KP544, a nerve growth factor amplifier: Pharmacokinetics,safety, and efficacy in the rat

In cultured cells, KP544 [2‐amino‐5‐(4‐chlorophenylethynyl)‐4‐(4‐trans‐hydroxycyclohexyl amino) pyrimidine] amplifies differentiation initiated by nerve growth factor (NGF) or cAMP. This report describes the pharmacokinetics, safety, and neuroprotective efficacy of KP544 in rats. After an oral dose of 10 mg/kg KP544 was 25% bioavailable with a plasma half‐life of 1.3 h and brain levels 6‐fold higher than plasma levels at 4 and 8 h post‐dose. In a safety study, daily oral dosing for 30 days at 10 and 100 mg/kg was well tolerated. The favorable pharmacokinetic and safety profiles, together with its amplification of NGF in vitro, prompted evaluation of KP544 in two models involving NGF deficiencies. In the first model, brains were lesioned with intrastriatal injections of quinolinic acid. KP544 at oral doses of 0.02 to 1.0 mg/kg/day almost completely prevented the resulting learning deficits as evaluated using a radial‐arm‐water maze. At the lowest dose, there was a slower onset of functional improvement. These effects were accompanied by reductions (16–34%) in the striatal lesion size that were greatest at the highest dose and comparable to those seen with NGF therapy. The second model involved a peripheral neuropathy induced by taxol that is associated with decreases in NGF. KP544 at oral doses of 0.1–10 mg/kg/day decreased the severity of the neuropathy as measured by caudal nerve conduction velocities (30–70% return to control values). In both models, KP544 had a large therapeutic index suggesting its potential as a new approach for treating clinical disorders involving deficiencies in NGF. Drug Dev. Res. 62:60–70, 2004. © 2004 Wiley‐Liss, Inc.     

Enhancement of neurite outgrowth in PC12 cells stimulated with cyclic AMP and NGF by 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), novel lipophilic ascorbate derivatives

It has been shown that ascorbate (AsA) and its stable derivative, ascorbic acid 2-O-alpha-glucoside (AA-2G), do not elicit neurite outgrowth in PC12 cells. However, these ascorbates are synergistically enhanced by both dibutyryl cyclic AMP (Bt(2)cAMP)- and nerve growth factor (NGF)-induced neurite outgrowth in this model. In the present study, the effects of a series of novel lipophilic ascorbate derivatives, 6-acylated ascorbic acid 2-O-alpha-glucosides (6-Acyl-AA-2G), on neurite outgrowth induced by Bt(2)cAMP and NGF were examined in PC12 cells. We found that all the tested acylated ascorbate derivatives enhanced neurite formation induced by both agents in a dose-dependent manner. Of the 6-Acyl-AA-2G derivatives, 6-octanoyl ascorbic acid 2-O-alpha-glucoside (6-Octa-AA-2G) enhanced the Bt(2)cAMP-induced phosphorylated MAPK p44 and p42 expression. A alpha-glucosidase inhibitor, castanospermine, completely abrogated the promotion of neurite outgrowth and MAPK expression by 6-Octa-AA-2G. Addition of 6-Octa-AA-2G (0.5 mM) to PC12 cells caused a rapid and significant increase in intracellular AsA content, which reached a maximum and was maintained from 12 to 24 h after the culture. These findings suggest that 6-Acyl-AA-2G is rapidly hydrolyzed to AsA within the cell and enhances neurite differentiation through the interaction with the inducer-activated MAPK pathway.     

Donepezil potentiates nerve growth factor-induced neurite outgrowth in PC12 cells

Donepezil is a potent and selective acetylcholinesterase inhibitor developed for the treatment of Alzheimer's disease. To elucidate whether donepezil causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Donepezil (10 microM) significantly potentiated the neurite outgrowth evoked by low (1 ng/ml) and high (50 ng/ml) concentrations of NGF. The effect of donepezil (1 - 10 microM) was concentration-dependent. The enhancement of neurite outgrowth caused by donepezil was not blocked by the acetylcholine receptor (AChR) antagonists mecamylamine and scopolamine. Furthermore, the AChR agonists nicotine and carbachol did not affect the neurite outgrowth induced by NGF. Donepezil (10 microM) also significantly potentiated neurite outgrowth evoked by dibutyryl cyclic AMP. Moreover, donepezil potentiated the NGF-induced phosphorylation of extracellular signal-regulated kinase (ERK). These results suggest that donepezil potentiated neuronal differentiation by enhancing the activation of ERK.     

Codonopsis pilosula （Franch） Nannftotal alkaloids potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

AIM:To explore the effect of Codonopsis pilosula (Franch) Nannf total alkaloids (DSA) on differentiation inducedby nerve growth factor (NGF) in PC12 cells. METHODS: After culturing PC12 cells with DSA in the presence orabsence of NGF, neurite outgrowth in PC12 cells and correlated protein kinases were assayed. RESULTS: DSAalone did not exhibit neuritogenic activity, but caused a significant enhancement of NGF (2 μg/L)-induced neuriteoutgrowth in PC12 cells, and increased the phosphorylation of mitogen-activated protein kinase (MAPK).Furthermore, this enhancing effect was completely blocked by a specific MAPK kinase inhibitor, PD98059.CONCLUSION: DSA enhanced the NGF-induced neurite outgrowth in PC12 cells by amplifying an up-streamstep of the MAPK-dependent signaling pathway.     

Potentiation of the NGF-mediated nerve fiber outgrowth by ginsenoside Rb1 in organ cultures of chicken dorsal root ganglia.

The fiber outgrowth induced by ACh, dibutyryl cyclic AMP and dibutyryl cyclic GMP in explanted chick embryonic dorsal root ganglia differed distinctly from that be nerve growth factor (NGF) and submandibular gland extract of adult male mice. Ginsenoside Rb1 potentiated the effects of NGF and submandibular extract at concentrations of 3 and 30 muM, but did not potentiate the effects of ACh, dibutyryl cyclic AMP and dibutyryl cyclic GMP. NGF-antibody inhibited the effects of NGF, but not the effects of ACh, dibutyryl cyclic AMP and dibutyryl cyclic GMP, Concanavalin A and KCL did not promote fiber outgrowth.     

Effects of (+)-eudesmin from the stem bark ofMagnolia kobus DC. var.borealis Sarg. on neurite outgrowth in PC12 cells

(+)-Eudesmin [4,8-bis(3,4-dimethoxyphenyl)-3,7-dioxabicyclo[3.3.0]octane] was isolated from the stem bark of Magnolia kobus DC. var. borealis Sarg. and found to have neuritogenic activity. 50 microM (+)-eudesmin induced neurite outgrowth and enhanced nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. At this concentration, (+)-eudesmin also enhanced NGF-induced neurite-bearing activity and this activity was partially blocked by various protein kinase inhibitors. These included PD98059, a mitogen-activated protein kinase (MAPK) kinase inhibitor. GF109203X, a protein kinase C (PKC) inhibitor and H89, a protein kinase A (PKA) inhibitor. These results suggest that (+)-eudesmin can induce neurite outgrowth from PC12 cells by stimulating up-stream MAPK, PKC and PKA pathways.     

Induction of neurite outgrowth by (-)-(7R, 8S)-dihydrodehyd-rodiconiferyl alcohol from PC12 Cells

A lignan derivative, (-)-(7R, 8S)-dihydrodehydrodiconiferyl alcohol (DHDA), was isolated from Kalopanax septemlobus L. and was observed to have neuritogenic activity. DHDA at 50 microM caused a marked induction of neurite outgrowth and an enhancement of nerve growth factor (NGF)-mediated neurite outgrowth from PC12 cells. However, it did not exhibit any neurotrophic action. At 50 microM, DHDA enhanced NGF-induced neurite-bearing activity. This activity was partially blocked by the mitogen-activated protein kinase (MAPK) inhibitor PD98059 and by GF109203X, a protein kinase C (PKC) inhibitor. These results suggest that DHDA can induce neurite outgrowth and enhance NGF-induced neurite outgrowth from PC12 cells by amplifying up-stream steps such as MAPK and PKC.     

Genipin exhibits neurotrophic effects through a common signaling pathway in nitric oxide synthase-expressing cells

We have reported previously that genipin, a natural iridoid compound, induces neuritogenesis through a nitric oxide (NO)-cyclic GMP (cGMP)-cGMP-dependent protein kinase (PKG) signaling pathway in PC12h cells and that neuronal NO synthase (nNOS) is one of the target molecules of genipin in vitro. Recently, it has been suggested that the neurotrophic effects of NO are due to its direct activation of receptor-tyrosine kinase, especially TrkA. In this study, we investigated whether mouse neuroblastoma Neuro2a cells, which express nNOS but not TrkA, respond to genipin with neurite outgrowth through the mechanism observed in PC12h cells, to assess the involvement of TrkA in the mechanism. Neuro2a cells expressed all three types of NO synthase (NOS), and nNOS was detectable as the main component in Western blot analysis. Genipin significantly induced neurite outgrowth and activation of NADPH-diaphorase, which were significantly blocked by a non-selective NOS inhibitor. Both a soluble guanylate cyclase inhibitor and a PKG inhibitor also inhibited the genipin-induced neuritogenesis. Genipin induced sustained phosphorylation of mitogen-activated protein kinase (MAPK). In fact, the genipin-induced neurite outgrowth was completely inhibited by a specific MAPK kinase inhibitor. Moreover, a NOS inhibitor abolished MAPK phosphorylation as well as neurite outgrowth in genipin-treated cells. These results suggest that genipin induces neurite outgrowth through an NO-cGMP-PKG signaling pathway followed by MAPK phosphorylation without TrkA activation in Neuro2a cells and that PKG downstream to NOSs, which may be mainly nNOS, is very important for the signaling molecule to induce neuritogenesis by genipin.     

Cyclic GMP-dependent neurite outgrowth by genipin and nerve growth factor in PC12h cells

We have demonstrated previously that a natural iridoid compound, genipin, induced neuritogenesis through activation of nitric oxide synthase (NOS) and mitogen-activated protein kinase (MAPK) in PC12h cells. In this paper, we investigated whether cyclic GMP (cGMP) and cGMP-dependent protein kinase (PKG) are involved in the neuritogenesis as a result of NOS activation. Furthermore, we also investigated the relationship between cGMP and MAPK activation in the signaling pathway. The genipin-induced neuritogenesis accompanied by induction of neurofilament was significantly inhibited by 1H-[1,2,4]oxadiazolo[4,3-a] quinoxalin-1-one (ODQ) and KT5823, inhibitors of soluble guanylate cyclase and PKG, respectively. Genipin-induced MAPK phosphorylation was also abolished by ODQ. These inhibitory effects of ODQ were similar to those observed for nerve growth factor (NGF)-induced neurite outgrowth and MAPK phosphorylation. The membrane-permeable cGMP analog, 8-Bromo-cGMP, had prominent neuritogenic activity, which was completely inhibited by a MAPK kinase inhibitor, PD98059. These results suggest that the soluble guanylate cyclase-PKG signaling pathway is important for MAPK activation by genipin as well as NGF during neuritogenesis in PC12h cells.     

Discovery of neurotrophic agents based on hydroxycinnamic acid scaffold

The number of people affected by neurodegenerative disorders such as Alzheimer's disease and Parkinson's disease is rapidly increasing owing to the global increase in life expectancy. Small molecules with neurotrophic effects have great potential for management of these neurological disorders. In this study, different (C1–C12) alkyl ester derivatives of hydroxycinnamic acids (HCAs) were synthesized (a total of 30 compounds). The neurotrophic capacity of the test compounds was examined by measuring promotion of survival in serum‐deprived conditions and enhancement of nerve growth factor (NGF)‐induced neurite outgrowth in PC12 neuronal cells. p‐Coumaric, ferulic, and sinapic acids and their esters did not alter cell survival, while caffeic acid and all its alkyl esters, especially decyl and dodecyl caffeate, significantly promoted neuronal survival at 25 μm . Methyl, ethyl, propyl, and butyl caffeate esters also significantly enhanced NGF‐induced neurite outgrowth, among which the most effective ones were propyl and butyl esters, which at 5 μm led to 25‐ and 22‐fold increases in the number of neurites, respectively. The findings of the docking study suggested phosphatidylinositol 3‐kinase (PI3K) as the potential molecular target. In conclusion, our findings demonstrate that alkyl esters of caffeic acid can be useful as scaffolds for the discovery of therapeutic agents for neurodegenerative diseases.     

FK506 potentiates NGF-induced neurite outgrowth via the Ras/Raf/MAP kinase pathway

Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons within the peripheral and central nervous systems. Neurophilin ligands, including FK506, potentiate NGF-induced neurite outgrowth in several experimental models, although the mechanism of this potentiation is unclear. Therefore, we tested which signaling pathways were involved in FK506-potentiated neurite outgrowth in SH-SY5Y neuroblastoma cells using specific pharmacological inhibitors of various signaling molecules. Inhibitors of Ras (lovastatin), Raf (GW5074), or MAP kinase (PD98059 and U0126) blocked FK506 activity, as did inhibitors of phospholipase C (U73122) and phosphatidylinositol 3' kinase (LY294002). Protein kinase C inhibitors (Go6983 and Ro31-8220) slightly but significantly inhibited neurite outgrowth, whereas inhibitors of p38 MAPK (SB203580) or c-Jun N-terminal kinase (SP600125) had no effect. These data suggest that FK506 potentiates neurite outgrowth through the Ras/Raf/MAP kinase signaling pathway downstream of phospholipase C and phosphatidylinositol 3' kinase.     

Na+/Ca2+ exchanger inhibitors inhibit neurite outgrowth in PC12 cells

To elucidate the role of Na(+)/Ca(2+) exchanger (NCX) in neurite outgrowth, we investigated the effects of NCX inhibitors on neurite outgrowth in PC12 cells. KB-R7943 and 3',4'-dichlorobenzamil, NCX inhibitors, inhibited the neurite outgrowth caused by nerve growth factor (NGF). NCX inhibitors inhibited the neurite outgrowth caused by dibutylyl cAMP, which rapidly reorganizes the cytoskeleton. KB-R7943 inhibited the neurite outgrowth caused by Y-27632, an inhibitor of Rho kinase (ROCK) that regulates actin. However, NCX inhibitors did not inhibit NGF-induced phosphorylation of extracellular signal-regulated kinase. These results suggest that NCX inhibitor affects downstream of the Rho-ROCK signal transduction pathways in neurite outgrowth.     

Induction of nerve growth factor by butanol fraction of Liriope platyphylla in C6 and primary astrocyte cells

Liriope platyphylla (LP) has been used as a tonic, antitussive, and expectorant in Korea for many years. In this study, we found that the buthanol fraction of Liriope platyphylla (BLP)-conditioned media of C6 and primary astrocyte induced the neurite outgrowth of PC12 cells, and that the effect was reversed by addition of nerve growth factor (NGF)-antibody and GF109203X, an inhibitor of protein kinase (PKC). Furthermore, we demonstrated that BLP increased the expression and secretion of NGF. GF109203X also decreased NGF expression in C6 cells. Taken together, our results suggest that astroglial NGF enhanced by BLP in a PKC-dependent pathway contributed to the induction of neurite outgrowth of PC12 cells.     

Effect of ethanol and docosahexaenoic acid on nerve growth factor-induced neurite formation and neuron specific growth-associated protein gene expression in PC12 cells.

It is known that prenatal and postnatal exposure to ethanol can result in hyperactive behavior and learning disturbance in offspring. We have previously shown that docosahexaenoic acid (DHA) ameliorated hyperactivity induced by in utero ethanol exposure in rat pups. The present study is designed to clarify the effects of DHA on neurite outgrowth and gene expression of GAP-43 and SCG10, neuron specific growth-associated proteins (GAPs) in PC12 cells treated with ethanol. Ethanol seemed to enhance the neurite outgrowth induced by nerve growth factor (NGF). DHA administration further increased neurite outgrowth in both NGF alone and the combination of NGF and ethanol treated PC12 cells. DHA treatment increased the levels of both GAP-43 and SCG10 mRNAs, and simultaneous administration of ethanol suppressed the elevation of GAP-43 and SCG10 mRNA enhanced by DHA. The present study has demonstrated, for the first time, the effect of ethanol and DHA on GAPs' gene expression. Interaction of ethanol and docosahexaenoic acid in NGF-induced neurite formation, as well as the mechanisms by which DHA ameliorate the hyperactivity induced by in utero ethanol exposure, are to be elucidated.     

Stimulatory effects of substance P and nerve growth factor (NGF) on neurite outgrowth in embryonic chick dorsal root ganglia.

Synthetic Substance P at 10⁴ M and Nerve Growth Factor (NGF) at 2 $\text{ng}\text{ml}$ produced a 3-fold increase in the concentration of cyclic AMP 10 min after addition to cultured chick dorsal root ganglia. Subsequently, increased fiber outgrowth was observed 24 hrs after these additions. Dibutyryl cyclic AMP at 10^?3 M also produced a similar induction of fiber outgrowth.These studies suggest that Substance P and NGF might act by increasing cyclic AMP levels, which may then act as an initial “second messenger”, inducing the morphological differentiation at a later time.     

Effects of methylmercury and mercuric chloride on differentiation and cell viability in PC12 cells.

The effects of methylmercury (CH3Hg) or mercuric chloride (HgCl(2)) on neurite outgrowth and cell viability were quantified using undifferentiated (unprimed) and differentiated (primed) pheochromocytoma (PC12) cells. In unprimed cells, following 24-h exposure, CH3Hg significantly decreased NGF-stimulated neurite outgrowth at concentrations of 0.3-3 microM. However, HgCl(2) significantly increased both neurite outgrowth and the number of branch points, a component of neurite outgrowth. In primed PC12 cells, following 24-h exposure, both CH3Hg and HgCl(2) inhibited NGF-stimulated neurite outgrowth with an EC(50) of approximately 0.03 microM; however, there was a difference between CH3Hg and HgCl(2) effects on the subcomponents of total neurite outgrowth. CH3Hg significantly decreased both the number of branch points (0.3 microM) and fragment length (0.01 microM), while HgCl(2) only decreased fragment length (0.03 microM). Cell viability was assessed in the same cultures by trypan-blue exclusion. In unprimed cells, the EC(50) for cytotoxicity of CH3Hg in the presence and absence of NGF was 0.21 +/- 0.04 and 0.87 +/- 0.12 microM, respectively, and for HgCl(2) in the presence and absence of NGF was 8.18 +/- 1.52 and 5.02 +/- 0.74 microM, respectively. In primed cells, the EC(50) for cytotoxicity of CH3Hg in the presence or absence of NGF was 1.17 +/- 0.38 and 0.73 +/- 0.14 microM, respectively, and for HgCl(2) in the presence or absence of NGF was 3.96 +/- 0.82 and 3.81 +/- 0.91 microM, respectively. In the primed PC12 model, cytotoxicity occurred at concentrations that were at least 30-fold higher than the EC(50) for neurite outgrowth, suggesting that the mercurial compounds can act selectively on the process of differentiation.     

Nardosinone enhances nerve growth factor-induced neurite outgrowth in a mitogen-activated protein kinase- and protein kinase C-dependent manner in PC12D cells

The mechanism to enhance nerve growth factor (NGF, 2 ng/ml)-induced neurite outgrowth from PC12D cells by nardosinone isolated from Nardostachys chinensis was examined. It was shown that the potentiation of the NGF-induced neurite outgrowth by nardosinone was mitogen-activated protein (MAP) kinase-dependent, but was not accompanied by stimulation of NGF-induced increase in MAP kinase phosphorylation. Furthermore, this augmentation of NGF-induced neurite outgrowth was abolished by GF109203X, a protein kinase C (PKC) inhibitor. These results suggest that the enhancement of NGF-induced neurite outgrowth from PC12D cells by nardosinone involves activation of a down-stream step of the MAP kinase-dependent cascade of NGF coupled with PKC.     

c-Jun N-terminal kinases mediate Fas-induced neurite regeneration in PC12 cells

In response to injury, peripheral neuronal cells initiate complex signalling cascades to promote survival and regeneration. In the present study, we used a model of experimental injury in the rat pheochromocytoma cell line PC12 to investigate receptor signals that lead to neurite outgrowth. Nerve growth factor (NGF) dose-dependently induced sprouting and the expression of the NGF receptors Trk tyrosine kinase receptor (TrkA) and p75 neurotrophin receptor (p75^NTR) as well as Fas and Fas ligand. Neurite regeneration was decreased by chemical inhibition of TrkA, but not p75^NTR, and by the Fas inhibitor protein Fas-Fc. The mitogen-activated protein kinases (MAPKs) extracellular signal-regulated kinase 1/2 (ERK1/2) and c-Jun N-terminal kinases (JNKs) were activated in response to NGF and both significantly contributed to neurite re-growth. Interestingly, otherwise apoptotic Fas ligation supported neuronal recovery exclusively via JNKs and promoted sprouting parallel to NGF. These findings suggest a novel signal integration from the NGF and Fas pathways in the JNK axis of MAPK signalling, where JNKs function as “physiological” mediators of normally apoptotic signals.     

Cytotoxic effects of acrylamide in nerve growth factor or fibroblast growth factor 1-induced neurite outgrowth in PC12 cells

Acrylamide is a neurological and reproductive toxicant in humans and laboratory animals; however, the neuron developmental toxicity of acrylamide remains unclear. The aims of this study are to investigate the cytotoxicity and neurite outgrowth inhibition of acrylamide in nerve growth factor (NGF)- or fibroblast growth factor 1 (FGF1)-mediated neural development of PC12 cells. MTS assay showed that acrylamide treatment suppresses NGF- or FGF1-induced PC12 cell proliferation in a time- and dose-dependent manner. Quantification of neurite outgrowth demonstrated that 0.5 mM acrylamide treatment resulted in significant decrease in differentiation of NGF- or FGF1-stimulated PC12 cells. This decrease is accompanied with the reduced expression of growth-associated protein-43, a neuronal marker. Moreover, relative levels of pERK, pAKT, pSTAT3 and pCREB were increased within 5–10 min when PC12 cells were treated with NGF or FGF1. Acrylamide (0.5 mM) decreases the NGF-induced activation of AKT–CREB but not ERK–STAT3 within 20 min. Similarly, acrylamide (0.5 mM) decreases the FGF1-induced activation of AKT–CREB within 20 min. In contrast to the NGF treatment, the ERK–STAT3 activation that was induced by FGF1 was slightly reduced by 0.5 mM acrylamide. We further showed that PI3K inhibitor (LY294002), but not MEK inhibitor (U0126), could synergize with acrylamide (0.5 mM) to reduce the cell viability and neurite outgrowth in NGF- or FGF1-stimulated PC12 cells. Moreover, acrylamide (0.5 mM) increased reactive oxygen species (ROS) activities in NGF- or FGF1-stimulated PC12 cells. This increase was reversed by Trolox (an ROS scavenging agent) co-treatment. Together, our findings reveal that NGF- or FGF1-stimulation of the neuronal differentiation of PC12 cells is attenuated by acrylamide through the inhibition of PI3K–AKT–CREB signaling, along with the production of ROS.