NGF-induced Neurite Outgrowth Research Articles

Mechanisms controlling neurite outgrowth in a pheochromocytoma cell line: The role of TRPC channels

Transient Receptor Potential Canonical (TRPC) channels are implicated in modulating neurite outgrowth. The expression pattern of TRPCs changes significantly during brain development, suggesting that fine-tuning TRPC expression may be important for orchestrating neuritogenesis. To study how alterations in the TRPC expression pattern affect neurite outgrowth, we used nerve growth factor (NGF)-differentiated rat pheochromocytoma 12 (PC12) cells, a model system for neuritogenesis. In PC12 cells, NGF markedly up-regulated TRPC1 and TRPC6 expression, but down-regulated TRPC5 expression while promoting neurite outgrowth. Overexpression of TRPC1 augmented, whereas TRPC5 overexpression decelerated NGF-induced neurite outgrowth. Conversely, shRNA-mediated knockdown of TRPC1 decreased, whereas shRNA-mediated knockdown of TRPC5 increased NGF-induced neurite extension. Endogenous TRPC1 attenuated the anti-neuritogenic effect of overexpressed TRPC5 in part by forming the heteromeric TRPC1-TRPC5 channels. Previous reports suggested that TRPC6 may facilitate neurite outgrowth. However, we found that TRPC6 overexpression slowed down neuritogenesis, whereas dominant negative TRPC6 (DN-TRPC6) facilitated neurite outgrowth in NGF-differentiated PC12 cells. Consistent with these findings, hyperforin, a neurite outgrowth promoting factor, decreased TRPC6 expression in NGF-differentiated PC12 cells. Using pharmacological and molecular biological approaches, we determined that NGF up-regulated TRPC1 and TRPC6 expression via a p75(NTR)-IKK(2)-dependent pathway that did not involve TrkA receptor signaling in PC12 cells. Similarly, NGF up-regulated TRPC1 and TRPC6 via an IKK(2) dependent pathway in primary cultured hippocampal neurons. Thus, our data suggest that a balance of TRPC1, TRPC5, and TRPC6 expression determines neurite extension rate in neural cells, with TRPC6 emerging as an NGF-dependent "molecular damper" maintaining a submaximal velocity of neurite extension.

K+-Cl-Cotransporter 1 (KCC1) Negatively Regulates NGF-induced Neurite Outgrowth in PC12 Cells

Potassium chloride cotransporters (KCCs) mediate electroneutrally-coupled transport of K⁺ and Cl^-, and play crucial roles in various cell functions including regulation of cell volume and homeostasis of cellular Cl^-content. Four isoforms of KCCs (KCC1, 2, 3, and 4) have been identified. KCC1 is ubiquitously expressed, whereas KCC2 is mainly expressed in neuronal cells of central nervous system. KCC3 is highly expressed in heart, skeletal muscle, kidney, lung and placenta. KCC4 is mainly expressed in epithelial cells. In this study, we investigated roles of KCCs in NGF-induced neurite outgrowth of rat pheochromocytoma PC12 cells. The most abundantly expressed isoform in PC12 cells was KCC1. Inhibition of KCCs using [(dihydronindenyl)oxy] alkanoic acid (DIOA), an inhibitor of KCCs, enhanced the NGF-induced neurite outgrowth of PC12 cells in a dose-dependent manner. Treatment of PC12 cells with NGF significantly decreased mRNA expression of KCC1, whereas other isoforms, KCC2-4, showed no changes in their mRNA expression in response to NGF treatment. Knockdown of KCC1 using small interfering RNA (siRNA) enhanced the NGF-induced neurite outgrowth. These results suggest that KCC1 negatively regulates the NGF-induced neurite outgrowth of PC12 cells.

NGF-induced neurite outgrowth in PC12 cells is independent of calcium entry through L-type calcium channels

Neuronal growth factor (NGF) induces neurodifferentiation of PC12 cells into cholinergic neurons-like cells. It was shown that intracellular Ca2+ ions participate in regulation of the differentiation of PC12 cells. We tested whether L-type calcium channels contribute to Ca2+ entry which supports neurite outgrowth accompanying NGF-activated differentiation process. Development of morphological changes did correlate with increase of functional expression of L-type calcium channels. However, inhibition of L-type calcium channels by 1 μM of isradipine did not affect significantly an NGF-activated neurite outgrowth.

Rab35 regulates Arf6 activity through centaurin β2/ACAP2 during neurite outgrowth

Two small GTPases, Rab and Arf, are well-known molecular switches that function in diverse membrane-trafficking routes in a coordinated manner; however, very little is known about the direct crosstalk between Rab and Arf. Although Rab35 and Arf6 were independently reported to regulate the same cellular events, including endocytic recycling, phagocytosis, cytokinesis and neurite outgrowth, the molecular basis that links them remains largely unknown. Here we show that centaurin-β2 (also known as ACAP2) functions both as a Rab35 effector and as an Arf6-GTPase-activating protein (GAP) during neurite outgrowth of PC12 cells. We found that Rab35 accumulates at Arf6-positive endosomes in response to nerve growth factor (NGF) stimulation and that centaurin-β2 is recruited to the same compartment in a Rab35-dependent manner. We further showed by knockdown and rescue experiments that after the Rab35-dependent recruitment of centaurin-β2, the Arf6-GAP activity of centaurin-β2 at the Arf6-positive endosomes was indispensable for NGF-induced neurite outgrowth. These findings suggest a novel mode of crosstalk between Rab and Arf: a Rab effector and Arf-GAP coupling mechanism, in which Arf-GAP is recruited to a specific membrane compartment by its Rab effector function.

Heparanase enhances nerve-growth-factor-induced PC12 cell neuritogenesis via the p38 MAPK pathway

Heparanase is involved in the cleavage of the HS (heparan sulfate) chain of HSPGs (HS proteoglycans) and hence participates in remodelling of the ECM (extracellular matrix) and BM (basement membrane). In the present study we have shown that NGF (nerve growth factor) promoted nuclear enrichment of EGR1 (early growth response 1), a transcription factor for heparanase, and markedly induced heparanase expression in rat adrenal pheochromocytoma (PC12) cells. K252a, an antagonist of the NGF receptor TrkA (tyrosine kinase receptor A), decreased heparanase protein expression induced by NGF in PC12 cells. Suramin, a heparanase inhibitor, decreased heparanase in PC12 cells and blocked NGF-induced PC12 neuritogenesis. Stable overexpression of heparanase activated p38 MAPK (mitogen-activated protein kinase) by phosphorylation and enhanced the neurite outgrowth induced by NGF, whereas knock down of heparanase impaired this process. However, overexpression of latent pro-heparanase with a Y156A mutation still led to enhanced NGF-induced neurite outgrowth and increased p38 MAPK phosphorylation. Inhibition of p38 MAPK by SB203580 suppressed the promotion of NGF-induced neuritogenesis by the wild-type and mutant heparanase. The impaired differentiation by knock down of heparanase could be restored by transfection of wild-type or mutant heparanase in PC12 cells. The results of the present study suggest that heparanase, at least in the non-enzymatic form, may promote NGF-induced neuritogenesis via the p38 MAPK pathway.

The Adaptor Protein SH2B3 (Lnk) Negatively Regulates Neurite Outgrowth of PC12 Cells and Cortical Neurons

SH2B adaptor protein family members (SH2B1-3) regulate various physiological responses through affecting signaling, gene expression, and cell adhesion. SH2B1 and SH2B2 were reported to enhance nerve growth factor (NGF)-induced neuronal differentiation in PC12 cells, a well-established neuronal model system. In contrast, SH2B3 was reported to inhibit cell proliferation during the development of immune system. No study so far addresses the role of SH2B3 in the nervous system. In this study, we provide evidence suggesting that SH2B3 is expressed in the cortex of embryonic rat brain. Overexpression of SH2B3 not only inhibits NGF-induced differentiation of PC12 cells but also reduces neurite outgrowth of primary cortical neurons. SH2B3 does so by repressing NGF-induced activation of PLCγ, MEK-ERK1/2 and PI3K-AKT pathways and the expression of Egr-1. SH2B3 is capable of binding to phosphorylated NGF receptor, TrkA, as well as SH2B1β. Our data further demonstrate that overexpression of SH2B3 reduces the interaction between SH2B1β and TrkA. Consistent with this finding, overexpressing the SH2 domain of SH2B3 is sufficient to inhibit NGF-induced neurite outgrowth. Together, our data demonstrate that SH2B3, unlike the other two family members, inhibits neuronal differentiation of PC12 cells and primary cortical neurons. Its inhibitory mechanism is likely through the competition of TrkA binding with the positive-acting SH2B1 and SH2B2.

Identification of a potent and selective σ1 receptor agonist potentiating NGF-induced neurite outgrowth in PC12 cells

Herein we report the synthesis, drug-likeness evaluation, and in vitro studies of new sigma (σ) ligands based on arylalkenylaminic scaffold. For the most active olefin the corresponding arylalkylamine was studied. Novel arylalkenylamines generally possess high σ1 receptor affinity (Ki values <25nM) and good σ1/σ2 selectivity (Kiσ2 >100). Particularly, the piperidine derivative (E)-17 and its arylalkylamine analog (R,S)-33 were observed to be excellent σ1 receptor ligands (Ki=0.70 and 0.86nM, respectively) and to display significantly high selectivity over σ2, μ-, and κ-opioid receptors and phencyclidine (PCP) binding site of the N-methyl-d-aspartate (NMDA) receptors. Moreover in PC12 cells (R,S)-33 promoted the nerve growth factor (NGF)-induced neurite outgrowth and elongation. Co-administration of the selective σ1 receptor antagonist BD-1063 totally counteracted this effect, confirming that σ1 receptors are involved in the (R,S)-33 modulation of the NGF effect in PC12 cells and suggesting a σ1 agonist profile. As a part of our work, a threedimensional σ1 pharmacophore model was also developed employing GALAHAD methodology. Only active compounds were used for deriving this model. The model included two hydrophobes and a positive nitrogen as relevant features and it was able to discriminate between molecules with and without affinity toward σ1 receptor subtype.

Evaluation of sigma (σ) receptors in the antidepressant-like effects of ketamine in vitro and in vivo

Ketamine is an NMDA antagonist and dissociative anesthetic that has been shown to display rapid acting and prolonged antidepressant activity in small-scale human clinical trials. Ketamine also binds to σ receptors, which are believed to be protein targets for a potential new class of antidepressant medications. The purpose of this study was to determine the involvement of σ receptors in the antidepressant-like actions of ketamine. Competition binding assays were performed to assess the affinity of ketamine for σ1 and σ2 receptors. The antidepressant-like effects of ketamine were assessed in vitro using a neurite outgrowth model and PC12 cells, and in vivo using the forced swim test. The σ receptor antagonists, NE-100 and BD1047, were evaluated in conjunction with ketamine in these assays to determine the involvement of σ receptors in the antidepressant-like effects of ketamine. Ketamine bound to both σ1 and σ2 receptors with μM affinities. Additionally, ketamine potentiated NGF-induced neurite outgrowth in PC12 cells and this effect was attenuated in the presence of NE-100. Ketamine also displayed antidepressant-like effects in the forced swim test; however, these effects were not attenuated by pretreatment with NE-100 or BD1047. Taken together, these data suggest that σ receptor-mediated neuronal remodeling may contribute to the antidepressant effects of ketamine.

Rab22 controls NGF signaling and neurite outgrowth in PC12 cells

Rab22 is a small GTPase that is localized on early endosomes and regulates early endosomal sorting. This study reports that Rab22 promotes nerve growth factor (NGF) signaling-dependent neurite outgrowth and gene expression in PC12 cells by sorting NGF and the activated/phosphorylated receptor (pTrkA) into signaling endosomes to sustain signal transduction in the cell. NGF binding induces the endocytosis of pTrkA into Rab22-containing endosomes. Knockdown of Rab22 via small hairpin RNA (shRNA) blocks NGF-induced pTrkA endocytosis into the endosomes and gene expression (VGF) and neurite outgrowth. Overexpression of human Rab22 can rescue the inhibitory effects of the Rab22 shRNA, suggesting a specific Rab22 function in NGF signal transduction, rather than off-target effects. Furthermore, the Rab22 effector, Rabex-5, is necessary for NGF-induced neurite outgrowth and gene expression, as evidenced by the inhibitory effect of shRNA-mediated knockdown of Rabex-5. Disruption of the Rab22-Rabex-5 interaction via overexpression of the Rab22-binding domain of Rabex-5 in the cell also blocks NGF-induced neurite outgrowth, suggesting a critical role of Rab22-Rabex-5 interaction in the biogenesis of NGF-signaling endosomes to sustain the signal for neurite outgrowth. These data provide the first evidence for an early endosomal Rab GTPase as a positive regulator of NGF signal transduction and cell differentiation.

Phosphorylation controls a dual-function polybasic nuclear localization sequence in the adapter protein SH2B1β to regulate its cellular function and distribution

An intriguing question in cell biology is what targets proteins to, and regulates their translocation between, specific cellular locations. Here we report that the polybasic nuclear localization sequence (NLS) required for nuclear entry of the adapter protein and candidate human obesity gene product SH2B1β, also localizes SH2B1β to the plasma membrane (PM), most probably via electrostatic interactions. Binding of SH2B1β to the PM also requires its dimerization domain. Phosphorylation of serine residues near this polybasic region, potentially by protein kinase C, releases SH2B1β from the PM and enhances nuclear entry. Release of SH2B1β from the PM and/or nuclear entry appear to be required for SH2B1β enhancement of nerve growth factor (NGF)-induced expression of urokinase plasminogen activator receptor gene and neurite outgrowth of PC12 cells. Taken together, our results provide strong evidence that the polybasic NLS region of SH2B1 serves the dual function of localizing SH2B1 to both the nucleus and the PM, the latter most probably through electrostatic interactions that are enhanced by SH2B1β dimerization. Cycling between the different cellular compartments is a consequence of the phosphorylation and dephosphorylation of serine residues near the NLS and is important for physiological effects of SH2B1, including NGF-induced gene expression and neurite outgrowth.

Nerve growth factor-induced neurite outgrowth is potentiated by stabilization of TrkA receptors

Exogenous stimuli such as nerve growth factor (NGF) exert their effects on neurite outgrowth via Trk neurotrophin receptors. TrkA receptors are known to be ubiquitinated via proteasome inhibition in the presence of NGF. However, the effect of proteasome inhibition on neurite outgrowth has not been studied extensively. To clarify these issues, we investigated signaling events in PC12 cells treated with NGF and the proteasome inhibitor MG132. We found that MG132 facilitated NGF-induced neurite outgrowth and potentiated the phosphorylation of the extracellular signal-regulated kinase/mitogen- activated protein kinase (ERK/MAPK) and phosphatidylinositol- 3-kinase (PI3K)/AKT pathways and TrkA receptors. MG132 stimulated internalization of surface TrkA receptor and stabilized intracellular TrkA receptor, and the Ub(K63) chain was found to be essential for stability. These results indicate that the ubiquitin-proteasome system potentiated neurite formation by regulating the stability of TrkA receptors.

Neurite outgrowth mediated by translation elongation factor eEF1A1: a target for antiplatelet agent cilostazol.

Cilostazol, a type-3 phosphodiesterase (PDE3) inhibitor, has become widely used as an antiplatelet drug worldwide. A recent second Cilostazol Stroke Prevention Study demonstrated that cilostazol is superior to aspirin for prevention of stroke after an ischemic stroke. However, its precise mechanisms of action remain to be determined. Here, we report that cilostazol, but not the PDE3 inhibitors cilostamide and milrinone, significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Furthermore, specific inhibitors for the endoplasmic reticulum protein inositol 1,4,5-triphosphate (IP3) receptors and several common signaling pathways (PLC-γ, PI3K, Akt, p38 MAPK, and c-Jun N-terminal kinase (JNK), and the Ras/Raf/ERK/MAPK) significantly blocked the potentiation of NGF-induced neurite outgrowth by cilostazol. Using a proteomics analysis, we identified that levels of eukaryotic translation elongation factor eEF1A1 protein were significantly increased by treatment with cilostazol, but not cilostamide, in PC12 cells. Moreover, the potentiating effects of cilostazol on NGF-induced neurite outgrowth were significantly antagonized by treatment with eEF1A1 RNAi, but not the negative control of eEF1A1. These findings suggest that eEF1A1 and several common cellular signaling pathways might play a role in the mechanism of cilostazol-induced neurite outgrowth. Therefore, agents that can increase the eEF1A1 protein may have therapeutic relevance in diverse conditions with altered neurite outgrowth.

Potentiation of NGF-induced neurite outgrowth in PC12 cells by papaverine: Role played by PLC-γ, IP 3 receptors

Papaverine, an inhibitor of phosphodiesterase (PDE) 10A, is gaining attention for its potential in the treatment of neuropsychiatric diseases such as schizophrenia. However, the precise mechanisms underlying the putative neuroprotective/neurotrophic actions of papaverine remain unclear. Thus, we investigated the effects of papaverine on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Papaverine potentiated NGF-induced neurite outgrowth in PC12 cells in a concentration-dependent manner. In contrast, the selective PDE10A inhibitor MP-10 had no effect on NGF-induced neurite outgrowth. The potentiation of NGF-induced neurite outgrowth by papaverine was blocked by the PLC-γ inhibitor U73122. Furthermore, papaverine's potentiation of NGF-induced neurite outgrowth was also blocked by the co-administration of inositol 1,4,5-trisphosphate (IP 3) receptor antagonists (xestospongin C and 2-aminoethoxydiphenyl borate (2-APB)) and by reduced expression of IP 3 receptor gene (i.e., itpr1 and itpr3) by siRNA. Our findings suggest that papaverine could potentiate NGF-induced neurite outgrowth, and that activation of PLC-γ and IP 3 receptors might be involved in the mechanism underlying papaverine's potentiation of neurite outgrowth in PC12 cells.

Quercetin Stimulates NGF-induced Neurite Outgrowth in PC12 Cells via Activation of Na+/K+/2Cl- Cotransporter

We have recently reported that Na⁺/K⁺/2Cl^- cotransporter isoform 1 (NKCC1) plays an essential role in nerve growth factor (NGF)-induced neurite outgrowth in PC12D cells. On the other hand, it has been reported that dietary flavonoids, such as quercetin, apigenin, and luteolin, stimulate various ion transporters. In the present report, we investigated the effect of quercetin, a flavonoid, on NGF-induced neurite outgrowth in PC12 cells (the parental strain of PC12D cells). Quercetin stimulated the NGF-induced neurite outgrowth in a dose-dependent manner. Knockdown of NKCC1 by RNAi methods abolished the stimulatory effect of flavonoid. Quercetin stimulated NKCC1 activity (measured as bumetanide-sensitive ⁸⁶Rb influx) without any increase in the expression level of NKCC1 protein. The stimulatory effect of quercetin on neurite outgrowth was dependent upon extracellular Cl^-. These observations indicate that quercetin stimulates the NGF-induced neurite outgrowth via an increase in Cl^- incorporation into the intracellular space by activating NKCC1 in PC12 cell.

Genistein enhances the NGF-induced neurite outgrowth

In the present report, we studied if an isoflavone, genistein, enhances the nerve growth factor (NGF)-induced neurite outgrowth of PC12 cells. Application of genistein enhanced the NGF-induced neurite outgrowth. Knockdown of Na+/K+/2Cl- cotransporter isoform 1 (NKCC1) abolished the stimulatory effect of genistein on the neurite outgrowth. These observations indicate that NKCC1 is essential for genistein to stimulate the NGF-induced neurite outgrowth, although genistein had no effect on the protein expression of NKCC1. On the other hand, genistein activates NKCC1 as shown in our previous study. Taken together, these observations indicate that genistein enhanced the NGF-induced neurite outgrowth in PC12 cells via activation of NKCC1.

A Rapid, Inexpensive High Throughput Screen Method for Neurite Outgrowth

Neurite outgrowth assays are the most common phenotypic screen to assess chemical effects on neuronal cells. Current automated assays involve expensive equipment, lengthy sample preparation and handling, costly reagents and slow rates of data acquisition and analysis. We have developed a high throughput screen (HTS) for neurite outgrowth using a robust neuronal cell model coupled to fast and inexpensive visualization methods, reduced data volume and rapid data analysis. Neuroscreen-1 (NS-1) cell, a subclone of PC12, possessing rapid growth and enhanced sensitivity to NGF was used as a model neuron. This method reduces preparation time by using cells expressing GFP or native cells stained with HCS CellMask™ Red in a multiplexed 30 min fixation and staining step. A 2x2 camera binning process reduced both image data files and analysis times by 75% and 60% respectively, compared to current protocols. In addition, eliminating autofocus steps during montage generation reduced data collection time. Pharmacological profiles for stimulation and inhibition of neurite outgrowth by NGF and SU6656 were comparable to current standard method utilizing immunofluorescence detection of tubulin. Potentiation of NGF-induced neurite outgrowth by members of a 1,120-member Prestwick compound library as assayed using this method identified six molecules, including etoposide, isoflupredone acetate, fludrocortisone acetate, thioguanosine, oxyphenbutazone and gibberellic acid, that more than doubled the neurite mass primed by 2 ng/ml NGF. This simple procedure represents an important routine approach in high throughput screening of large chemical libraries using the neurite outgrowth phenotype as a measure of the effects of chemical molecules on neuronal cells.

Mono-(2-ethylhexyl) phthalate impairs neurodevelopment: Inhibition of proliferation and promotion of differentiation in PC12 cells

Di-(2-ethylhexyl)-phthalate (DEHP) is the most widely used plasticizers in daily-life products. In this study we evaluated the influence of mono-(2-ethylhexyl) phthalate (MEHP), the active metabolite of DEHP, on the neurodevelopment in vitro. After neuronotypic PC12 cells were exposed to MEHP (0.25, 2.5, 25, and 250 μM), the effects of which on cell proliferation and differentiation were investigated. In undifferentiated PC12 cells, MEHP inhibited cell proliferation in a dose-dependent manner. After 24 h of MEHP treatment, there was a dose-dependent G2/M cell cycle arrest as well as a sharp drop of DNA synthesis. During the process of NGF-induced differentiation of the cell line, 4 days of MEHP exposure (2.5–250 μM) increased membrane and cytoskeletal protein contents, enhanced NGF-induced neurite outgrowth, up-regulated the choline acetyl transferase (ChAT) mRNA and down-regulated tyrosine hydroxylase (TH) mRNA levels, which suggested the promoted differentiation towards the acetylcholine (ACh) phenotype at the expense of the dopamine (DA) phenotype. Take together, our results indicate that MEHP has a potential to disturb neurodevelopment by suppressing cell proliferation and promote cell differentiation in neurocytes.

Correction: A Novel Target of Action of Minocycline in NGF-Induced Neurite Outgrowth in PC12 Cells: Translation Initiator Factor eIF4AI

Correction: A Novel Target of Action of Minocycline in NGF-Induced Neurite Outgrowth in PC12 Cells: Translation Initiator Factor eIF4AI

Long-term Delivery of Nerve Growth Factor by Encapsulated Cell Biodelivery in the Göttingen Minipig Basal Forebrain

Nerve growth factor (NGF) prevents cholinergic degeneration in Alzheimer's disease (AD) and improves memory in AD animal models. In humans, the safe delivery of therapeutic doses of NGF is challenging. For clinical use, we have therefore developed an encapsulated cell (EC) biodelivery device, capable of local delivery of NGF. The clinical device, named NsG0202, houses an NGF-secreting cell line (NGC-0295), which is derived from a human retinal pigment epithelial (RPE) cell line, stably genetically modified to secrete NGF. Bioactivity and correct processing of NGF was confirmed in vitro. NsG0202 devices were implanted in the basal forebrain of Göttingen minipigs and the function and retrievability were evaluated after 7 weeks, 6 and 12 months. All devices were implanted and retrieved without associated complications. They were physically intact and contained a high number of viable and NGF-producing NGC-0295 cells after explantation. Increased NGF levels were detected in tissue surrounding the devices. The implants were well tolerated as determined by histopathological brain tissue analysis, blood analysis, and general health status of the pigs. The NsG0202 device represents a promising approach for treating the cognitive decline in AD patients.

A Novel Target of Action of Minocycline in NGF-Induced Neurite Outgrowth in PC12 Cells: Translation Initiation Factor eIF4AI

BackgroundMinocycline, a second-generation tetracycline antibiotic, has potential activity for the treatment of several neurodegenerative and psychiatric disorders. However, its mechanisms of action remain to be determined.Methodology/Principal FindingsWe found that minocycline, but not tetracycline, significantly potentiated nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells, in a concentration dependent manner. Furthermore, we found that the endoplasmic reticulum protein inositol 1,4,5-triphosphate (IP3) receptors and several common signaling molecules (PLC-γ, PI3K, Akt, p38 MAPK, c-Jun N-terminal kinase (JNK), mammalian target of rapamycin (mTOR), and Ras/Raf/ERK/MAPK pathways) might be involved in the active mechanism of minocycline. Moreover, we found that a marked increase of the eukaryotic translation initiation factor eIF4AI protein by minocycline, but not tetracycline, might be involved in the active mechanism for NGF-induced neurite outgrowth.Conclusions/SignificanceThese findings suggest that eIF4AI might play a role in the novel mechanism of minocycline. Therefore, agents that can increase eIF4AI protein would be novel therapeutic drugs for certain neurodegenerative and psychiatric diseases.