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• W2004207754 abstract "Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase β2 (CTβ2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988–44994). CTβ2 mRNA is highly expressed in the brain. We show that CTβ2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTβ2 expression in PC12 cells differentiated by nerve growth factor. In CTβ2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTβ2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased, and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CTβ2-deficient cells. CTβ2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTβ2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTβ2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions. Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase β2 (CTβ2), an isoform of a key phosphatidylcholine (PC) biosynthetic enzyme, accompanies neurite outgrowth (Carter, J. M., Waite, K. A., Campenot, R. B., Vance, J. E., and Vance, D. E. (2003) J. Biol. Chem. 278, 44988–44994). CTβ2 mRNA is highly expressed in the brain. We show that CTβ2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTβ2 expression in PC12 cells differentiated by nerve growth factor. In CTβ2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTβ2 facilitates neurite outgrowth and branching. However, the length of individual neurites was significantly increased, and the total amount of neuronal membrane was unchanged. Neurite branching of PC12 cells is known to be inhibited by activation of Akt and promoted by the Akt inhibitor LY294002. Our experiments showed that LY294002 increases neurite sprouting and branching in control PC12 cells but not in CTβ2-deficient cells. CTβ2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTβ2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTβ2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions. In response to nerve growth factor (NGF), 6The abbreviations used are:NGFnerve growth factorCTCTP:phosphocholine cytidylyltransferaseGFPgreen fluorescent proteinHAhemagglutininPCphosphatidylcholinesiRNAsmall interfering RNA rat pheochromocytoma (PC12) cells stop proliferating and differentiate into sympathetic neuron-like cells (1Greene L.A. Tischler A.S. Proc. Natl. Acad. Sci. U. S. A. 1976; 73: 2424-2428Crossref PubMed Scopus (4873) Google Scholar). The morphological hallmark of neuronal differentiation is neurite sprouting and elongation and subsequent maturation of neurites into axons and dendrites. These processes increase the demand for membrane components. Accordingly, the biosynthesis of the predominant membrane phospholipid, phosphatidylcholine (PC), is accelerated during neurite outgrowth in response to NGF (2Araki W. Wurtman R.J. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 11946-11950Crossref PubMed Scopus (75) Google Scholar, 3Carter J.M. Waite K.A. Campenot R.B. Vance J.E. Vance D.E. J. Biol. Chem. 2003; 278: 44988-44994Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). nerve growth factor CTP:phosphocholine cytidylyltransferase green fluorescent protein hemagglutinin phosphatidylcholine small interfering RNA PC is synthesized in neurons by the CDP-choline pathway (4Vance J.E. Pan D. Campenot R.B. Bussiere M. Vance D.E. J. Neurochem. 1994; 62: 329-337Crossref PubMed Scopus (94) Google Scholar), in which the rate-limiting reaction is catalyzed by CTP: phosphocholine cytidylyltransferase (CT) (5Vance D.E. Pelech S. Trends Biochem. Sci. 1984; 9: 17-20Abstract Full Text PDF Scopus (101) Google Scholar). Three CT isoforms, encoded by two genes, have been identified in rodents. CTα is encoded by the Pcyt1a gene, whereas CTβ2 and CTβ3 are derived from the Pcyt1b gene (6Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar, 7Lykidis A. Baburina I. Jackowksi S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). CTα and CTβ2 share considerable sequence identity, but CTα contains a nuclear localization signal (8Wang Y. Sweitzer T.D. Weinhold P.A. Kent C. J. Biol. Chem. 1993; 268: 5899-5904Abstract Full Text PDF PubMed Google Scholar), whereas CTβ2 does not (7Lykidis A. Baburina I. Jackowksi S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). Immunofluorescence studies have found that CTα is primarily located in the nucleus and that CTβ2 resides in the cytosol and on the endoplasmic reticulum (9Ridsdale R. Tseu I. Wang J. Post M. J. Biol. Chem. 2001; 276: 49148-49155Abstract Full Text Full Text PDF PubMed Scopus (63) Google Scholar). The subcellular localization of CTβ3 has not yet been reported. Although CTα is expressed in all tissues, CTβ2 and CTβ3 mRNAs are predominantly expressed in the brain (6Lykidis A. Murti K.G. Jackowski S. J. Biol. Chem. 1998; 273: 14022-14029Abstract Full Text Full Text PDF PubMed Scopus (114) Google Scholar). In neurons, PC is synthesized not only in cell bodies but also in distal axons (4Vance J.E. Pan D. Campenot R.B. Bussiere M. Vance D.E. J. Neurochem. 1994; 62: 329-337Crossref PubMed Scopus (94) Google Scholar, 10Strosznajder J. Radominska-Pyrek A. Horrocks L.A. Biochim. Biophys. Acta. 1979; 574: 48-56Crossref PubMed Scopus (24) Google Scholar, 11Tanaka T. Yamaguchi H. Kishimoto Y. Gould R.M. Biochim. Biophys. Acta. 1987; 922: 85-94Crossref PubMed Scopus (13) Google Scholar, 12Vance J.E. Pan D. Vance D.E. Campenot R.B. J. Cell Biol. 1991; 115: 1061-1068Crossref PubMed Scopus (68) Google Scholar). During neurite outgrowth of PC12 cells and Neuro2a cells, CTβ2 expression and CT activity are increased, whereas CTα expression is unchanged, indicating a link between CTβ2 expression and neurite outgrowth (3Carter J.M. Waite K.A. Campenot R.B. Vance J.E. Vance D.E. J. Biol. Chem. 2003; 278: 44988-44994Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar). Neurites are categorized by their location relative to the cell body. Primary neurites project directly from the cell body. Secondary neurites (i.e. branches) project from primary neurites. The sprouting of primary neurites and branch formation are distinct processes that are independently regulated (13Markus A. Zhong J. Snider W.D. Neuron. 2002; 35: 65-76Abstract Full Text Full Text PDF PubMed Scopus (275) Google Scholar). During neuronal differentiation, NGF activates phosphatidylinositol-3-kinase, which activates Akt (14Jackson T. Blader I. Hammonds-Odie L. Burga C. Cooke F. Hawkins P. Wolf A. Heldman K. Theibert A. J. Cell Sci. 1996; 109: 289-300Crossref PubMed Google Scholar). Neurite extension and branching are promoted in some populations of neurons by activation of Akt, whereas in other neurons activation of Akt impairs neurite elongation and branching (15Namikawa K. Honma M. Abe K. J. Neurosci. 2000; 20: 2875-2886Crossref PubMed Google Scholar). In PC12 cells, Akt activation inhibits neurite branching (16Higuchi M. Onishi K. Masuyama N. Gotoh Y. Genes Cells. 2003; 8: 657-669Crossref PubMed Scopus (43) Google Scholar). On the basis of these findings, we hypothesized that neurons require CTβ2 in axons to provide a localized source of PC for neurite outgrowth, extension, and/or branching. We show that CTβ2 is abundant in PC12 cells and in distal axons of primary cultures of neurons. RNA silencing of CTβ2 in PC12 cells reduced the number of primary neurites and markedly reduced the number of branches but increased the length of individual neurites. Moreover, stimulation of neurite branching in PC12 cells, a process that is normally induced by inhibition of Akt, was abrogated in CTβ2-deficient cells. These data provide important new insights into the role of CTβ2 and PC biosynthesis in neurite sprouting and branch formation. Cell Culture—Rat pheochromocytoma cells (PC12) were obtained from the American Type Cell Culture Collection. Cells were maintained in F12-K medium supplemented with 15% heat-inactivated horse serum and 2.5% fetal bovine serum at 37 °C in a humidified atmosphere containing 5% CO2. For differentiation and transfection experiments, cells were seeded on collagen-coated 35-mm dishes at a density of 2 × 105 cells/dish. The cells were incubated overnight and then treated with medium containing 0.5% horse serum and 50 ng/ml 2.5 S NGF (Alomone Laboratories, Jerusalem, Israel), and cells were grown for up to 5 days. Compartmented Cultures of Rat Sympathetic Neurons and Retinal Ganglion Cells—Sympathetic neurons from the superior cervical ganglia of 1-day-old rats were isolated as previously described (17Campenot R. Dev. Biol. 1982; 93: 1-12Crossref PubMed Scopus (197) Google Scholar). Briefly, following dissection, the ganglia were mechanically and enzymatically dissociated and plated into the center compartment of compartmented culture dishes. Initially, all compartments contained 2.5 S NGF (100 ng/ml). NGF was withdrawn from the center compartment (containing cell bodies and proximal axons) after 7 days. The side compartments (containing distal axons alone) contained 100 ng/ml NGF throughout the experiments. Retinal ganglion cells were isolated from 1-day-old rats and cultured in compartmented culture dishes as previously described (18Hayashi H. Campenot R.B. Vance D.E. Vance J.E. J. Biol. Chem. 2004; 279: 14009-14015Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar). Proximal axons/cell bodies, distal axons of sympathetic neurons, and distal axons of retinal ganglion cells were harvested from 2-week-old cultures (18Hayashi H. Campenot R.B. Vance D.E. Vance J.E. J. Biol. Chem. 2004; 279: 14009-14015Abstract Full Text Full Text PDF PubMed Scopus (148) Google Scholar, 19Posse de Chaves E. Bussiere M. MacInnis B. Vance D.E. Campenot R.B. Vance J.E. J. Biol. Chem. 2001; 276: 36207-36214Abstract Full Text Full Text PDF PubMed Scopus (36) Google Scholar). Antibodies—Anti-M rabbit polyclonal antibodies that recognize CTβ2 were generated against the conserved membrane domain of rat liver CT (amino acids 256–288) and were a generous gift from Dr. R. Cornell (Simon Fraser University, Vancouver, Canada). Anti-human CTβ2 rabbit polyclonal antibodies were a generous gift from Dr. S. Jackowski (St. Jude Children's Research Hospital, Memphis, TN). The CTβ2-specific antibody was raised against a peptide corresponding to amino acids 347–365 of CTβ2 (7Lykidis A. Baburina I. Jackowksi S. J. Biol. Chem. 1999; 274: 26992-27001Abstract Full Text Full Text PDF PubMed Scopus (134) Google Scholar). The mouse anti-tubulin monoclonal antibodies were from Sigma, and the anti-phospho-Akt (Ser-473) and anti-Akt antibodies were from Cell Signaling Technology (Beverly, MA). The Cdk5 (cylcin-dependent kinase 5) antibody was purchased from Chemicon International, Inc. (Temecula, CA). Transfection of PC12 Cells and Measurement of Neurite Length and Branching—PC12 cells were plated at a density of 2 × 105 cells/35-mm dish for 12 h, after which the medium was changed to F12-K medium without serum. Cells were transfected with 2.5 μg of DNA/35-mm dish. For RNA silencing, cells were co-transfected with a 3.7-kb mammalian expression vector encoding recombinant green fluorescent protein (GFP) (phrGFP) (Stratagene, Cedar Creek, TX) and pSILENCER 4.0 encoding rat CTβ2. PC12 cells were transfected with equimolar amounts of phrGFP and either the silencing construct (pSiCTβ2) or pSILENCER 4.0 containing a scrambled, noncoding insert. For CTβ2 overexpression studies, cells were transfected with a cDNA encoding a hemagglutinin (HA)-tagged CTβ2 (pCI[CTβ2-HA]) and phrGFP. The cells were transfected in the presence of Lipofectamine 2000 according to the manufacturer's protocol (Invitrogen). Briefly, pSiCTβ2, pSILENCER 4.0 containing the scrambled insert, or pCI[CTβ2-HA] and phrGFP were added in a 3:1 ratio to Lipofectamine 2000 for 20 min to allow formation of DNA complexes, which were then applied to PC12 cells. After 12 h, the cells were given serum-free F12-K medium containing 50 ng/ml NGF to promote differentiation and neurite outgrowth. After 2, 3, and 4 days, cells were viewed by fluorescence microscopy. Cells that had been co-transfected with phr-GFP and pSiCTβ2 were identified by their fluorescence at 520 nm. Cells bearing at least one neurite longer than 20 pixels were considered to be differentiated, and all neurites and branches were counted and/or measured. At each time point, at least 20 random fields of view were sampled. Immunoprecipitation and Immunoblotting—Two dishes of PC12 cells or six dishes of sympathetic neurons and retinal ganglion cells were rinsed with ice-cold phosphate-buffered saline and harvested into 1 ml of immunoprecipitation lysis buffer containing 50 mm Tris-HCl (pH 7.4), 1% Triton X-100, and a protease inhibitor mixture (Sigma). Cell lysates were centrifuged at 10,000 × g for 15 min at 4 °C, and then supernatants were incubated for 1 h with 3 μl of anti-M antibodies on a rotating shaker at 4 °C. A 50% slurry of Protein A-Sepharose beads (40 μl) was added, and the mixtures were incubated for 1 h at 4 °C. Protein A-Sepharose bead conjugates containing immunoprecipitated CTβ2 were washed three times with buffer containing 50 mm Tris-HCl (pH 7.4) and 0.1% Triton X-100. Proteins were eluted from the Protein A-Sepharose beads using SDS-PAGE sample buffer (62.5 mm Tris-HCl (pH 8.3), 10% (v/v) glycerol, 1% SDS, and 0.04% bromphenol blue) and heated at 70 °C for 3 min. Immunoprecipitated proteins were separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS and then transferred to Immobilon-P transfer membranes (Millipore, Cambridge, Canada). Ponceau S staining was used to compare protein loading in lanes of the gel. The membranes were blocked for 2 h with 5% skimmed milk in Tris-buffered saline (20 mm Tris-HCl (pH 7.5) containing 500 mm NaCl and 0.1% Tween 20) and then incubated overnight with anti-M (membrane domain) (dilution 1:1,000), anti-CTβ2 (dilution 1:1,000), or mouse anti-tubulin (dilution 1:1,000) antibodies. All blots were washed for 1 h and subsequently incubated with anti-rabbit, or anti-mouse, IgG conjugated to horseradish peroxidase (1:2,500 dilution) for 1 h. Immunoreactivity was detected using Westdura or Westfemto ECL reagent (Amersham Biosciences). In Vitro Measurement of CT Activity—PC12 cells from two 35-mm dishes were collected in 1 ml of homogenization buffer containing 50 mm Tris-HCl (pH 7.5), 150 mm NaCl, 2 mm EDTA, 1 mm dithiothreitol, 0.1 mm phenylmethylsulfonyl fluoride, and 100 μg/ml each of leupeptin and aprotinin. The cells were sonicated for 20 s at 4 °C. Cell lysates were centrifuged at 7,000 × g for 5 min to remove nuclei and unbroken cells. Aliquots of the supernatant were used for measurement of CT activity and immunoblot analysis. CT activity was determined in the presence of PC/oleate vesicles by monitoring the conversion of phospho-[3H]choline to CDP-[3H]choline (20Pelech S. Pritchard P. Vance D. J. Biol. Chem. 1981; 256: 8283-8286Abstract Full Text PDF PubMed Google Scholar). Incubation of PC12 Cells with [3H]Choline—PC12 cells were transfected with control plasmid or phrGFP + pSiCTβ2. Six h later, the medium was changed to serum-free medium that contained 50 ng/ml NGF. After 48 h, cells were incubated with [3H]choline (5 μCi/35-mm dish) for 3 h. Cells were harvested, the amount of cellular protein was determined, and radioactivity in PC was measured. In Vivo Phosphorylation of CTβ2—PC12 cells were incubated with 50 ng/ml NGF for 4 days, after which the cells were incubated for 48 h with LY294002 or for 1 h with 25 μm roscovitine or an equivalent volume of vehicle (dimethyl sulfoxide) in the presence of 100 μm [32P]orthophosphate (Amersham Biosciences). Cells were rinsed with ice-cold phosphate-buffered saline and harvested into immunoprecipitation lysis buffer. The cell lysates were then centrifuged at 10,000 × g for 15 min at 4 °C, after which supernatants were incubated with 3 μlof anti-M antibody on a rotating shaker at 4 °C. After 1 h, 40 μlof a 50% slurry of protein A-Sepharose beads were added, and the mixture was incubated for an additional 1 h at 4 °C. Protein A-Sepharose bead conjugates containing immunoprecipitated CTβ2 were washed three times. CTβ2 was eluted from the beads by heating for 3 min in SDS-PAGE sample buffer at 70 °C. Proteins were separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS. The gel was dried overnight and then exposed to a PhosphorImager screen (Eastman Kodak Co.) for up to 5 days. Quantification of [32P]orthophosphate-labeled CT protein was performed with a Bio-Rad PhosphorImager. In Vitro Phosphorylation Assays—PC12 cells were harvested into 1 ml of immunoprecipitation buffer. CTβ2 was immunoprecipitated using anti-M antibody as described above. Tubulin was immunoprecipitated using anti-tubulin antibodies. Cdk5 was immunoprecipitated using 10 μlof anti-Cdk5 antibodies. Following immunoprecipitation, Cdk5-bound protein A-Sepharose beads were warmed at 30 °C for 10 min. CTβ2 protein was eluted from the beads with 40 μlof buffer containing 50 mm Tris-HCl, pH 7.4, 10 mm MgCl2, and 2 mm dithiothreitol and boiled for 5 min. The eluted CTβ2 protein was added to Cdk5-protein A-Sepharose beads in the presence of 1 mCi of [32P]ATP and incubated for 15 min at 30 °C. The kinase reaction was terminated by heating at 100 °C for 3 min in the presence of 5× SDS-sample buffer. Proteins were separated by electrophoresis on 10% polyacrylamide gels containing 0.1% SDS. The gels were dried overnight and then exposed to a PhosphorImager screen (Eastman Kodak Co.) for up to 3 days. Phospholipid Extraction and Quantification—Cell lysates were centrifuged at 7,000 × g for 5 min to remove nuclei and unbroken cells. Phospholipids were extracted with chloroform/methanol (2:1, v/v) and separated by thin layer chromatography on silica gel G60 plates with chloroform/methanol/acetic acid/formic acid/water (70:30:12:4:1) as developing solvent. Phospholipids were visualized by exposure of the plate to iodine vapor and identified by comparison with phospholipid standards. The relevant spots were scraped from the plate, and amounts of phospholipids were determined by phosphorus analysis (21Zhou X. Arthur G. J. Lipid Res. 1992; 33: 1233-1236Abstract Full Text PDF PubMed Google Scholar). CTβ2-silencing RNA Oligonucleotides—An oligonucleotide specific for CTβ2 mRNA (ACA GGT ATC CCA AAA TCCC) was designed using a sequence finder algorithm (Ambion, Austin, TX) and synthesized at the core facility (Department of Biochemistry, University of Alberta). The sequence showed no homology to other sequences in the NCBI data base; nor was the siCTβ2 oligonucleotide homologous to any other mRNA listed in GenBank™. The siCTβ2 oligonucleotide was inserted into pSILENCER 4.1CMV (Ambion, Austin, TX), and the plasmid was named pSiCTβ2. Insertion of the cDNA was confirmed by sequencing. Cloning and Hemagglutinin Modification of CTβ2 cDNA—A cDNA encoding the entire open reading frame of CTβ2 was cloned from a cDNA population generated from PC12 cell mRNA using the following primers: 5′-GCCATGCCAGTAGTTACCACT-3′ (forward) and 3′-GCTAAGGTTTGTGTGGGTTGTC-5′ (reverse). The amplicon was inserted into TOPO 2.1 vector (Invitrogen) and used as a template for the addition of an HA tag to the 3′-end of the open reading frame. The following sequence encoded the HA tag: 3′-TCAAGCATAATCTGGAACATCATATGGATACTTCTCATCCTCATCCCCCTCACTCAT-5′. The amplicon was cloned into the mammalian expression vector pCI (Invitrogen) and named pCI[CTβ2-HA]. Expression of CTβ2-HA protein was confirmed by immunoblotting of proteins from lysates of cells using anti-HA and anti-CTβ2 antibodies. Other Methods—Protein concentrations were measured using the Bio-Rad protein assay with bovine serum albumin as a standard. LY294002 and roscovitine were purchased from Sigma and dissolved in dimethyl sulfoxide. CTβ2 Is Present in Axons of Sympathetic Neurons and Retinal Ganglion Cells—In light of our previous observation that NGF increases the expression of CTβ2 in PC12 cells (3Carter J.M. Waite K.A. Campenot R.B. Vance J.E. Vance D.E. J. Biol. Chem. 2003; 278: 44988-44994Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), we reasoned that the subcellular localization of CT isoforms within neurons might provide insight into their contributions to PC biosynthesis and neurite growth. Our laboratory has previously demonstrated that PC biosynthetic enzyme activities, including CT, are present in distal axons of rat sympathetic neurons (4Vance J.E. Pan D. Campenot R.B. Bussiere M. Vance D.E. J. Neurochem. 1994; 62: 329-337Crossref PubMed Scopus (94) Google Scholar). The finding that in most cell types CTα is predominantly nuclear, whereas CTβ is cytoplasmic, suggested that CTβ2, rather than CTα, is a functionally important CT isoform in axons. We therefore immunoprecipitated CT from PC12 cells, rat sympathetic neurons, and retinal ganglion cells using an antibody (anti-M) raised against the membrane domain of rat liver CT. We confirmed that CTβ2 was immunoprecipitated by the anti-M antibody by immunoblotting the immunoprecipitated proteins from retinal ganglion cells with anti-CTβ2 antibodies (Fig. 1). Based on these immunoblots, and consistent with the molecular mass of CTβ2 (43 kDa), we identified the upper band in Fig. 1 as CTβ2. Immunoblotting of the immunoprecipitated proteins in the absence of primary antibody identified the other major band in Fig. 1 as the IgG heavy chain of the anti-M antibody. CTβ2 was also detected in PC12 cells and rat sympathetic neurons (Fig. 1, right-hand panels). To determine whether or not axons contain CTβ2, we immunoprecipitated CT from distal axons of compartmented cultures of sympathetic neurons and retinal ganglion cells. In this culture system, cell bodies and proximal axons are physically separated from distal axons by a silicone grease barrier and a Teflon divider (17Campenot R. Dev. Biol. 1982; 93: 1-12Crossref PubMed Scopus (197) Google Scholar). Thus, distal axons can be harvested independently from cell bodies/proximal axons. As shown in Fig. 1 (left), immunoblotting of anti-M-immunoprecipitated proteins with anti-M or anti-CTβ2 antibodies revealed that CTβ2 is abundant in both distal axons and cell bodies/proximal axons of sympathetic neurons and also in distal axons of retinal ganglion cells. These immunoblotting experiments demonstrate that CTβ2 is present in PC12 cells as well as in distal axons of rat sympathetic neurons and rat retinal ganglion cells. CTβ2 Facilitates Sprouting of Neurites and Branch Formation—Since CTβ2 is present in axons (Fig. 1), and since the amount of CTβ2, but not CTα, is increased during neurite outgrowth and/or extension (3Carter J.M. Waite K.A. Campenot R.B. Vance J.E. Vance D.E. J. Biol. Chem. 2003; 278: 44988-44994Abstract Full Text Full Text PDF PubMed Scopus (42) Google Scholar), we considered that CTβ2 might regulate neurite growth. We therefore hypothesized that suppression of CTβ2 expression in PC12 cells would impair neurite growth. To test this hypothesis, we used a RNA silencing strategy to “knock down” expression of CTβ2 in PC12 cells. PC12 cells were co-transfected with a GFP-encoding mammalian expression vector (phrGFP) and either a vector encoding small interfering RNA targeted to CTβ2 mRNA (pSiCTβ2) or a vector containing a scrambled insert (designated “control”). The transfected cells were incubated with NGF to induce differentiation and neurite outgrowth. We estimated that transfection efficiency was ∼30% according to the proportion of cells that expressed GFP in several random fields of view. In pSiCTβ2-transfected cultures, the amount of CTβ2 protein, as detected by immunoblotting, was reduced compared with that in control cells (Fig. 2A). Immunoblotting of tubulin demonstrated that protein synthesis in general was not suppressed (Fig. 2A). We also measured the in vitro activity of CT in cellular homogenates. This activity represents the combined activities of CTα and CTβ. In pSiCTβ2-transfected cells, CT activity was 2.9 nmol/min/mg protein, whereas CT activity of control cells was 4.3 nmol/min/mg protein (Fig. 2B). A trend (p < 0.09) toward reduced CT activity (∼30%) in CTβ2-silenced cells was observed. However, since CT activity reflects the activities of both CTα (the predicted major isoform of CT) and CTβ, and since RNA interference-silencing of CTβ2 was operative in only ∼30% of the cells, it is likely that CTβ2 activity was reduced in the SiCTβ2-transfected cells. Consistent with the lack of reduction of total CT activity in SiCTβ2-transfected cells, the incorporation of [3H]choline into PC in PC12 cells over a 3-h time period was not significantly reduced (4.69 × 106 ± 0.37 × 106 dpm/mg of protein for control and 4.41 × 106 ± 0.19 × 106 dpm/mg of protein for SiCTβ2-transfected cells). To determine if suppression of CTβ2 expression altered the morphology of NGF-treated PC12 cells, the cells were co-transfected with phrGFP and pSiCTβ2, and the morphologies of cells transfected with pSiCTβ2 and those transfected with the empty vector were compared (Fig. 3). Transfected cells were identified by the presence of GFP fluorescence. Within 2 days of NGF treatment, the number of neurites (primary neurites plus branches) per cell was markedly less in CTβ2-deficient cells than in cells transfected with pSILENCER containing a scrambled insert or in control cells transfected with empty vector. After 4 days of NGF treatment, differences in the number of neurites per cell, the degree of neurite branching, and neurite length between SiCTβ2-transfected cells and control cells were more pronounced (Fig. 3, A and B versus C and D). Moreover, the neurites of siCTβ2-expressing cells grew in a more linear fashion, with fewer points of attachment to the collagen substratum and fewer directional changes. To quantify differences in numbers of primary neurites and branches, we counted the total number of primary neurites and branches per cell (Fig. 4A) in control and CTβ2-deficient cells over a 5-day period of differentiation. After 2 days of NGF treatment, control cells contained 34% more neurites per cell than did CTβ2-deficient cells (Fig. 4B) (3.09 ± 0.06 versus 2.03 ± 0.07 (p < 0.05)). During NGF treatment, control cells steadily produced primary neurites and branches. After 5 days of NGF treatment, control cells contained 5.41 ± 0.35 neurites/cell. In contrast, CTβ2-deficient cells had only marginally increased the number of neurites between day 2 (2.03 ± 0.07 neurites/cell) and day 5 (2.60 ± 0.27 neurites/cell). As a result, after 5 days of exposure to NGF, CTβ2-deficient cells contained fewer than half the neurites of control cells (2.60 ± 0.27 versus 5.41 ± 0.35 neurites/cell (p < 0.003)) (Fig. 4B). Since primary neurites and branches are distinct neurite populations (Fig. 4A) that are regulated by separate signaling pathways (22Acebes A. Ferrus A. Trends Neurosci. 2000; 23: 557-565Abstract Full Text Full Text PDF PubMed Scopus (82) Google Scholar), we determined if a decrease in the amount of CTβ2 affected each of these neurite populations. Within 2 days of NGF treatment, the extent of neurite sprouting was significantly less in CTβ2-deficient cells than in control cells (2.52 ± 0.09 versus 1.83 ± 0.01 neurites/cell (p < 0.01)) (Fig. 4C). The difference in the number of primary neurites was still apparent after 4 days of NGF-induced differentiation (control versus siCTβ2-expressing cells: 3.48 ± 0.12 versus 2.46 ± 0.18 neurites/cell (p < 0.01)) (Fig. 4C). Furthermore, CTβ2-deficient cells had far fewer branches than did control cells after 2 and 4 days of NGF treatment (Fig. 4D). Thus, CTβ2 deficiency impairs neurite sprouting and branching. CTβ2 Is Not Required for Neurite Extension—When a primary neurite has sprouted from the cell body, either the neurite continues to elongate or neurite extension pauses for branch formation. Since CTβ2-deficient PC12 cells contained fewer neurites (both primary neurites and branches) than did control cells (Figs. 3 and 4), we measured the lengths of primary neurites and branches to determine whether or not CTβ2 is required for neurite extension. In control cells, mean neurite length (including both primary neurites and branches) did not increase between days 2 and 5: 84 ± 1.5 pixels at day 2 and 100 ± 37 pixels at day 5 (Fig. 5A). Typically, once a neurite attained a length of ∼80 pixels, it had produced at least one branch, and further elongation of the primary neurite was limited. However, extension of primary neurites of CTβ2-deficient cells continued throughout the period of NGF treatment. Consequently," @default.
• W2004207754 created "2016-06-24" @default.
• W2004207754 creator A5006782806 @default.
• W2004207754 creator A5019669813 @default.
• W2004207754 creator A5057746394 @default.
• W2004207754 creator A5073998071 @default.
• W2004207754 creator A5089223424 @default.
• W2004207754 date "2008-01-01" @default.
• W2004207754 modified "2023-10-14" @default.
• W2004207754 title "Phosphatidylcholine Biosynthesis via CTP:Phosphocholine Cytidylyltransferase β2 Facilitates Neurite Outgrowth and Branching" @default.
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