Inhibition of Rho kinase (ROCK) increases neurite outgrowth on chondroitin sulphate proteoglycan in vitro and axonal regeneration in the adult optic nerve in vivo

Inhibitory molecules derived from CNS myelin and glial scar tissue are major causes for insufficient functional regeneration in the mammalian CNS. A multitude of these molecules signal through the Rho/Rho kinase (ROCK) pathway. We evaluated three inhibitors of ROCK, Y- 27632, Fasudil (HA-1077), and Dimethylfasudil (H-1152), in models of neurite outgrowth in vitro. We show, that all three ROCK inhibitors partially restore neurite outgrowth of Ntera-2 neurons on the inhibitory chondroitin sulphate proteoglycan substrate. In the rat optic nerve crush model Y-27632 dose-dependently increased regeneration of retinal ganglion cell axons in vivo. Application of Dimethylfasudil showed a trend towards increased axonal regeneration in an intermediate concentration. We demonstrate that inhibition of ROCK can be an effective therapeutic approach to increase regeneration of CNS neurons. The selection of a suitable inhibitor with a broad therapeutic window, however, is crucial in order to minimize unwanted side effects and to avoid deleterious effects on nerve fiber growth.     

Neurite outgrowth resistance to rho kinase inhibitors in PC12 Adh cell

Rho kinase (ROCK) inhibitor is a promising agent for neural injury disorders, which mechanism is associated with neurite outgrowth. However, neurite outgrowth resistance occurred when PC12 Adh cell was treated with ROCK inhibitors for a longer time. PC12 Adh cells were treated with ROCK inhibitor Y27632 or NGF for different durations. Neurite outgrowth resistance occurred when PC12 Adh cell exposed to Y27632 (33 µM) for 3 or more days, but not happen when exposed to nerve growth factor (NGF, 100 ng/mL). The gene expression in the PC12 Adh cells treated with Y27632 (33 µM) or NGF (100 ng/mL) for 2 or 4 days was assayed by gene microarray, and the reliability of the results were confirmed by real‐time RT‐PCR. Cluster analysis proved that the gene expression profile of PC12 Adh cell treated with Y27632 for 4 days was different from that treated with Y27632 for 2 days and those treated with NGF for 2 and 4 days, respectively. Pathway analysis hinted that the neurite outgrowth resistance could be associated with up‐regulation of inflammatory pathways, especially rno04610 (complement and coagulation cascades), and down‐regulation of cell cycle pathways, especially rno04110.     

Picrosides I and II,selective enhancers of the mitogen-activated protein kinase-dependent signaling pathway in the action of neuritogenic substances on PC12D cells

Picrosides I and II caused a concentration-dependent (> 0.1 microM) enhancement of basic fibroblast growth factor (bFGF, 2 ng/ml)-, staurosporine (10 nM)- and dibutyryl cyclic AMP (dbcAMP, 0.3 mM)-induced neurite outgrowth from PC12D cells. PD98059 (20 microM), a potent mitogen-activated protein (MAP) kinase kinase inhibitor, blocked the enhancement of bFGF (2 ng/ml)-, staurosporine (10 nM)- or dbcAMP (0.3 mM)-induced neurite outgrowth by picrosides, suggesting that picrosides activate MAP kinase-dependent signaling pathway. However, PD98059 did not affect the bFGF (2 ng/ml)-, staurosporine (10 nM)- and dbcAMP (0.3 mM)-induced neurite outgrowth in PC12D cells, indicating the existence of two components in neurite outgrowth induced by bFGF, staurosporine and dbcAMP, namely the MAP kinase-independent and the masked MAP kinase-dependent one. Furthermore, picrosides-induced enhancements of the bFGF-action were markedly inhibited by GF109203X (0.1 microM), a protein kinase C inhibitor. The expression of phosphorylated MAP kinase was markedly increased by bFGF (2 ng/ml) and dbcAMP (0.3 mM), whereas that was not enhanced by staurosporine (10 nM). Picrosides had no effect on the phosphorylation of MAP kinase induced by bFGF or dbcAMP and also unaffected it in the presence of staurosporine. These results suggest that picrosides I and II enhance bFGF-, staurosporine- or dbcAMP-induced neurite outgrowth from PC12D cells, probably by amplifying a down-stream step of MAP kinase in the intracellular MAP kinase-dependent signaling pathway. Picrosides I and II may become selective pharmacological tools for studying the MAP kinase-dependent signaling pathway in outgrowth of neurites induced by many kinds of neuritogenic substances including bFGF.     

Sonic Hedgehog Promotes Neurite Outgrowth of Primary Cortical Neurons Through Up-Regulating BDNF Expression

Sonic hedgehog (Shh), a secreted glycoprotein factor, can activate the Shh pathway, which has been implicated in neuronal polarization involving neurite outgrowth. However, little evidence is available about the effect of Shh on neurite outgrowth in primary cortical neurons and its potential mechanism. Here, we revealed that Shh increased neurite outgrowth in primary cortical neurons, while the Shh pathway inhibitor (cyclopamine, CPM) partially suppressed Shh-induced neurite outgrowth. Similar results were found for the expressions of Shh and Patched genes in Shh-induced primary cortical neurons. Moreover, Shh increased the levels of brain-derived neurotrophic factor (BDNF) not only in lysates and in culture medium but also in the longest neurites of primary cortical neurons, which was partially blocked by CPM. In addition, blocking of BDNF action suppressed Shh-mediated neurite elongation in primary cortical neurons. In conclusion, these findings suggest that Shh promotes neurite outgrowth in primary cortical neurons at least partially through modulating BDNF expression.     

Ceramide influences neurite outgrowth and neuroblastoma cell apoptosis regulated by novel protein kinase C isoforms

We have previously seen that protein kinase C (PKC) epsilon induces neurite outgrowth and that PKCdelta and PKCtheta elicit apoptosis in neuroblastoma cells. In this study we investigate the effects of cell-permeable C(2)-ceramide on these events in SK-N-BE(2) neuroblastoma cells. C(2)-ceramide abolishes neurite formation induced by overexpression of PKCepsilon and, in cells overexpressing PKCdelta or PKCtheta, ceramide treatment leads to apoptosis. Exposure to C(2)-ceramide also suppressed neurite outgrowth induced by retinoic acid, but ceramide did not abrogate neurite induction by treatment with the ROCK inhibitor Y-27632, demonstrating that C(2)-ceramide is not a general inhibitor of neurite outgrowth. The neurite-suppressing effect occurs independently of cell-death. Furthermore, C(2)-ceramide relocated PKCepsilon and the isolated regulatory domain of PKCepsilon from the cytosol to the perinuclear region. In contrast, neither the localization of PKCdelta nor of PKCtheta was affected by C(2)-ceramide. Taken together, the data indicate that the neurite-inhibiting effect of C(2)-ceramide treatment may be caused by a re-localization of PKCepsilon and thus identify a functional consequence of ceramide effects on PKCepsilon localization.     

The FGFR1 inhibitor PD 173074 selectively and potently antagonizes FGF-2 neurotrophic and neurotropic effects

Basic fibroblast growth factor (FGF-2) promotes survival and/or neurite outgrowth from a variety of neurons in cell culture and regenerative processes in vivo. FGFs exert their effects by activating cell surface receptor tyrosine kinases. FGF receptor (FGFR) inhibitors have not been characterized on neuronal cell behaviors to date. In the present study, we show that the FGFR1 inhibitor PD 173074 potently and selectively antagonized the neurotrophic and neurotropic actions of FGF-2. Nanomolar concentrations of PD 173074 prevented FGF-2, but not insulin-like growth factor-1, support of cerebellar granule neuron survival under conditions of serum/K(+) deprivation; another FGF-2 inhibitor, SU 5402, was effective only at a 1,000-fold greater concentration. Neither PD 173074 nor SU 5402, at 100 times their IC(50) values, interfered with the survival of dorsal root ganglion neurons promoted by nerve growth factor, ciliary neurotrophic factor, or glial cell line-derived neurotrophic factor. PD 173074 and SU 5402 displayed 1,000-fold differential IC(50) values for inhibition of FGF-2-stimulated neurite outgrowth in PC12 cells and in granule neurons, and FGF-2-induced mitogen-activated protein kinase (p44/42) phosphorylation. The two inhibitors failed to disturb downstream signalling stimuli of FGF-2. PD 173074 represents a valuable tool for dissecting the role of FGF-2 in normal and pathological nervous system function without compromising the actions of other neurotrophic factors.     

p116Rip targets myosin phosphatase to the actin cytoskeleton and is essential for RhoA/ROCK-regulated neuritogenesis

Activation of the RhoA-Rho kinase (ROCK) pathway stimulates actomyosin-driven contractility in many cell systems, largely through ROCK-mediated inhibition of myosin II light chain phosphatase. In neuronal cells, the RhoA-ROCK-actomyosin pathway signals cell rounding, growth cone collapse, and neurite retraction; conversely, inhibition of RhoA/ROCK promotes cell spreading and neurite outgrowth. The actin-binding protein p116(Rip), whose N-terminal region bundles F-actin in vitro, has been implicated in Rho-dependent neurite remodeling; however, its function is largely unknown. Here, we show that p116(Rip), through its C-terminal coiled-coil domain, interacts directly with the C-terminal leucine zipper of the regulatory myosin-binding subunits of myosin II phosphatase, MBS85 and MBS130. RNA interference-induced knockdown of p116(Rip) inhibits cell spreading and neurite outgrowth in response to extracellular cues, without interfering with the regulation of myosin light chain phosphorylation. We conclude that p116(Rip) is essential for neurite outgrowth and may act as a scaffold to target the myosin phosphatase complex to the actin cytoskeleton.     

Fasudil protects hippocampal neurons against hypoxia-reoxygenation injury by suppressing microglial inflammatory responses in mice

Rho kinase (ROCK) may play an important role in regulating biological events of cells, including proliferation, differentiation and survival/death. Blockade of ROCK promotes axonal regeneration and neuron survival in vivo and in vitro, thereby exhibiting potential clinical applications in spinal cord damage and stroke. Our previous studies have demonstrated that Fasudil, a selective ROCK inhibitor, induced neuroprotection in vitro. Here we used an in vivo model of hypoxia/reoxygenation (H/R) injury to examine the neuroprotective effect of Fasudil, and explore its possible mechanism(s) in vivo. H/R resulted in the loss of hippocampal neurons, accompanied by increased apoptosis of neurons in hippocampus. The expression of ROCK II and activity of ROCK in the brain were increased after H/R, and located only in microglia, but not in astrocytes and neurons. The administration of Fasudil inhibited the activity of ROCK in brain tissue and cultured microglia, and protected hippocampal neurons against H/R injury. Further immunohistochemical analysis and cytokine determination revealed that Fasudil inhibited inducible nitric oxide synthase immunoreactivity in microglia and pro-inflammatory factors in brain tissue after H/R, which is consistent with the observation wherein Fasudil reduced the pro-inflammatory factors nitric oxide, IL-1β, IL-6 and TNF-, and increased anti-inflammatory factor IL-10 in cultured microglia under normoxic or hypoxic conditions. Our results indicate that inhibition of ROCK by Fasudil may represent a useful therapeutic perspective by inhibiting microglial inflammatory responses in the CNS.     

Neurite Outgrowth Effect of 4-O-methylhonokiol by Induction of Neurotrophic Factors Through ERK Activation

Compounds isolated from Magnolia officinalis such as magnolol, honokiol and obovatol exhibit several pharmacological effects on CNS including depressant, anxiolytic and anticonvulsant effects, as well as neuroprotective effects against chemical and heat damages. Recently, honokiol was found to have a neurotrophic effect in fetal rat cortical neurons. In the present study, we show that 4-O-methylhonokiol, a novel compound from Magnolia officinalis, promotes neurite outgrowth in a concentration-dependent manner in rat embryonic neuronal cells. In parallel with the neurite outgrowth activity, the expression of neurite outgrowth marker proteins is also increased by treatment with 4-O-methylhonokiol. We also found that 4-O-methylhonokiol promotes the release of NGF and BDNF into cell culture medium. In addition, lower concentration of 4-O-methylhonokiol (1 and 2 μM) further enhanced neurite outgrowth and expression of neurite outgrowth marker proteins in the presence of NGF (50 ng/ml) or BDNF (10 ng/ml). Subsequently, we found that 4-O-methylhonokiol activates ERK in a concentration-dependent manner. However, the neurite outgrowth activity and the NGF and BDNF release induced by 4-O-methylhonokiol are suppressed by an ERK-specific inhibitor. These results suggest that 4-O-methylhonokiol has the ability to induce neurite outgrowth via the increase of neurotrophic factor levels through ERK activation.     

Induction of neurites by the regulatory domains of PKCdelta and epsilon is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) epsilon, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCepsilon resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCepsilon-overexpressing cells and led to neurite induction in cells overexpressing PKCdelta. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCdelta induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCdelta-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCdelta can be counteracted by PKCdelta kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.     

Phospholipase D1 is an Important Regulator of bFGF-Induced Neurotrophin-3 Expression and Neurite Outgrowth in H19-7 Cells

The purpose of this study was to examine the role of phospholipase D1 (PLD1) in basic fibroblast growth factor (bFGF)-induced neurotrophin-3 (NT-3) expression and neurite outgrowth in H19-7 rat hippocampal neuronal progenitor cells. Overexpression of PLD1 increased bFGF-induced NT-3 expression, and dominant-negative-PLD1 or PLD1 siRNA abolished bFGF-induced NT-3 expression and neurite outgrowth. Treatment with bFGF activated the RhoA/Rho-associated kinase (ROCK)/c-jun N-terminal kinase (JNK) pathway, and bFGF-induced NT-3 expression was blocked by a dominant-negative RhoA as well as by a specific Rho-kinase inhibitor (Y27632) and a SAPK/JNK inhibitor (SP600125). Furthermore, bFGF-induced JNK activation was also blocked by Y27632. These results indicate that the RhoA/ROCK/JNK pathway acts as an upstream signaling pathway in bFGF-induced NT-3 expression. Also, phosphatidic acid, the product of PLD, increased NT-3 expression. We found that PLD regulated the RhoA/ROCK/JNK pathway, which then led to Elk-1 transactivation. When Elk-1 activity was blocked by Elk-1 siRNA, bFGF-induced NT-3 expression and neurite outgrowth decreased. NT-3 overexpression increased neurite outgrowth, indicating that NT-3 is important for neurite outgrowth. Taken together, these results suggest that PLD1 is an important regulator of bFGF-induced NT-3 expression and neurite outgrowth, which are mediated by the RhoA/ROCK/JNK pathway via Elk-1 in H19-7 cells.     

Evaluation of the role of Disabled-2 in nerve growth factor-mediated neurite outgrowth and cellular signalling

Disabled-2 (DAB2) is an adapter protein that plays a key role in cell proliferation and differentiation. We reported here that DAB2 is expressed in various regions of rat central nervous system and is most abundant in the olfactory bulb. The up-regulation of DAB2 upon 5,7-dihydroxytryptamine-induced spinal cord lesion implicates that DAB2 may participate in the regulation of neuronal plasticity. To investigate DAB2 function in the regulation of neurite outgrowth, the rat p59 and p82 form of DAB2 was individually and stably expressed in the PC12 cells. Both p59 and p82 inhibited nerve growth factor (NGF)-induced neurite outgrowth concomitantly with a decrease in the expression of neuron-specific cytoskeleton protein beta-tubulin III. To unveil the molecular mechanism of DAB2 in NGF signaling, we found that RhoA-GTPase activity was up-regulated in DAB2 stable lines whereas the Ras/MAPK and PI3-kinase/Akt signaling was not affected. The inhibitory effect of DAB2 on NGF-mediated neurite outgrowth was reversed by the pretreatment of Rho-kinase (ROCK) inhibitor Y27632, implicating that DAB2 modulates RhoA/ROCK signaling. Together, this study defines a role of DAB2 in the control of neuronal plasticity and demonstrates for the first time that DAB2 is a negative regulator in NGF-mediated neurite outgrowth.     

The interplay of cyclic stretch and vascular endothelial growth factor in regulating the initial steps for angiogenesis

Angiogenesis is regulated by chemical and mechanical factors in vivo. The regulatory role of mechanical factors and how chemical and mechanical angiogenic regulators work in concert remains to be explored. We investigated the effect of cyclic uniaxial stretch (20%, 1 Hz), with and without the stimulation of vascular endothelial growth factor (VEGF), on sprouting angiogenesis by employing a stretchable three‐dimensional cell culture model. When compared to static controls, stretch alone significantly increased the density of endothelial sprouts, and these sprouts aligned perpendicular to the direction of stretch. The Rho‐associated kinase (ROCK) inhibitor Y27632 suppressed stretch‐induced sprouting angiogenesis and associated sprout alignment. While VEGF is a potent angiogenic stimulus through ROCK‐dependent pathways, the combination of VEGF and stretch did not have an additive effect on angiogenesis. In the presence of VEGF stimulation, the ROCK inhibitor suppressed stretch‐induced sprout alignment but did not affect stretch‐induced sprout density; in contrast, the receptor tyrosine kinase (RTK) inhibitor sunitinib had no effect on stretch‐induced alignment but trended toward suppressed stretch‐induced sprout density. Our results suggest that the formation of sprouts and their directionality do not have completely identical regulatory pathways, and thus it is possible to separately manipulate the number and pattern of new sprouts. © 2014 American Institute of Chemical Engineers Biotechnol. Prog., 31:248–257, 2015     

Prevention of activity-dependent neuronal death: Vasoactive intestinal polypeptide stimulates astrocytes to secrete the thrombin-inhibiting neurotrophic serpin,protease nexin I

Neuronal cell death occurs as a programmed, naturally occurring mechanism and is the primary regressive event in central nervous system development. Death of neurons also occurs on an injury-induced basis after trauma and in human neurodegenerative diseases. Classical neurotrophic factors can reverse this phenomenon in experimental models prompting initiation of clinical trials in conditions such as amyotrophic lateral sclerosis and Alzheimer's disease. The glial-derived protease nexin I (PNI), a known promoter of neurite outgrowth in cell culture and a potent inhibitor of serine proteases, also enhances neuronal cell survival. PNI, in nanomolar concentrations, rescues spinal cord motor neurons from both naturally-occurring programmed cell death in the chick embryo as well as following injury in the neonatal mouse. The potent neuromodulator, vasoactive intestinal polypeptide (VIP), influences neuronal survival through glial-mediated factors and also induces secretion of newly synthesized astrocyte PNI. We now report that subnanomolar amounts of PNI enhance neuronal survival in mixed spinal cord cell culture, especially when neuronal cells were made electrically silent by administration of tetrodotoxin. The mediation of this effect is by inhibition of the multifunctional serine protease, thrombin, because hirudin, a thrombin-specific inhibitor, has the same effect. In addition, spinal cord neurons are exquisitely sensitive to thrombin because picomolar and lower levels of the coagulation factor causes neuronal death. Thus, PNI is an astrocyte-derived, thrombin-inhibiting, activity-dependent neurotrophic agent, enhanced secretion of which by VIP may be one approach to treat neurological disorders. © 1996 John Wiley & Sons, Inc.     

Ciliary neurotrophic factor,unlike nerve growth factor,supports neurite outgrowth but not synapse formation by adult Lymnaea neurons

The nerve growth factor (NGF) family and ciliary neurotrophic factor (CNTF) support survival and/or neurite outgrowth of many cell types. However, it is not known whether the neurite outgrowth induced by neurotrophic factors results in the formation of synapses. We tested NGF and CNTF for their ability to induce neurite outgrowth and synapse formation in vitro by interneurons from the mollusc Lymnaea. Dopaminergic and peptidergic interneurons survived in the absence of neurotrophic factors but exhibited robust outgrowth in response to both NGF and CNTF. Chemical synapses formed between these interneurons and their target neurons cultured in NGF, but synapses were absent in CNTF. Survival, neurite outgrowth, and synaptogenesis are therefore differentially regulated in these neurons. © 1996 John Wiley & Sons, Inc.     

Neurite outgrowth from progeny of epidermal growth factor–responsive hippocampal stem cells is significantly less robust than from fetal hippocampal cells following grafting onto organotypic hippocampal slice cultures: Effect of brain‐derived neurotrophic factor

Epidermal growth factor (EGF)–responsive stem cells from both developing and adult central nervous system (CNS) can be expanded and induced to differentiate into neurons and glia in vitro. Because of their self‐renewal and multipotent properties, these cells can potentially provide an unlimited tissue source for neural grafting in neurodegenerative disorders. However, the capability of neurons derived from these stem cells to project axons to distant targets following grafting, thereby enabling the restoration of damaged CNS circuitry, remains unknown. We hypothesize that grafted EGF‐responsive stem cells and their progeny are not competent to project axons into distant target sites unless exposed to specific neurotrophic factors. We compared neurite outgrowth between gestation day 14 primary mouse hippocampal cells and EGF‐generated secondary neurospheres of postnatal mouse hippocampal stem cells, following grafting onto the CA3 region of organotypic hippocampal slice cultures prepared from postnatal rats. Neurite outgrowth from grafted cells was visualized using immunohistochemical staining for the mouse specific antigen M6. Fetal hippocampal cells showed extensive and specific neurite outgrowth into many regions of the slice, including the CA1 region and distant subiculum, by 7 days after grafting. In contrast, neurite outgrowth from neurosphere cells was nonspecific and restricted to the immediate surrounding region after either 7 or even 15 days following grafting. Application of brain‐derived neurotrophic factor (BDNF) (5 ng in 0.5 μL) to slices on day 1 after grafting significantly enhanced neurite outgrowth from neurosphere cells, but overall neurite outgrowth from neurosphere cells remained decreased compared to that from fetal hippocampal cells. These results underscore that EGF‐responsive stem cell‐derived neurons possess limited intrinsic capability for long‐distance neurite outgrowth compared to fetal neurons. However, neurite outgrowth from EGF‐responsive stem cell–derived neurons can be enhanced by treating with specific neurotrophic factors such as BDNF. © 1999 John Wiley & Sons, Inc. J Neurobiol 38: 391–413, 1999     

miR-124-regulated RhoG reduces neuronal process complexity via ELMO/Dock180/Rac1 and Cdc42 signalling

The small GTPase RhoG plays a central role in actin remodelling during diverse biological processes such as neurite outgrowth, cell migration, phagocytosis of apoptotic cells, and the invasion of pathogenic bacteria. Although it is known that RhoG stimulates neurite outgrowth in the rat pheochromocytoma PC12 cell line, neither the physiological function nor the regulation of this GTPase in neuronal differentiation is clear. Here, we identify RhoG as an inhibitor of neuronal process complexity, which is regulated by the microRNA miR-124. We find that RhoG inhibits dendritic branching in hippocampal neurons in vitro and in vivo. RhoG also inhibits axonal branching, acting via an ELMO/Dock180/Rac1 signalling pathway. However, RhoG inhibits dendritic branching dependent on the small GTPase Cdc42. Finally, we show that the expression of RhoG in neurons is suppressed by the CNS-specific microRNA miR-124 and connect the regulation of RhoG expression by miR-124 to the stimulation of neuronal process complexity. Thus, RhoG emerges as a cellular conductor of Rac1 and Cdc42 activity, in turn regulated by miR-124 to control axonal and dendritic branching.