Vasoactive Intestinal Peptide and Forskolin Stimulate Interleukin 6 Production by Rat Cortical Astrocytes in Culture via a Cyclic AMP-Dependent, Prostaglandin-Independent Mechanism

Abstract: In this study we analyzed the involvement of the cyclic AMP (cAMP)-protein kinase A system in the regulation of interleukin 6 production by cultured cortical astrocytes. Vasoactive intestinal peptide strongly increased, in a dose-dependent manner, interleukin 6 production. This effect was reduced when protein kinase A was blocked by KT-5720; it was not affected by calphostin C, a protein kinase C inhibitor. Forskolin caused a concentration-dependent increase in interleukin 6 release that was also inhibited by KT-5720. Because prostaglandins are believed to play a role in interleukin 6 production, we tried to determine whether the stimulatory effects of vasoactive intestinal peptide and forskolin on cytokine release might be mediated by stimulation of prostaglandin production in cortical astrocytes. Vasoactive intestinal peptide did not increase the production of either prostaglandin E₂ or F_2α. Conversely, forskolin concentration-dependently stimulated the production of both prostaglandins, an effect that was blocked by indomethacin. Indomethacin did not affect either vasoactive intestinal peptide- or forskolin-stimulated interleukin 6 production. To exclude the possibility that prostaglandins participate in interleukin 6 production induced by forskolin, we tested prostaglandins E₂ and F_2α. The former was completely ineffective in eliciting the cytokine production, whereas prostaglandin F_2α slightly increased interleukin 6 production only at the highest concentrations. 8-Bromo-cAMP and dibutyryl-cAMP stimulated interleukin 6 production to a lesser extent than vasoactive intestinal peptide and forskolin. In conclusion, we provide evidence that vasoactive intestinal peptide increases interleukin 6 production by astrocytes through the stimulation of the cAMP-protein kinase A pathway, an effect that is reproduced by cAMP analogues. In addition, we point out that prostaglandins are not involved in vasoactive intestinal peptide- and forskolin-mediated induction of interleukin 6 production in cultured astrocytes.     

Pituitary adenylate cyclase-activating polypeptide is up-regulated in cortical pyramidal cells after focal ischemia and protects neurons from mild hypoxic/ischemic damage

The protective effect of pituitary adenylate cyclase-activating polypeptide (PACAP) in stroke models is poorly understood. We studied patterns of PACAP, vasoactive intestinal peptide, and the PACAP-selective receptor PAC1 after middle cerebral artery occlusion and neuroprotection by PACAP in cortical cultures exposed to oxygen/glucose deprivation (OGD). Within hours, focal ischemia caused a massive, NMDA receptor (NMDAR)-dependent up-regulation of PACAP in cortical pyramidal cells. PACAP expression dropped below the control level after 2 days and was normalized after 4 days. Vasoactive intestinal peptide expression was regulated oppositely to that of PACAP. PAC1 mRNA showed ubiquitous expression in neurons and astrocytes with minor changes after ischemia. In cultured cortical neurons PACAP27 strongly activated Erk1/2 at low and p38 MAP kinase at higher nanomolar concentrations via PAC1. In astrocyte cultures, effects of PACAP27 on Erk1/2 and p38 were weak. During OGD, neurons showed severely reduced Erk1/2 activity and dephosphorylation of Erk1/2-regulated Ser112 of pro-apoptotic Bad. PACAP27 stimulation counteracted Erk1/2 inactivation and Bad dephosphorylation during short-term OGD but was ineffective after expanded OGD. Consistently, PACAP27 caused MEK-dependent neuroprotection during mild but not severe hypoxic/ischemic stress. While PACAP27 protected neurons at 1–5 nmol/L, full PAC1 activation by 100 nmol/L PACAP exaggerated hypoxic/ischemic damage. PACAP27 stimulation of astrocytes increased the production of Akt-activating factors and conferred ischemic tolerance to neurons. Thus, ischemia-induced PACAP may act via neuronal and astroglial PAC1. PACAP confers protection to ischemic neurons by maintaining Erk1/2 signaling via neuronal PAC1 and by increasing neuroprotective factor production via astroglial PAC1.     

Protein Kinase Inhibitors Block Neurite Outgrowth from Explants of Goldfish Retina

A role for protein phosphorylation in the process of neurite outgrowth has been inferred from many studies of the effects of protein kinase inhibitors and activators on cultured neurotumor cells and primary neuronal cells from developing brain or ganglia. Here we re-examine this issue, using a culture system derived from a fully differentiated neuronal system undergoing axonal regeneration—the explanted goldfish retina following optic nerve crush. Of the relatively non-selective protein kinase inhibitors employed, H7, staurosporine and K_252a were found to block neurite outgrowth, whereas HA1004 had no effect, a result which appears to rule out a critical role for protein kinase A. The more selective protein kinase C inhibitors, sphingosine, calphostin C and Ro-31-8220 were all inhibitory, as was prolonged treatment with phorbol ester and the protein phosphatase inhibitor okadaic acid. These results are in support of a role for protein kinase C in axonal regrowth.     

Neuronal differentiation is triggered by oleic acid synthesized and released by astrocytes

Unlike in the adult brain, the newborn brain specifically takes up serum albumin during the postnatal period, coinciding with the stage of maximal brain development. Here we report that albumin stimulates oleic acid synthesis by astrocytes from the main metabolic substrates available during brain development. Oleic acid released by astrocytes is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones. Oleic acid promotes axonal growth, neuronal clustering, and expression of the axonal growth-associated protein-43, GAP-43; all these observations indicating neuronal differentiation. The effect of oleic acid on GAP-43 synthesis is brought about by the activation of protein kinase C, since it was prevented by inhibitors of this kinase, such as H-7, polymyxin or sphingosine. The expression of GAP-43 was significantly increased in neurons co-cultured with astrocytes by the presence of albumin indicating that neuronal differentiation takes place in the presence of oleic acid synthesized and released by astrocytes in situ. In conclusion, during brain development the presence of albumin could play an important role by triggering the synthesis and release of oleic acid by astrocytes, which induces neuronal differentiation.     

Calcium/calmodulin-dependent protein kinase II expression in motor neurons: Effect of axotomy

Although Ca²⁺/calmodulin-dependent (CaM) protein kinase II isoforms are present in the nervous system in high amounts, many aspects of in vivo expression, localization, and function remain unexplored. During development, CaM kinase IIα and IIβ are differentially expressed. Here, we examined CaM kinase II isoforms in Sprague-Dawley rat sciatic motor neurons before and after axotomy. We cut the L4-5 spinal nerves unilaterally and exposed the proximal nerve stumps to a fluoroprobe, to retrogradely label the neurons of origin. Anti-CaM kinase IIβ antibody showed immunoreactivity in motor neurons, which decreased to low levels by 4 days after axotomy. We found a similar response by in situ hybridization with riboprobes. The decrease in expression of mRNA and protein was confined to fluorescent motor neurons. For CaM kinase IIα, in situ hybridization showed that the mRNA was in sciatic motor neurons, with a density unaffected by axotomy. However, these neurons were also enlarged, suggesting an up-regulation of expression. Northern blots confirmed an mRNA increase. We were unable to find CaM kinase IIα immunoreactivity before or after axotomy in sciatic motor neuron cell bodies, suggesting that CaM kinase IIα is in the axons or dendrites, or otherwise unavailable to the antibody. Using rats with crush lesions, we radiolabeled axonal proteins being synthesized in the cell body and used two-dimensional polyacrylamide gel electrophoresis with Western blots to identify CaM kinase IIα as a component of slow axonal transport. This differential regulation and expression of kinase isoforms suggests separate and unique intracellular roles. Because we find CaM kinase IIβ down-regulates during axonal regrowth, its role in these neurons may be related to synaptic transmission. CaM kinase IIα appears to support axonal regrowth. © 1997 John Wiley & Sons, Inc. J Neurobiol 33: 796–810, 1997     

Nocturnal expression of phosphorylated-ERK1/2 in gastrin-releasing peptide neurons of the rat suprachiasmatic nucleus

Extracellular regulated kinase (ERK) signalling is believed to play roles in various aspects of circadian clock mechanisms. In this study, we show in rat that the nuclear versus cytoplasmic intracellular distribution of the phosphorylated forms of ERK1/2 (P-ERK1/2) in the central clock, namely the suprachiasmatic nucleus (SCN), is proportionally constant across the light/dark cycle while the spatial distribution and neurochemical phenotype of cells expressing these activated forms are time-regulated according to a daily rhythm and light-regulated. P-ERK1/2 was exclusively found in neuronal elements. At daytime, it was detected throughout the dorsoventral extent of the SCN, partly within neurons synthesizing either arginine-vasopressin or vasoactive intestinal peptide (VIP). At night time, it was segregated in the ventrolateral aspect of the nucleus, within a cluster of cells 45% of which were gastrin-releasing peptide (GRP) neurons with or without co-localization with VIP. After a light pulse at night, expression of P-ERK1/2 increased in GRP neurons but also appeared in a population of neurons that stained for VIP only. These data show that the GRP neurons are closely associated with ERK1/2 activation at night and point to the importance of ERK1/2 signalling not only in intra-SCN transmission of photic information but also in maintenance of neuronal rhythms in the SCN.     

GLT1, glial glutamate transporter, is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain.

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high-affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin-like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin-positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development.     

Astrocyte-synthesized oleic acid behaves as a neurotrophic factor for neurons.

Unlike in the adult brain, the newborn brain specifically takes up serum albumin during the postnatal period, coinciding with the stage of maximal brain development. Here we shall summarize our knowledge about the role played by albumin in brain development. The role of this protein in brain development is intimately related to its ability to carry fatty acids. Thus, albumin stimulates oleic acid synthesis by astrocytes from the main metabolic substrates available during brain development. Astrocytes internalize albumin in vesicle-like structures by receptor-mediated endocytosis, which is followed by transcytosis, including passage through the endoplasmic reticulum (ER). The presence of albumin in the ER activates the sterol regulatory element-binding protein-1 (SREBP-1) and increases stearoyl-CoA 9-desaturase (SCD) mRNA, the key enzyme in oleic acid synthesis. Oleic acid released by astrocytes is used by neurons for the synthesis of phospholipids and is specifically incorporated into growth cones. In addition, oleic acid promotes axonal growth, neuronal clustering, and the expression of the axonal growth associated protein, GAP-43. All of these observations indicate neuronal differentiation. The effect of oleic acid on GAP-43 synthesis is brought about by the activation of protein kinase C. The expression of GAP-43 is significantly increased by the presence of albumin in neurons co-cultured with astrocytes, indicating that neuronal differentiation takes place by the presence of oleic acid synthesized and released by astrocytes in situ. In conclusion, during brain development the presence of albumin could play an important role by triggering the synthesis and release of oleic acid by astrocytes, thereby inducing neuronal differentiation.     

Epidermal growth factor induces oxidative neuronal injury in cortical culture

Recently, we have demonstrated that certain neurotrophic factors can induce oxidative neuronal necrosis by acting at the cognate tyrosine kinase-linked receptors. Epidermal growth factor (EGF) has neurotrophic effects via the tyrosine kinase-linked EGF receptor (EGFR), but its neurotoxic potential has not been studied. Here, we examined this possibility in mouse cortical culture. Exposure of cortical cultures to 1-100 ng/ml EGF induced gradually developing neuronal death, which was complete in 48-72 h; no injury to astrocytes was noted. Electron microscopic findings of EGF-induced neuronal death were consistent with necrosis; severe mitochondrial swelling and disruption of cytoplasmic membrane occurred, whereas nuclei appeared relatively intact. The EGF-induced neuronal death was accompanied by increased free radical generation and blocked by the anti-oxidant Trolox. Suggesting mediation by the EGFR, an EGFR tyrosine kinase-specific inhibitor, C56, attenuated EGF-induced neuronal death. In addition, inhibitors of extracellular signal-regulated protein kinase 1/2 (Erk-1/2) (PD98056), protein kinase A (H89), and protein kinase C (GF109203X) blocked EGF-induced neuronal death. A p38 mitogen-activated protein kinase inhibitor (SB203580) or glutamate antagonists (MK-801 and 6-cyano-7-nitroquinoxaline-2,3-dione) showed no protective effect. The present results suggest that prolonged activation of the EGFR may trigger oxidative neuronal injury in central neurons.     

Chemical coding of neurons projecting to pelvic viscera in the male guinea pig: a study by retrograde transport and immunohistochemistry

Summary Retrograde transport studies using Fast Blue dye demonstrated that the ductus deferens, seminal vesicle, prostate and rectum, but not the urinary bladder of the male guinea pig are at least in part innervated by the anterior major pelvic ganglion. In the ductus deferens, seminal vesicle and prostate innervation is derived from ipsilateral and contralateral ganglia. In addition to retrograde studies, dye-filled neurons were analysed immunohistochemically for neuronal markers and associations with specifically identified neuronal projections. Neurons of the ganglion projecting to the ductus deferens either contained tyrosine hydroxylase alone, tyrosine hydroxylase and neuropeptide Y, neuropeptide Y alone, neuropeptide Y and vasoactive intestinal peptide, or vasoactive intestinal peptide alone. These neurons were associated with three classes of neuronal projections, substance P-, leucine-enkephalin-, and methionine-enkephalin-immunoreactive. Neurons projecting to the seminal vesicles were similar to the neurons supplying the ductus deferens, except none of the seminal vesicle-specific neurons exhibited vasoactive intestinal peptide immunoreactivity. Neurons supplying the prostate were immunoreactive for either tyrosine hydroxylase or neuropeptide Y; these neurons were infrequently associated with the three classes of varicose neuronal projections. Neurons projecting to the rectum contained neuropeptide Y and were only associated with methionine-enkephalin immunoreactive neuronal projections in one animal.     

Regulation of heat shock protein 70 release in astrocytes: role of signaling kinases

The ability to mount a successful stress response in the face of injury is critical to the long-term viability of individual cells and to the organism in general. The stress response, characterized in part by the upregulation of heat shock proteins, is compromised in several neurodegenerative disorders and in some neuronal populations, including motoneurons (MNs). Because astrocytes have a greater capacity than neurons to survive metabolic stress, and because they are intimately associated with the regulation of neuronal function, it is important to understand their stress response, so that we may to better appreciate the impact of stress on neuronal viability during injury or disease. We show that astrocytes subjected to hyperthermia upregulate Hsp/c70 in addition to intracellular signaling components including activated forms of extracellular-signal-regulated kinase (ERK1/2), Akt, and c-jun N-terminal kinase/stress activated protein kinase (JNK/SAPK). Furthermore, astrocytes release increasing amounts of Hsp/c70 into the extracellular environment following stress, an event that is abrogated when signaling through the ERK1/2 and phosphatidylinositol-3 kinase (PI3K) pathways is compromised and enhanced by inhibition of the JNK pathway. Last, we show that the Hsp/c70 is released from astrocytes in exosomes. Together, these data illustrate the diverse regulation of stress-induced Hsp/c70 release in exosomes, and the way in which the balance of activated signal transduction pathways affects this release. These data highlight how stressful insults can alter the microenvironment of an astrocyte, which may ultimately have implications for the survival of neighboring neurons.     

Specific Proteolysis of Neuronal Protein GAP-43 by Calpain: Characterization,Regulation, and Physiological Role

The mechanism of specific proteolysis of the neuronal protein GAP-43 in axonal terminals has been investigated. In synaptic terminals in vivo and in synaptosomes in vitro GAP-43 is cleaved only at the single peptide bond formed by Ser41; this is within the main effector domain of GAP-43. Proteolysis at this site involves the cysteine calcium-dependent neutral protease calpain. The following experimental evidences support this conclusion: 1) calcium-dependent proteolysis of GAP-43 in synaptosomes is insensitive to selective inhibitor of micro-calpain (PD151746), but it is completely blocked by micro- and m-calpain inhibitor PD150606; 2) GAP-43 proteolysis in the calcium ionophore A23187-treated synaptosomes is activated by millimolar concentration of calcium ions; 3) the pattern of fragmentation of purified GAP-43 by m-calpain (but not by micro-calpain) is identical to that observed in synaptic terminals in vivo. GAP-43 phosphorylated at Ser41 by protein kinase C (PKC) is resistant to the cleavage by calpain. In addition, calmodulin binding to GAP-43 decreases the rate of calpain-mediated GAP-43 proteolysis. Our results indicate that m-calpain-mediated GAP-43 proteolysis regulated by PKC and calmodulin is of physiological relevance, particularly in axonal growth cone guidance. We suggest that the function of the N-terminal fragment of GAP-43 (residues 1-40) formed during cleavage by m-calpain consists in activation of neuronal heterotrimeric GTP-binding protein G(o); this results in growth cone turning in response to repulsive signals.     

The activation loop phosphorylation of protein kinase D is an early marker of neuronal DNA damage

In neurons, DNA damage induces protein synthesis-dependent apoptosis mediated by the mitochondrial intrinsic cell-death pathway. Signal transduction cascades activated by genotoxic stress upstream of the mitochondria are largely unknown. We identified protein kinase D (PKD) as one of the earliest markers of neuronal DNA damage. Phosphorylation of the PKD-activation domain could be detected within 15 min of genotoxic stress and was concurrent with ataxia telangiectasia-mutated (ATM) activation. PKD stimulation was selective to DNA damage and did not occur with other stress stimuli examined. In vivo, both young and adult rats showed increased levels of phosphorylated PKD in neuronal tissues after injection of DNA-toxin etoposide. These results indicate that PKD activation is an early neuronal response to DNA damage, suggesting that signaling downstream of PKD may be critical for neuronal survival after genotoxic stress.     

Cellular distribution and developmental expression of AMP-activated protein kinase isoforms in mouse central nervous system

The mammalian AMP-activated protein kinase is a heterotrimeric serine/threonine protein kinase with multiple isoforms for each subunit (alpha, beta, and gamma) and is activated under conditions of metabolic stress. It is widely expressed in many tissues, including the brain, although its expression pattern throughout the CNS is unknown. We show that brain mRNA levels for the alpha2 and beta2 subunits were increased between embryonic days 10 and 14, whereas expression of alpha1, beta1, and gamma1 subunits was consistent at all ages examined. Immunostaining revealed a mainly neuronal distribution of all isoforms. The alpha2 catalytic subunit was highly expressed in neurons and activated astrocytes, whereas the alpha1 catalytic subunit showed low expression in neuropil. The gamma1 noncatalytic subunit was highly expressed by neurons, but not by astrocytes. Expression of the beta1 and beta2 noncatalytic subunits varied, but some neurons, such as granule cells of olfactory bulb, did not express detectable levels of either beta isoform. Preferential nuclear localization of the alpha2, beta1, and gamma1 subunits suggests new functions of the AMP-activated protein kinase, and the different expression patterns and cellular localization between the two catalytic subunits alpha1 and alpha2 point to different physiological roles.     

Effects of neurotransmitters and peptides on phospholipid hydrolysis in sympathetic and sensory neurons

The effects of neurotransmitters and peptides on phosphoinositide hydrolysis were studied by measuring [3H]inositol monophosphate ([3H]IP) and protein kinase C (PKC) activity in the sympathetic and sensory neuronal cultures of the chick embryo. [3H]IP was increased in sympathetic neurons by acetylcholine (ACh), muscarine, serotonin (5-HT), and vasoactive intestinal polypeptide. ACh, muscarine, 5-HT, and bradykinin increased [3H]IP in sensory neuronal cultures. Dopamine, norepinephrine, histamine, and nerve growth factor did not stimulate [3H]IP formation in both cultures. ACh and phorbol 12,13-dibutyrate (PDB) increased the PKC activity by two- to sevenfold in the particulate fraction of both cultures. In sympathetic neurons, PKC activity was increased in the particulate fraction; activity in the cytosolic fraction was not affected. There was a 50% decline in the protein kinase C activity of the cytosolic fraction after PDB and ACh treatment of sensory cultures. The decline in PKC activity in the cytosolic fraction was attributed to the presence of nonneuronal cells in sensory cultures. To confirm this, the enzyme activity was determined in tissues that contain a heterogeneous population of cells. PDB activated PKC in the adrenal medulla and the brain of the rat. In both tissues there was a 65% decline in the PKC activity of the cytosolic fraction and about a 75% increase in the particulate fraction. We conclude that the mechanism of activation of protein kinase C in pure cultures of sympathetic neurons is different than in tissues containing a mixed population of neurons and nonneuronal cells.     

Calcium/Diacylglycerol-Dependent Protein Kinase and Its Major 87-Kilodalton Protein Substrate Are Differentially Distributed in Rat Basal Ganglia

When brain tissue is subjected to subcellular fractionation, both calcium/diacylglycerol-dependent protein kinase (protein kinase C) and an 87-kilodalton (kDa) protein substrate for this enzyme are enriched in the crude nerve terminal fraction. The present study, using chemical and surgical lesions of neurons in the rat neostriatum and substantia nigra, has examined whether the 87-kDa protein is colocalized with protein kinase C in identified neurons and nerve terminals. Our results show that, in the basal ganglia, protein kinase C is highly enriched in local striatal neurons and the striatonigral fibers and terminals. In contrast, the 87-kDa protein appears to be widely and evenly distributed in both neuronal and nonneuronal cells. The 87-kDa protein may therefore mediate functions of protein kinase C not restricted to nerve terminals.     

GLT1, glial glutamate transporter,is transiently expressed in neurons and develops astrocyte specificity only after midgestation in the ovine fetal brain

Glutamate transport is a primary mechanism for regulating extracellular levels of glutamate in the central nervous system. GLT1, the most abundant of the known high‐affinity glutamate transporters, is found exclusively in astrocytes in adult brain of several species, but we and others have recently identified neurons that transiently express GLT1 protein in the developing brain. We now demonstrate the development of cell type specificity for GLT1 expression at 60, 71, and 136 days' gestation in the developing sheep brain (term = 145 days). At 60 and 71 days of gestation, GLT1 colocalizes with calbindin in Purkinje cells in the cerebellum, and this expression pattern has a novel distribution that is reminiscent of the parasagittal zebrin‐like bands. GLT1 immunoreactivity simultaneously occurs in periventricular white matter, anterior commissure, and striatal white matter, dissipating by 136 days. GLT1 protein expression within astrocytes is developmentally regulated, appearing first in vimentin positive radial glia at 60 and 71 days and then switching to GFAP positive parenchymal and perivascular astrocytes at 136 days. Expression of GLT1 in subsets of vimentin‐positive astrocytes persists in white matter but not in cortex. These results identify a novel compartmentation within cerebellar cortex and neuronal and axonal pathway localization of GLT1, suggesting the participation of this glutamate transporter in the development of the topographic organization of cerebellar cortex and a transient neuronal function for GLT1 in developing brain. In addition, GLT1 expression is highly plastic, being neither exclusively astroglial nor uniformly expressed in different populations of astrocytes during brain development. © 1999 John Wiley & Sons, Inc. J Neurobiol 39: 515–526, 1999     

P311 accelerates nerve regeneration of the axotomized facial nerve

In axotomized adult neurons, a process of axonal regrowth and re-establishment of the neuronal function has to be activated. Developmentally regulated factors may be reactivated during neuronal regeneration. Here we identify a gene, previously designated P311, that is up-regulated in the axotomized facial motoneurons. Ectopically expressed P311 localizes in the cytoplasm and the nucleus. Over-expression of P311 induces p21(WAF1/Cip1) expression, leading PC12 cells to differentiate and to have neuron-like morphologies. Adenovirus-mediated P311 gene transfer promotes neurite outgrowth of postnatal dorsal root ganglion neurons and embryonic hippocampal neurons in vitro. This effect is abolished by the activation of Rho kinase. P311 also facilitates nerve regeneration following facial nerve injury in vivo. Our data provide evidence that genes involved in the differentiation process contribute to the regeneration of injured mature neurons, and may provide a practical molecular target.