulates the stability of RNA transcripts (13, 15), which may DHS by RNAi both block neurite outgrowth and markedly mediate cell growth and differentiation (46). Additionally, eIF5A reduce cell survival. regulates p53 and p53-dependent apoptosis (9). The importance of The polyamines spermidine and spermine and their precursor p53 for survival of many types of cells including neurons suggests putrescine have been studied extensively for many years as potential that this pathway may mediate the neuronal alterations we have regulators of nucleic acid disposition, protein formation, and tissue observed. Which of these proposed actions of eIF5A is critical for growth and regeneration (19, 20). Because of their marked positive the tissue restorative influences is unclear. Regardless of the exact charges, polyamines bind nucleic acids and other negatively charged molecular mechanism, our findings indicate a requirement of macromolecules. They have been implicated in gene transcription, hypusinated eIF5A for the growth and survival of neurons and mRNA translation (34), synaptic plasticity (35, 36), and tumori- presumably many other cell types. Agents selectively influencing its genesis (20, 22). In neuronal systems, polyamines regulate gluta- disposition may have therapeutic relevance in diverse conditions mate receptors (35). Conceivably, incorporation of spermidine into with altered cell growth and survival. hypusine alters its availability for regulation of glutamate receptors, which might affect neurotransmission and/or neuronal growth. Materials and Methods Despite abundant investigation of these multiple mechanisms, Cell Cultures. The preparation for primary hippocampal neurons is none have been definitively linked to the regulation by polyamines as described (47). Briefly, primary neurons were prepared from rat of tissue growth and cellular survival. Our study provides substan- embryonic day 18–19 brains. Neurons were plated in 35-mm Petri tial evidence that the transformation of spermidine to hypusine in dishes with glass coverslips, which were coated with poly-D-lysine the formation of eIF5A mediates the effects of polyamines on (40 ␮g/ml), at a density of Ϸ2–4 ϫ 105 per well in neurobasal neuronal process extension and survival. Other influences of poly- medium with B27 supplement (Invitrogen, Carlsbad, CA). PC12 amines on tissue growth may also involve hypusinated eIF5A. Thus, cells (ATCC, Manassas, VA) were maintained in DMEM with 10% deletion of eIF5A or DHS in yeast reduces cell growth and is lethal heat-inactivated horse serum, 10% FBS, and penicillin– (37–40). Moreover in C. elegans (41) and Drosophila (42), deletion streptomycin (100 units/ml and 100 ␮g/ml, respectively) (Invitro- of eIF5A, DHS, or DOHH is lethal. gen). The primed PC12 cells were prepared as described (43). The PC12 cells used in this study were primed with NGF, then Briefly, PC12 cells were plated on collagen IV (Roche, Indianap- stripped off the culture dish and replated, a procedure that models olis, IN)-coated 10-cm dishes and treated with 50 ng/ml NGF

Huang et al. PNAS ͉ March 6, 2007 ͉ vol. 104 ͉ no. 10 ͉ 4197 Downloaded by guest on September 23, 2021 (NGF-7S; Sigma, St. Louis, MO) for 5 days at 37°C in a humidified sociated from the dishes by trypsin treatment and counted. In the atmosphere with 5% CO2. Half of the medium was replaced with DHS RNAi experiment, Ϸ70–90% of primed PC12 cells were the same fresh medium every 2 days with a constant NGF con- GFP-positive cells before plating. After a 24 h treatment, GFP- centration. The NGF-primed cells were washed four times with positive cells were counted by using a fluorescent microscope. regular PC12 culture medium and dissociated from the culture Among the cells that remained attached to the dishes after shaking, dishes with forceful trituration in culture medium. Cells were spun 60% were GFP-positive, whereas 96% of cells that floated in ϫ down at 500 g for 5 min and resuspended in fresh medium medium were GFP-positive. This indicates that the Trypan blue- containing 10% DMSO and stored in liquid nitrogen. An aliquot of positive cells are largely transfected with DHS RNAi, whereas NGF-primed cells was washed once in serum-free DMEM, resus- untransfected cells contribute little, if any, to the Trypan blue- pended in serum-free medium, and plated on collagen IV-coated positive staining. six-well plates or 35-mm dishes with coverslips (MatTek Corp., Ashland, MA). NGF (50 ng/ml) (Sigma), 25 ␮M BEC (Calbiochem, Arginase Activity Assay. Primed PC12 cells were plated onto six-well San Diego, CA), 2 mM DMFO (Sigma), and 1 ␮M GC-7 were used in all experiments. plates in serum-free DMEM (Invitrogen) or in medium with 50 ng/ml NGF and then treated with 25 ␮M BEC, 2 mM DMFO, or ␮ DHS RNAi Construct. Double-stranded oligos containing RNAi se- 1 M GC-7 for 14 h. Before the arginase activity assay, PC12 cells quence derived from rat DHS gene (GCCCAUAAGAACCA- cultured in six-well plates were gently rinsed twice in 2 ml of washing CAUAC) were subcloned into pSUPER neo ϩ GFP vector buffer (124 mM NaCl/3 mM KCl/1.25 mM Na2HPO4/1.6 mM (OligoEngine, Seattle, WA) linearized with HindIII and BglII. The CaCl2/1.8 mM MgSO4/10 mM D-glucose/10 mM Hepes, pH 7.4) expression cassette for short hairpin RNA is driven by the H1 and then incubated for 45 min at 37°C in 0.5 ml of washing buffer promoter, and EGFP expression cassette is under control of a CMV with 5 ␮M unlabeled L-arginine plus 0.25 ␮M L-[guanido-14C]- promoter. PC12 cells were transfected with plasmids by using arginine (5 ␮Ci/ml; NEN, Boston, MA). After incubation, 1.5 ml of Lipofectamine 2000 (Invitrogen). ice-cold methanol was directly added into wells. The plates were incubated on ice for 30 min with gentle shaking. Samples were Immunoblots. Cells were lysed in 1ϫ sample buffer (Invitrogen) transferred to Eppendorf tubes. Cell debris was removed by a brief with 5% 2-mercaptoethanol, EDTA-free protease inhibitors spin. The supernatants were collected and concentrated by speed (Roche), 1.5 mM Na3VO4, and 10 mM NaF. The primary GAPDH vacuum. Then, 10 ␮l of the concentrated sample was spotted onto (Sigma) and arginase I (BD Bioscience, San Jose, CA) antibodies TLC plates (Merck, Darmstadt, Germany), dried for 30 min at were diluted in blocking buffer (3% BSA/0.05% Tween 20 in PBS). room temperature, and developed in a solvent system composed of The blots were developed by using supersignal west pico chemilu- chloroform/methanol/ammonium hydroxide/water in a ratio of minescent substrate (Pierce, Rockford, IL). 0.5/4.5/2.0/1.0 (vol/vol). 14C-labeled urea was used as a standard. TLC plates were dried and exposed to a film. Urea spots were cut Quantification of Neurite Growth. Primed PC12 cells were resus- from TLC plates and analyzed by scintillation counting. pended in serum-free DMEM and plated onto collagen-coated 35-mm dishes with coverslips at the bottom at a low-density of 1 ϫ ODC Activity Assay. After treatment, PC12 cells cultured in six-well 105 per well. Cells were treated with 50 ng/ml NGF alone or plus other agents (25 ␮M BEC, 2 mM DMFO, or 1 ␮M GC-7, plates were gently rinsed twice in 2 ml of washing buffer (124 mM respectively). Approximately 14–18 h later, cells were fixed in 4% NaCl/3 mM KCl/1.25 mM Na2HPO4/1.6 mM CaCl2/1.8 mM paraformaldehyde in PBS at room temperature. Neuronal pro- MgSO4/10 mM D-glucose/10 mM Hepes, pH 7.4). One milliliter of ␮ ␮ cesses were visualized by expression of GFP with a plasmid (pSU- washing buffer with 5 M unlabeled L-arginine plus 0.25 M PER) transiently transfected into PC12 cells during priming. In the L-14C(U)-arginine (5 ␮Ci/ml; NEN) was added into each well. DHS RNAi experiments, the RNAi plasmid was transiently trans- Whatman 3 MM paper (Ϸ0.5 ϫ 0.5 cm in size) was soaked with 100 fected into PC12 cells 24 h before harvesting the primed cells. ␮l of 1 M KOH and put in an Eppendorf tube cap, and then Individual images were acquired with a Meta 510 confocal micro- carefully placed into six-well plates. Each well was sealed with a scope. PC12 cells were divided into two groups: one group of cells rubber stopper. Forty-five minutes after incubation, the 3 MM having processes longer than the diameter of their cell body and the paper was removed from the plates and immediately placed into a other group of cells having shorter or no processes. Approximately vial for scintillation counting. 200 cells were counted for each experiment. Primary hippocampal neurons in vitro day two were treated with 1 ␮M GC-7 for 2–3 days. Measurement of Hypusine by Ion-Exchange Chromatography and Neurons were then fixed in 4% paraformaldehyde in PBS at room Detection of Hypusinated eIF5A by Fluorogram. 3H-hypusine was temperature. Neuronal processes were visualized by phalloidin- monitored as described (31). Briefly, primed PC12 cells were plated FITC staining for F-actin. on 6- to 10-cm dishes in serum-free DMEM for 2 h. NGF (50 ng/ml), GC-7 (0.1, 1, and 10 ␮M, respectively), and 1,8- Trypan Blue Staining and Cell Counting. PC12 cells primed as de- [3H]spermidine⅐HCl (30–40 ␮Ci) (20–36 Ci/mmol; PerkinElmer scribed above were resuspended in serum-free DMEM and then Life Sciences, Boston, MA) were applied afterward. After incuba- plated onto collagen-coated 35-mm dishes at a low density of 1 ϫ tion at 37°C for 14–18 h, PC12 cells were scraped and pelleted by 105 per well. Cells were incubated either with 50 ng/ml NGF alone a brief spin. Protein was precipitated with 10% trichloroacetic acid or plus other agents (25 ␮M BEC, 2 mM DMFO, or 1 ␮M GC-7, respectively). Twenty-four hours after treatment, cells were gently plus 1 mM polyamines and washed in the same buffer 3–4 times. rinsed once with PBS and detached from the dish by trypsin The precipitated proteins were hydrolyzed in 6 M HCl at 110°C for treatment. Cells were pelleted by a brief spin, resuspended in 0.5 ml 18 h. Samples were dried down by a speed vacuum and resuspended ␮ 3 of culture medium, and manually counted by using a hemocytom- in 100 l of water. The content of [ H]hypusine was determined eter. Dying cells were counted by Trypan blue staining as follows. after ion-exchange chromatographic separation. For DHS RNAi Culture dishes were sealed with parafilm and placed in a shaker at experiments, PC12 cells were transfected with a control or a DHS 37°C at 30 rpm for 30 min. Detached cells were collected by a brief RNAi plasmid for 24 h and then labeled with [3H]spermidine (60 spin, and the total number of cells and Trypan blue-positive cells ␮Ci) in serum-free medium with 50 ng/ml NGF for 18 h. The were counted. Because 99% of cells still attached to the dishes after hypusinated eIF5A was also monitored by a fluorogram. Briefly, shaking were viable (Trypan blue-negative), these cells were dis- proteins from trichloroacetic acid precipitation were separated by

4198 ͉ www.pnas.org͞cgi͞doi͞10.1073͞pnas.0611609104 Huang et al. Downloaded by guest on September 23, 2021 SDS/PAGE. Gels were treated with sodium salicylate (1 M) for 30 This work was supported by U.S. Public Health Service Grant MH 18501 min, dried down, and exposed to a film at Ϫ70°C for 4 days. and Research Scientist Award DA-00074 (to S.H.S.).

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