Functional regulation of FEZ1 by the U-box-type ubiquitin ligase E4B contributes to neuritogenesis

E4B (also known as UFD2a) is a mammalian homolog of Saccharomyces cerevisiae Ufd2, which was originally described as a ubiquitin chain assembly factor (E4). E4B is a U-box-type ubiquitin-protein isopeptide ligase (E3) and likely functions as either an E3 or an E4. With a yeast two-hybrid screen, we have now identified FEZ1 (fasciculation and elongation protein zeta 1) as a protein that interacts with E4B. FEZ1 is implicated in neuritogenesis when phosphorylated by protein kinase Czeta (PKCzeta). Interaction between E4B and FEZ1 in mammalian cells was enhanced by coexpression of constitutively active PKCzeta. E4B mediated the polyubiquitylation of FEZ1 but did not affect its intracellular stability, suggesting that such modification of FEZ1 is not a signal for its proteolysis. Polyubiquitylation of FEZ1 by E4B required Lys(27) of ubiquitin. Expression of a dominant-negative mutant of E4B in rat pheochromocytoma PC12 cells resulted in inhibition of neurite extension induced either by nerve growth factor or by coexpression of FEZ1 and constitutively active PKCzeta. These findings indicate that E4B serves as a ubiquitin ligase for FEZ1 and thereby regulates its function but not its degradation.     

Fasciculation and elongation protein zeta-1 (FEZ1) participates in the polarization of hippocampal neuron by controlling the mitochondrial motility

The fasciculation and elongation protein zeta-1 (FEZ1), a mammalian orthologue of Caenorhabditis elegans UNC-76 protein, is a 45-kDa protein with four coiled-coiled domains and efficiently promotes the neurite elongation in the rat phaeochromocytoma PC12 cells. UNC-76 proteins of C. elegans and Drosophila have been genetically demonstrated to be involved in the axonal guidance. We here show that FEZ1 RNA interference (RNAi) represses the formation of axon in rat embryo hippocampal neurons. An anterograde mitochondrial movement is also retarded in neurites of the RNAi-treated hippocampal neurons. Moreover, the size of mitochondria is considerably elongated by the RNAi treatment. The transport of mitochondria from soma to axon or dendrites is essential for the neuronal differentiation. Therefore, our results strongly suggest that FEZ1 participates in the establishment of neuronal polarity by controlling the mitochondrial motility along axon.     

Identification of FEZ1 as a protein that interacts with JC virus agnoprotein and microtubules: role of agnoprotein-induced dissociation of FEZ1 from microtubules in viral propagation

The human polyomavirus JC virus (JCV) is the causative agent of a fatal demyelinating disease, progressive multifocal leukoencephalopathy, and encodes six major proteins, including agnoprotein. Agnoprotein colocalizes with microtubules in JCV-infected cells, but its function is not fully understood. We have now identified fasciculation and elongation protein zeta 1 (FEZ1) as a protein that interacted with JCV agnoprotein in a yeast two-hybrid screen of a human brain cDNA library. An in vitro binding assay showed that agnoprotein interacted directly with FEZ1 and microtubules. A microtubule cosedimentation assay revealed that FEZ1 also associates with microtubules and that agnoprotein induces the dissociation of FEZ1 from microtubules. Agnoprotein inhibited the promotion by FEZ1 of neurite outgrowth in PC12 cells. Conversely, overexpression of FEZ1 suppressed JCV protein expression and intracellular trafficking in JCV-infected cells. These results suggest that FEZ1 promotes neurite extension through its interaction with microtubules, and that agnoprotein facilitates JCV propagation by inducing the dissociation of FEZ1 from microtubules.     

Mammalian Homologue of the Caenorhabditis elegans UNC-76 Protein Involved in Axonal Outgrowth Is a Protein Kinase C ζ–interacting Protein

By the yeast two-hybrid screening of a rat brain cDNA library with the regulatory domain of protein kinase C ζ (PKCζ) as a bait, we have cloned a gene coding for a novel PKCζ-interacting protein homologous to the Caenorhabditis elegans UNC-76 protein involved in axonal outgrowth and fasciculation. The protein designated FEZ1 (fasciculation and elongation protein zeta-1) consisting of 393 amino acid residues shows a high Asp/Glu content and contains several regions predicted to form amphipathic helices. Northern blot analysis has revealed that FEZ1 mRNA is abundantly expressed in adult rat brain and throughout the developmental stages of mouse embryo. By the yeast two-hybrid assay with various deletion mutants of PKC, FEZ1 was shown to interact with the NH₂-terminal variable region (V1) of PKCζ and weakly with that of PKCε. In the COS-7 cells coexpressing FEZ1 and PKCζ, FEZ1 was present mainly in the plasma membrane, associating with PKCζ and being phosphorylated. These results indicate that FEZ1 is a novel substrate of PKCζ. When the constitutively active mutant of PKCζ was used, FEZ1 was found in the cytoplasm of COS-7 cells. Upon treatment of the cells with a PKC inhibitor, staurosporin, FEZ1 was translocated from the cytoplasm to the plasma membrane, suggesting that the cytoplasmic translocation of FEZ1 is directly regulated by the PKCζ activity. Although expression of FEZ1 alone had no effect on PC12 cells, coexpression of FEZ1 and constitutively active PKCζ stimulated the neuronal differentiation of PC12 cells. Combined with the recent finding that a human FEZ1 protein is able to complement the function of UNC-76 necessary for normal axonal bundling and elongation within axon bundles in the nematode, these results suggest that FEZ1 plays a crucial role in the axon guidance machinery in mammals by interacting with PKCζ.     

Isotypes of alpha-tubulin are differentially regulated during neuronal maturation

The mRNAs for two isotypes of alpha-tubulin, termed T alpha 1 and T26, are known to be expressed in the rat nervous system. We have compared the expression of these two alpha-tubulin mRNAs during neural development, using RNA blotting and in situ hybridization techniques with probes directed against unique sequences of each mRNA. T alpha 1 mRNA is highly enriched in the embryonic nervous system but is markedly less abundant in the adult brain; T26 mRNA is expressed in many embryonic tissues with little change in abundance during development. Within the nervous system, T alpha 1 mRNA is enriched in regions with neurons actively undergoing neurite extension, such as the cortical plate, whereas T26 mRNA is relatively homogeneous in distribution, with some enrichment in proliferative zones. Expression of T alpha 1 mRNA is also increased in PC12 cells induced to differentiate and extend neurite processes by nerve growth factor. Taken together, the data indicate that T alpha 1-tubulin mRNA is expressed at high levels during the extension of neuronal processes. The abundant expression of T alpha 1-tubulin mRNA may therefore reflect either a means to increase the available pool of alpha-tubulin or a specific requirement for the T alpha 1 isotype for neurite extension.     

Expression and regulation of phospholipase D isoforms in mammalian cell lines

Phospholipase D (PLD) is activated in mammalian cells in response to diverse stimuli that include growth factors, activators of protein kinase C, and agonists binding to G-protein-coupled receptors. Two forms of mammalian PLD, PLD1 and PLD2, have been identified. Expression of mRNA and protein for PLD1 and PLD2 was analyzed in the following cell lines: A7r5 (rat vascular smooth muscle); EL4 (mouse thymoma); HL-60 (human myeloid leukemia); Jurkat (human leukemia); PC-3 (human prostate adenocarcinoma); PC-12K (rat phaeochromocytoma); and Rat-1 HIR (rat fibroblast). All, with the exception of EL4, express agonist-activated PLD activity. PLD1 is expressed in A7r5, HL-60, PC-3, and Rat-1, while PLD2 is expressed in A7r5, Jurkat, PC12K, PC-3, and Rat-1. Neither isoform is expressed in EL4. Guanine nucleotide-independent PLD activity is present in membranes from all cells expressing PLD2. In PC12K cells, which express only PLD2, treatment with nerve growth factor causes neurite outgrowth and increases expression of PLD2 mRNA and protein within 6-12 h. A corresponding increase is observed in membrane PLD activity and in phorbol-12-myristate-13-acetate (PMA)-stimulated PLD activity in intact cells. These results show that PLD2 can be regulated both pretranslationally and posttranslationally by agonists.     

Inhibition of neurite extension by overexpression of individual domains of LIM kinase 1

Lin-11, Isl-1 and Mec-3 (LIM) kinases are serine/threonine kinases that phosphorylate cofilin, an actin depolymerizing protein. LIM kinases have a highly modular structure composed of two N-terminal LIM domains (LIM 1/2), a PSD-95, Dlg and ZO-1 (PDZ) domain and a C-terminal protein kinase domain. Here, we overexpressed individual domains of mouse LIM kinase 1 (LIMK1) in PC12 cells and investigated their effects on neurite outgrowth. Although none of the LIMK1 domains had an effect on spontaneous neurite outgrowth, the N-terminal LIM 1/2 domains strongly inhibited differentiation of PC12 cells after stimulation with both nerve growth factor (NGF) and the Rho-kinase inhibitor Y-27632. In contrast, the overexpressed PDZ domain reduced neurite outgrowth only when differentiation had been induced by Y-27632, but not by NGF. Our data suggest that the different non-catalytic N-terminal domains of LIMK1 contribute to the regulation of neurite extension by using distinct signal transduction pathways.     

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: the role of TRPC channels

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1-TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75(NTR)-IKK(2)-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK(2) dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent "molecular damper" maintaining a submaximal velocity of neurite extension.     

Saccharin enhances neurite extension by regulating organization of the microtubules

Aims

In the present study, we found that saccharin, an artificial calorie-free sweetener, promotes neurite extension in the cultured neuronal cells. The purposes of this study are to characterize the effect of saccharine on neurite extension and to determine how saccharin enhances neurite extension.

Main methods

The analyses were performed using mouse neuroblastoma N1E-115 cells and rat pheochromocytoma PC12 cells. Neurite extension was evaluated by counting the cells bearing neurites and measuring the length of neurites. Formation, severing and transportation of the microtubules were evaluated by immunostaining and western blotting analysis.

Key findings

Deprivation of glucose increased the number of N1E-115 cells bearing long processes. And the effect was inhibited by addition of glucose. Saccharin increased the number of these cells bearing long processes in a dose-dependent manner and total neurite length and longest neurite length in each cell. Saccharin also had a similar effect on NGF-treated PC12 cells. Saccharin increased the amount of the microtubules reconstructed after treatment with nocodazole, a disruptor of microtubules. The effect of saccharin on microtubule reconstruction was not influenced by dihydrocytochalasin B, an inhibitor of actin polymerization, indicating that saccharin enhances microtubule formation without requiring actin dynamics. In the cells treated with vinblastine, an inhibitor of microtubule polymerization, after microtubule reorganization, filamentous microtubules were observed more distantly from the centrosome in saccharin-treated cells, indicating that saccharin enhances microtubule severing and/or transportation.

Significance

These results suggest that saccharin enhances neurite extension by promoting microtubule organization.     

Simultaneous suppression of cdc2 and cdk2 activities induces neuronal differentiation of PC12 cells

The involvement of cdc2 and cdk2 during neuronal differentiation in rat pheochromocytoma PC12 cells was examined. When PC12 cells were cultured with nerve growth factor (NGF), expression of cdc2 decreased significantly after day 5, while expression of cdk2 decreased gradually after day 7. Cells overexpressing cdc2 or cdk2 were resistant to NGF-induced differentiation and growth suppression, and maintained high cdc2 or cdk2 kinase activity, respectively, during NGF treatment. In contrast, the NGF-treated parental cells showed a marked decline in these kinase activities after day 3. When PC12 cells were treated with specific inhibitors of cdc2/cdk2 (butyrolactone-I, olomoucin), they showed marked neurite extension and up-regulation of microtubule-associated protein 2 expression. In addition, treatment with mixtures of antisense oligonucleotides for cdc2 and cdk2 resulted in down-regulation of both cdc2 and cdk2 kinase activities as well as significant neurite outgrowth and up-regulation of microtubule-associated protein 2 expression. However, neurite outgrowth was not observed in cells treated with either single antisense oligonucleotide, or antisense cdc2 + cdk4 or cdk2 + cdk4 oligonucleotide mixtures. These results suggest that simultaneous down-regulation of cdc2 and cdk2 activity is sufficient and necessary for neuronal differentiation in PC12 cells.     

LIM kinase and slingshot are critical for neurite extension

Cofilin and its closely related protein, actin-depolymerizing factor (ADF), are key regulators of actin cytoskeleton dynamics that have been implicated in growth cone motility and neurite extension. Cofilin/ADF are inactivated by LIM kinase (LIMK)-catalyzed phosphorylation and reactivated by Slingshot (SSH)-catalyzed dephosphorylation. Here we examined the roles of cofilin/ADF, LIMKs (LIMK1 and LIMK2), and SSHs (SSH1 and SSH2) in nerve growth factor (NGF)-induced neurite extension. Knockdown of cofilin/ADF by RNA interference almost completely inhibited NGF-induced neurite extension from PC12 cells, and double knockdown of SSH1/SSH2 significantly suppressed both NGF-induced cofilin/ADF dephosphorylation and neurite extension from PC12 cells, thus indicating that cofilin/ADF and their activating phosphatases SSH1/SSH2 are critical for neurite extension. Interestingly, NGF stimulated the activities of both LIMK1 and LIMK2 in PC12 cells, and suppression of LIMK1/LIMK2 expression or activity significantly reduced NGF-induced neurite extension from PC12 cells or chick dorsal root ganglion (DRG) neurons. Inhibition of LIMK1/LIMK2 activity reduced actin filament assembly in the peripheral region of the growth cone of chick DRG neurons. These results suggest that proper regulation of cofilin/ADF activities through control of phosphorylation by LIMKs and SSHs is critical for neurite extension and that LIMKs regulate actin filament assembly at the tip of the growth cone.     

Enhanced expression and activation of CTP:phosphocholine cytidylyltransferase beta2 during neurite outgrowth

During differentiation neurons increase phospholipid biosynthesis to provide new membrane for neurite growth. We studied the regulation of phosphatidylcholine (PC) biosynthesis during differentiation of two neuronal cell lines: PC12 cells and Neuro2a cells. We hypothesized that in PC12 cells nerve growth factor (NGF) would up-regulate the activity and expression of the rate-limiting enzyme in PC biosynthesis, CTP:phosphocholine cytidylyltransferase (CT). During neurite outgrowth, NGF doubled the amount of cellular PC and CT activity. CTbeta2 mRNA increased within 1 day of NGF application, prior to the formation of visible neurites, and continued to increase during neurite growth. When neurites retracted in response to NGF withdrawal, CTbeta2 mRNA, protein, and CT activity decreased. NGF specifically activated CTbeta2 by promoting its translocation from cytosol to membranes. In contrast, NGF did not alter CTalpha expression or translocation. The increase in both CTbeta2 mRNA and CT activity was inhibited by U0126, an inhibitor of mitogen-activated kinase/extracellular signal-regulated kinase kinase 1/2 (MEK1/2). In Neuro2a cells, retinoic acid significantly increased CT activity (by 54%) and increased CTbeta2 protein, coincident with neurite outgrowth but did not change CTalpha expression. Together, these data suggest that the CTbeta2 isoform of CT is specifically up-regulated and activated during neuronal differentiation to increase PC biosynthesis for growing neurites.     

Phospholipase D2 activation by p38 MAP kinase is involved in neurite outgrowth

p38 mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth. However, the underlying molecular mechanism(s) remains unclear. Here, we demonstrate that phospholipase D2 (PLD2) mediates p38 signaling in neurite outgrowth. Stimulation of rat pheochromocytoma PC12 cells with nerve growth factor activated PLD2 and augmented neurite outgrowth, both of which were inhibited by pharmacological suppression of p38. Overexpression of constitutively active MAP kinase kinase 6 (MKK6-CA) activated coexpressed PLD2 in PC12 and mouse neuroblastoma N1E-115 cells. Overexpression of wild-type PLD2 in these cells strongly augmented the neurite outgrowth induced by MKK6-CA, whereas lipase-deficient PLD2 suppressed it. These findings provide evidence that PLD2 functions as a downstream molecule of p38 in the neurite outgrowth signaling cascade.     

Dominant Negative ATF1 Blocks Cyclic AMP-Induced Neurite Outgrowth in PC12D Cells

Abstract: Extension of the neuronal process is a crucial step for establishment of the neuronal network. As CREB preferentially forms heterodimers with ATF1 in PC12D cells, we examined the roles of the CREB/ATF1 heterodimer on cyclic AMP (cAMP)-induced neurite extension, using originally constructed ATF1RL, which has a point mutation at the DNA binding domain of ATF1. Transient expression of ATF1RL suppressed the protein kinase A/CREB-induced expression of the CRE reporter gene as expected. Treatment with forskolin elicited a relatively poor mRNA induction for immediate early genes in PC12D-ATF1RL cells, a PC12D cell line stably expressing ATF1RL, in comparison with the parental PC12D cells. Furthermore, the PC12D-ATF1RL cells were proved to be defective at cAMP-induced neurite outgrowth. In contrast, both the gene expression and the differentiation after nerve growth factor treatment noted in PC12D-ATF1RL cells were at the same levels as those in the parental cells. These data provide us the first evidence that links CREB/ATF1 to the cAMP-induced differentiation of PC12 cells.     

Neuronal functions of the novel serine/threonine kinase Ndr2

We have identified a novel member of the Ndr subfamily of serine/threonine protein kinases, Ndr2, as a gene product that is induced in the mouse amygdala during fear memory consolidation and examined a possible function of this kinase in neural differentiation. Expression of Ndr2 mRNA was detected in various cortical and subcortical brain regions, as well as non-neuronal tissues. Its expression in the amygdala was increased 6 h after Pavlovian fear conditioning training and returned to control levels within 24 h. To study intracellular localization and functions of Ndr2, EGFP::Ndr2 fusion proteins were expressed in rat pheochromocytoma (PC12) cells and acutely isolated cortical neurons, thereby revealing an association of Ndr2 with the actin cytoskeleton in somata, neurites and filopodia, in spines and at sites of cell contact. Co-precipitation and pull-down experiments support this finding. Evidence for an involvement of Ndr2 in actin-mediated cellular functions further comes from the observation of decreased cell spreading and changes in neurite outgrowth that were associated with protein serine phosphorylation in transfected PC12 cells. Together, our data suggest that Ndr2 is an interesting candidate gene for the regulation of structural processes in differentiating and mature neuronal cells.     

Thiazolidinediones inhibit the growth of PC12 cells both in vitro and in vivo

Thiazolidinediones (TZDs) have recently been proposed as a therapy for PPARγ-expressing tumors. Pheochromocytoma (PHEO) is associated with high morbidity and mortality due to excess catecholamine production, and few effective drug therapies currently exist. We investigated the effects of TZDs on PHEO both in vitro and in vivo. PPARγ protein was expressed in human adrenal PHEO tissues as well as in rat PHEO cells, PC12. TZDs, including rosiglitazone (RGZ) and pioglitazone (PGZ), inhibited proliferation of PC12 cells in a dose-dependent manner and increased casapse-3 expression of PC12 cells. TZDs also reduced expression of cyclin E and cyclin-dependent kinase2. RGZ inhibited nerve growth factor-induced neurite outgrowth and reduced expression of catecholamine-synthesizing enzymes. Finally, rat PHEO growth generated by subcutaneous injection of PC12 cells was slowed in an RGZ-treated mouse. These data suggest that TZDs may be a promising therapeutic approach for medical treatment for PHEO.     

Prohormone-converting enzymes: regulation and evaluation of function using antisense RNA.

Several putative peptide-processing endoproteases have been identified by homology to the yeast Kex2 endoprotease, including furin, PC2, and PC1. However, the question is still open as to which might be involved in peptide posttranslational processing. To enable detailed comparisons of physiological changes in peptide processing with biochemical and molecular biological studies, we cloned rat pituitary cDNAs for PC1 and PC2. The amino acid sequence homologies among rat, human, and mouse PC1, PC2, and furin are consistent with each being a highly conserved but distinct member of a larger family of mammalian subtilisin-like proteases. PC1 and PC2 mRNAs show a restricted distribution among rat tissues and cultured cell lines, consistent with a role in tissue-specific peptide processing; the occurrence of furin mRNA among these tissues and cell lines is much more widespread, being high in many nonneuroendocrine tissues. In the neurointermediate pituitary, PC1 and PC2 mRNAs are strikingly regulated in response to dopaminergic agents, in parallel with mRNAs for POMC, peptidylglycine alpha-amidating monooxygenase, and carboxypeptidase-H. In AtT-20 cells, PC1 mRNA is coregulated with POMC and peptidylglycine alpha-amidating monooxygenase mRNAs in response to CRH and glucocorticoids. When the endogenous PC1 mRNA level in AtT-20 cells is significantly and specifically decreased by stable expression of antisense RNA to PC1, biosynthetic labeling of newly synthesized POMC-derived peptides shows a substantial blockade of normal POMC processing. These data are consistent with a role for PC1 protein in endoproteolysis, either as a processing endoprotease or as the activator of the actual processing endoprotease(s).     

KLHL1/MRP2 mediates neurite outgrowth in a glycogen synthase kinase 3beta-dependent manner

The actin-based cytoskeleton is essential for the generation and maintenance of cell polarity, cellular motility, and the formation of neural cell processes. MRP2 is an actin-binding protein of the kelch-related protein family. While MRP2 has been shown to be expressed specifically in brain, its function is still unknown. Here, we report that in neuronal growth factor (NGF)-induced PC12 cells, MRP2 was expressed along the neurite processes and colocalized with Talin at the growth cones. MRP2 mRNA and protein levels were up-regulated in PC12 cells following NGF stimulation. Moreover, treatment of PC12 cells with interfering RNAs for MRP2 and glycogen synthase kinase 3beta (GSK3beta) resulted in the inhibition of neurite outgrowth. A significant decrease in MRP2 expression levels was observed following GSK3beta inhibition, which was correlated with the inhibited neurite outgrowth, while GSK3beta overexpression was found to increase MRP2 expression levels. MRP2 interacted with GSK3beta through its NH2 terminus containing the BTB domain, and these molecules colocalized along neurite processes and growth cones in differentiated PC12 cells and rat primary hippocampal neurons. Additionally, increased associations of MRP2 with GSK3beta and MRP2 with actin were observed in the NGF-treated PC12 cells. Thus, this study provides, for the first time, insights into the involvement of MRP2 in neurite outgrowth, which occurs in a GSK3beta-dependent manner.     

Phosphatidylcholine biosynthesis via CTP:phosphocholine cytidylyltransferase 2 facilitates neurite outgrowth and branching

Hallmarks of neuronal differentiation are neurite sprouting, extension, and branching. We previously showed that increased expression of CTP:phosphocholine cytidylyltransferase beta2 (CTbeta2), 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). CTbeta2 mRNA is highly expressed in the brain. We show that CTbeta2 is abundant in axons of rat sympathetic neurons and retinal ganglion cells. We used RNA silencing to decrease CTbeta2 expression in PC12 cells differentiated by nerve growth factor. In CTbeta2-silenced cells, numbers of primary and secondary neurites were markedly reduced, suggesting that CTbeta2 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 CTbeta2-deficient cells. CTbeta2 was not phosphorylated in vitro by Akt. However, inhibition of Cdk5 by roscovitine blocked CTbeta2 phosphorylation and reduced neurite outgrowth and branching. These results highlight the importance of CTbeta2 in neurons for promoting neurite outgrowth and branching and represent the first identification of a lipid biosynthetic enzyme that facilitates these functions.