Rho-associated kinase (ROCK) inhibitor, Y27632, promotes neurite outgrowth in PC12 cells in the absence of NGF

Neurotrophins support neuronal survival and axonal regeneration after injury. To test whether local expression of Neurotrophin-3 (NT-3) would elicit axonal regeneration we lesioned the corticospinal tract (CST) at the level of the hindbrain and measured the number of axons that would grow from the unlesioned CST to the contralateral side where NT-3 was over expressed at the lumbar level of the spinal cord. An adenoviral vector that carried the rat NT-3 gene and the NGF signal peptide driven by the EF1α promoter (Adv.EF-NT-3) was used. This model enabled us to test the effects of NT-3 on axonal regeneration without confounding injury processes. Biotinylated dextran amine (BDA) was injected into the rat cortex on unlesioned side to mark CST axons 10 days postlesion. Adenoviral vectors (1 × 10⁹ pfu, Adv.EF-NT-3 or Adv.EF-LacZ) were delivered to lumbar spinal cord by retrograde transport from the sciatic nerve 4 days later. Histological examination 3 weeks later revealed that more BDA-labelled axons had grown from the unlesioned CST to the denervated side at the lumbar level. Morphometric measurements showed that a significantly larger number of BDA-labelled CST axons ( p  < 0.001) were present in the animals that were treated with Adv.EF-NT-3 than those treated with Adv.EF-LacZ. These data demonstrate that local expression of NT-3 will support axonal regeneration in the injured spinal cord without adverse effects and suggest that gene delivery of neurotrophins may be an effective strategy for nervous system repair after injury.
Acknowledgements:   Funded by NIH Grant NS35280 and by Mission Connect of the TIRR Foundation.     

ErbB-4 activation promotes neurite outgrowth in PC12 cells

Neu differentiation factor (NDF; also known as neuregulin) induces a pleiotropic cellular response that is cell type-dependent. NDF and its receptor ErbB-4 are highly expressed in neurons, implying important roles in neuronal cell functions. In the present study we demonstrate that ErbB-4 receptors expressed in PC12 cells mediate NDF-induced signals and neurite outgrowth that are indistinguishable from those mediated by the nerve growth factor-activated Trk receptors. In PC12-ErbB-4 cells but not in PC12 cells, NDF induced an initial weak mitogenic signal and subsequently neurite outgrowth. The NDF-induced differentiation in PC12-ErbB-4 cells was mimicked by the pan-ErbB ligand betacellulin but not by other epidermal growth factor-like ligands. Thus, NDF and betacellulin mediate similar activities through the ErbB-4 receptor. Indeed, only these ligands induced strong phosphorylation of the ErbB-4 receptors. Neurite outgrowth induced by NDF in PC12-ErbB-4 cells was accompanied by sustained activation of mitogen-activated protein kinase (MAPK) and induction of the neural differentiation marker GAP-43. Inhibition of the MAPK kinase MEK or of protein kinase C (PKC) blocked NDF-induced differentiation, whereas elevation of cyclic AMP levels enhanced the response. Taken together, these results indicate that neurite outgrowth induced by ErbB-4 in PC12 cells requires MAPK and PKC signaling networks.     

Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.     

Potentiation of nerve growth factor(NGF)-mediated neurite outgrowth in high K+ medium is associated with increased binding of iodinated NGF in PC12 cells

Nerve growth factor(NGF)-mediated neurite outgrowth of PC12 pheochromocytoma cells was potentiated in medium containing high concentrations of extracellular K+. The binding of iodinated NGF to the cells was also enhanced by raising the concentration of K+ in medium up to 100 mM; the enhancement was saturated at 50 mM K+. Although the mechanism by which NGF-mediated neurite outgrowth is potentiated in high K+ medium remains to be largely unknown, high K+-induced alterations in the NGF binding are suggested to play a role in this phenomenon.     

TMC-95A, a reversible proteasome inhibitor, induces neurite outgrowth in PC12 cells

TMC-95A has been characterized as a potent proteasome inhibitor that binds to enzymes non-covalently at low nanomolar concentrations. Herein, the neuritogenic activity of TMC-95A in PC12 rat pheochromocytoma cells is reported for the first time. TMC-95A induced a positive neurite initiation of PC12 cells at concentration ranging from 1 to 20 microM.     

Scribble controls NGF-mediated neurite outgrowth in PC12 cells

Neurite outgrowth is mediated by dynamic changes of the cytoskeleton and is largely controlled by Rho GTPases and their regulators. Here, we show that the polarity protein Scribble controls PC12 cell neurite outgrowth in response to nerve growth factor. Scribble knockdown decreases neurite numbers and increases neurite length. This effect is linked to TrkA the cognate receptor for NGF as pharmacological inhibition of phosphorylated TrkA (pTrkA) reduces Scribble expression. Moreover, Scribble forms a complex with the MAPK components ERK1/2 in a growth factor dependent manner. In RNAi experiments where Scribble expression is efficiently depleted sustained ERK1/2 phosphorylation is reduced. Conversely, siRNA with intermediate Scribble silencing efficiency fails to match this effect indicating that ERK1/2 activation depends on basic Scribble protein levels. Finally, Scribble translocates to the plasma membrane in response to growth factor where it complexes with HRas and Rac1 suggesting that the phenotype activated by loss of Scribble may be a result of altered GTPase activity. Together, these results demonstrate a novel role for Scribble in neurite outgrowth of PC12 cells.     

Clusterin interacts with SCLIP (SCG10-like protein) and promotes neurite outgrowth of PC12 cells

Clusterin has been known as a chaperone-like molecule capable of interacting with various proteins. In this study, we show that clusterin interacts with the microtubule-destabilizing stathmin family protein SCLIP by GST pull-down and co-immunoprecipitation assays. Interestingly, SCLIP interacts with 80 kDa mature form of clusterin in the cytosolic fraction of PC12 cells permeabilized by low concentration of a weak nonionic detergent digitonin, but not with intracellular variants of clusterin known as binding isoforms of Ku70 or TGF-beta receptors. Both clusterin and SCLIP are co-localized at the perinuclear region and growth cone of PC12 cells. In addition, we show that the minimal domains for the interaction are mapped to the C-terminal valine-rich region (367-447) of clusterin and the N-terminal palmitoylation and membrane attachment site (1-34) of SCLIP. Finally, we demonstrate that ectopic expression of clusterin in PC12 cells elongates neurite-formation triggered by NGF and induces spontaneous neurite outgrowth even in the absence of NGF. Taken together, these results suggest that the clusterin interacts with SCLIP and the interaction may act as an important modulator during neuronal differentiation.     

Iron enhances NGF-induced neurite outgrowth in PC12 cells

Rat pheochromocytoma 12 (PC12) cells undergo neuronal differentiation in response to nerve growth factor (NGF). NGF-induced differentiation involves a number of protein kinases, including extracellular signal-regulated kinase (ERK). We studied the effect of iron on neuronal differentiation, using as model the neurite outgrowth of PC12 cells triggered by NGF when the cells are plated on collagen-coated dishes in medium containing 1% serum. The addition of iron enhanced NGF-mediated cell adhesion, spreading and neurite outgrowth. The differentiation-promoting effect of iron seems to depend on intracellular iron, since nitrilotriacetic acid (an efficient iron-uptake mediator) enhanced the response to iron. In agreement with this, intracellular, but not extracellular, iron enhanced NGF-induced neurite outgrowth in pre-spread PC12 cells, and this was correlated with increased ERK activity. Taken together, these data suggest that intracellular iron promotes NGF-stimulated differentiation of PC12 cells by increasing ERK activity.     

rSac3, a novel Sac domain phosphoinositide phosphatase, promotes neurite outgrowth in PC12 cells

Sac domain-containing proteins belong to a newly identified family of phosphoinositide phosphatases （the PIPPase family）. Despite well-characterized enzymatic activity, the biological functions of this mammalian Sac domain PIPPase family remain largely unknown. We identified a novel Sac domain-containing protein, rat Sac3 （rSac3）, which is widely expressed in various tissues and localized to the endoplasmic reticulum, Golgi complex and recycling endosomes, rSac3 displays PIPPase activity with PI（3）P, PI（4）P and PI（3,5）P2 as substrates in vitro, and a mutation in the catalytic core of the Sac domain abolishes its enzymatic activity. The expression of rSac3 is upregulated during nerve growth factor （NGF）-stimulated PC 12 cell neuronal differentiation, and overexpression of this protein promotes neurite outgrowth in PC 12 cells. Conversely, inhibition ofrSac3 expression by antisense oligonucleotides reduces neurite outgrowth of NGF- stimulated PC 12 cells, and the active site mutation of rSac3 eliminates its neurite-outgrowth-promoting activity. These results indicate that rSac3 promotes neurite outgrowth in differentiating neurons through its PIPPase activity, suggesting that Sac domain PIPPase proteins may participate in forward membrane trafficking from the endoplasmic reticulum and Golgi complex to the plasma membrane, and may function as regulators of this crucial process of neuronal cell growth and differentiation.[第一段]     

Csk-homologous kinase interacts with SHPS-1 and enhances neurite outgrowth of PC12 cells

SHPS-1 is an immunoglobulin superfamily protein with four immunoreceptor tyrosine-based inhibitory motifs (ITIMs) in its cytoplasmic region. Various neurotrophic factors induce the tyrosine phosphorylation of SHPS-1 and the association of SHPS-1 with the protein tyrosine phosphatase SHP-2. Using a yeast two-hybrid screen, we identified a protein tyrosine kinase, Csk-homologous kinase (CHK), as an SHPS-1-interacting protein. Immunoprecipitation and pull-down assays using glutathione S -transferase (GST) fusion proteins containing the Src homology 2 (SH2) domain of CHK revealed that CHK associates with tyrosine-phosphorylated SHPS-1 via its SH2 domain. HIS3 assay in a yeast two-hybrid system using the tyrosine-to-phenylalanine mutants of SHPS-1 indicated that the first and second ITIMs of SHPS-1 are required to bind CHK. Over-expression of wild-type CHK, but not a kinase-inactive CHK mutant, enhanced the phosphorylation of SHPS-1 and its subsequent association with SHP-2. CHK phosphorylated each of four tyrosines in the cytoplasmic region of SHPS-1 in vitro . Co-expression of SHPS-1 and CHK enhanced neurite outgrowth in PC12 cells. Thus, CHK phosphorylates and associates with SHPS-1 and is involved in neural differentiation via SHP-2 activation.     

Gelsolin overexpression enhances neurite outgrowth in PC12 cells.

The rational design of therapies for treating nerve injuries requires an understanding of the mechanisms underlying neurite extension. Neurite motility is driven by actin polymerization; however, the mechanisms are not clearly understood. One actin accessory protein, gelsolin, is involved with remodeling the cytoskeleton, although its role in cell motility is unclear. We report a two-fold upregulation of gelsolin upon differentiation with nerve growth factor. Cells that were genetically modified to overexpress gelsolin have longer neurites and a greater neurite motility rate compared to controls. These data suggest that gelsolin plays an important role in neurite outgrowth.     

Nitric oxide mediates laminin-induced neurite outgrowth in PC12 cells

Laminin is a potent stimulator of neurite outgrowth in a variety of primary neurons and neuronal cell lines. Here, we investigate the role of nitric oxide in the signaling mechanism of laminin-mediated neurite outgrowth in the PC12 cell line. Within 8 s of exposure to laminin, PC12 cells produce nitric oxide. Peak laminin-induced nitric oxide levels reach 8 nM within 12 s of exposure to laminin and constitutive nitric oxide production is sustained for 1 min. A neurite outgrowth promoting synthetic peptide (AG73), derived from the laminin-1-alpha globular domain, also stimulated nitric oxide release. The nitric oxide synthase inhibitor, 1-NAME, prevents the formation of nitric oxide and here, 1-NAME inhibited both laminin-mediated and AG73-mediated neurite outgrowth by 88 and 95%, respectively. In contrast, C16, a synthetic peptide derived from the laminin-1-gamma chain, is shown here to promote PC12 cell attachment, but not neurite outgrowth. Interestingly, the C16 peptide did not activate nitric oxide release, suggesting that laminin-induced nitric oxide release in PC12 cells is associated only with neurite outgrowth promoting laminin domains and signals. In addition, the data here show that the nitric oxide released by PC12 cells in response to laminin is required as a part of the mechanism of laminin-mediated neurite outgrowth.     

Protein deacetylase SIRT1 in the cytoplasm promotes nerve growth factor-induced neurite outgrowth in PC12 cells

SIRT1, a NAD⁺-dependent protein deacetylase, is known to have neural functions. However, despite its cytoplasmic expression in some neural cells, its cytoplasmic function, if any, is unknown. Here we found that PC12 (pheochromocytoma) cells expressed SIRT1 in the cytoplasm. Nerve growth factor (NGF)-induced neurite outgrowth of these cells was promoted by activators of SIRT1, while inhibitors of SIRT1 or SIRT1-siRNA significantly inhibited it. The overexpression of a mutant SIRT1 that localised to the cytoplasm but not the nucleus enhanced the NGF-dependent neurite outgrowth, and a cytoplasmic dominant-negative SIRT1 suppressed it. Thus, cytoplasmic SIRT1 increases the NGF-induced neurite outgrowth of PC12 cells.     

Immunosuppressant FK506 induces sustained activation of MAP kinase and promotes neurite outgrowth in PC12 mutant cells incapable of differentiating

During the continuous culturing of neural PC12 cells, a drug hypersensitive PC12 mutant cell line (PC12m3) was obtained, which demonstrated high neurite outgrowth when stimulated by various drugs. When the immunosuppressant drug FK506 and nerve growth factor (NGF) were introduced to the PC12m3 cells, the frequency of neurite outgrowth increased approximately 40-fold for NGF alone. However, the effect of FK506 on neuritogenesis in PC12 parental and drug insensitive PC12m1 mutant cells was much lower than in PC12m3 cells. The sustained activation of mitogen-activated protein (MAP) kinase plays an important role in neurite outgrowth of PC12 cells. Interestingly, the drug hypersensitive PC12m3 cells exhibited the sustained activation of MAP kinase with FK506 in comparison to low or no activities in PC12 parental or drug insensitive PC12m1 cells. These results indicate that PC12m3 cells have a novel FK506-induced MAP kinase pathway for neuritogenesis.     

Nuclear Rac1 regulates the bFGF-induced neurite outgrowth in PC12 cells

Rac1 plays a key role in neurite outgrowth via reorganization of the actin cytoskeleton. The molecular mechanisms underlying Rac1-mediated actin dynamics in the cytosol and plasma membrane have been intensively studied, but the nuclear function of Rac1 in neurite outgrowth has not yet been addressed. Using subcellular fractionation and immunocytochemistry, we sought to explore the role of nuclear Rac1 in neurite outgrowth. bFGF, a strong agonist for neurite outgrowth in PC12 cells, stimulated the nuclear accumulation of an active form of Rac1. Rac1-PBR (Q) mutant, in which six basic residues in the polybasic region at the C-terminus were replaced by glutamine, didn’t accumulate in the nucleus. In comparison with control cells, cells expressing this mutant form of Rac1 displayed a marked defect in extending neurites that was concomitant with reduced expression of MAP2 and MEK-1. These results suggest that Rac1 translocation to the nucleus functionally correlates with bFGF-induced neurite outgrowth. [BMB Reports 2013; 46(12): 617-622]     

Cyclic AMP potentiates bFGF-induced neurite outgrowth in PC12 cells.

We report here that basic fibroblast growth factor (bFGF)-elicited neurite outgrowth in PC12 cells is potentiated by dibutyryl cyclic adenosine monophosphate (dbcAMP) or forskolin. This property was also described for nerve growth factor (NGF), suggesting that both NGF and bFGF may share common intracellular events leading to neurite outgrowth and synergism with dbcAMP and forskolin. The synergistic effect of dbcAMP and forskolin is specific, since treatment of PC12 cells with bFGF and dibutyryl cyclic guanosine monophosphate (dbcGMP) or phorbol ester did not change the neurite outgrowth response of cells treated with bFGF alone. Furthermore, neurite outgrowth depends on cellular adhesion. Increasing adhesion by plate treatment with poly-d-lysine increases the neurite outgrowth elicited by bFGF alone or bFGF plus dbcAMP. On the other hand, decreasing cellular adhesiveness by plating PC12 cells in semi-solid agarose renders the cells unable to develop neuritic processes. In addition, 3H-methylthymidine incorporation studies showed that bFGF-treated PC12 cells cease growth only when they become fully differentiated after 3-5 days of treatment. In contrast, dbcAMP, which is a poor differentiation factor, is able to block cellular growth after 24 hour treatment. These results suggest that when PC12 cells become differentiated, they stop growing. However, growth inhibition does not necessarily lead to differentiation.     

Neuroserpin regulates neurite outgrowth in nerve growth factor-treated PC12 cells

Neuroserpin is a serine protease inhibitor widely expressed in the developing and adult nervous systems and implicated in the regulation of proteases involved in processes such as synaptic plasticity, neuronal migration and axogenesis. We have analysed the effect of neuroserpin on growth factor-induced neurite outgrowth in PC12 cells. We show that small changes in neuroserpin expression result in changes to the number of cells extending neurites and total neurite length following NGF treatment. Increased expression of neuroserpin resulted in a decrease in the number of cells extending neurites and a reduction in total free neurite length whereas reduced levels of neuroserpin led to a small increase in the number of neurite extending cells and a significant increase in total free neurite length compared to the parent cell line. Neuroserpin also altered the response of PC12 cells to bFGF and EGF treatment. Neuroserpin was localised to dense cored secretory vesicles in PC12 cells but was unable to complex with its likely enzyme target, tissue plasminogen activator at the acidic pH found in these vesicles. These data suggest that modulation of neuroserpin levels at the extending neurite growth cone may play an important role in regulating axonal growth.     

Green tea polyphenol (-)-epigallocatechin-3-gallate induces neurorescue of long-term serum-deprived PC12 cells and promotes neurite outgrowth

Our previous studies have shown that the green tea polyphenol (-)-epigallocatechin-3-gallate (EGCG) prevents neuronal cell death caused by several neurotoxins. The present study sought to determine the neuroprotective effect of EGCG when it is administered after the induction of cell damage ('neurorescue'). In an attempt to imitate a progressive mode of death, PC12 cells were initially subjected to serum-starvation conditions for a period of 1 or 3 days before administration of EGCG (0.1-10 microM) for up to 3 days. In spite of the high percentage of cell death, single or repetitive administration of EGCG (1 microM) significantly attenuated cell death. The neurorescue effect of EGCG was abolished by pre-treatment with the protein kinase C inhibitor GF109203X (2.5 microM), suggesting the involvement of the protein kinase C pathway in neurorescue by the drug. This is consistent with the rapid (15 min) translocation of the protein kinase C alpha isoform to the cell membrane in response to EGCG. The correlative neurite outgrowth activity of EGCG on PC12 cells may also contribute to its neurorescue effect. The present findings suggest that EGCG may have a positive impact on aging and neurodegenerative diseases to retard or perhaps even reverse the accelerated rate of neuronal degeneration.     

Membrane targeting of p21-activated kinase 1 (PAK1) induces neurite outgrowth from PC12 cells.

Rho-family GTPases regulate cytoskeletal dynamics in various cell types. p21-activated kinase 1 (PAK1) is one of the downstream effectors of Rac and Cdc42 which has been implicated as a mediator of polarized cytoskeletal changes in fibroblasts. We show here that the extension of neurites induced by nerve growth factor (NGF) in the neuronal cell line PC12 is inhibited by dominant-negative Rac2 and Cdc42, indicating that these GTPases are required components of the NGF signaling pathway. While cytoplasmically expressed PAK1 constructs do not cause efficient neurite outgrowth from PC12 cells, targeting of these constructs to the plasma membrane via a C-terminal isoprenylation sequence induced PC12 cells to extend neurites similar to those stimulated by NGF. This effect was independent of PAK1 ser/thr kinase activity but was dependent on structural domains within both the N- and C-terminal portions of the molecule. Using these regions of PAK1 as dominant-negative inhibitors, we were able to effectively inhibit normal neurite outgrowth stimulated by NGF. Taken together with the requirement for Rac and Cdc42 in neurite outgrowth, these data suggest that PAK(s) may be acting downstream of these GTPases in a signaling system which drives polarized outgrowth of the actin cytoskeleton in the developing neurite.