The present study was designed to investigate the role of quercetin on neurite growth in N1E-115 cells and the underlying mechanisms. Quercetin was evaluated for its effects on cell numbers of neurites, neurite length, intracellular cAMP content, and Gap-43 expression in N1E-115 cells in vitro by use of microscopy, LANCE(tm) cAMP 384 kit, and Western blot analysis, respectively. Our results showed that quercetin could increase the neurite length in a concentration-dependent manner, but had no effect on the numbers of cells. Quercetin significantly increased the expression of cellular cAMP in a time- and concentration-dependent manner. The Gap-43 expression was up-regulated in a time-dependent manner. In conclusion, quercetin could promote neurite growth through increasing the intracellular cAMP level and Gap-43 expression.
Cell Line ; Cyclic AMP ; metabolism ; GAP-43 Protein ; metabolism ; Nerve Regeneration ; Neurites ; drug effects ; Plant Extracts ; pharmacology ; Quercetin ; pharmacology ; Signal Transduction

Neurite outgrowth, a cell differentiation process involving membrane morphological changes, is critical for neuronal network and development. The membrane lipid, phosphatidylinositol (PI) 4,5-bisphosphate (PIP2), is a key regulator of many important cell surface events of membrane signaling, trafficking and dynamics. This lipid is produced mainly by the type I PI 4-phosphate 5-kinase (PIP5K) family members. In this study, we addressed whether PIP5Kalpha, an isoform of PIP5K, could have a role in neurite outgrowth induced by nerve growth factor (NGF). For this purpose, we knocked down PIP5Kalpha in PC12 rat pheochromocytoma cells by stable expression of PIP5Kalpha microRNA that significantly reduced PIP5Kalpha expression and PIP2 level. Interestingly, NGF-induced neurite outgrowth was more prominent in PIP5Kalpha-knockdown (KD) cells than in control cells. Conversely, add-back of PIP5Kalpha into PIP5Kalpha KD cells abrogated the effect of NGF on neurite outgrowth. NGF treatment activated PI 3-kinase (PI3K)/Akt pathway, which seemed to be associated with reactive oxygen species generation. Similar to the changes in neurite outgrowth, the PI3K/Akt activation by NGF was potentiated by PIP5Kalpha KD, but was attenuated by the reintroduction of PIP5Kalpha. Moreover, exogenously applied PIP2 to PIP5Kalpha KD cells also suppressed Akt activation by NGF. Together, our results suggest that PIP5Kalpha acts as a negative regulator of NGF-induced neurite outgrowth by inhibiting PI3K/Akt signaling pathway in PC12 cells.
Animals ; Enzyme Activation/drug effects ; Gene Knockdown Techniques ; Mice ; Nerve Growth Factor/*pharmacology ; Neurites/drug effects/*enzymology ; PC12 Cells ; Phosphatidylinositol 3-Kinases/metabolism ; Phosphatidylinositol 4,5-Diphosphate/metabolism ; Phosphorylation/drug effects ; Phosphotransferases (Alcohol Group Acceptor)/*metabolism ; Proto-Oncogene Proteins c-akt/metabolism ; Rats ; Reactive Oxygen Species/metabolism ; Signal Transduction/drug effects

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 its γ subunit, but does not interact with DCC (deleted in colorectal cancer), another major receptor for netrin-1. Netrin-treatment of cultured cortical neurons leads to increased phosphorylation of AMPK. Both AMPK mutant 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.
AMP-Activated Protein Kinases ; antagonists & inhibitors ; genetics ; metabolism ; Animals ; Cell Adhesion Molecules ; genetics ; metabolism ; Cells, Cultured ; HEK293 Cells ; Humans ; Mice ; Nerve Growth Factors ; pharmacology ; Netrin-1 ; Neurites ; physiology ; Neurons ; cytology ; drug effects ; metabolism ; Phosphorylation ; Protein Binding ; Protein Kinase Inhibitors ; pharmacology ; RNA Interference ; RNA, Small Interfering ; Recombinant Fusion Proteins ; biosynthesis ; genetics ; Signal Transduction ; drug effects ; Transfection ; Tumor Suppressor Proteins ; pharmacology

To investigate the potential ability of the nitrite to induce neuronal differentiation of PC12 cells, cultured PC12 cells planted on matrigel in the presence or absence of sodium nitrite were employed as model, nerve growth factor (NGF) served as a positive control. After 48 h, sodium nitrite enhanced cell viability and vascular endothelial growth factor (VEGF) secretion. Same as the effect of NGF, sodium nitrite (1.4 mmol x L(-1)) treated cultures contained a greater proportion of cells bearing neurites and neurites were much longer than those found in negative control cultures (P < 0.05). Compared with the negative control, sodium nitrite (1.4 mmol x L(-1)) also upregulated the expression of VEGF mRNA (P < 0.05) and hypoxia inducible factor 1 alpha (HIF-1 alpha) or VEGF protein expression (P < 0.05) in cultures of PC12 cells. On the other hand, these effects of the sodium nitrite were likely mediated by HIF-1alpha, since their effects were antagonized by addition of HIF-1alpha inhibitor YC-1. Taken together, these results suggest that low doses of sodium nitrite could induce neurite outgrowth in PC12 cells by activating the HIF-1alpha-VEGF pathway.
Animals ; Cell Differentiation ; drug effects ; Cell Survival ; drug effects ; Food Preservatives ; pharmacology ; Hypoxia-Inducible Factor 1, alpha Subunit ; genetics ; metabolism ; Neurites ; drug effects ; PC12 Cells ; RNA, Messenger ; metabolism ; Rats ; Sodium Nitrite ; pharmacology ; Up-Regulation ; Vascular Endothelial Growth Factor A ; genetics ; secretion

One common feature of glaucoma, optic neuritis and some other optic nerve diseases is sustained and irreversible apoptosis of retinal ganglion cells (RGCs). Ginkgolide B is believed to protect neurons in brain and contribute to neurite outgrowth and synapse formation. The aim of the present study was to explore the effects of Ginkgo biloba extract (EGB761) and ginkgolide B on axonal growth of RCGs. Retina explants were cultured in three-dimensional tissue culture system, and the number and length of neurites were analyzed. Immunohistochemistry staining was performed to confirm that the neurite observed was axon of RGCs. TUNEL and activated caspase-3 staining were also applied to observe RGCs apoptosis. The result shows that neurites of RGCs treated with EGB761 or ginkgolide B were more and longer than those in control. The neurite is proved to be the axon of RGCs by immunostaining. Furthermore, compared with control group, RGCs treated with ginkgolide B showed decreased cellular apoptosis and inhibited caspase-3 activation. These results suggest ginkgolide B can promote RGCs axon growth by protecting RGCs against apoptosis.
Animals ; Apoptosis ; Axons ; drug effects ; Caspase 3 ; metabolism ; Ginkgolides ; pharmacology ; Lactones ; pharmacology ; Neurites ; drug effects ; Organ Culture Techniques ; Plant Extracts ; pharmacology ; Rats ; Retina ; Retinal Ganglion Cells ; cytology ; drug effects

OBJECTIVETo search for low-molecular-weight neuritogenic compounds from the traditional Chinese medicine (TCM).

METHODAn extract library of TCM was prepared. Targeted isolation guided by biological screening led to the discovery of compound 1, and its structure was elucidated by analysis of spectroscopic methods and comparison of spectroscopic data with these reported from the literature.

RESULTA neuritogenic compound 1, 3-O-beta-D-glucopyranosyl-22E, 24R-5alpha, 8alpha-epidioxyergosta-6, 22-diene, was isolated and identified from the methanol extract of T. fuciformis. This compound showed a significant neuritogenic activity against PC12 cells at 3 micromol x L(-1)).

CONCLUSIONMethonal extract of T. fuciformis and targeted compound 1 both showed significant neuritogenic activity against PC12 cells. These results suggested that the extract and compound 1 might be used to prevent and treat neurodegenerative disease such as Alzheimer's disease.

Animals ; Basidiomycota ; chemistry ; Drugs, Chinese Herbal ; chemistry ; isolation & purification ; pharmacology ; Molecular Structure ; Nerve Growth Factor ; chemistry ; isolation & purification ; pharmacology ; Neurites ; drug effects ; PC12 Cells ; Rats

We previously reported that nidogen is an extracellular matrix protein regulating Schwann cell proliferation and migration. Since Schwann cells play a critical role in peripheral nerve regeneration, nidogen may play a role in it via regulation of Schwann cells. Here, we demonstrate direct evidence that nidogen induces elongation of regenerative axon growth of adult sensory neurons, and that the effect is Schwann cell dependent. Continuous infusion of recombinant ectodomain of tumor endothelial marker 7, which specifically blocks nidogen function in Schwann cells, suppressed regenerative neurite growth in a sciatic nerve axotomy model. Taken together, it is likely that nidogen is required for proper regeneration of peripheral nerves after injury.
Animals ; Axotomy ; Cell Movement ; Cell Proliferation ; Male ; Membrane Glycoproteins/*physiology ; Membrane Proteins/pharmacology ; *Nerve Regeneration ; Nerve Tissue Proteins/pharmacology ; Neurites/drug effects/*physiology/ultrastructure ; Rats ; Rats, Sprague-Dawley ; Recombinant Proteins/pharmacology ; Schwann Cells/cytology/*physiology ; Sensory Receptor Cells/*physiology

OBJECTIVETo study the role of adaptor protein Dok6 in neurite outgrowth in PC12 cells.

METHODSSeries of fusion clones were constructed by fusing different domains of Dok6 into mutant TrkC/Y516F. These constructs were transiently transfected into PC12 cells separately and the expression levels of fusion proteins were detected by Western blot. Neurite outgrowth in these PC12 cells was tested after stimulation of NT-3.

RESULTSEach fusion clone was stably expressed in PC12 cells. The fusion clones that fused both TrkC/Y516F-Dok6 (PTB+C) and TrkC/Y516F-Dok6C rescued the loss of neurite outgrowth in PC12 cells resulting from the mutation in tyrosine 516, while fusion clones that fused with single TrkC/Y516F-Dok6PTB did not show such effect.

CONCLUSIONDok6 can promote neurite outgrowth induced by NT-3 stimulation through its C-terminal in TrkC-positive PC12 cells.

Adaptor Proteins, Signal Transducing ; genetics ; metabolism ; Animals ; Neurites ; drug effects ; physiology ; Neurotrophin 3 ; pharmacology ; PC12 Cells ; Rats ; Receptor, trkC ; metabolism ; Transfection

OBJECTIVETo study the effects of androgen on the expression of phosphacan and NG2 proteoglycan (NG2) and neurite regeneration in neonatal rats with hypoxic-ischemic brain damage (HIBD) and the potential mechanism underlying the protective effect of androgen against HIBD.

METHODSOne hundred and twenty neonatal Sprague-Dawley rats were randomly divided into three groups: sham-operated, HIBD and androgen treatment. HIBD was induced by the ligation of left common carotid artery and hypoxia exposure. The androgen treatment group rats were injected with testosterone propionate (25 mg/kg) immediately after HIBD. Phosphacan and NG2 expression in the cortex and the hippocampus was detected with the immunohistochemical method 24 and 72 hrs and 7 and 10 days after hypoxia-ischemia (HI). The ultrastructure and neurite regeneration of neurons in the cortex and the hippocampus were observed under a transmission electron microscope.

RESULTSThe neurite regeneration was obvious in the sham-operated group, but seldom in the HIBD group. The androgen treatment group showed increased neurite regeneration compared with the HIBD group. There were fewer phosphacan and NG2 positive cells in the cortex and the hippocampus in the sham-operated group. Phosphacan and NG2 expression in the cortex and the hippocampus was observed at 24 hrs, increased at 72 hrs, and peaked at 7 days after HI in the HIBD group and remained at a higher expression 10 days after HI than in the sham-operated group. The levels of phosphacan and NG2 expression in the cortex and the hippocampus in the androgen treatment group were significantly reduced compared with those in the HIBD group 24 and 72 hrs and 7 and 10 days after HI (P<0.01).

CONCLUSIONSPhosphacan and NG2 may be important inhibitory factors for neurite regeneration following HIBD in neonatal rats. The neuroprotection of androgen against neonatal HIBD is produced possibly through an inhibition of phosphacan and NG2 expression.

Animals ; Animals, Newborn ; Antigens ; analysis ; Brain Chemistry ; drug effects ; Female ; Hypoxia-Ischemia, Brain ; physiopathology ; Immunohistochemistry ; Male ; Microscopy, Electron, Transmission ; Nerve Regeneration ; drug effects ; Neurites ; physiology ; ultrastructure ; Proteoglycans ; analysis ; Random Allocation ; Rats ; Rats, Sprague-Dawley ; Receptor-Like Protein Tyrosine Phosphatases, Class 5 ; analysis ; Testosterone Propionate ; pharmacology

OBJECTIVETo observe the neuroprotective and neurotrophic effects ofliquiritin (LQ) on rats primary cultured hippocampal neuronal damage induced by Abeta25-35.

METHODThe rats hippocampal neuronal damage model by Abeta25-35 was established. The protective effects of LQ to the cells were observed through the MTU assay. The apoptosis of neurons was detected by flow cytometer and the concentration of intracellular calcium by fluorescence probe dying. LQ' s neurotrophic effects were observed through measuring the neurite outgrowth induced by LQ in primary cultured hippocampal neurons and the differentiation of LQ on hippocampal stem cells to cholinergic neurons was assayed by flow cytometry.

RESULTTreatment of the cells with 10 micromol x L(-1) Abeta25-35 could induce a significant decrease of cell viability, enhance the level of intracellular [Ca2+] and increase the percentage of apoptosis to 28%. However, pretreatment with LQ (0.1, 1, and 10 micromol x L(-1)) for 6 hours exhibited cytoprotective effects, inhibited the cells' s death induced by Abeta25-35, prevented the accumulation of [Ca2+], and decreased the apoptosis neurons significantly to 10%, 15% and 9%, which meaned that LQ could antagonize Abeta25-35 induced apoptosis. LQ together with NGF had a dramatic prolonged effect on the neurite of the primary cultured hippocampal neurons, which was blocked by a specific MAPK kinase inhibitor to some extent. In addition, LQ could induce the differentiation of hippocampal stem cells to cholinergic neurons in vitro.

CONCLUSIONThese results demonstrate that LQ has the neuroprotective capacity to cell damage iduced by Abeta25-35 in primary cultured hippocampal neurons, and also has the neurotrophic effects.

Amyloid beta-Peptides ; adverse effects ; Animals ; Apoptosis ; drug effects ; Calcium ; metabolism ; Cell Differentiation ; drug effects ; Cell Movement ; drug effects ; Cell Survival ; drug effects ; Cells, Cultured ; Female ; Flavanones ; pharmacology ; Glucosides ; pharmacology ; Hippocampus ; cytology ; pathology ; Male ; Neurites ; drug effects ; pathology ; Neurons ; cytology ; drug effects ; metabolism ; pathology ; Neuroprotective Agents ; pharmacology ; Peptide Fragments ; adverse effects ; Rats ; Stem Cells ; pathology