Rat PC12 Cells Research Articles

Recombinant human TAT-OP1 to enhance NGF neurogenic potential: preliminary studies on PC12 cells

Osteogenic protein 1 (OP1), also known as bone morphogenic protein-7 (BMP7), is a multifunctional cytokine with demonstrated neurogenic potential. As the recombinant OP1 (rhOP1) was shown to provide axonal guidance cues and to prevent the reduction of dendritic growth in the injury-induced cortical cultures, it was suggested that an in vivo efficient rhOP1 delivery could enhance neurite growth and functional reconnectivity in the damaged brain. In the present work, we engineered a chimeric molecule in which rhBMP7 was fused to a protein transduction domain derived from HIV-1 TAT protein to deliver the denatured recombinant BMP7 into cells and obtain its chaperone-mediated folding, circumventing the expensive and not much efficient in vitro refolding procedures. When tested on rat PC12 cells, a widely used in vitro neurogenic differentiation model, the resulting fusion protein (rhTAT-OP1) demonstrated to enter fastly into the cells, lose HIV-TAT sequence and interact with membrane receptors activating BMP pathway by SMAD 1/5/8 phosphorylation. In comparison with nerve growth factor (NGF) and BMP7, it proved itself effective to induce the formation of more organized H and M neurofilaments. Moreover, if used in combination with NGF, it stimulated a significant (P < 0.05) and more precocious dendritic outgrowth with respect to NGF alone. These results indicate that rhTAT-OP1 fused with TAT transduction domain shows neurogenic activity and may be a promising enhancer factor in NGF-based therapies.

ChemInform Abstract: Imidazodiazepinediones: A New Class of Adenosine Receptor Antagonists.

A series of imidazo[4,5-e][1-4]diazepine-5,8-diones were synthesized from hypoxanthines. Certain of these cyclic homologues of caffeine, theophylline, theobromine, 3-isobutyl-1-methylxanthine, and enprofylline were inhibitors of binding of adenosine analogues to rat brain A1 and A2 adenosine receptors and were antagonists of A2 adenosine receptors stimulatory to adenylate cyclase in rat PC12 cell membranes. Activity at adenosine receptors was lower than the corresponding xanthines, perhaps because imidazodiazepinediones contain a boat-shaped seven-membered ring rather than the planar heteroaryl ring system of the xanthines. The imidazodiazepinediones had low affinity for brain benzodiazepine sites.

Dual actions of lindane (γ-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells

The persistent organochlorine pesticide lindane is still abundantly found in the environment and in human and animal tissue samples. Lindane induces a wide range of adverse health effects, which are at least partially mediated via the known inhibition of GABA A and glycine receptors. Additionally, lindane has been reported to increase the basal intracellular Ca 2+ concentration ([Ca 2+] i). As Ca 2+ triggers many cellular processes, including cell death and vesicular neurotransmitter release (exocytosis), we investigated whether lindane affects exocytosis, Ca 2+ homeostasis, production of reactive oxygen species (ROS) and cytotoxicity in neuroendocrine PC12 cells. Amperometric recordings and [Ca 2+] i imaging experiments with fura-2 demonstrated that lindane (≥ 10 μM) rapidly increases basal exocytosis and basal [Ca 2+] i. Additional imaging and electrophysiological recordings revealed that this increase was largely due to a lindane-induced membrane depolarization and subsequent opening of N- and P/Q-type voltage-gated Ca 2+ channels (VGCC). On the other hand, lindane (≥ 3 μM) induced a concentration-dependent but non-specific inhibition of VGCCs, thereby limiting the lindane-induced increase in basal [Ca 2+] i and exocytosis. Importantly, the non-specific inhibition of VGCCs also reduced stimulation-evoked exocytosis and Ca 2+ influx. Though lindane exposure concentration-dependently increased ROS production, cell viability was not affected indicating that the used concentrations were not acute cytotoxic. These combined findings indicate that lindane has two, partly counteracting effects. Lindane causes membrane depolarization, thereby increasing basal [Ca 2+] i and exocytosis. In parallel, lindane inhibits VGCCs, thereby limiting the basal effects and reducing stimulation-evoked [Ca 2+] i and exocytosis. This study further underlines the need to consider presynaptic, non-receptor-mediated effects in human risk assessment.

Involvement of cAMP in nerve growth factor-triggered p35/Cdk5 activation and differentiation in PC12 cells

The signaling mechanisms underlying cell differentiation have been extensively studied with the use of rat PC12 cells as a model system. Nerve growth factor (NGF) is a trophic factor inducing PC12 cell differentiation through the activation of the p35/cyclin-dependent kinase 5 (Cdk5) complex. It has been reported that adenylyl cyclase activation and cAMP production may be involved in NGF-dependent actions. Our previous results indicate that cAMP activates the p35/Cdk5 complex in reproductive cells. Therefore, the role of cAMP in NGF-triggered p35/Cdk5 activation and PC12 differentiation was interesting to explore. Our results indicate that roscovitine, a molecular inhibitor of Cdk5, blocks cAMP-triggered PC12 differentiation, which was evaluated by neurite initiation, a decrease in proliferation, and cell cycle G(1) arrest. The following data show that cAMP treatment increased Cdk5 activity through p35 upregulation. cAMP downstream components, protein kinase A (PKA) and phosphorylated cAMP response element binding protein (CREB), are involved in this regulation. The immunocytochemical results indicate that PKA inhibition disrupted cAMP-triggered p35/Cdk5 localization in PC12 cells. In addition, adenylyl cyclase inhibition was found to diminish NGF-induced intracellular cAMP production, CREB phosphorylation, and p35 expression. The cAMP antagonist and the PKA inhibitors reduced NGF-induced p35 expression. Finally, NGF-triggered PC12 differentiation was partially decreased by adenylyl cyclase or PKA inhibitors. In conclusion, these results demonstrate that cAMP may play a role in NGF-p35/Cdk5-dependent PC12 differentiation.

Dual effects of lindane (gamma-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells

Dual effects of lindane (gamma-hexachlorocyclohexane) on calcium homeostasis and exocytosis in rat PC12 cells

Deviant Nicotinic Acid Adenine Dinucleotide Phosphate (NAADP)-mediated Ca2+ Signaling upon Lysosome Proliferation

Accumulating evidence suggests that the endolysosomal system is a novel intracellular Ca(2+) pool mobilized by the second messenger, nicotinic acid adenine dinucleotide phosphate (NAADP). Although lysosomes in neurons are known to proliferate in numerous neurodegenerative diseases and during the normal course of aging, little is known concerning the effect of lysosomal proliferation on Ca(2+) homeostasis. Here, we induce proliferation of lysosomes in primary cultures of rat hippocampal neurons and PC12 cells through chronic treatment with the cathepsin inhibitor, Z-Phe-Ala-diazomethylketone. We demonstrate that lysosome proliferation increases the size of the lysosomal Ca(2+) pool and enhances Ca(2+) signals in response to direct cellular delivery of NAADP and glutamate, an identified NAADP-producing agonist. Our data suggest that deregulated lysosomal Ca(2+) signaling through NAADP may contribute to neuronal dysfunction and highlight the usefulness of lysosomal hydrolase inhibition in probing NAADP action.

Reserpine-induced reduction in norepinephrine transporter function requires catecholamine storage vesicles

Treatment of rats with reserpine, an inhibitor of the vesicular monoamine transporter (VMAT), depletes norepinephrine (NE) and regulates NE transporter (NET) expression. The present study examined the molecular mechanisms involved in regulation of the NET by reserpine using cultured cells. Exposure of rat PC12 cells to reserpine for a period as short as 5 min decreased [ 3H]NE uptake capacity, an effect characterized by a robust decrease in the V max of the transport of [ 3H]NE. As expected, reserpine did not displace the binding of [ 3H]nisoxetine from the NET in membrane homogenates. The potency of reserpine for reducing [ 3H]NE uptake was dramatically lower in SK-N-SH cells that have reduced storage capacity for catecholamines. Reserpine had no effect on [ 3H]NE uptake in HEK-293 cells transfected with the rat NET (293-hNET), cells that lack catecholamine storage vesicles. NET regulation by reserpine was independent of trafficking of the NET from the cell surface. Pre-exposure of cells to inhibitors of several intracellular signaling cascades known to regulate the NET, including Ca 2+/Ca 2+–calmodulin dependent kinase and protein kinases A, C and G, did not affect the ability of reserpine to reduce [ 3H]NE uptake. Treatment of PC12 cells with the catecholamine depleting agent, α-methyl- p-tyrosine, increased [ 3H]NE uptake and eliminated the inhibitory effects of reserpine on [ 3H]NE uptake. Reserpine non-competitively inhibits NET activity through a Ca 2+-independent process that requires catecholamine storage vesicles, revealing a novel pharmacological method to modify NET function. Further characterization of the molecular nature of reserpine's action could lead to the development of alternative therapeutic strategies for treating disorders known to be benefitted by treatment with traditional competitive NET inhibitors.

Inhibition of heat shock protein 90 attenuates adenylate cyclase sensitization after chronic morphine treatment

Cellular adaptations to chronic opioid treatment result in enhanced responsiveness of adenylate cyclase and an increase in forskolin- or agonist-stimulated cAMP production. It is, however, not known whether chaperone molecules such as heat shock proteins contribute to this adenylate cyclase sensitization. Here, we report that treatment of cells with geldanamycin, an inhibitor of heat shock protein 90 (Hsp90), led to effective attenuation of morphine-induced adenylate cyclase sensitization. In SK-N-SH human neuroblastoma cells, morphine significantly increased RNA transcript and protein levels of type I adenylate cyclase, leading to sensitization. Whole-genome tiling array analysis revealed that cAMP response element-binding protein, an important mediator for cellular adaptation to morphine, associated with the proximal promoter of Hsp90AB1 not only in SK-N-SH cells but also in rat PC12 and human embryonic kidney cells. Hsp90AB1 transcript and protein levels increased significantly during morphine treatment, and co-application of geldanamycin (0.1–10 nM) effectively suppressed the increase in forskolin-activated adenylate cyclase activation by 56%. Type I adenylate cyclase, but not Hsp90AB1, underwent significant degradation during geldanamycin treatment. These results indicate that Hsp90 is a new pharmacological target for the suppression of adenylate cyclase sensitization induced by chronic morphine treatment.

Mitochondrial Ca2+ Uptake Increases Ca2+ Release from Inositol 1,4,5-Trisphosphate Receptor Clusters in Smooth Muscle Cells

Smooth muscle activities are regulated by inositol 1,4,5-trisphosphate (InsP(3))-mediated increases in cytosolic Ca2+ concentration ([Ca2+](c)). Local Ca2+ release from an InsP(3) receptor (InsP(3)R) cluster present on the sarcoplasmic reticulum is termed a Ca2+ puff. Ca2+ released via InsP(3)R may diffuse to adjacent clusters to trigger further release and generate a cell-wide (global) Ca2+ rise. In smooth muscle, mitochondrial Ca2+ uptake maintains global InsP(3)-mediated Ca2+ release by preventing a negative feedback effect of high [Ca2+] on InsP(3)R. Mitochondria may regulate InsP(3)-mediated Ca2+ signals by operating between or within InsP(3)R clusters. In the former mitochondria could regulate only global Ca2+ signals, whereas in the latter both local and global signals would be affected. Here whether mitochondria maintain InsP(3)-mediated Ca2+ release by operating within (local) or between (global) InsP(3)R clusters has been addressed. Ca2+ puffs evoked by localized photolysis of InsP(3) in single voltage-clamped colonic smooth muscle cells had amplitudes of 0.5-4.0 F/F(0), durations of approximately 112 ms at half-maximum amplitude, and were abolished by the InsP(3)R inhibitor 2-aminoethoxydiphenyl borate. The protonophore carbonyl cyanide 3-chloropheylhydrazone and complex I inhibitor rotenone each depolarized DeltaPsi(M) to prevent mitochondrial Ca2+ uptake and attenuated Ca2+ puffs by approximately 66 or approximately 60%, respectively. The mitochondrial uniporter inhibitor, RU360, attenuated Ca2+ puffs by approximately 62%. The "fast" Ca2+ chelator 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid acted like mitochondria to prolong InsP(3)-mediated Ca2+ release suggesting that mitochondrial influence is via their Ca2+ uptake facility. These results indicate Ca2+ uptake occurs quickly enough to influence InsP(3)R communication at the intra-cluster level and that mitochondria regulate both local and global InsP(3)-mediated Ca2+ signals.

The ELAV Protein HuD Stimulates Cap-Dependent Translation in a Poly(A)- and eIF4A-Dependent Manner

The RNA-binding protein HuD promotes neuronal differentiation by an unknown mechanism. Here we identify an enhancer function of HuD in translation. Translation stimulation by HuD requires both a 3' poly(A) tail and a 5' m(7)G cap structure. We also show that HuD directly interacts with eIF4A. This interaction and the poly(A)-binding activity of HuD are critical for its translational enhancer function because HuD-eIF4A- and HuD-poly(A)-binding mutants fail to stimulate translation. We show that translation of HCV IRES mRNA, which is eIF4A independent, is not stimulated by HuD. We also find that the eIF4A and poly(A)-binding activities of HuD are not only important for stimulating translation but also are essential for HuD-induced neurite outgrowth in PC12 cells. This example of cap-dependent translational regulation might explain at least in part how HuD triggers the induction of neuronal differentiation.

Role of Varp, a Rab21 exchange factor and TI‐VAMP/VAMP7 partner, in neurite growth

The vesicular soluble N-ethylmaleimide-sensitive factor attachment protein receptor (SNARE) tetanus neurotoxin-insensitive vesicle-associated membrane protein (TI-VAMP/VAMP7) was previously shown to mediate an exocytic pathway involved in neurite growth, but its regulation is still largely unknown. Here we show that TI-VAMP interacts with the Vps9 domain and ankyrin-repeat-containing protein (Varp), a guanine nucleotide exchange factor (GEF) of the small GTPase Rab21, through a specific domain herein called the interacting domain (ID). Varp, TI-VAMP and Rab21 co-localize in the perinuclear region of differentiating hippocampal neurons and transiently in transport vesicles in the shaft of neurites. Silencing the expression of Varp by RNA interference or expressing ID or a form of Varp deprived of its Vps9 domain impairs neurite growth. Furthermore, the mutant form of Rab21, defective in GTP hydrolysis, enhances neurite growth. We conclude that Varp is a positive regulator of neurite growth through both its GEF activity and its interaction with TI-VAMP.

Simulated microgravity promotes cellular senescence via oxidant stress in rat PC12 cells

Microgravity has a unique effect on biological organisms. Organs exposed to microgravity display cellular senescence, a change that resembles the aging process. To directly investigate the influence of simulated microgravity on neuronal original rat PC12 cells, we used a rotary cell culture system that simulates the microgravity environment on the earth. We found that simulated microgravity induced partial G1 phase arrest, upregulated senescence-associated β-galactosidase (SA-β-gal) activity, and activated both p53 and p16 protein pathways linked to cell senescence. The amount of reactive oxygen species (ROS) was also increased. The activity of intracellular antioxidant enzymes, such as superoxide dismutase (SOD), glutathione peroxidase (GSH-Px), and catalase (CAT), was all significantly increased at 12 h after the microgravity onset, yet decreased at 96 h. Furthermore, concomitant block of ROS by the antioxidant N-acetylcysteine significantly inhibited the microgravity-induced upregulation of SA-β-gal activity. These results suggest that exposure to simulated microgravity induces cellular senescence in PC12 cells via an increased oxidant stress.

Exocytosis of Post-Golgi Vesicles Is Regulated by Components of the Endocytic Machinery

Post-Golgi vesicles target and deliver most biosynthetic cargoes to the cell surface. However, the molecules and mechanisms involved in fusion of these vesicles are not well understood. We have employed a system to simultaneously monitor release of luminal and membrane biosynthetic cargoes from individual post-Golgi vesicles. Exocytosis of these vesicles is not calcium triggered. Release of luminal cargo can be accompanied by complete, partial, or no release of membrane cargo. Partial and no release of membrane cargo of a fusing vesicle are fates associated with kiss-and-run exocytosis, and we find that these are the predominant mode of post-Golgi vesicle exocytosis. Partial cargo release by post-Golgi vesicles occurs because of premature closure of the fusion pore and is modulated by the activity of clathrin, actin, and dynamin. Our results demonstrate that these components of the endocytic machinery modulate the nature and extent of biosynthetic cargo delivery by post-Golgi vesicles at the cell membrane.

A novel inducible tyrosine kinase receptor to regulate signal transduction and neurite outgrowth

Nervous system growth factor gene delivery can promote axonal growth and prevent cell death in animal models of CNS trauma and neurodegenerative diseases. The ability to regulate growth factor expression or signaling pathways downstream from growth factor receptors remains a desirable goal for in vivo gene transfer. To achieve precise pharmacological modulation of neurotrophin activity, we have generated a chimeric trkA receptor (ItrkA) by fusing the entire intracellular domain of the trkA high-affinity NGF receptor to two intracellular, modified FK506 binding domains for the synthetic small molecule dimerization ligand AP20187. Rat PC12 cells were transduced with lentiviral vectors containing ItrkA and green fluorescent protein (GFP; via an internal ribosome entry site). Treatment of ItrkA-expressing PC12 cells with AP20187 induced neurite outgrowth and differentiation in a time- and dose-dependent fashion, with a half-maximal response at a concentration of 1 nM AP20187. Seventy percent of cells responded to AP20187 by day 3. Western blots demonstrated that AP20187 treatment resulted in phosphorylation of Erk1/2 and Akt in ItrkA-transduced PC12 cells but not in nontransduced, naïve cells. Phosphorylation levels were comparable to levels obtained with 50 ng/ml nerve growth factor (NGF). In addition, ItrkA lentiviral transduction of primary E15 dorsal root ganglion neurons significantly increased neurite growth three- to fourfold in the presence of AP20187 compared with control GFP transduced and naïve neurons. These results demonstrate that small ligand-induced dimerization of the intracellular domain of trkA can efficiently simulate the biological activity of NGF and provide a means to regulate intracellular neurotrophin receptor signaling.

A selective small molecule glucagon-like peptide-1 secretagogue acting via depolarization-coupled Ca2+ influx

Glucagon-like peptide-1 (GLP-1) is an incretin hormone that potentiates insulin secretion in a glucose-dependent manner. Selective GLP-1 secretagogue would be one of the potential therapeutic targets for type 2 diabetes. Here, we describe a newly identified small molecule compound (compound A) that stimulates secretion of GLP-1 in murine enteroendocrine cell lines, STC-1 and GLUTag cells, and in primary cultured fetal rat intestinal cells (FRIC). The underlying mechanism by which compound A stimulated GLP-1 secretion was also examined. Compound A stimulated GLP-1 secretion from STC-1 cells in a concentration-dependent manner, and also from GLUTag cells and FRIC. The action of compound A was selective against other tested endocrine functions such as secretion of insulin from rat islets, growth hormone from rat pituitary gland cells, and norepinephrine from rat PC-12 cells. In STC-1 cells, the compound A-stimulated GLP-1 secretion was neither due to cyclic AMP production nor to Ca(2+) release from intracellular stores, but to extracellular Ca(2+) influx. The response was inhibited by the presence of either L-type Ca(2+) channel blockers or K(+) ionophore. Perforated-patch clamp study revealed that compound A induces membrane depolarization. These results suggest that neither Galphas- nor Galphaq-coupled signaling account for the mechanism of action, but depolarization-coupled Ca(2+) influx from extracellular space is the primary cause for the GLP-1 secretion stimulated by compound A. Identifying a specific target molecule for compound A will reveal a selective regulatory pathway that leads to depolarization-mediated GLP-1 secretion.

Neuroendocrine regulatory peptide-1 and -2: novel bioactive peptides processed from VGF.

Neuroendocrine regulatory peptides (NERP)-1 and NERP-2 are derived from distinct regions of VGF, a neurosecretory protein that was originally identified as a product of a nerve growth factor-responsive gene in rat PC12 cells. The amino acid length of human NERP-1 is 26, and that of rat NERP-1 is 25. Human and rat NERP-2 are both 38 amino acid peptides. NERPs colocalize with vasopressin in the storage granules of the paraventricular and supraoptic nuclei in the hypothalamus of both rats and humans. Administration of NERPs suppresses hypertonic saline- or angiotensin II-induced vasopressin release from the hypothalamus and pituitary. Thus, VGF is a precursor of multiple bioactive peptides with diverse neuroendocrine functions, and NERPs are novel hypothalamic peptides involved in the control of body fluid homeostasis by regulating vasopressin release.

Characterization of transcriptional regulation of 5-HT3B in rat PC-12 cells: the role of glucocorticoids

Characterization of transcriptional regulation of 5-HT3B in rat PC-12 cells: the role of glucocorticoids

Kaempferol Derivatives Prevent Oxidative Stress–Induced Cell Death in a DJ-1–Dependent Manner

DJ-1, a causative gene product of a familial form of Parkinson's disease (PD), PARK7, plays a role in anti-oxidative stress, and loss of its function is thought to result in the onset of PD. Superfluous oxidation of cysteine at amino acid 106 (C106) of DJ-1 renders DJ-1 inactive, and such oxidized DJ-1 was observed in patients with the sporadic form of PD. In this study, we examined the relationship between DJ-1 and compounds extracted from traditional Chinese medicines possessing anti-oxidant activity. Of the 12 compounds tested, 5 were found to specifically bind to the C106 region by using a quartz crystal microbalance. Although 4 compounds prevented rat PC12 and primary neuronal cells from undergoing H2O2-induced cell death, the protective activity of 2 compounds, kaempferol 3-O-beta-rutinoside and 6-hydroxykaempferol 3,6-di-O-beta-D-glucoside, was diminished in cells transfected with siRNA targeting DJ-1, indicating DJ-1-dependent reaction of these compounds. Furthermore, these compounds reduced the level of reactive oxygen species and restored tyrosine hydroxylase activity that had been induced and compromised, respectively, by treatment of cells with H2O2. The results suggest that these compounds are useful lead compounds for PD therapy.

Toponomics Analysis of Functional Interactions of the Ubiquitin Ligase PAM (Protein Associated with Myc) during Spinal Nociceptive Processing

Protein associated with Myc (PAM) is a giant E3 ubiquitin ligase of 510 kDa. Although the role of PAM during neuronal development is well established, very little is known about its function in the regulation of synaptic strength. Here we used multiepitope ligand cartography (MELC) to study protein network profiles associated with PAM during the modulation of synaptic strength. MELC is a novel imaging technology that utilizes biomathematical tools to describe protein networks after consecutive immunohistochemical visualization of up to 100 proteins on the same sample. As an in vivo model to modulate synaptic strength we used the formalin test, a common model for acute and inflammatory pain. MELC analysis was performed with 37 different antibodies or fluorescence tags on spinal cord slices and led to the identification of 1390 PAM-related motifs that distinguish untreated and formalin-treated spinal cords. The majority of these motifs related to ubiquitin-dependent processes and/or the actin cytoskeleton. We detected an intermittent colocalization of PAM and ubiquitin with TSC2, a known substrate of PAM, and the glutamate receptors mGluR5 and GLUR1. Importantly these complexes were detected exclusively in the presence of F-actin. A direct PAM/F-actin interaction was confirmed by colocalization and cosedimentation. The binding of PAM toward F-actin varied strongly between the PAM splice forms found in rat spinal cords. PAM did not ubiquitylate actin or alter actin polymerization and depolymerization. However, F-actin decreased the ubiquitin ligase activity of purified PAM. Because PAM activation is known to involve its translocation, the binding of PAM to F-actin may serve to control its subcellular localization as well as its activity. Taken together we show that defining protein network profiles by topological proteomics analysis is a useful tool to identify previously unknown protein/protein interactions that underlie synaptic processes.

M-Ras evolved independently of R-Ras and its neural function is conserved between mammalian and ascidian, which lacks classical Ras

The Ras family small GTPases play a variety of essential roles in eukaryotes. Among them, classical Ras (H-Ras, K-Ras, and N-Ras) and its orthologues are conserved from yeast to human. In ascidians, which phylogenetically exist between invertebrates and vertebrates, the fibroblast growth factor (FGF)–Ras–MAP kinase signaling is required for the induction of neural system, notochord, and mesenchyme. Analyses of DNA databases revealed that no gene encoding classical Ras is present in the ascidians, Ciona intestinalis and Halocynthia roretzi, despite the presence of classical Ras-orthologous genes in nematode, fly, amphioxus, and fish. By contrast, both the ascidians contain single genes orthologous to Mras, Rras, Ral, Rap1, and Rap2. A single Mras orthologue exists from nematode to mammalian. Thus, Mras evolved in metazoans independently of other Ras family genes such as Rras. Whole-mount in situ hybridization showed that C. intestinalis Mras orthologue ( Ci-Mras) was expressed in the neural complex of the ascidian juveniles after metamorphosis. Knockdown of Ci-Mras with morpholino antisense oligonucleotides in the embryos and larvae resulted in undeveloped tails and neuronal pigment cells, abrogation of the notochord marker brachyury expression, and perturbation of the neural marker Otx expression, as has been shown in the experiments of the FGF–Ras–MAP kinase signaling inhibition. Mammalian Ras and M-Ras mediate nerve growth factor-induced neuronal differentiation in rat PC12 cells by activating the ERK/MAP kinase pathway transiently and sustainedly, respectively. Activated Ci-M-Ras bound to target proteins of mammalian M-Ras and Ras. Exogenous expression of an activated Ci-M-Ras in PC12 cells caused ERK activation and induced neuritogenesis via the ERK pathway as do mammalian M-Ras and Ras. These results suggest that the ascidian M-Ras orthologue compensates for lacked classical Ras and plays essential roles in neurogenesis in the ascidian.