Small molecule inhibitor DDQ-treated hippocampal neuronal cells show improved neurite outgrowth and synaptic branching

The process of neurite outgrowth and branching is a crucial aspect of neuronal development and regeneration.Axons and dendrites,sometimes referred to as neurites,are extensions of a neuron's cellular body that are used to start networks.Here we explored the effects of diethyl(3,4-dihydroxyphenethylamino)(quinolin-4-yl)methylphosphonate(DDQ)on neurite developmental features in HT22 neuronal cells.In this work,we examined the protective effects of DDQ on neuronal processes and synaptic outgrowth in differentiated HT22cells expressing mutant Tau(mTau)cDNA.To investigate DDQ chara cteristics,cell viability,biochemical,molecular,western blotting,and immunocytochemistry were used.Neurite outgrowth is evaluated through the segmentation and measurement of neural processes.These neural processes can be seen and measured with a fluorescence microscope by manually tracing and measuring the length of the neurite growth.These neuronal processes can be observed and quantified with a fluorescent microscope by manually tracing and measuring the length of the neuronal HT22.DDQ-treated mTau-HT22 cells(HT22 cells transfected with cDNA mutant Tau)were seen to display increased levels of synaptophysin,MAP-2,andβ-tubulin.Additionally,we confirmed and noted reduced levels of both total and p-Tau,as well as elevated levels of microtubule-associated protein 2,β-tubulin,synaptophysin,vesicular acetylcholine transporter,and the mitochondrial biogenesis protein-pe roxisome prolife rator-activated receptor-gamma coactivator-1α.In mTa u-expressed HT22 neurons,we observed DDQ enhanced the neurite characteristics and improved neurite development through increased synaptic outgrowth.Our findings conclude that mTa u-HT22(Alzheimer's disease)cells treated with DDQ have functional neurite developmental chara cteristics.The key finding is that,in mTa u-HT22 cells,DDQ preserves neuronal structure and may even enhance nerve development function with mTa u inhibition.

Modulation of the Nogo signaling pathway to overcome amyloid-β-mediated neurite inhibition in human pluripotent stem cell-derived neurites

Neuronal cell death and the loss of connectivity are two of the primary pathological mechanisms underlying Alzheimer's disease.The accumulation of amyloid-βpeptides,a key hallmark of Alzheimer's disease,is believed to induce neuritic abnormalities,including reduced growth,extension,and abnormal growth cone morphology,all of which contribute to decreased connectivity.However,the precise cellular and molecular mechanisms governing this response remain unknown.In this study,we used an innovative approach to demonstrate the effect of amyloid-βon neurite dynamics in both two-dimensional and three-dimensional cultu re systems,in order to provide more physiologically relevant culture geometry.We utilized various methodologies,including the addition of exogenous amyloid-βpeptides to the culture medium,growth substrate coating,and the utilization of human-induced pluripotent stem cell technology,to investigate the effect of endogenous amyloid-βsecretion on neurite outgrowth,thus paving the way for potential future applications in personalized medicine.Additionally,we also explore the involvement of the Nogo signaling cascade in amyloid-β-induced neurite inhibition.We demonstrate that inhibition of downstream ROCK and RhoA components of the Nogo signaling pathway,achieved through modulation with Y-27632(a ROCK inhibitor)and Ibuprofen(a Rho A inhibitor),respectively,can restore and even enhance neuronal connectivity in the presence of amyloid-β.In summary,this study not only presents a novel culture approach that offers insights into the biological process of neurite growth and inhibition,but also proposes a specific mechanism for reduced neural connectivity in the presence of amyloid-βpeptides,along with potential intervention points to restore neurite growth.Thereby,we aim to establish a culture system that has the potential to serve as an assay for measuring preclinical,predictive outcomes of drugs and their ability to promote neurite outgrowth,both generally and in a patient-specific manner.

Outgrowth endothelial cells form a functional cerebral barrier and restore its integrity after damage

Breakdown of blood-brain barrier,formed mainly by brain microvascular endothelial cells(BMECs),represents the major cause of mortality during early phases of ischemic strokes.Hence,discovery of novel agents that can effectively replace dead or dying endothelial cells to restore blood-brain barrier integrity is of paramount importance in stroke medicine.Although endothelial progenitor cells(EPCs)represent one such agents,their rarity in peripheral blood severely limits their adequate isolation and therapeutic use for acute ischemic stroke which necessitate their ex vivo expansion and generate early EPCs and outgrowth endothelial cells(OECs)as a result.Functional analyses of these cells,in the present study,demonstrated that only OECs endocytosed DiI-labelled acetylated low-density lipoprotein and formed tubules on matrigel,prominent endothelial cell and angiogenesis markers,respectively.Further analyses by flow cytometry demonstrated that OECs expressed specific markers for sternness(CD34),immaturity(CD133)and endothelial cells(CD31)but not for hematopoietic cells(CD45).Like BMECs,OECs established an equally tight in vitro model of human BBB with astrocytes and pericytes,suggesting their capacity to form tight junctions.Ischemic injury mimicked by concurrent deprivation of oxygen and glucose(4 hours)or deprivation of oxygen and glucose followed by reperfusion(20 hours)affected both barrier integrity and function in a similar fashion as evidenced by decreases in transendothelial electrical resistance and increases in paracellular flux,respectively.Wound scratch assays comparing the vasculoreparative capacity of cells revealed that,compared to BMECs,OECs possessed a greater proliferative and directional migratory capacity.In a triple culture model of BBB established with astrocytes,pericytes and BMEC,exogenous addition of OECs effectively repaired the damage induced on endothelial layer in serum-free conditions.Taken together,these data demonstrate that OECs may effectively home to the site of vascular injury and repair the damage to maintain(neuro)vascular homeostasis during or after a cerebral ischemic injury.

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.

Proteoglycans: Road Signs for Neurite Outgrowth

Proteoglycans in the central nervous system play integral roles as ＂traffic signals＂ for the direction of neurite outgrowth. This attribute of proteoglycans is a major factor in regeneration of the injured central nervous system. In this review, the structures of proteoglycans and the evidence suggesting their involvement in the response following spinal cord injury are presented. The review further describes the methods routinely used to determine the effect proteoglycans have on neurite outgrowth. The effects of proteoglycans on neurite outgrowth are not completely understood as there is disagreement on what component of the molecule is interacting with growing neurites and this ambiguity is chronicled in an historical context. Finally, the most recent findings suggesting possible receptors, interactions, and sulfation patterns that may be important in eliciting the effect of proteoglycans on neurite outgrowth are discussed. A greater understanding of the proteoglycan-neurite interaction is necessary for successfully promoting regeneration in the iniured central nervous system.

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.

PAd-shRNA-PTN reduces pleiotrophin of pancreatic cancer cells and inhibits neurite outgrowth of DRG

AIM:To investigate the silencing effects of pAdshRNA-pleiotrophin(PTN) on PTN in pancreatic cancer cells,and to observe the inhibition of pAd-shRNA-PTN on neurite outgrowth from dorsal root ganglion(DRG) neurons in vitro.METHODS:PAd-shRNA-PTN was used to infect pancreatic cancer BxPC-3 cells;assays were conducted for knockdown of the PTN gene on the 0th,1st,3rd,5th,7th and 9th d after infection using immunocytochemistry,real-time quantitative polymerase chain reaction(PCR),and Western blotting analysis.The morphologic changes of cultured DRG neurons were observed by mono-culture of DRG neurons and co-culture with BXPC-3 cells in vitro.RESULTS:The real-time quantitative PCR showed that the inhibition rates of PTN mRNA expression in the BxPC-3 cells were 20%,80%,50% and 25% on the 1st,3rd,5th and 7th d after infection.Immunocytochemistry and Western blotting analysis also revealed the same tendency.In contrast to the control,the DRG neurons co-cultured with the infected BxPC-3 cells shrunk;the number and length of neurites were significantly decreased.CONCLUSION:Efficient and specific knockdown of PTN in pancreatic cancer cells and the reduction in PTN expression resulted in the inhibition of neurite outgrowth from DRG neurons.

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.

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.

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.

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.

JAK/STAT signaling regulates tissue outgrowth and male germline stem cell fate in Drosophila

In multicellular organisms, biological activities are regulated by cell signaling. The various signal transduction path- ways regulate cell fate, proliferation, migration, and polarity. Miscoordination of the communicative signals will lead to disasters like cancer and other fatal diseases. The JAK/STAT signal transduction pathway is one of the pathways, which was first identified in vertebrates and is highly conserved throughout evolution. Studying the JAK/STAT signal transduc- tion pathway in Drosophila provides an excellent opportunity to understand the molecular mechanism of the cell regu- lation during development and tumor formation. In this review, we discuss the general overview of JAK/STAT signaling in Drosophila with respect to its functions in the eye development and stem cell fate determination.

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.

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.

Safflower Yellow Compounds Alleviate Okadaic Acid-Induced Impairment of Neurite Outgrowth in Differentiated SH-SY5Y Cells

The dynamic assembly of microtubules plays a key role in maintaining structural and functional integrity of eukaryotic cel s,especial y with regard to neuronal differentiation or neurite outgrowth and synaptic plasticity,which contribute to the development of the nervous system and memory formation.

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.

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）.

Prokaryotic expression of recombinant human p75NTR-Fc fusion protein and its effect on the neurite outgrowth of dorsal root ganglia neuron

Objective： To clone, express, and identify the extracellular domain gene of human p75 neurotrophin receptor with IgG-Fe （hp75NTR-Fc） in prokaryotic expression system, and investigate the effect of the recombinant protein on dorsal root ganglia （DRG） neuron neurites. Methods： The hp75NTR-Fc coding sequence was amplified from pcDNA-hp75NTR-Fc by polymerase chain reaction （PCR） and subcloned into vector pET30a （＋）, in which hp75NTR-Fc expression was controlled under the T7 promoter. The recombinant vectors were amplified in E. coli DH5α and identified by PCR, enzyme digestion and sequencing, and then transformed into E. coli BL21 （DE3）. The expression product was analyzed with SDS-PAGE and Western blot. Then after the recombinant protein purified with Protein A affinity chromatograph, and renaturated with dialysis, respectively, the effect of the recombinant protein on DRG neuron neuritis was further investigated. Results： The results of PCR, enzyme digestion, and sequencing demonstrated the success of inserting the hp75NTR-Fc fragment into vector pET30a （＋）. SDS-PAGE and Western blot showed a positive protein band with molecular weight about 50 kD in the expression product, which is accordant with the interest protein, and this band could be specifically recognized by rabbit anti-NGFRp75 antibody. The purified infusion protein following dialysis could promote neurite outgrowth of DRG neurons cultured with myelin-associated glycoprotein （MAG）. Conclusion： The hp75NTR-Fc coding sequence was subcloned into the expression vector pET30a （＋） correctly and expressed successfully in the prokaryotie expression system. The infusion protein could promote neurite outgrowth of DRG neurons cultured with MAG.

Enhancing Effect of Aged Garlic Extract on Induction of Morphological Differentiation with Neurite Outgrowth in NGF-treated PC12 Cells

Background: We have been searching effective compounds that can stimulate the growth and differentiation of nerve cells. We found previously that fulleren derivatives enhanced induction of morphological differentiation with neurite outgrowth in nerve growth factor (NGF)-treated PC12 cells. In the course of our further search for other effective compounds, we found the aged garlic extract (AGE) has the activity similar to fulleren. Methods: PC12 cells were used to examine the effectiveness of test compound. Results: AGE enhanced the stimulating effect of NGF to induce morphological differentiation with neurite outgrowth in PC12 cells. In order to examine the active constituents of AGE, it was fractionated into several components. The activity was mainly localized in the F1 fraction that contains low molecular weight polar compounds. S-Allymercaptocysteine (SAMC) is one of the sulfur components of AGE present in F1 fraction and found to exhibit the enhancing effect similar to AGE. Conclusion: AGE had the ability to induce morphological differentiation with neurite outgrowth in NGF-treated PC12 cell and SAMC was one of its active constituents.

The role of cell surface β1， 4－galactosyltransferase duringectoplacentacone outgrowth on laminin

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