The Mixture of Gotu Kola, Cnidium Fruit, and Goji Berry Enhances Memory Functions by Inducing Nerve-Growth-Factor-Mediated Actions Both In Vitro and In Vivo.

Nerve growth factor (NGF), a typical neurotrophin, has been characterized by the regulation of neuronal cell differentiation and survival involved in learning and memory functions. NGF has a main role in neurite extension and synapse formation by activating the cyclic adenosine monophosphate-response-element-binding protein (CREB) in the hippocampus. The purpose of this study was to determine whether a mixture of Gotu Kola, Cnidium fruit, and Goji berry (KYJ) enhances memory function by inducing NGF-mediated actions both in vitro and in vivo. The KYJ combination increased NGF concentration and neurite length in C6 glioma and N2a neuronal cells, respectively. Additionally, we discovered memory-enhancing effects of KYJ through increased NGF-mediated synapse maturation, CREB phosphorylation, and cell differentiation in the mouse hippocampus. These findings suggest that this combination may be a potential nootropic cognitive enhancer via the induction of NGF and NGF-dependent activities.

Modelling acute and lasting effects of tDCS on epileptic activity.

Transcranial Direct brain stimulation (tDCS) is commonly used in order to modulate cortical networks activity during physiological processes through the application of weak electrical fields with scalp electrodes. Cathodal stimulation has been shown to decrease brain excitability in the context of epilepsy, with variable success. However, the cellular mechanisms responsible for the acute and the long-lasting effect of tDCS remain elusive. Using a novel approach of computational modeling that combines detailed but functionally integrated neurons we built a physiologically-based thalamocortical column. This model comprises 10,000 individual neurons made of pyramidal cells, and 3 types of gamma-aminobutyric acid (GABA) -ergic cells (VIP, PV, and SST) respecting the anatomy, layers, projection, connectivity and neurites orientation. Simulating realistic electric fields in term of intensity, main results showed that 1) tDCS effects are best explained by modulation of the presynaptic probability of release 2) tDCS affects the dynamic of cortical network only if a sufficient number of neurons are modulated 3)VIP GABAergic interneurons of the superficial layer of the cortex are especially affected by tDCS 4) Long lasting effect depends on glutamatergic synaptic plasticity.

Postischemic supplementation of folic acid improves neuronal survival and regeneration in vitro.

Supplementation of folic acid (FA) is beneficial to several neurological diseases because it promotes notch signaling and neurogenesis and reduces blood homocysteine levels. We hypothesized that postischemic supplementation of FA is beneficial for neuronal survival and regeneration. The objective of the present study was to determine the postischemic neuroprotective and neuroregenerative efficacy of FA supplementation and its effects on various cellular processes in vitro. This work benefited from the use of FA and glucose-free media to better assess the ischemic neuroprotection provided by FA supplementation. The postischemic supplementation of FA significantly improved cell viability, and the improvement was primarily by obstructing the oxygen-glucose deprivation (OGD)-activated apoptosis. Furthermore, postischemic treatment with FA significantly reduced the mitochondrial membrane depolarization and the formation of acidic organelles triggered by OGD. Moreover, FA's effect on neuroregeneration following OGD was evaluated by measuring the cell proliferation and neurite outgrowth length. Treatment with FA enhanced cell proliferation and neurite outgrowth significantly. Thus, these results revealed some of the mechanisms by which FA supplementation provided neuroprotection and neuroregeneration following ischemic injury and highlighted the need for further research into the potential of folic acid as a clinical drug for ischemic stroke.

Comparative Neuroprotective, Anti-Inflammatory and Neurite Outgrowth Activities of Extracts of King Oyster Mushroom, Pleurotus eryngii (Agaricomycetes).

Pleurotus eryngii (king oyster mushroom) is a renowned culinary mushroom with various medicinal properties that may be beneficial for health maintenance and disease prevention. However, its effect on the nervous system remains elusive. In this study, hot water (PE-HWA) and ethanol (PE-ETH) extracts of P. eryngii were investigated and compared for their neuroprotective, anti-inflammatory, and neurite outgrowth activities in vitro. Based on the results, both extracts up to 400 µg/mL were nontoxic to PC12 cells and BV2 microglia (p > 0.05). Treatment with 250 µM hydrogen peroxide (H2O2) markedly (p < 0.0001) reduced the PC12 cell viability to 67.74 ± 6.47%. Coincubation with 200 µg/mL and 400 µg/mL of PE-ETH dose-dependently increased the cell viability to 85.34 ± 1.91% (p < 0.001) and 98.37 ± 6.42% (p < 0.0001) respectively, while PE-HWA showed no activity. Nitric oxide (NO) released by BV2 microglia was notably (p < 0.0001) increased by 1 µg/mL lipopolysaccharides (LPS) from 7.46 ± 0.73 µM to 80.00 ± 3.78 µM indicating an inflammatory reaction. However, coincubation with 200 and 400 µg/mL of PE-ETH significantly (p < 0.0001) reduced the NO level to 58.57 ± 6.19 µM and 52.86 ± 3.43 µM respectively, while PE-HWA was noneffective. PE-ETH and PE-HWA at 40 µg/mL significantly increased the neurite-bearing cells from 4.70 ± 3.36% to 13.12 ± 2.82% (p < 0.01) and 20.93 ± 5.37% (p < 0.0001) respectively. Pleurotus eryngii, particularly the ethanol extract (PE-ETH) and its potentially bioactive compounds, could be explored as a neurohealth promoting agent, due to its collective neuroprotective, anti-inflammatory, and neurite outgrowth activities.

Neurite regrowth stimulation by a red-light spot focused on the neuronal cell soma following blue light-induced retraction.

The interaction of light with biological tissues has been considered for various therapeutic applications. Light-induced neurite growth has the potential to be a clinically useful technique for neuron repair. However, most previous studies used either a large illumination area to accelerate overall neurite growth or employed a light spot to guide a growing neurite. It is not clear if optical stimulation can induce the regrowth of a retracted neurite. In the present work, we used blue light (wavelength: 473 nm) to cause neurite retraction, and we proved that using a red-light (wavelength: 650 nm) spot to illuminate the soma near the junction of the retracted neurite could induce neurite regrowth. As a comparison, we found that green light (wavelength 550 nm) had a 62% probability of inducing neurite regrowth, while red light had a 75% probability of inducing neurite regrowth at the same power level. Furthermore, the neurite regrowth length induced by red light was increased by the pre-treatment with inhibitors of myosin functions. We also observed actin propagation from the soma to the tip of the re-growing neurite following red-light stimulation of the soma. The red light-induced extension and regrowth were abrogated in the calcium-free medium. These results suggest that illumination with a red-light spot on the soma may trigger the regrowth of a neurite after the retraction caused by blue-light illumination.

L-NBP, a multiple growth factor activator, attenuates ischemic neuronal impairments possibly through promoting neuritogenesis.

In China, L-3-n-butylphthalide (L-NBP) showed promising pharmacological actions in stroke treatment. Analyzing the characteristics of L-NBP might provide valuable hints for new drug design. The current study is aimed to determine the effects of L-NBP on neuritogenesis and further to elucidate the neuronal protection against stroke impairment in vitro. L-NBP was applied to rat pheochromocytoma PC12 cells and cultured rat cortical neurons under the normoxic condition and the oxygen-glucose deprivation/reoxygenation (OGD/R) insults, respectively. Immunofluorescence staining, western blot analysis, Sholl analysis, lactate dehydrogenase (LDH) release assay, 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) reduction assay and enzyme-linked immunosorbent assay (ELISA) were performed. L-NBP could concentration-dependently stimulate the development of growth cones, enhance the neuritic branches and synapse formation. It indicated that L-NBP possibly promoted the neuritogenic activity in a stage-dependent manner. Further research proved that L-NBP could promptly activate epidermal growth factor (EGF) receptor, up-regulate the expressions of extracellular signal-regulated kinase1/2 (ERK1/2), cAMP response element-binding protein (CREB) and E-26-like protein 1 (ELK-1). In addition, L-NBP enhanced the sustained expressions of brain derived neurotrophic factor (BDNF) and nerve growth factor (NGF). The inhibition to the receptors of EGF, NGF,BDNF could attenuate L-NBP induced neuritogenic and neuronal survival after the OGD/R toxicity. Basing on these investigations, we concluded that L-NBP might reconstruct the impaired neuronal network and improved the neuronal complexity after the ischemic insults through multiple pathways which at least were via the activations of EGF receptor, BDNF and NGF related signals.

Lactucopicrin potentiates neuritogenesis and neurotrophic effects by regulating Ca2+/CaMKII/ATF1 signaling pathway.

ETHNO-PHARMACOLOGICAL RELEVANCE: Lactucopicrin is one of constitutes in Cichorium intybus L, which is commonly known as chicory in worldwide. It has been used for traditional usage such as antianalgesics, antidepressants and antihyperglycemics AIM OF STUDY: We investigated the neurotrophin-mediated neuroprotective effect of lactucopicrin in in vitro and examined for the underlying mechanism. MATERIALS AND METHOD: To verify the neuroprotective effect of lactucopicrin, we investigated the inhibitory AChE activity, neurite outgrowth-related downstream signaling in murine neuroblastoma N2a and neurotrophins secretion in rat C6 glioma cells. RESULTS: Lactucopicrin inhibited the AChE activity and increased intracellular Ca2+ levels with a substantial rise in muscarinic acetylcholine receptor M1 (CHRM1) expression in N2a cells. Moreover, lactucopicrin actively promoted neurite outgrowth via Ca2+-mediated activation of Ca2+/calmodulin-dependent protein kinase-II (CaMKII). It further activates transcription factor 1 (ATF1) along with modulating the levels of tropomyosin receptor kinase A, extracellular signal-regulated kinase 1 and 2, AKT, and synaptophysin 1 in N2a cells. Additionally, the levels of neurotrophins including NGF, BDNF, and NT3 were increased by treatment of lactucopicrin in C6 cells. The effects of lactucopicrin on NGF secretion and neuritogenesis were maintained even in the presence of phosphatidylinositol-3-kinase (PI3K) inhibitor LY294002, indicating that lactucopicrin exerts its effect on neuritogenesis in a PI3K-independent manner. CONCLUSION: Our results suggest that the natural compound lactucopicrin may be a promising neurotrophin-mediated neuroprotective candidate for neurodegenerative diseases.

P2X1 Receptor-Mediated Ca2+ Influx Triggered by DA-9801 Potentiates Nerve Growth Factor-Induced Neurite Outgrowth.

Nerve growth factor (NGF)-induced neuronal regeneration has emerged as a strategy to treat neuronal degeneration-associated disorders. However, direct NGF administration is limited by the occurrence of adverse effects at high doses of NGF. Therefore, development of a therapeutic strategy to promote the NGF trophic effect is required. In view of the lack of understanding of the mechanism for potentiating the NGF effect, this study investigated molecular targets of DA-9801, a well-standardized Dioscorea rhizome extract, which has a promoting effect on NGF. An increase in intracellular calcium ion level was induced by DA-9801, and chelation of extracellular calcium ions with ethylene-bis(oxyethylenenitrilo)tetraacetic acid (EGTA) suppressed the potentiating effect of DA-9801 on NGF-induced neurite outgrowth. In addition, EGTA treatment reduced the DA-9801-induced phosphorylation of extracellular signal-regulated kinase1/2 (ERK1/2), the major mediators of neurite outgrowth. To find which calcium ion-permeable channel contributes to the calcium ion influx induced by DA-9801, we treated PC12 cells with various inhibitors of calcium ion-permeable channels. NF449, a P2X1 receptor selective antagonist, significantly abolished the potentiating effect of DA-9801 on NGF-induced neurite outgrowth and abrogated the DA-9801-induced ERK1/2 phosphorylation. In addition, transfection with siRNA of P2X1 receptor significantly reduced the DA-9801-enhanced neurite outgrowth. In conclusion, calcium ion influx through P2X1 receptor mediated the promoting effect of DA-9801 on NGF-induced neurite outgrowth via ERK1/2 phosphorylation.

Design and Synthesis of Dimeric Securinine Analogues with Neuritogenic Activities.

Neurite outgrowth is crucial during neuronal development and regeneration, and strategies that aim at promoting neuritogenesis are beneficial for reconstructing synaptic connections after neuronal degeneration and injury. Using a bivalent analogue strategy as a successful approach, the current study identifies a series of novel dimeric securinine analogues as potent neurite outgrowth enhancers. Compounds 13, 14, 17-19, and 21-23, with different lengths of carbon chain of N,N-dialkyl substituting diacid amide linker between two securinine molecules at C-15 position, exhibited notable positive effects on both neuronal differentiation and neurite extension of neuronal cells. Compound 14, one of the most active compounds, was used as a representative compound for mechanistic studies. Its action on neurite outgrowth was through phosphorylation/activation of multiple signaling molecules including Ca2+/calmodulin-dependent protein kinase II (CaMKII), extracellular signal-regulated kinase (ERK) and Akt. These findings collectively identify a new group of beneficial compounds for neuritogenesis, and may provide insights on drug discovery of neural repair and regeneration.

Fabrication of Aligned Conducting PPy-PLLA Fiber Films and Their Electrically Controlled Guidance and Orientation for Neurites.

Electrically conductive biomaterial scaffolds have great potential in neural tissue regeneration. In this work, an aligned conductive fibrous scaffold was prepared by electrospinning PLLA on rotating collector and chemical oxidation polymerization of pyrrole (PPy) codoped with poly(glutamic acid)/dodecyl benzenesulfonic acid sodium. The characterization results of composition, structure and mechanics of fiber films show that the existence of weak polar van der Waals' force between PPy coating and PLLA fibers. The resistivity of aligned rough PPy-PLLA fiber film (about 800 nm of fiber diameter) at the perpendicular and parallel directions is 0.971 and 0.874 Ω m, respectively. Aligned rough PPy-PLLA fiber film could guide the extension of 68% PC12 neurites along the direction of fiber axis. Under electrostimulation (ES) of 100, 200, and 400 mV/cm, median neurite lengths of differentiated PC12 on aligned fiber-films are 128, 149, and 141 µm, respectively. Furthermore, under ES of 100, 200, and 400 mV/cm, the alignment rate of neurite along the electropotential direction (angle between neurite and electropotential direction ≤10°) on random fibers film are 17, 23, and 28%, respectively, and the alignment rate of neurites along the fiber axis (angle between neurite and fiber axis ≤10°) on aligned fibers film reach to 76, 83, and 79%, respectively, indicating that the combination of ES and rough conducting aligned structure could adjust the alignment of cellular neurites along the direction of the fiber axis or electropotential.

Negletein as a neuroprotectant enhances the action of nerve growth factor and induces neurite outgrowth in PC12 cells.

Negletein has been shown to have therapeutic potential for inflammation-associated diseases, but its effect on neurite outgrowth is still unknown. The present study showed that negletein alone did not trigger PC12 cells to differentiate and extend neurites. When compared with the cells in the untreated control, a significant (P < 0.05) induction and a higher neurite outgrowth activity was observed when the cells were cotreated with negletein (10 µM) and a low dose of nerve growth factor (NGF; 5 ng/mL). The neurite outgrowth process was blocked by the tyrosine kinase receptor (Trk) inhibitor, K252a, suggesting that the neuritogenic effect was NGF-dependent. Negletein (10 µM) together with NGF (5 ng/mL) enhanced the phosphorylation of extracellular signal-regulated kinases (ERKs), protein kinase B (Akt), and cAMP response element-binding protein (CREB). The growth associated protein-43 (GAP-43) and the NGF level were also upregulated by negletein (10 µM) and a low dose of NGF (5 ng/mL). Negletein at nanomolar concentration also was found to be sufficient to mediate the survival of serum-deprived PC12 cells up to 72 h. Taken together, negletein might be useful as an efficient bioactive compound to protect neurons from cell death and promote neuritogenesis. © 2016 BioFactors, 42(6):591-599, 2016.

Prostaglandin D2 elicits the reversible neurite retraction in hypothalamic cell line.

Prostaglandins (PGs) play important roles in diverse physiological processes in the central nervous system. PGD2 is the most abundant PG in the brain and acts through specific receptors, DP1 and CRTH2. We investigated the effects of PGD2 on the morphology of the hypothalamic cell line mHypoE-N37 (N37). In N37 cells, serum starvation induced neurite outgrowth and PGD2 elicited neurite retraction, although we failed to detect transcripts for DP1 and CRTH2. Such an effect of PGD2 was efficiently mimicked by its metabolite, 15-deoxy-Δ(12,14)-prostaglandin J2. N-acetyl cysteine completely abolished the effect of PGD2, and reactive oxygen species (ROS) were considered to be important. Notably, neurite outgrowth was restored by PGD2 removal. These results suggest that PGD2 induces reversible neurite retraction in a ROS-mediated mechanism that does not involve any known receptor.

Induction of neurite outgrowth in 3D hydrogel-based environments.

The ability of peripheral nervous system (PNS) axons to regenerate and re-innervate their targets after an injury has been widely recognized. However, despite the considerable advances made in microsurgical techniques, complete functional recovery is rarely achieved, especially for severe peripheral nerve injuries (PNIs). Therefore, alternative therapies that can successfully repair peripheral nerves are still essential. In recent years the use of biodegradable hydrogels enriched with growth-supporting and guidance cues, cell transplantation, and biomolecular therapies have been explored for the treatment of PNIs. Bearing this in mind, the aim of this study was to assess whether Gly-Arg-Gly-Asp-Ser synthetic peptide (GRGDS)-modified gellan gum (GG) based hydrogels could foster an amenable environment for neurite/axonal growth. Additionally, strategies to further improve the rate of neurite outgrowth were also tested, namely the use of adipose tissue derived stem cells (ASCs), as well as the glial derived neurotrophic factor (GDNF). In order to increase its stability and enhance its bioactivity, the GDNF was conjugated covalently to iron oxide nanoparticles (IONPs). The impact of hydrogel modification as well as the effect of the GDNF-IONPs on ASC behavior was also screened. The results revealed that the GRGDS-GG hydrogel was able to support dorsal root ganglia (DRG)-based neurite outgrowth, which was not observed for non-modified hydrogels. Moreover, the modified hydrogels were also able to support ASCs attachment. In contrast, the presence of the GDNF-IONPs had no positive or negative impact on ASC behavior. Further experiments revealed that the presence of ASCs in the hydrogel improved axonal growth. On the other hand, GDNF-IONPs alone or combined with ASCs significantly increased neurite outgrowth from DRGs, suggesting a beneficial role of the proposed strategy for future applications in PNI regenerative medicine.

[Search of Neurotrophin-mimic Natural Products for Prevention and Treatment of Neurodegenerative Disease].

As part of our continuing studies on neurotrophin-mimic active compounds in natural products, we investigated the chemical constituents of the pericarps of Illicium jiadifengpi and the roots of Indonesian ginger Zingiber purpureum, resulting in the isolation of new seco-prezizaane-type sesquiterpenoid 1 and phenylbutenoid dimer 3-4 and two new curcuminoids 5-6. The MeOH extract of I. jiadifengpi was fractionated, leading to the isolation of compound 1. Compound 1 significantly enhanced neurite outgrowth in primary cell cultures of fetal rat cortical neurons. It is noteworthy that compound 1 has potential significantly to promote differentiation of multipotent neural stem cell line (MEB5 cells) into neurons. Additionally, we investigated the MeOH extract of the root of Bangle (Z. purpureum) that exhibited neuritogenesis activity in PC12 cells at 25 µg/mL, resulting in the isolation of neurotrophic phenylbutenoid dimers 3-4 and new compounds 5-6. Compounds 3 and 4 were found not only significantly to induce neurite sprouting of PC12 cells but also to increase the neurite length and number of neurites in primary cultured rat cortical neurons, and also showed protective activity against cell death caused by deprivation of serum. Furthermore, chronic treatment with these compounds enhanced hippocampal neurogenesis in dementia model olfactory bulbectomized (OBX) mice. Compounds 5 and 6 had significant NGF-potentiating effects on PC12 cells whereas compound 5 enhanced prevention of amyloid ß (Aß) 42 aggregation.

Phenotypic assays to identify agents that induce reactive gliosis: a counter-screen to prioritize compounds for preclinical animal studies.

Astrocyte phenotypes change in a process called reactive gliosis after traumatic central nervous system (CNS) injury. Astrogliosis is characterized by expansion of the glial fibrillary acidic protein (GFAP) cytoskeleton, adoption of stellate morphologies, and differential expression of some extracellular matrix molecules. The astrocytic response immediately after injury is beneficial, but in the chronic injury phase, reactive astrocytes produce inhibitory factors (i.e., chondroitin sulfate proteoglycans [CSPGs]) that limit the regrowth of injured axons. There are no drugs that promote axon regeneration or functional recovery after CNS trauma in humans. To develop novel therapeutics for the injured CNS, we screened various libraries in a phenotypic assay to identify compounds that promote neurite outgrowth. However, the effects these compounds have on astrocytes are unknown. Specifically, we were interested in whether compounds could alter astrocytes in a manner that mimics the glial reaction to injury. To test this hypothesis, we developed cell-based phenotypic bioassays to measure changes in (1) GFAP morphology/localization and (2) CSPG expression/immunoreactivity from primary astrocyte cultures. These assays were optimized for six-point dose-response experiments in 96-well plates. The GFAP morphology assay is suitable for counter-screening with a Z-factor of 0.44±0.03 (mean±standard error of the mean; N=3 biological replicates). The CSPG assay is reproducible and informative, but does not satisfy common metrics for a "screenable" assay. As proof of principle, we tested a small set of hit compounds from our neurite outgrowth bioassay and identified one that can enhance axon growth without exacerbating the deleterious characteristics of reactive gliosis.

N-Propargyl Caffeate Amide (PACA) Potentiates Nerve Growth Factor (NGF)-Induced Neurite Outgrowth and Attenuates 6-Hydroxydopamine (6-OHDA)-Induced Toxicity by Activating the Nrf2/HO-1 Pathway.

Insufficient production of neurotrophic factors is implicated in the pathogenesis of various neurodegenerative disorders. The aim of the present study was to evaluate the potential of N-propargyl caffeate amide (PACA) to enhance nerve growth factor (NGF)-induced neurite outgrowth and the underlying mechanisms. We discovered that PACA not only potentiated NGF-induced neurite outgrowth but also attenuated 6-hydroxydopamine (6-OHDA) neurotoxicity in dopaminergic PC12 cells and primary rat midbrain neurons. To identify the PACA-binding proteins, we introduced a biotin tag to the covalent PACA-protein adducts via "click chemistry" alkyne-azido cycloaddition. As a result, kelch-like ECH-associated protein 1 (Keap1) was isolated as the predominant protein from PACA treated PC12 cells. We demonstrated that the formation of PACA-Keap1 conjugates induced the nuclear translocation of transcription factor Nrf2 and the expression of antioxidant heme oxygenase-1 (HO-1). Importantly, specific HO-1 inhibitor SnPP diminished the neuroprotective and neuritogenic activities of PACA. Moreover, PACA attenuated 6-OHDA-induced production of neurotoxic reactive oxygen species and reactive nitrogen species. PACA also preserved mitochondrial membrane integrity and enhanced the cellular resistance against 6-OHDA neurotoxicity. These results suggest that PACA may exhibit neuroprotective and neuritogenic activities via activating the Nrf2/HO-1 antioxidant pathway.

The neuritogenic and synaptogenic effects of the ethanolic extract of radix Puerariae in cultured rat hippocampal neurons.

ETHNOPHARMACOLOGICAL RELEVANCE: Radix Puerariae, the root of Pueraria montana var. lobata (Willd.) Sanjappa & Pradeep, is used in Korean traditional medicine to treat neuronal disorders including Parkinson's disease, and its active constituent, puerarin has been reported to have a neuroprotective effect in experimental models of Parkinson's and Alzheimer's disease. AIMS OF THE STUDY: To investigate the neurotrophic effects of these ethnomedicines on the development of central nervous system neurons and the molecular bases of these activities. MATERIALS AND METHODS: Rat embryonic (E19) brain neurons were cultured in the absence or presence of the ethanolic extract of Radix Puerariae (RPE) or puerarin. At predetermined times, cells were fixed and immunostained to visualize neuronal morphologies, or lysed for protein harvesting. Morphometric analyses of neurite outgrowths and synaptogenesis were performed using Image J software. RPE or puerarin-mediated changes in the protein profiles of cultured neurons were assessed by MALDI-TOF-MS/PMF and measuring immunofluorescent intensities. RESULTS: RPE and puerarin alone promoted maximum neurite outgrowths at concentrations of 1µg/ml and 5µM, respectively. At these optimal concentrations, RPE and puerarin provided neurotrophic support by promoting axo-dendritic arbors and synapse formation in cultured neurons. Proteomic study revealed that RPE and puerarin both up-regulated a number of proteins, including dynein light chain 2 (DLC2) and elongation factor 2 (EF2), which are associated with neuritogenesis and synaptic potentiation, respectively. Immunofluorescence intensity measurements confirmed the expressions of the DLC2 and Dync1h1 subunits of dynein in RPE or puerarin treated hippocampal neurons were up-regulated when RPE or puerarin induced changes in neuronal cytoarchitecture. CONCLUSIONS: Our study demonstrates that RPE and puerarin should be considered potentially valuable preventative therapeutics for brain disorders due to their abilities to promote the neuronal cytoarchitecture and the synaptic functionality, which are possibly associated with dynein-dependent regulation of cytoskeletal structures and up-regulation of translation machinery.

A small linear peptide encompassing the NGF N-terminus partly mimics the biological activities of the entire neurotrophin in PC12 cells.

Ever since the discovery of its neurite growth promoting activity in sympathetic and sensory ganglia, nerve growth factor (NGF) became the prototype of the large family of neurotrophins. The use of primary cultures and clonal cell lines has revealed several distinct actions of NGF and other neurotrophins. Among several models of NGF activity, the clonal cell line PC12 is the most widely employed. Thus, in the presence of NGF, through the activation of the transmembrane protein TrkA, these cells undergo a progressive mitotic arrest and start to grow electrically excitable neuritis. A vast number of studies opened intriguing aspects of NGF mechanisms of action, its biological properties, and potential use as therapeutic agents. In this context, identifying and utilizing small portions of NGF is of great interest and involves several human diseases including Alzheimer's disease. Here we report the specific action of the peptide encompassing the 1-14 sequence of the human NGF (NGF(1-14)), identified on the basis of scattered indications present in literature. The biological activity of NGF(1-14) was tested on PC12 cells, and its binding with TrkA was predicted by means of a computational approach. NGF(1-14) does not elicit the neurite outgrowth promoting activity, typical of the whole protein, and it only has a moderate action on PC12 proliferation. However, this peptide exerts, in a dose and time dependent fashion, an effective and specific NGF-like action on some highly conserved and biologically crucial intermediates of its intracellular targets such as Akt and CREB. These findings indicate that not all TrkA pathways must be at all times operative, and open the possibility of testing each of them in relation with specific NGF needs, biological actions, and potential therapeutic use.

Effect of enriching the diet with menhaden oil or daily treatment with resolvin D1 on neuropathy in a mouse model of type 2 diabetes.

The purpose of this study was to determine the effect of supplementing the diet of a mouse model of type 2 diabetes with menhaden (fish) oil or daily treatment with resolvin D1 on diabetic neuropathy. The end points evaluated included motor and sensory nerve conduction velocity, thermal sensitivity, innervation of sensory nerves in the cornea and skin, and the retinal ganglion cell complex thickness. Menhaden oil is a natural source for n-3 polyunsaturated fatty acids, which have been shown to have beneficial effects in other diseases. Resolvin D1 is a metabolite of docosahexaenoic acid and is known to have anti-inflammatory and neuroprotective properties. To model type 2 diabetes, mice were fed a high-fat diet for 8 wk followed by a low dosage of streptozotocin. After 8 wk of hyperglycemia, mice in experimental groups were treated for 6 wk with menhaden oil in the diet or daily injections of 1 ng/g body wt resolvin D1. Our findings show that menhaden oil or resolvin D1 did not improve elevated blood glucose, HbA1C, or glucose utilization. Untreated diabetic mice were thermal hypoalgesic, had reduced motor and sensory nerve conduction velocities, had decreased innervation of the cornea and skin, and had thinner retinal ganglion cell complex. These end points were significantly improved with menhaden oil or resolvin D1 treatment. Exogenously, resolvin D1 stimulated neurite outgrowth from primary cultures of dorsal root ganglion neurons from normal mice. These studies suggest that n-3 polyunsaturated fatty acids derived from fish oil could be an effective treatment for diabetic neuropathy.

Emodin induces neurite outgrowth through PI3K/Akt/GSK-3ß-mediated signaling pathways in Neuro2a cells.

In this study, a neurite outgrowth-inducing substance was isolated from the ethylacetate extract of the Polygonum multiflorum roots and identified as emodin by gas-liquid chromatography-mass spectrometry and (1)H NMR and (13)C NMR. Emodin displayed remarkable neurite outgrowth-inducing activity in Neuro2a cells, as demonstrated by morphological changes and immunocytochemistry for class III ß-tubulin. Emodin exhibited a stronger neutrophic activity than retinoic acid (RA) known as inducer of neurite outgrowth in Neuro2a cells. Emodin treatment resulted in marked increases in phosphorylation of Akt a direct downstream signaling molecule of phosphatidylinositol 3-kinase (PI3K), but upstream of glycogen synthase kinase-3ß (GSK-3ß) and cAMP response element-binding protein (CREB). These augmentations and neurite-bearing cells induced by emodin were remarkably reduced by the addition of PI3K inhibitor LY294002. These results demonstrate that emodin induces neuronal differentiation of Neuro2a cells via PI3K/Akt/GSK-3ß pathway.