Neuroprotective effects of ginsenoside Rb1 on hippocampal neuronal injury and neurite outgrowth

Ginsenoside Rb1 has been reported to exert anti-aging and anti-neurodegenerative effects. In the present study, we investigate whether ginsenoside Rb1 is involved in neurite outgrowth and neuroprotection against damage induced by amyloid beta（25–35） in cultured hippocampal neurons, and explore the underlying mechanisms. Ginsenoside Rb1 significantly increased neurite outgrowth in hippocampal neurons, and increased the expression of phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2. These effects were abrogated by API-2 and PD98059, inhibitors of the signaling proteins Akt and MEK. Additionally, cultured hippocampal neurons were exposed to amyloid beta（25–35） for 30 minutes; ginsenoside Rb1 prevented apoptosis induced by amyloid beta（25–35）, and this effect was blocked by API-2 and PD98059. Furthermore, ginsenoside Rb1 significantly reversed the reduction in phosphorylated-Akt and phosphorylated extracellular signal-regulated kinase 1/2 levels induced by amyloid beta（25–35）, and API-2 neutralized the effect of ginsenoside Rb1. The present results indicate that ginsenoside Rb1 enhances neurite outgrowth and protects against neurotoxicity induced by amyloid beta（25–35） via a mechanism involving Akt and extracellular signal-regulated kinase 1/2 signaling.

Ginsenoside Rg1 protects against neurodegeneration by inducing neurite outgrowth in cultured hippocampal neurons

Ginsenoside Rg1（Rg1） has anti-aging and anti-neurodegenerative effects. However, the mechanisms underlying these actions remain unclear. The aim of the present study was to determine whether Rg1 affects hippocampal survival and neurite outgrowth in vitro after exposure to amyloid-beta peptide fragment 25–35（Aβ_（25–35））, and to explore whether the extracellular signal-regulated kinase（ERK） and Akt signaling pathways are involved in these biological processes. We cultured hippocampal neurons from newborn rats for 24 hours, then added Rg1 to the medium for another 24 hours, with or without pharmacological inhibitors of the mitogen-activated protein kinase（MAPK） family or Akt signaling pathways for a further 24 hours. We then immunostained the neurons for growth associated protein-43, and measured neurite length. In a separate experiment, we exposed cultured hippocampal neurons to Aβ_（25–35） for 30 minutes, before adding Rg1 for 48 hours, with or without Akt or MAPK inhibitors, and assessed neuronal survival using Hoechst 33258 staining, and phosphorylation of ERK1/2 and Akt by western blot analysis. Rg1 induced neurite outgrowth, and this effect was blocked by API-2（Akt inhibitor） and PD98059（MAPK/ERK kinase inhibitor）, but not by SP600125 or SB203580（inhibitors of c-Jun N-terminal kinase and p38 MAPK, respectively）. Consistent with this effect, Rg1 upregulated the phosphorylation of Akt and ERK1/2; these effects were reversed by API-2 and PD98059, respectively. In addition, Rg1 significantly reversed Aβ_（25–35）-induced apoptosis; this effect was blocked by API-2 and PD98059, but not by SP600125 or SB203580. Finally, Rg1 significantly reversed the Aβ_（25–35）-induced decrease in Akt and ERK1/2 phosphorylation, but API-2 prevented this reversal. Our results indicate that Rg1 enhances neurite outgrowth and protects against Aβ_（25–35）-induced damage, and that its mechanism may involve the activation of Akt and ERK1/2 signaling.

Chemical components of Dendrobium crepidatum and their neurite outgrowth enhancing activities

15 compounds,including two new ones crepidatuols A(1)and B(2)were isolated from the stems of Dendrobium crepidatum.The planar structures of these compounds were elucidated by spectroscopic methods(NMR,MS,UV,and IR)and comparison with those from literatures.10 compounds were send for enhancing activities on nerve growth factor(NGF)medicated neurite outgrowth in PC12 cells and the results indicated that crepidatuol A(1),confusarin and 3-(2-acetoxy-5-methoxy)-phenylpropanol showed enhancing activities at the concentration of 10.0μM.

ROCK inhibition enhances neurite outgrowth in neural stem cells by upregulating YAP expression in vitro

Spontaneous axonal regeneration of neurons does not occur after spinal cord injury because of inhibition by myelin and other inhibitory factors. Studies have demonstrated that blocking the Rho/Rho-kinase （ROCK） pathway can promote neurite outgrowth in spinal cord injury models. In the present study, we investigated neurite outgrowth and neuronal differentiation in neural stem cells from the mouse subventricular zone after inhibition of ROCK in vitro. Inhibition of ROCK with Y-27632 increased neurite length, enhanced neuronal differentiation, and upregulated the expression of two major signaling pathway effectors, phospho-Akt and phospho-mitogen-activated protein kinase, and the Hippo pathway effector YAP. These results suggest that inhibition of ROCK mediates neurite outgrowth in neural stem cells by activating the Hippo signaling pathway.

Inhibition of neurite outgrowth using commercial myelin associated glycoprotein-Fc in neuro-2a cells

Myelin-associated glycoprotein（MAG） inhibits the growth of neurites from nerve cells. Extraction and purification of MAG require complex operations; therefore, we attempted to determine whether commercially available MAG-Fc can replace endogenous MAG for research purposes. Immunofluorescence using specific antibodies against MAG, Nogo receptor（NgR） and paired immunoglobulin-like receptor B（PirB） was used to determine whether MAG-Fc can be endocytosed by neuro-2a cells. In addition, neurite outgrowth of neuro-2a cells treated with different doses of MAG-Fc was evaluated. Enzyme linked immunosorbent assays were used to measure RhoA activity. Western blot assays were conducted to assess Rho-associated protein kinase（ROCK） phosphorylation. Neuro-2a cells expressed NgR and PirB, and MAG-Fc could be endocytosed by binding to NgR and PirB. This activated intracellular signaling pathways to increase RhoA activity and ROCK phosphorylation, ultimately inhibiting neurite outgrowth. These findings not only verify that MAG-Fc can inhibit the growth of neural neurites by activating RhoA signaling pathways, similarly to endogenous MAG, but also clearly demonstrate that commercial MAG-Fc is suitable for experimental studies of neurite outgrowth.

Methyl 3,4-dihydroxybenzoate promotes neurite outgrowth of cortical neurons cultured in vitro

Cerebral cortical neurons from neonatal rats were cultured in the presence of methyl 3,4-dihydroxybenzoate （MDHB; 2, 4, and 8 IJM）. Results showed that MDHB significantly promoted neurite outgrowth and microtubule-associated protein 2 mRNA expression, and increased neuronal survival in a dose-dependent manner. Moreover, MDHB induced brain-derived neurotrophic factor expression. These findings suggest that MDHB has a neurotrophic effect, which may be due to its ability to increase brain-derived neurotrophic factor expression.

Low-frequency electrical stimulation improves neurite outgrowth of dorsal root ganglion neurons in vitro via upregulating Ca^(2+)-mediated brain-derived neurotrophic factor expression

Short-term, low-frequency electrical stimulation of neural tissues significantly enhances axonal regeneration of peripheral nerves following injury. However, little is known about the mechanisms of electrical stimulation to induce neurite outgrowth. In the present study, short-term, low-frequency electrical stimulation, using identical stimulation parameters of in vivo experiments, was administered to in vitro dorsal root ganglion （DRG） neurons. Enhanced neurite outgrowth, as well as synthesis and release of brain-derived neurotrophic factor （BDNF）, were examined in electrical stimulation-treated DRG neuronal cultures. Because the effects of electrical stimulation on neuronal intracellular signaling molecules are less reported, classic calcium intracellular signals are directly or indirectly involved in electrical stimulation effects on neurons. Cultured DRG neurons were pretreated with the calcium channel blocker nifedipine, followed by electrical stimulation. Results suggested that electrical stimulation not only promoted in vitro neurite outgrowth, but also enhanced BDNF expression. However, nifedipine reduced electrical stimulation-enhanced neurite outgrowth and BDNF biosynthesis. These results suggest that the promoting effects of electrical stimulation on DRG neurite outgrowth could be associated with altered calcium influx, which is involved induction of neuronal BDNF expression and secretion.

Valproic acid as a micro RNA modulator to promote neurite outgrowth

Valproic acid （VPA） has been a first-choice drug for clinical treatment of epilepsy and manic disorder. For decades, its phar- macological action was believed to act on inhibition of gam- ma-aminobutyric acid （GABA） transaminase, in turn, increas- ing GABA in inhibitory synapses. However, in recent years, VPA has been investigated on other therapeutic actions. Those investigations demonstrate that VPA shows neuroprotective ef- fects by promoting neurogenesis, neuronal differentiation, and neuroregeneration （Foti et al., 2013）.

Peritoneal macrophages attenuate retinal ganglion cell survival and neurite outgrowth

Inflammation is a critical pathophysiological process that modulates neuronal survival in the central nervous system after disease or injury.However,the effects and mechanisms of macrophage activation on neuronal survival remain unclear.In the present study,we co-cultured adult Fischer rat retinas with primary peritoneal macrophages or zymosan-treated peritoneal macrophages for 7 days.Immunofluorescence analysis revealed that peritoneal macrophages reduced retinal ganglion cell survival and neurite outgrowth in the retinal explant compared with the control group.The addition of zymosan to peritoneal macrophages attenuated the survival and neurite outgrowth of retinal ganglion cells.Conditioned media from peritoneal macrophages also reduced retinal ganglion cell survival and neurite outgrowth.This result suggests that secretions from peritoneal macrophages mediate the inhibitory effects of these macrophages.In addition,increased inflammationand oxidation-related gene expression may be related to the enhanced retinal ganglion cell degeneration caused by zymosan activation.In summary,this study revealed that primary rat peritoneal macrophages attenuated retinal ganglion cell survival and neurite outgrowth,and that macrophage activation further aggravated retinal ganglion cell degeneration.This study was approved by the Animal Ethics Committee of the Joint Shantou International Eye Center of Shantou University and the Chinese University of Hong Kong,Shantou,Guangdong Province,China,on March 11,2014(approval no.EC20140311(2)-P01).

Releasing Nrf2 to promote neurite outgrowth

Roles of Keap1-Nrf2 pathway in brain：Neuronal survival and neurogenesis are impaired in neurodegenerative diseases such as Parkinson＇s disease and Alzheimer＇s disease（Winner et al.,2011）.Genetic up-regulation of growth factors enhanced neuronal survival and neurogenesis.

Emerging roles of the neural adaptor FE65 in neurite outgrowth

The brain is the third largest organ in the human body and consists of over80 billion neurons（Herculano-Houzel,2009）.Neurons are interconnected by neurite to form a complex neural network that allows the communication of neurons to regulate different body functions and activities.Neurites,body.

GIT1 enhances neurite outgrowth by stimulating microtubule assembly

GIT1,a G-protein-coupled receptor kinase interacting protein,has been reported to be involved in neurite outgrowth.However,the neurobiological functions of the protein remain unclear.In this study,we found that GIT1 was highly expressed in the nervous system,and its expression was maintained throughout all stages of neuritogenesis in the brain.In primary cultured mouse hippocampal neurons from GIT1 knockout mice,there was a significant reduction in total neurite length per neuron,as well as in the average length of axon-like structures,which could not be prevented by nerve growth factor treatment.Overexpression of GIT1 significantly promoted axon growth and fully rescued the axon outgrowth defect in the primary hippocampal neuron cultures from GIT1 knockout mice.The GIT1 N terminal region,including the ADP ribosylation factor-GTPase activating protein domain,the ankyrin domains and the Spa2 homology domain,were sufficient to enhance axonal extension.Importantly,GIT1 bound to many tubulin proteins and microtubule-associated proteins,and it accelerated microtubule assembly in vitro.Collectively,our findings suggest that GIT1 promotes neurite outgrowth,at least partially by stimulating microtubule assembly.This study provides new insight into the cellular and molecular pathogenesis of GIT1-associated neurological diseases.

Overexpression of Tau Rescues Nogo-66-Induced Neurite Outgrowth Inhibition In Vitro

Abstract Nogo-66 plays a central role in the myelin- mediated inhibition of neurite outgrowth. Tau is a micro- tubule-associated protein involved in microtubule assembly and stabilization. It remains unverified whether tau inter- acts directly with growth factor receptors, or engages in cross-talk with regeneration inhibitors like Nogo-66. Here, we report that plasmid overexpression of tau significantly elevated the protein levels of total tau, phosphorylated tau, and microtubule-affinity regulating kinase （MARK）. Nogo- 66 transiently elevated the total tau protein level and per- sistently reduced the level of p-s262 tau （tau phosphory- lated at serine 262）, whereas it had little influence on the level of p-T205 tau （tau phosphorylated at threonine 205）. Nogo-66 significantly decreased the protein level of MARK. Hymenialdisine, an inhibitor of MARK, signifi- cantly reduced the level of p-S262 tau. Overexpression of tau rescued the Nogo-66-induced inhibition of neurite outgrowth in neuroblastoma cortical neurons. However, 2a （N2a） cells and primary concomitant inhibition ofMARK abolished the rescue of neurite outgrowth by tan in N2a cells. We conclude that dephosphorylation of tau at S262 is able to regulate Nogo-66 signaling, and that overexpression of tau can rescue the Nogo-66-induced inhibition of neurite outgrowth in vitro.

MiR-130a regulates and dendritic spine MeCP2 neurite outgrowth density by targeting

MicroRNAs （miRNAs） are critical for both development and function of the central nervous system. Significant evidence suggests that abnormal expression of miRNAs is associated with neurodevelopmental disorders. MeCP2 protein is an epigenetic regulator repressing or activating gene transcription by binding to methylated DNA. Both loss-of-function and gain-of-function muta- tions in the MECP2 gene lead to neurodevelopmental disorders such as Rett syndrome, autism and MECP2 duplication syndrome. In this study, we demonstrate that miR-130a inhibits neurite outgrowth and reduces dendritic spine density as well as dendritic complexity. Bioinformatics analyses, cell cultures and biochemical experiments indicate that miR-130a targets MECP2 and down-regulates MeCP2 protein expression. Further- more, expression of the wild-type MeCP2, but not a loss- of-function mutant, rescues the miR-130a-induced phe- notype. Our study uncovers the MECP2 gene as a pre- vious unknown target for miR-130a, supporting that miR-130a may play a role in neurodevelopment by reg- ulating MeCP2. Together with data from other groups,our work suggests that a feedback regulatory mecha- nism involving both miR-130a and MeCP2 may serve to ensure their appropriate expression and function in neural development.

AMPK interacts with DSCAM and plays an important role in Netrin-1 induced neurite outgrowth

Down syndrome cell adhesion molecule(DSCAM)acts as a netrin-1 receptor and mediates attractive response of axons to netrin-1 in neural development.However,the signaling mechanisms of netrin-DSCAM remain unclear.Here we report that AMP-activated protein kinase(AMPK)interacts with DSCAM through itsγsubunit,but does not interact with DCC(deleted in co-lorectal cancer),another major receptor for netrin-1.Netrin-treatment of cultured cortical neurons leads to increased phosphorylation of AMPK.Both AMPK mu-tant with dominant-negative effect and AMPK inhibitor can significantly suppress netrin-1 induced neurite outgrowth.Together,these findings demonstrate that AMPK interacts with DSCAM and plays an important role in netrin-1 induced neurite outgrowth.Our study uncovers a previously unknown component,AMPK,in netrin-DSCAM signaling pathway.

Progranulin promotes neurite outgrowth and neuronal differentiation by regulating GSK-3β

Progranulin(PGRN)has recently emerged as a key player in a subset of frontotemporal dementias(FTD).Numerous mutations in the progranulin gene have been identified in patients with familial or sporadic frontotemporal lobar degeneration(FTLD).In order to understand the molecular mechanisms by which PGRN deficiency leads to FTLD,we examined activity of PGRN in mouse cortical and hippocampal neurons and in human neuroblastoma SH-SY5Y cells.Treatment of mouse neurons with PGRN protein resulted in an increase in neurite outgrowth,supporting the role of PGRN as a neurotrophic factor.PGRN treatment stimulated phosphorylation of glycogen synthase kinase-3 beta(GSK-3β)in cultured neurons.Knockdown of PGRN in SH-SY5Y cells impaired retinoic acid induced differentiation and reduced the level of phosphorylated GSK-3β.PGRN knockdown cells were also more sensitized to staurosporineinduced apoptosis.These results reveal an important role of PGRN in neurite outgrowth and involvement of GSK-3βin mediating PGRN activity.Identification of GSK-3βactivation as a downstream event for PGRN signaling provides a mechanistic explanation for PGRN activity in the nervous system.Our work also suggest that loss of axonal growth stimulation during neural injury repair or deficits in axonal repair may contribute to neuronal damage or axonal loss in FTLD associated with PGRN mutations.Finally,our study suggests that modulating GSK-3βor similar signaling events may provide therapeutic benefits for FTLD cases associated with PGRN mutations.

Promotion of Neurite Outgrowth and Extension Using Injectable Welded Nanofibers

We report a general strategy to develop injectable welded nanofibers to facilitate the outgrowth and extension of neurites.In this case,nonwoven mats of uniaxially aligned poly(caprolactone)(PCL)nanofibers were firstly cut into several small pieces with fixed fiber lengths of 25,50 and 100µm,respectively,using a cryotome.A tissuelyser was employed to homogenize and disperse the short nanofibers to a homogeneous suspension.By tuning treatment duration from 100 s to 400 s,the temperature of the suspension was brought close to the melting point of PCL.As such,the short nanofibers were welded at their cross points while the fibers far away from the cross points remain the original structures.We showed that the viability of neuroblastoma SH-SY5Y cells and their neurite outgrowth and extension were enhanced with the use of such welded short nanofibers.Taken together,this study provides a simple way to generate injectable welded nanofibers,holding potential in affecting neurite outgrowth and extension for nerve repair,in particular,in the central nervous system.

Rho-associated protein kinase modulates neurite extension by regulating microtubule remodeling and vinculin distribution

Rho-associated protein kinase is an essential regulator of cytoskeletal dynamics during the process of neurite extension. However, whether Rho kinase regulates microtubule remodeling or the distri- bution of adhesive proteins to mediate neurite outgrowth remains unclear. By specifically modulat- ing Rho kinase activity with pharmacological agents, we studied the morpho-dynamics of neurite outgrowth. We found that lysophosphatidic acid, an activator of Rho kinase, inhibited neurite out- growth, which could be reversed by Y-27632, an inhibitor of Rho kinase. Meanwhile, reorganization of microtubules was noticed during these processes, as indicated by their significant changes in the soma and growth cone. In addition, exposure to lysophosphatidic acid led to a decreased mem- brane distribution of vinculin, a focal adhesion protein in neurons, whereas Y-27632 recruited vin- culin to the membrane. Taken together, our data suggest that Rho kinase regulates rat hippocampal neurite growth and microtubule formation via a mechanism associated with the redistribution of vinculin.

Enteric glia mediate neuronal outgrowth through release of neurotrophic factors

Previous studies have shown that transplanted enteric glia enhance axonal regeneration, reduce tissue damage, and promote functional recovery following spinal cord injury. However, the mechanisms by which enteric glia mediate these beneficial effects are unknown. Neurotrophic factors can promote neuronal differentiation, survival and neurite extension. We hypothesized that enteric glia may exert their protective effects against spinal cord injury partially through the secretion of neurotrophic factors. In the present study, we demonstrated that primary enteric glia cells release nerve growth factor, brain-derived neurotrophic factor and glial cell line-derived neurotrophic factor over time with their concentrations reaching approximately 250, 100 and 50 pg/mL of culture medium respectively after 48 hours. The biological relevance of this secretion was assessed by incubating dissociated dorsal root ganglion neuronal cultures in enteric glia-conditioned medium with and/or without neutralizing antibodies to each of these proteins and evaluating the differences in neurite growth. We discovered that conditioned medium enhances neurite outgrowth in dorsal root ganglion neurons. Even though there was no detectable amount of neurotrophin-3 secretion using ELISA analysis, the neurite outgrowth effect can be attenuated by the antibody-mediated neutralization of each of the aforementioned neurotrophic factors. Therefore, enteric glia secrete nerve growth factor, brain-derived neurotrophic factor, glial cell line-derived neurotrophic factor and neurotrophin-3 into their surrounding environment in concentrations that can cause a biological effect.

Axonal growth inhibitors and their receptors in spinal cord injury:from biology to clinical translation

Axonal growth inhibitors are released during traumatic injuries to the adult mammalian central nervous system, including after spinal cord injury. These molecules accumulate at the injury site and form a highly inhibitory environment for axonal regeneration. Among these inhibitory molecules, myelinassociated inhibitors, including neurite outgrowth inhibitor A, oligodendrocyte myelin glycoprotein, myelin-associated glycoprotein, chondroitin sulfate proteoglycans and repulsive guidance molecule A are of particular importance. Due to their inhibitory nature, they represent exciting molecular targets to study axonal inhibition and regeneration after central injuries. These molecules are mainly produced by neurons, oligodendrocytes, and astrocytes within the scar and in its immediate vicinity. They exert their effects by binding to specific receptors, localized in the membranes of neurons. Receptors for these inhibitory cues include Nogo receptor 1, leucine-rich repeat, and Ig domain containing 1 and p75 neurotrophin receptor/tumor necrosis factor receptor superfamily member 19(that form a receptor complex that binds all myelin-associated inhibitors), and also paired immunoglobulin-like receptor B. Chondroitin sulfate proteoglycans and repulsive guidance molecule A bind to Nogo receptor 1, Nogo receptor 3, receptor protein tyrosine phosphatase σ and leucocyte common antigen related phosphatase, and neogenin, respectively. Once activated, these receptors initiate downstream signaling pathways, the most common amongst them being the Rho A/ROCK signaling pathway. These signaling cascades result in actin depolymerization, neurite outgrowth inhibition, and failure to regenerate after spinal cord injury. Currently, there are no approved pharmacological treatments to overcome spinal cord injuries other than physical rehabilitation and management of the array of symptoms brought on by spinal cord injuries. However, several novel therapies aiming to modulate these inhibitory proteins and/or their receptors are under investigation in ongoing clinical trials. Investigation has also been demonstrating that combinatorial therapies of growth inhibitors with other therapies, such as growth factors or stem-cell therapies, produce stronger results and their potential application in the clinics opens new venues in spinal cord injury treatment.