Matsumi Yamazaki

Activation of the mitogen-activated protein kinase cascade through nitric oxide synthesis as a mechanism of neuritogenic effect of genipin in PC12h cells.

Prominent neurite outgrowth induced by genipin, a plant‐derived iridoid, was substantially inhibited by addition of NG‐nitro‐l‐arginine methyl ester (l‐NAME), a nitric oxide (NO) synthase (NOS) inhibitor, and carboxy‐PTIO, an NO scavenger, in PC12h cells. Increases of the NADPH‐diaphorase activity and neuronal and inducible NOS proteins in cells preceded the neurite outgrowth after addition of genipin to medium. NO donors could induce the neurite outgrowth dose‐dependently in the cells. On the other hand, an inhibitor of soluble guanylate cyclase (SGC), which is known to be a stimulatory target of NO, abolished greatly the genipin‐induced neurite outgrowth. Addition of extracellular signal‐regulated kinase (ERK) kinase inhibitors could almost completely abolish the neurite induction. l‐NAME remarkably depressed genipin‐stimulated phosphorylation of ERK‐1 and ‐2. A neuritogenic effect of nerve growth factor (NGF) in PC12h cells was also remarkably inhibited by the NOS inhibitor, NO scavenger and SGC inhibitor. These findings suggest that induced NO production followed by cyclic GMP‐mediated stimulation of the mitogen‐activated protein kinase (MAPK) cascade is implicated in the neuritogenesis by genipin and NGF in PC12h cells.

Long-acting genipin derivative protects retinal ganglion cells from oxidative stress models in vitro and in vivo through the Nrf2/ antioxidant response element signaling pathway

J. Neurochem. (2010) 115, 79–91.

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.

Fundamental role of nitric oxide in neuritogenesis of PC12h cells.

We investigated the neuritogenic action of nitric oxide (NO)‐generating agents and their mechanisms of action in a subclone of rat pheochromocytoma, PC12h cells. NO donors such as sodium nitroprusside (SNP, 0.05–1 μM), NOR1 (5–100 μM), NOR2 (5–20 μM), NOR3 (5–20 μM), NOR4 (5–100 μM), or S‐nitroso‐N‐acetyl‐DL‐penicillamine (SNAP, 10–100 μM) significantly induced neurite outgrowth. NOR4‐induced neurite outgrowth was accompanied by expression of neurofilament 200 kDa subunit (NF200) protein, an axonal marker, and was significantly inhibited by an NO scavenger, a soluble GC inhibitor, and a PKG inhibitor: 2‐(4‐carboxyphenyl)‐4,4,5,5‐tetramethylimidazole‐1‐oxyl‐3‐oxide (carboxy‐PTIO, 20–100 μM), 1H‐[1,2,4]oxadiazolo[4,3‐a] quinoxalin‐1‐one (ODQ, 100 μM) and KT5823 (0.2–1 μM), respectively. The intracellular cGMP concentration of cells was markedly increased by treatment with NOR4 (100 μM). A mitogen‐activated protein kinase (MAPK) kinase inhibitor, PD98059 (10–50 μM), abolished the NOR4‐induced neurite outgrowth. In agreement with this observation, NOR4 did phosphorylate extracellular signal‐regulated kinase (ERK) 1 and 2, substrates of MAPK kinase. A membrane‐permeable cGMP analog, 8‐Br‐cGMP (1 mM) also induced significant neurite outgrowth. The 8‐Br‐cGMP‐induced neurite outgrowth was almost completely inhibited by both KT5823 (0.5 μM) and PD98059 (50 μM). Moreover, sustained ERK phosphorylation was observed in the 8‐Br‐cGMP‐treated PC12h cells. These results suggest that NO itself has the ability to induce neurite outgrowth and that NO‐induced ERK activation involves the NO‐cGMP‐PKG signaling pathway in PC12h cells.

Differences in neuritogenic response to nitric oxide in PC12 and PC12h cells

We have demonstrated that a natural iridoid compound, genipin, induces neurite outgrowth through the nitric oxide (NO)-cGMP-protein kinase G signaling pathway in PC12h cells. PC12 cells, the parental cell line of PC12h cells, have been shown to carry out neurite extension that accompanies NO production in response to nerve growth factor (NGF). This neurite outgrowth was significantly inhibited by NG-nitro-L-arginine methyl ester (L-NAME), an NO synthase inhibitor, in both PC12 and PC12h cells, suggesting that the neuritogenesis is NO-dependent in both cells. In this report, we investigated whether genipin also induces neurite outgrowth in PC12 cells in order to determine the NO-dependent neurotrophic action of genipin in more than just one cell type. Genipin induced marked neurite outgrowth in PC12h cells but not in PC12 cells. The genipin-induced neurite outgrowth was significantly inhibited by L-NAME in PC12h cells. An NO donor, NOR4, also significantly induced neurite outgrowth in a concentration-dependent manner in PC12h cells but not in PC12 cells. On the other hand, NGF-primed PC12 cells exhibited significant neurite extension, which was inhibited by L-NAME, in response to genipin. Interestingly, NGF-primed PC12 cells responded to NOR4 extending neurites and expressed detectable neuronal NO synthase protein which is not detected in naive PC12 cells. These results suggest that genipin exerts a neuritogenic action on neuronal cells which are responsive to NO itself. Furthermore, the results also suggest that PC12h cells are more suitable for the study of NO-dependent neuronal function than PC12 cells which were not responsive to NO.

Neuritogenic activity of a genipin derivative in retinal ganglion cells is mediated by retinoic acid receptor β expression through nitric oxide/S-nitrosylation signaling.

J. Neurochem. (2011) 10.1111/j.1471‐4159.2011.07533.x

Requirement of Retinoic Acid Receptor β for Genipin Derivative-Induced Optic Nerve Regeneration in Adult Rat Retina

Like other CNS neurons, mature retinal ganglion cells (RGCs) are unable to regenerate their axons after nerve injury due to a diminished intrinsic regenerative capacity. One of the reasons why they lose the capacity for axon regeneration seems to be associated with a dramatic shift in RGCs’ program of gene expression by epigenetic modulation. We recently reported that (1R)-isoPropyloxygenipin (IPRG001), a genipin derivative, has both neuroprotective and neurite outgrowth activities in murine RGC-5 retinal precursor cells. These effects were both mediated by nitric oxide (NO)/S-nitrosylation signaling. Neuritogenic activity was mediated by S-nitrosylation of histone deacetylase-2 (HDAC2), which subsequently induced retinoic acid receptor β (RARβ) expression via chromatin remodeling in vitro. RARβ plays important roles of neural growth and differentiation in development. However, the role of RARβ expression during adult rat optic nerve regeneration is not clear. In the present study, we extended this hypothesis to examine optic nerve regeneration by IPRG001 in adult rat RGCs in vivo. We found a correlation between RARβ expression and neurite outgrowth with age in the developing rat retina. Moreover, we found that IPRG001 significantly induced RARβ expression in adult rat RGCs through the S-nitrosylation of HDAC2 processing mechanism. Concomitant with RARβ expression, adult rat RGCs displayed a regenerative capacity for optic axons in vivo by IPRG001 treatment. These neuritogenic effects of IPRG001 were specifically suppressed by siRNA for RARβ. Thus, the dual neuroprotective and neuritogenic actions of genipin via S-nitrosylation might offer a powerful therapeutic tool for the treatment of RGC degenerative disorders.

New physiological function of secoiridoids: neuritogenic activity in PC12h cells

Previously, we have reported that geniposide isolated from an extract of Gardenia fructus has neuritogenic activity in PC12h cells, a subclone of rat pheochromocytoma cells. Furthermore, we have indicated that several geniposide-related iridoid compounds also had similar potent neuritogenic activity. In this study, we have examined the effects of various secoiridoid compounds [K-1, sweroside; K-2, swertiamarin; K-3, gentiopicroside; K-4, 6′-O-β-d-glucopyranosylsweroside; K-5, 6′-O-β-d-glucopyranosylgentiopicroside; K-6, 6′-O-β-d-glucopyranosylswertiamarin; K-7, 5′-O-β-d-glucopyranosylamarogentin; K-8, 5′-O-β-d-glucopyranosylamaroswertin; H-1, n-butyl vogeloside; H-2, n-butyl epivogeloside; H-3, (7S)-secologanin butyl methyl acetal; H-4, (7R)-secologanin butyl methyl acetal; H-5, secologanin dimethyl acetal] isolated from various medicinal herbs. The secoiridoids H-1, H-2, H-3, H-4, and H-5 induced significant neurite outgrowth. Among these H-series compounds, H-2 was the most potent neuritogenic compound. Among the K-series compounds, K-1, K-2, K-3, and K-8 showed the most potent activity. These results suggest that secoiridoids have neuritogenic activity in PC12h cells and that these secoiridoid compounds are promising starting compounds for the development of neurotrophic factor-like and iridoid compounds.

The Neuroprotective and Neurotrophic Effects of Tremella fuciformis in PC12h Cells

We examined the neuroprotective and neurotrophic effects of Tremella fuciformis. The neurotrophic effects of the hot water extract of T. fuciformis was evaluated by microscopically monitoring its potency to induce neurite outgrowth in PC12h cells. The hot water extract of T. fuciformis promoted neurite outgrowth in PC12h cells in this study, superior to other natural substances which was reported previously. When cells were treated with the hot water extract of T. fuciformis prior to β-amyloid peptide treatment (active domain of A peptide 25~35 treated), toxicity was significantly diminished (p < 0.01). These results suggest that T. fuciformis might potentially be used as a precautionary agent in neurodegenerative disease, such as Alzheimer’s disease, etc.