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1.  Uridine from Pleurotus giganteus and Its Neurite Outgrowth Stimulatory Effects with Underlying Mechanism 
PLoS ONE  2015;10(11):e0143004.
Neurodegenerative diseases are linked to neuronal cell death and impairment of neurite outgrowth. An edible mushroom, Pleurotus giganteus was found to stimulate neurite outgrowth in vitro but the chemical constituents and the underlying mechanism is yet to be elucidated. The chemical constituents of P. giganteus (linoleic acid, oleic acid, cinnamic acid, caffeic acid, p-coumaric acid, succinic acid, benzoic acid, and uridine) were tested for neurite outgrowth activity. Uridine (100 μM) was found to increase the percentage of neurite-bearing cells of differentiating neuroblastoma (N2a) cells by 43.1±0.5%, which was 1.8-fold higher than NGF (50 ng/mL)-treated cells. Uridine which was present in P. giganteus (1.80±0.03 g/100g mushroom extract) increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Further, phosphorylation of the mammalian target of rapamycin (mTOR) was also increased. MEK/ERK and PI3K-Akt-mTOR further induced phosphorylation of cAMP-response element binding protein (CREB) and expression of growth associated protein 43 (GAP43); all of which promoted neurite outgrowth of N2a cells. This study demonstrated that P. giganteus may enhance neurite outgrowth and one of the key bioactive molecules responsible for neurite outgrowth is uridine.
PMCID: PMC4643974  PMID: 26565787
2.  Potentiation of neuritogenic activity of medicinal mushrooms in rat pheochromocytoma cells 
Senescence of the neurons is believed to be a focal factor in the development of age-related neurodegenerative diseases such as Alzheimer’s disease. Diminutions in the levels of nerve growth factor (NGF) lead to major declines in brain cell performance. Functional foods, believed to mitigate this deficiency, will be reaching a plateau in the near future market of alternative and preventive medicine. In the search for neuroactive compounds that mimic the NGF activity for the prevention of neurodegenerative diseases, the potential medicinal values of culinary and medicinal mushrooms attract intense interest.
Cytotoxic effects of aqueous extracts of three medicinal mushrooms basidiocarps, Ganoderma lucidum, Ganoderma neo-japonicum and Grifola frondosa towards rat pheochromocytoma (PC-12) cells were determined by 3-(4,5-dimethythiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The potentiation of neuritogenic activity was assessed by neurite outgrowth stimulation assay. Involvement of cellular signaling pathways, mitogen-activated protein kinase kinase/extracellular signal-regulated kinase (MEK/ERK1/2) and phosphoinositide-3-kinase/protein kinase B (PI3K/Akt) in mushrooms-stimulated neuritogenesis were examined by using specific pharmacological inhibitors. Alteration of neuronal morphology by inhibitors was visualized by immunofluorescence staining of the neurofilament.
All the aqueous extracts tested caused a marked stimulation of neuritogenesis with no detectable cytotoxic effects towards PC-12 cells. The aqueous extract of G. neo-japonicum triggered maximal stimulation of neurite outgrowth at a lower concentration (50 μg/ml) with 14.22 ± 0.43% of neurite-bearing cells, compared to G. lucidum and G. frondosa that act at a higher concentration (75 μg/ml), with 12.61 ± 0.11% and 12.07 ± 0.46% of neurite-bearing cells, respectively. The activation of MEK/ERK1/2 and PI3K/Akt signaling pathways were necessary for the NGF and aqueous extracts to promote neuritogenesis.
Ganoderma lucidum, G. neo-japonicum and G. frondosa may contain NGF-like bioactive compound(s) for maintaining and regenerating the neuronal communications network. The present study reports the first evidence of the neuritogenic effects of aqueous extracts of basidiocarps of G. neo-japonicum in-vitro and showed the involvement of MEK/ERK1/2 and P13K/Akt signaling pathways for neuritogenesis in PC-12 cells.
PMCID: PMC3720279  PMID: 23822837
Ganoderma lucidum; Ganoderma neo-japonicum; Grifola frondosa; Neuritogenesis; Neurodegenerative disease; Nerve growth factor; MEK/ERK signaling pathway; PI3K/Akt signaling pathway
3.  Intrastrain Comparison of the Chemical Composition and Antioxidant Activity of an Edible Mushroom, Pleurotus giganteus, and Its Potent Neuritogenic Properties 
The Scientific World Journal  2014;2014:378651.
Two strains of Pleurotus giganteus (commercial and wild) were tested for their ability to induce neurite outgrowth in rat pheochromocytoma (PC12) and mouse neuroblastoma-2a (N2a) cells. Treatment with the mushroom extracts resulted in neuronal differentiation and neuronal elongation, but not nerve growth factor (NGF) production. Linoleic acid (4.5–5.0%, w/w) which is a major fatty acid present in the ethanol extract promoted NGF biosynthesis when augmented with low concentration of NGF (5 ng/mL). The two strains of mushroom were found to be high in protein (154–192 g kg−1), total polysaccharides, phenolics, and flavonoids as well as vitamins B1, B2, and B3. The total phenolics present in the mushroom extracts were positively correlated to the antioxidant activity (free radical scavenging, ferric reducing power, and lipid peroxidation inhibition). To conclude, P. giganteus could potentially be used in well-balanced diet and as a source of dietary antioxidant to promote neuronal health.
PMCID: PMC4121195  PMID: 25121118
4.  Neurite outgrowth stimulatory effects of culinary-medicinal mushrooms and their toxicity assessment using differentiating Neuro-2a and embryonic fibroblast BALB/3T3 
Mushrooms are not only regarded as gourmet cuisine but also as therapeutic agent to promote cognition health. However, little toxicological information is available regarding their safety. Therefore, the aim of this study was to screen selected ethno-pharmacologically important mushrooms for stimulatory effects on neurite outgrowth and to test for any cytotoxicity.
The stimulatory effect of mushrooms on neurite outgrowth was assessed in differentiating mouse neuroblastoma (N2a) cells. Neurite length was measured using Image-Pro Insight processor system. Neuritogenesis activity was further validated by fluorescence immunocytochemical staining of neurofilaments. In vitro cytotoxicity was investigated by using mouse embryonic fibroblast (BALB/3T3) and N2a cells for any embryo- and neuro-toxic effects; respectively.
Aqueous extracts of Ganoderma lucidum, Lignosus rhinocerotis, Pleurotus giganteus and Grifola frondosa; as well as an ethanol extract of Cordyceps militaris significantly (p < 0.05) promoted the neurite outgrowth in N2a cells by 38.4 ± 4.2%, 38.1 ± 2.6%, 33.4 ± 4.6%, 33.7 ± 1.5%, and 35.8 ± 3.4%; respectively. The IC50 values obtained from tetrazolium (MTT), neutral red uptake (NRU) and lactate dehydrogenase (LDH) release assays showed no toxic effects following 24 h exposure of N2a and 3T3 cells to mushroom extracts.
Our results indicate that G. lucidum, L. rhinocerotis, P. giganteus, G. frondosa and C. militaris may be developed as safe and healthy dietary supplements for brain and cognitive health.
PMCID: PMC3852280  PMID: 24119256
Culinary-medicinal mushrooms; Neurite outgrowth; Cytotoxicity; Mouse neuroblastoma N2a cell; Mouse 3T3 embryonic fibroblast; Neurofilament
5.  Neuronal Health – Can Culinary and Medicinal Mushrooms Help? 
Hericium erinaceus a culinary and medicinal mushroom is a well established candidate for brain and nerve health. Ganoderma lucidum, Grifola frondosa and Sarcodon scabrosus have been reported to have neurite outgrowth and neuronal health benefits. The number of mushrooms, however, studied for neurohealth activity are few compared to the more than 2 000 species of edible and / or medicinal mushrooms identified. In the on-going search for other potent culinary and / or medicinal mushrooms, indigenous mushrooms used in traditional medicines such as Lignosus rhinocerotis and Ganoderma neo-japonicum are also being investigated. Further, the edible mushroom, Pleurotus giganteus can be a potential candidate, too. Can these edible and medicinal mushrooms be tapped to tackle the health concerns of the aging population which is projected to be more than 80-90 million of people age 65 and above in 2050 who may be affected by age-related neurodegenerative disorders. Scientific validation is needed if these mushrooms are to be considered and this can be achieved by understanding the molecular and biochemical mechanisms involved in the stimulation of neurite outgrowth. Though it is difficult to extrapolate the in vitro studies to what may happen in the human brain, studies have shown that there can be improvement in cognitive abilities of the aged if the mushroom is incorporated in their daily diets.
PMCID: PMC3924982  PMID: 24716157
Neuronal health; Mushrooms; Hericium erinaceus; Pleurotus giganteus; Lignosus rhinocerotis; Ganoderma neo-japonicum; Neurite outgrowth; Nerve regeneration
6.  Lignosus rhinocerus (Cooke) Ryvarden: A Medicinal Mushroom That Stimulates Neurite Outgrowth in PC-12 Cells 
A national treasure mushroom, Lignosus rhinocerus, has been used to treat variety of ailments by local and indigenous communities in Malaysia. The aim of this study was to investigate the potential of the most valuable part of L. rhinocerus, the sclerotium, on neurite outgrowth activity by using PC-12Adh cell line. Differentiated cells with one thin extension at least double the length of the cell diameter were scored positive. Our results showed that aqueous sclerotium L. rhinocerus extract induced neurite outgrowths of 24.4% and 42.1% at 20 μg/mL (w/v) of aqueous extract alone and a combination of 20 μg/mL (w/v) aqueous extract and 30 ng/mL (w/v) of NGF, respectively. Combination of NGF and sclerotium extract had additive effects and enhanced neurite outgrowth. Neuronal differentiation was demonstrated by indirect immunofluorescence of neurofilament protein. Aqueous sclerotium extract contained neuroactive compounds that stimulated neurite outgrowth in vitro. To our knowledge this is the first report on neurite-stimulating activities of L. rhinocerus.
PMCID: PMC3235797  PMID: 22203867
7.  Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells 
Cytotechnology  2009;60(1-3):125-132.
Neurite outgrowth and neuronal differentiation play a crucial role in the development of the nervous system. Understanding of neurotrophins induced neurite outgrowth was important to develop therapeutic strategy for axon regeneration in neurodegenerative diseases as well as after various nerve injuries. It has been reported that extension of neurite and differentiation of sympathetic neuron-like phenotype was modulated by nerve growth factor (NGF) in PC12 cells. In this study, NGF mediated neurite outgrowth was investigated in PC12 cells after liquiritin exposure. Liquiritin is a kind of flavonoids that is extracted from Glycyrrhizae radix, which is frequently used to treat injury or swelling for its life-enhancing properties as well as detoxification in traditional Oriental medicine. The result showed that liquiritin significantly promotes the neurite outgrowth stimulated by NGF in PC12 cells in dose dependant manners whereas the liquiritin alone did not induce neurite outgrowth. Oligo microarray and RT-PCR analysis further clarified that the neurotrophic effect of liquiritin was related to the overexpression of neural related genes such as neurogenin 3, neurofibromatosis 1, notch gene homolog 2, neuromedin U receptor 2 and neurotrophin 5. Thus, liquiritin may be a good candidate for treatment of various neurodegenerative diseases such as Alzheimer’s disease or Parkinson’s disease.
PMCID: PMC2780551  PMID: 19789989
Liquiritin; PC12 cells; Neurite; NGF; Microarray
8.  Hepatoprotective Effects of Panus giganteus (Berk.) Corner against Thioacetamide- (TAA-) Induced Liver Injury in Rats 
Panus giganteus, a culinary and medicinal mushroom consumed by selected indigenous communities in Malaysia, is currently being considered for large scale cultivation. This study was undertaken to investigate the hepatoprotective effects of P. giganteus against thioacetamide- (TAA-) induced liver injury in Sprague-Dawley rats. The rats were injected intraperitoneally with TAA thrice weekly and were orally administered freeze-dried fruiting bodies of P. giganteus (0.5 or 1 g/kg) daily for two months, while control rats were given vehicle or P. giganteus only. After 60 days, rats administered with P. giganteus showed lower liver body weight ratio, restored levels of serum liver biomarkers and oxidative stress parameters comparable to treatment with the standard drug silymarin. Gross necropsy and histopathological examination further confirmed the hepatoprotective effects of P. giganteus. This is the first report on hepatoprotective effects of P. giganteus. The present study showed that P. giganteus was able to prevent or reduce the severity of TAA-induced liver injury.
PMCID: PMC3357533  PMID: 22649470
9.  Lignosus rhinocerotis (Cooke) Ryvarden mimics the neuritogenic activity of nerve growth factor via MEK/ERK1/2 signaling pathway in PC-12 cells 
Scientific Reports  2015;5:16349.
The traditional application of the sclerotium of Lignosus rhinocerotis (tiger’s milk mushroom) by the indigenous folks as tonic and remedy to treat a variety of ailments has been documented in Malaysia. Indigenous communities claimed to have consumed the decoction to boost their alertness during hunting. Mental alertness is believed to be related to neuronal health and neuroactivity. In the present study, the cell viability and neuritogenic effects of L. rhinocerotis sclerotium hot aqueous and ethanolic extracts, and crude polysaccharides on rat pheochromocytoma (PC-12) cells were studied. Interestingly, the hot aqueous extract exhibited neuritogenic activity comparable to NGF in PC-12 cells. However, the extracts and crude polysaccharides stimulated neuritogenesis without stimulating the production of NGF in PC-12 cells. The involvements of the TrkA receptor and MEK/ERK1/2 pathway in hot aqueous extract-stimulated neuritogenesis were examined by Trk (K252a) and MEK/ERK1/2 (U0126 and PD98059) inhibitors. There was no significant difference in protein expression in NGF- and hot aqueous extract-treated cells for both total and phosphorylated p44/42 MAPK. The neuritogenic activity in PC-12 cells stimulated by hot aqueous and ethanolic extracts, and crude polysaccharides of L. rhinocerotis sclerotium mimicking NGF activity via the MEK/ERK1/2 signaling pathway is reported for the first time.
PMCID: PMC4635385  PMID: 26542212
10.  Kinase/phosphatase overexpression reveals pathways regulating hippocampal neuron morphology 
Kinases and phosphatases that regulate neurite number versus branching versus extension are weakly correlated.The kinase family that most strongly enhances neurite growth is a family of non-protein kinases; sugar kinases related to NADK.Pathway analysis revealed that genes in several cancer pathways were highly active in enhancing neurite growth.
In neural development, neuronal precursors differentiate, migrate, extend long axons and dendrites, and finally establish connections with their targets. Clinical conditions such as spinal cord injury, traumatic brain injury, stroke, multiple sclerosis, Parkinson's disease, Huntington's disease, and Alzheimer's disease are often associated with a loss of axon and/or dendrite connectivity and treatment strategies would be enhanced by new therapies targeting cell intrinsic mechanisms of axon elongation and regeneration.
Phosphorylation controls most cellular processes, including the cell cycle, proliferation, metabolism, and apoptosis. Neuronal differentiation, including axon formation and elongation, is also regulated by a wide range of kinases and phosphatases. For example, the non-receptor tyrosine kinase Src is required for cell adhesion molecule-dependent neurite outgrowth. In addition to individual kinases and phosphatases, signaling pathways like the MAPK, growth factor signaling, PIP3, cytoskeletal, and calcium-dependent pathways have been shown to impinge on or control neuronal process development. Recent results have implicated GSK3 and PTEN as therapeutically relevant targets in axonal regeneration after injury. However, these and other experiments have studied only a small fraction of the total kinases and phosphatases in the genome. Because of recent advances in genomic knowledge, large-scale cDNA production, and high-throughput phenotypic analysis, it is now possible to take a more comprehensive approach to understanding the functions of kinases and phosphatases in neurons.
We performed a large, unbiased set of experiments to answer the question ‘what effect does the overexpression of genes encoding kinases, phosphatases, and related proteins have on neuronal morphology?' We used ‘high-content analysis' to obtain detailed results about the specific phenotypes of neurons. We studied embryonic rat hippocampal neurons because of their stereotypical development in vitro (Dotti et al, 1988) and their widespread use in studies of neuronal differentiation and signaling. We transfected over 700 clones encoding kinases and phosphatases into hippocampal neurons and analyzed the resulting changes in neuronal morphology.
Many known genes, including PP1a, ERK1, ErbB2, atypical PKC, Calcineurin, CaMK2, IGF1R, FGFR, GSK3, and PIK3 were observed to have significant effects on neurite outgrowth in our system, consistent with earlier findings in the literature.
We obtained quantitative data for many cellular and neuronal morphological parameters from each neuron imaged. These included nuclear morphology (nuclear area and Hoechst dye intensity), soma morphology (tubulin intensity, area, and shape), and numerous parameters of neurite morphology (e.g. tubulin intensity along the neurites, number of primary neurites, neurite length, number of branches, distance from the cell body to the branches, number of crossing points, width and area of the neurites, and longest neurite; Supplementary Figure 1). Other parameters were reported on a ‘per well' basis, including the percentage of transfected neurons in a condition, as well as the percentage of neurons initiating neurite growth. Data for each treatment were normalized to a control (pSport CAT) within the same experiment, then aggregated across replicate experiments.
Correlations among the 19 normalized parameters were analyzed for neurons transfected with all kinase and phosphatase clones (Figure 2). On the basis of this analysis, the primary variables that define the neurite morphology are primary neurite count, neurite average length, and average branches. Interestingly, primary neurite count was not well correlated with neurite length or branching. The Pearson correlation coefficient (r2) between the number of primary neurites and the average length of the neurites was 0.3, and between the number of primary neurites and average branching was 0.2. In contrast, the correlation coefficient of average branching with neurite average length was 0.7. The most likely explanation is that signaling mechanisms underlying the neurite number determination are different than those controlling length/branching of the neurites.
Related proteins are often involved in similar neuronal functions. For example, families of receptor protein tyrosine phosphatases are involved in motor axon extension and guidance in both Drosophila and in vertebrates, and a large family of Eph receptor tyrosine kinases regulates guidance of retinotectal projections, motor axons, and axons in the corpus callosum. We therefore asked whether families of related genes produced similar phenotypes when overexpressed in hippocampal neurons. Our set of genes covered 40% of the known protein kinases, and many of the non-protein kinases and phosphatases.
Gene families commonly exhibit redundant function. Redundant gene function has often been identified when two or more knockouts are required to produce a phenotype. Our technique allowed us to measure whether different members of gene families had similar (potentially redundant) or distinct effects on neuronal phenotype.
To determine whether groups of related genes affect neuronal morphology in similar ways, we used sequence alignment information to construct gene clusters (Figure 6). Genes were clustered at nine different thresholds of similarity (called ‘tiers'). The functional effect for a particular parameter was then averaged within each cluster of a given tier, and statistics were performed to determine the significance of the effect. We analyzed the results for three key neurite parameters (average neurite length, primary neurite count, and average branching). Genes that perturbed each of these phenotypes are grouped in Figure 6. Eight families, most with only a few genes, produced significant changes for one or two parameters. A diverse family of non-protein kinases had a positive effect on neurite outgrowth in three of the four parameters analyzed. This family of kinases consisted of a variety of enzymes, mostly sugar and lipid kinases. A similar analysis was performed using pathway cluster analysis with pathways from the KEGG database, rather than sequence homology. Interestingly, pathways involved in cancer cell proliferation potentiated neurite extension and branching.
Our studies have identified a large number of kinases and phosphatases, as well as structurally and functionally defined families of these proteins, that affect neuronal process formation in specific ways. We have provided an analytical methodology and new tools to analyze functional data, and have implicated genes with novel functions in neuronal development. Our studies are an important step towards the goal of a molecular description of the intrinsic control of axodendritic growth.
Development and regeneration of the nervous system requires the precise formation of axons and dendrites. Kinases and phosphatases are pervasive regulators of cellular function and have been implicated in controlling axodendritic development and regeneration. We undertook a gain-of-function analysis to determine the functions of kinases and phosphatases in the regulation of neuron morphology. Over 300 kinases and 124 esterases and phosphatases were studied by high-content analysis of rat hippocampal neurons. Proteins previously implicated in neurite growth, such as ERK1, GSK3, EphA8, FGFR, PI3K, PKC, p38, and PP1a, were confirmed to have effects in our functional assays. We also identified novel positive and negative neurite growth regulators. These include neuronal-developmentally regulated kinases such as the activin receptor, interferon regulatory factor 6 (IRF6) and neural leucine-rich repeat 1 (LRRN1). The protein kinase N2 (PKN2) and choline kinase α (CHKA) kinases, and the phosphatases PPEF2 and SMPD1, have little or no established functions in neuronal function, but were sufficient to promote neurite growth. In addition, pathway analysis revealed that members of signaling pathways involved in cancer progression and axis formation enhanced neurite outgrowth, whereas cytokine-related pathways significantly inhibited neurite formation.
PMCID: PMC2925531  PMID: 20664637
bioinformatics; development; functional genomics; metabolic and regulatory networks; neuroscience
11.  Neurotrophic Effects of Mu Bie Zi (Momordica cochinchinensis) Seed Elucidated by High-Throughput Screening of Natural Products for NGF Mimetic Effects in PC-12 Cells 
Neurochemical research  2015;40(10):2102-2112.
Post-mitotic central nervous system (CNS) neurons have limited capacity for regeneration, creating a challenge in the development of effective therapeutics for spinal cord injury or neurodegenerative diseases. Furthermore, therapeutic use of human neurotrophic agents such as nerve growth factor (NGF) are limited due to hampered transport across the blood brain barrier (BBB) and a large number of peripheral side effects (e.g. neuro-inflammatory pain/tissue degeneration etc.). Therefore, there is a continued need for discovery of small molecule NGF mimetics that can penetrate the BBB and initiate CNS neuronal outgrowth/regeneration. In the current study, we conduct an exploratory high-through-put (HTP) screening of 1144 predominantly natural/herb products (947 natural herbs/plants/spices, 29 polyphenolics and 168 synthetic drugs) for ability to induce neurite outgrowth in PC12 dopaminergic cells grown on rat tail collagen, over 7 days. The data indicate a remarkably rare event-low hit ratio with only 1/1144 tested substances (<111.25 µg/mL) being capable of inducing neurite outgrowth in a dose dependent manner, identified as; Mu Bie Zi, Momordica cochinchinensis seed extract (MCS). To quantify the neurotrophic effects of MCS, 36 images (n = 6) (average of 340 cells per image), were numerically assessed for neurite length, neurite count/cell and min/max neurite length in microns (µm) using Image J software. The data show neurite elongation from 0.07 ± 0.02 µm (controls) to 5.5 ± 0.62 µm (NGF 0.5 μg/mL) and 3.39 ± 0.45 µm (138 μg/mL) in MCS, where the average maximum length per group extended from 3.58 ± 0.42 µm (controls) to 41.93 ± 3.14 µm (NGF) and 40.20 ± 2.72 µm (MCS). Imaging analysis using immunocytochemistry (ICC) confirmed that NGF and MCS had similar influence on 3-D orientation/expression of 160/200 kD neurofilament, tubulin and F-actin. These latent changes were associated with early rise in phosphorylated extracellular signal-regulated kinase (ERK) p-Erk1 (T202/Y204)/p-Erk2 (T185/Y187) at 60 min with mild changes in pAKT peaking at 5 min, and no indication of pMEK involvement. These findings demonstrate a remarkable infrequency of natural products or polyphenolic constituents to exert neurotrophic effects at low concentrations, and elucidate a unique property of MCS extract to do so. Future research will be required to delineate in depth mechanism of action of MCS, constituents responsible and potential for therapeutic application in CNS degenerative disease or injury.
PMCID: PMC4600633  PMID: 25862192
Nerve growth factor; High throughput screening; Neurotrophic; NGF mimetic; NGF signaling; Chinese herbs
12.  Inhibition of Nerve Growth Factor-Induced Neurite Outgrowth from PC12 Cells by Dexamethasone: Signaling Pathways through the Glucocorticoid Receptor and Phosphorylated Akt and ERK1/2 
PLoS ONE  2014;9(3):e93223.
Glucocorticoids are important mediators of the stress response and are commonly employed as drugs for the suppression of immune rejection after organ transplantation. Previous investigations uncovered the possibility of mood depression in patients undergoing long-term treatment with synthetic glucocorticoids, including dexamethasone (DEX). Exogenous glucocorticoids and their synthetic derivatives can also adversely affect the development of the central nervous system. Although neurite extension from rat pheochromocytoma-derived PC12 cells and a variety of primary neurons is stimulated by nerve growth factor (NGF), and signaling pathways triggered by the binding of NGF to tyrosine kinase receptor type 1 (TrkA) function in both neurite outgrowth and neuronal survival, the effect of DEX on the activation of regulatory proteins and pathways downstream of TrkA has not been well characterized. To analyze the influence of DEX on NGF-induced neurite outgrowth and signaling, PC12 cells, a widely utilized model of neuronal differentiation, were pretreated with the glucocorticoid prior to NGF induction. NGF-induced neurite outgrowth was attenuated by pretreatment with DEX, even in the absence of DEX after the addition of NGF. Moreover, DEX suppressed the phosphorylation of Akt and extracellular-regulated kinase 1/2 (ERK1/2) in the neurite outgrowth signaling cascade initiated by NGF. Finally, the glucocorticoid receptor (GR) antagonist, RU38486, counteracted the inhibitory effect of DEX pretreatment, not only on the phosphorylation of Akt and ERK1/2, but also on neurite extension from PC12 cells. These results suggest that DEX binding to the GR impairs NGF-promoted neurite outgrowth by interfering with the activation/phosphorylation of Akt and ERK1/2. These novel findings are likely to be useful for elucidating the central nervous system depressive mechanism(s) of action of DEX and other glucocorticoids.
PMCID: PMC3965538  PMID: 24667984
13.  Rit Contributes to Nerve Growth Factor-Induced Neuronal Differentiation via Activation of B-Raf-Extracellular Signal-Regulated Kinase and p38 Mitogen-Activated Protein Kinase Cascades 
Molecular and Cellular Biology  2005;25(2):830-846.
Rit is one of the original members of a novel Ras GTPase subfamily that uses distinct effector pathways to transform NIH 3T3 cells and induce pheochromocytoma cell (PC6) differentiation. In this study, we find that stimulation of PC6 cells by growth factors, including nerve growth factor (NGF), results in rapid and prolonged Rit activation. Ectopic expression of active Rit promotes PC6 neurite outgrowth that is morphologically distinct from that promoted by oncogenic Ras (evidenced by increased neurite branching) and stimulates activation of both the extracellular signal-regulated kinase (ERK) and p38 mitogen-activated protein (MAP) kinase signaling pathways. Furthermore, Rit-induced differentiation is dependent upon both MAP kinase cascades, since MEK inhibition blocked Rit-induced neurite outgrowth, while p38 blockade inhibited neurite elongation and branching but not neurite initiation. Surprisingly, while Rit was unable to stimulate ERK activity in NIH 3T3 cells, it potently activated ERK in PC6 cells. This cell type specificity is explained by the finding that Rit was unable to activate C-Raf, while it bound and stimulated the neuronal Raf isoform, B-Raf. Importantly, selective down-regulation of Rit gene expression in PC6 cells significantly altered NGF-dependent MAP kinase cascade responses, inhibiting both p38 and ERK kinase activation. Moreover, the ability of NGF to promote neuronal differentiation was attenuated by Rit knockdown. Thus, Rit is implicated in a novel pathway of neuronal development and regeneration by coupling specific trophic factor signals to sustained activation of the B-Raf/ERK and p38 MAP kinase cascades.
PMCID: PMC543422  PMID: 15632082
14.  p190RhoGAP and Rap-dependent RhoGAP (ARAP3) inactivate RhoA in response to nerve growth factor leading to neurite outgrowth from PC12 cells 
Experimental & Molecular Medicine  2010;42(5):335-344.
Rat pheochromocytoma (PC12) cells have been used to investigate neurite outgrowth. Nerve growth factor (NGF) has been well known to induce neurite outgrowth from PC12 cells. RhoA belongs to Ras-related small GTP-binding proteins, which regulate a variety of cellular processes, including cell morphology alteration, actin dynamics, and cell migration. NGF suppressed GTP-RhoA levels after 12 h in PC12 cells and was consistently required for a long time to induce neurite outgrowth. Constitutively active (CA)-RhoA suppressed neurite outgrowth from PC12 cells in response to NGF, whereas dominant-negative (DN)-RhoA stimulated it, suggesting that RhoA inactivation is essential for neurite outgrowth. Here, we investigated the mechanism of RhoA inactivation. DN-p190RhoGAP abrogated neurite outgrowth, whereas wild-type (WT)-p190RhoGAP and WT-Src synergistically stimulated it along with accelerating RhoA inactivation, suggesting that p190RhoGAP, which can be activated by Src, is a major component in inhibiting RhoA in response to NGF in PC12 cells. Contrary to RhoA, Rap1 was activated by NGF, and DN-Rap1 suppressed neurite outgrowth, suggesting that Rap1 is also essential for neurite outgrowth. RhoA was co-immunoprecipitated with Rap1, suggesting that Rap1 interacts with RhoA. Furthermore, a DN-Rap-dependent RhoGAP (ARAP3) prevented RhoA inactivation, abolishing neurite formation from PC12 cells in response to NGF. These results suggest that NGF activates Rap1, which, in turn, up-regulates ARAP3 leading to RhoA inactivation and neurite outgrowth from PC12 cells. Taken together, p190RhoGAP and ARAP3 seem to be two main factors inhibiting RhoA activity during neurite outgrowth in PC12 cells in response to NGF.
PMCID: PMC2877255  PMID: 20200473
ARAP3 protein; nerve growth factor; neurites; p190RhoGAP; PC12 cells; Rap1; RhoA
15.  LAMTOR2-Mediated Modulation of NGF/MAPK Activation Kinetics during Differentiation of PC12 Cells 
PLoS ONE  2014;9(4):e95863.
LAMTOR2 (p14), a part of the larger LAMTOR/Ragulator complex, plays a crucial role in EGF-dependent activation of p42/44 mitogen-activated protein kinases (MAPK, ERK1/2). In this study, we investigated the role of LAMTOR2 in nerve growth factor (NGF)-mediated neuronal differentiation. Stimulation of PC12 (rat adrenal pheochromocytoma) cells with NGF is known to activate the MAPK. Pharmacological inhibition of MEK1 as well as siRNA–mediated knockdown of both p42 and p44 MAPK resulted in inhibition of neurite outgrowth. Contrary to expectations, siRNA–mediated knockdown of LAMTOR2 effectively augmented neurite formation and neurite length of PC12 cells. Ectopic expression of a siRNA-resistant LAMTOR2 ortholog reversed this phenotype back to wildtype levels, ruling out nonspecific off-target effects of this LAMTOR2 siRNA approach. Mechanistically, LAMTOR2 siRNA treatment significantly enhanced NGF-dependent MAPK activity, and this effect again was reversed upon expression of the siRNA-resistant LAMTOR2 ortholog. Studies of intracellular trafficking of the NGF receptor TrkA revealed a rapid colocalization with early endosomes, which was modulated by LAMTOR2 siRNA. Inhibition of LAMTOR2 and concomitant destabilization of the remaining members of the LAMTOR complex apparently leads to a faster release of the TrkA/MAPK signaling module and nuclear increase of activated MAPK. These results suggest a modulatory role of the MEK1 adapter protein LAMTOR2 in NGF-mediated MAPK activation required for induction of neurite outgrowth in PC12 cells.
PMCID: PMC3994133  PMID: 24752675
16.  K-252a, a potent protein kinase inhibitor, blocks nerve growth factor- induced neurite outgrowth and changes in the phosphorylation of proteins in PC12h cells 
The Journal of Cell Biology  1988;107(4):1531-1539.
Nerve growth factor (NGF) promotes neuronal differentiation of PC12 pheochromocytoma cells. One of the most prominent and distinguishing features of neuronal differentiation is neurite outgrowth. The mechanism by which NGF causes the cells to elaborate neurites is unknown. This study shows that K-252a, a potent protein kinase inhibitor, blocks NGF-induced neurite outgrowth and the changes in protein phosphorylation elicited by NGF. In the experiment with intact cells phosphorylated with 32P-orthophosphoric acid, an exposure of PC12h cells to NGF (50 ng/ml) caused an increase in the phosphorylation of tyrosine hydroxylase and a 35,000-D protein and a decrease in a 36,500-D protein. Pretreatment of PC12h cells with K-252a (100 nM) inhibited the effects of NGF on the phosphorylation of these three proteins. In the phosphorylation of cell-free extracts with [gamma-32P] ATP, treatment of PC12h cells with NGF (50 ng/ml) caused a decrease in the phosphorylation of Nsp100. Pretreatment of the cells with K-252a (30 nM) almost completely blocked the NGF effect on the phosphorylation of Nsp100 elicited by subsequent treatment of the cells with NGF. Treatment of PC12h cells with NGF promoted outgrowth of neurites. The addition of K-252a (100 nM) into the culture almost completely blocked the generation of neurites elicited by NGF. Earlier studies demonstrated that NGF-induced neurite outgrowth in PC12 cells involves at least two components: the first of these is transcription-dependent and the second is transcription-independent. To determine the component on which K-252a acts, experiments were carried out on NGF-induced priming or regeneration of neurites. When K-252a was present in the priming step, NGF induced only actinomycin D-sensitive neurites, showing that K-252a interferes with the transcription-dependent actions of NGF. When already primed cells were treated with NGF, actinomycin D- resistant neurites were formed and these were blocked by K-252a, showing that the inhibitor interferes with the transcription- independent actions of NGF as well. Although the exact mechanism of inhibition of NGF-promoted neurite formation by K-252a is unknown, the most probable explanation is that both transcription-dependent and - independent components are involved in at least one step of the activation of some specific protein kinase(s) that can be suppressed by K-252a.
PMCID: PMC2115256  PMID: 2844830
17.  A novel phenoxy thiophene sulphonamide molecule protects against glutamate evoked oxidative injury in a neuronal cell model 
BMC Neuroscience  2013;14:93.
Glutamate is one of the major neurotransmitters in the central nervous system. It is a potent neurotoxin capable of neuronal destruction through numerous signal pathways when present in high concentration. Glutamate-evoked excitotoxicity has been implicated in the etiology of many neurodegenerative diseases including Alzheimer’s disease (AD), Parkinson’s disease (PD), and ischemic stroke. Increasing evidence has shown that reactive oxygen species (ROS) provoked by glutamate-linked oxidative stress plays a crucial role in the pathogenesis of these disorders. We previously reported the discovery of an aryl thiophene compound, 4-chloro-N-(naphthalen-1-ylmethyl)-5-(3-(piperazin-1-yl)phenoxy)thiophene-2-sulfonamide (B355252) from a proprietary library of small molecules. We showed that this compound was capable of potentiating nerve growth factor (NGF)-primed neurite outgrowth in neuronal cell models in a low NGF environment. In the present study we investigated the neuroprotective effects and signaling pathways of B355252 on glutamate-evoked excitotoxicity in HT-22, a murine hippocampal neuronal cell line.
Glutamate significantly decreased HT-22 neuronal cell viability in a concentration-dependent manner as measured by the MTT assay. Co-treatment with 2, 4, and 8 μM B355252 protected against cell death caused by glutamate-induced toxicity by 9.1% (p<0.01), 26.0% (p<0.001), and 61.9% (p<0.001) respectively, compared to glutamate-treated control group. B355252 at a concentration of 8 μM fully rescued HT-22 from the neurototoxic effects of glutamate, and by itself increased cell viability by 16% (p<0.001) above untreated control. Glutamate enhanced reduction in glutathione (GSH) synthesis was reversed by 15% (p<0.01) in the presence of B355252. B355252 reduced the expression of apoptosis inducing factor (AIF) by 27%, while the proapoptotic Bcl-2 associated X protein (Bax) was strongly attenuated 3-fold. Glutamate-evoked increase in intracellular calcium (Ca2+) load and subsequent ROS production was inhibited by 71% (p<0.001) and 40% (p<0.001) respectively, to comparable level as untreated control in the presence of B355252. Glutamate significantly upregulated the phosphorylation of extracellular signal regulated kinase Erk1/2 (pERK1/2), while decreasing Erk3. In contrast, B355252 potently attenuated the glutamate-dependent activation of Erk1/2 and robustly increased the level of ERK3 in HT-22.
A novel phenoxy thiophene small molecule, B355252, suppresses glutamate-evoked oxidative stress in HT-22 neurons by blocking Ca2+ and ROS production, and altering the expression or phosphorylation states of Erk kinases. This molecule previously reported to enhance neurite outgrowth in the presence of sub-physiological concentrations of NGF appears to be a promising drug candidate for development as a potential therapeutic and neuroprotective agent for various neurodegenerative disorders.
PMCID: PMC3846642  PMID: 24004478
Glutamate; Neuroprotection; Excitotoxicity; Small molecule; Alzheimer’s disease; Oxidative stress; ERK3; Neurodegenerative disease; Phenoxy thiophene; HT-22
18.  Light-Mediated Kinetic Control Reveals the Temporal Effect of the Raf/MEK/ERK Pathway in PC12 Cell Neurite Outgrowth 
PLoS ONE  2014;9(3):e92917.
It has been proposed that differential activation kinetics allows cells to use a common set of signaling pathways to specify distinct cellular outcomes. For example, nerve growth factor (NGF) and epidermal growth factor (EGF) induce different activation kinetics of the Raf/MEK/ERK signaling pathway and result in differentiation and proliferation, respectively. However, a direct and quantitative linkage between the temporal profile of Raf/MEK/ERK activation and the cellular outputs has not been established due to a lack of means to precisely perturb its signaling kinetics. Here, we construct a light-gated protein-protein interaction system to regulate the activation pattern of the Raf/MEK/ERK signaling pathway. Light-induced activation of the Raf/MEK/ERK cascade leads to significant neurite outgrowth in rat PC12 pheochromocytoma cell lines in the absence of growth factors. Compared with NGF stimulation, light stimulation induces longer but fewer neurites. Intermittent on/off illumination reveals that cells achieve maximum neurite outgrowth if the off-time duration per cycle is shorter than 45 min. Overall, light-mediated kinetic control enables precise dissection of the temporal dimension within the intracellular signal transduction network.
PMCID: PMC3965503  PMID: 24667437
19.  Neurite outgrowth stimulatory effects of myco synthesized AuNPs from Hericium erinaceus (Bull.: Fr.) Pers. on pheochromocytoma (PC-12) cells 
Hericium erinaceus has been reported to have a wide range of medicinal properties such as stimulation of neurite outgrowth, promotion of functional recovery of axonotmetic peroneal nerve injury, antioxidant, antihypertensive, and antidiabetic properties. In recent years, the green synthesis of gold nanoparticles (AuNPs) has attracted intense interest due to the potential use in biomedical applications. The aim of this study was to investigate the effects of AuNPs from aqueous extract of H. erinaceus on neurite outgrowth of rat pheochromocytoma (PC-12) cells.
The formation of AuNPs was characterized by UV–visible spectrum, energy dispersive X-ray (EDX), field-emission scanning electron microscope (FESEM), transmission electron microscopy (TEM), particle size distribution, and Fourier transform-infrared spectroscopy (FTIR). Furthermore, the neurite extension study of synthesized AuNPs was evaluated by in vitro assay.
The AuNPs exhibited maximum absorbance between 510 and 600 nm in UV–visible spectrum. FESEM and TEM images showed the existence of nanoparticles with sizes of 20–40 nm. FTIR measurements were carried out to identify the possible biomolecules responsible for capping and efficient stabilization of the nanoparticles. The purity and the crystalline properties were confirmed by EDX diffraction analysis, which showed strong signals with energy peaks in the range of 2–2.4 keV, indicating the existence of gold atoms. The synthesized AuNPs showed significant neurite extension on PC-12 cells. Nerve growth factor 50 ng/mL was used as a positive control. Treatment with different concentrations (nanograms) of AuNPs resulted in neuronal differentiation and neuronal elongation. AuNPs induced maximum neurite outgrowth of 13% at 600 ng/mL concentration.
In this study, the AuNPs synthesis was achieved by a simple, low-cost, and rapid bioreduction approach. AuNPs were shown to have potential neuronal differentiation and stimulated neurite outgrowth. The water-soluble bioconstituents could be responsible for the neuroactivity. This is the first report for the biosynthesis of AuNPs using the hot aqueous extract of H. erinaceus.
PMCID: PMC4583117  PMID: 26425086
AuNPs; nanoparticles; Hericium erinaceus; PC-12; neurite outgrowth
20.  Amitriptyline protects against TNF‐α‐induced atrophy and reduction in synaptic markers via a Trk‐dependent mechanism 
Neuritic degeneration and synaptic loss are features of both neuroinflammation and neurodegenerative disease. The tricyclic antidepressant amitriptyline has neurotrophic and anti‐inflammatory properties and acts as a novel agonist of the neurotrophin Trk receptors. Primary cortical neurons were treated with amitriptyline, nortriptyline and NGF and tested for neuronal complexity by Sholl analysis, protein expression by Western immunoblotting, and synapse number by colocalization of pre and postsynaptic makers. Amitriptyline (500 nmol/L) and its active metabolite nortriptyline (50 nmol/L) are found to induce neurite outgrowth in rat primary cortical neurons. Amitriptyline‐induced neurite outgrowth is blocked by inhibition of Trk signaling using Trk antagonist K252a (200 nmol/L) but not by the neurotrophin inhibitor Y1036 (40 μmol/L), indicating that amitriptyline binds directly to the Trk receptor to initiate neurite outgrowth. MEK inhibitor PD98059 (10 μmol/L) also blocks amitriptyline‐induced neurite outgrowth, implicating activation of the MAPK signaling pathway downstream of Trk receptor activation. Furthermore, pretreatment of primary cortical neurons with amitriptyline and nortriptyline prevents the effects of the proinflammatory cytokine TNF‐α (10 ng/mL) on neurite outgrowth and colocalization of synaptic proteins. These findings suggest that amitriptyline and nortriptyline can exert neurotrophic effects in primary cortical neurons via activation of a Trk/MAPK signaling pathway. These compounds therefore have significant potential to be used in the treatment of neurodegenerative conditions where atrophy and loss of synaptic connections contribute to progression of disease.
PMCID: PMC4804321  PMID: 27069625
Amitriptyline; neurite outgrowth; neurotrophin; nortriptyline; PSD‐95; synaptogenesis; synaptophysin; Trk receptor
21.  Guanosine stimulates neurite outgrowth in PC12 cells via activation of heme oxygenase and cyclic GMP 
Purinergic Signalling  2005;1(2):161-172.
Undifferentiated rat pheochromocytoma (PC12) cells extend neurites when cultured in the presence of nerve growth factor (NGF). Extracellular guanosine synergistically enhances NGF-dependent neurite outgrowth. We investigated the mechanism by which guanosine enhances NGF-dependent neurite outgrowth. Guanosine administration to PC12 cells significantly increased guanosine 3-5-cyclic monophosphate (cGMP) within the first 24 h whereas addition of soluble guanylate cyclase (sGC) inhibitors abolished guanosine-induced enhancement of NGF-dependent neurite outgrowth. sGC may be activated either by nitric oxide (NO) or by carbon monoxide (CO). \documentclass[12pt]{minimal} \usepackage{amsmath} \usepackage{wasysym} \usepackage{amsfonts} \usepackage{amssymb} \usepackage{amsbsy} \usepackage{mathrsfs} \usepackage{upgreek} \setlength{\oddsidemargin}{-69pt} \begin{document} $$N^{\omega } $$ \end{document}-Nitro-l-arginine methyl ester (l-NAME), a non-isozyme selective inhibitor of nitric oxide synthase (NOS), had no effect on neurite outgrowth induced by guanosine. Neither nNOS (the constitutive isoform), nor iNOS (the inducible isoform) were expressed in undifferentiated PC12 cells, or under these treatment conditions. These data imply that NO does not mediate the neuritogenic effect of guanosine. Zinc protoporphyrin-IX, an inhibitor of heme oxygenase (HO), reduced guanosine-dependent neurite outgrowth but did not attenuate the effect of NGF. The addition of guanosine plus NGF significantly increased the expression of HO-1, the inducible isozyme of HO, after 12 h. These data demonstrate that guanosine enhances NGF-dependent neurite outgrowth by first activating the constitutive isozyme HO-2, and then by inducing the expression of HO-1, the enzymes responsible for CO synthesis, thus stimulating sGC and increasing intracellular cGMP.
PMCID: PMC2096532  PMID: 18404501
carbon monoxide; cyclic GMP; guanosine; heme oxygenase; neurite outgrowth; NGF; nitric oxide synthase; PC12 cells
22.  The Pseudophosphatase MK-STYX Induces Neurite-Like Outgrowths in PC12 Cells 
PLoS ONE  2014;9(12):e114535.
The rat pheochromocytoma PC12 cell line is a widely used system to study neuronal differentiation for which sustained activation of the extracellular signaling related kinase (ERK) pathway is required. Here, we investigate the function of MK-STYX [MAPK (mitogen-activated protein kinase) phosphoserine/threonine/tyrosine-binding protein] in neuronal differentiation. MK-STYX is a member of the MAPK phosphatase (MKP) family, which is generally responsible for dephosphorylating the ERKs. However, MK-STYX lacks catalytic activity due to the absence of the nucleophilic cysteine in the active site signature motif HC(X5)R that is essential for phosphatase activity. Despite being catalytically inactive, MK-STYX has been shown to play a role in important cellular pathways, including stress responses. Here we show that PC12 cells endogenously express MK-STYX. In addition, MK-STYX, but not its catalytically active mutant, induced neurite-like outgrowths in PC12 cells. Furthermore, MK-STYX dramatically increased the number of cells with neurite extensions in response to nerve growth factor (NGF), whereas the catalytically active mutant did not. MK-STYX continued to induce neurites in the presence of a MEK (MAP kinase kinase) inhibitor suggesting that MK-STYX does not act through the Ras-ERK/MAPK pathway but is involved in another pathway whose inactivation leads to neuronal differentiation. RhoA activity assays indicated that MK-STYX induced extensions through the Rho signaling pathway. MK-STYX decreased RhoA activation, whereas RhoA activation increased when MK-STYX was down-regulated. Furthermore, MK-STYX affected downstream players of RhoA such as the actin binding protein cofilin. The presence of MK-STYX decreased the phosphorylation of cofilin in non NGF stimulated cells, but increased its phosphorylation in NGF stimulated cells, whereas knocking down MK-STYX caused an opposite effect. Taken together our data suggest that MK-STYX may be a regulator of RhoA signaling, and implicate this pseudophosphatase as a regulator of neuronal differentiation.
PMCID: PMC4257672  PMID: 25479605
23.  Cortex Mori Radicis Extract induces neurite outgrowth in PC12 cells activating ERK signaling pathway via inhibiting Ca2+ influx 
Cortex Mori Radicis is a traditional Chinese herbal medicine which has a long history of use for the treatment of headaches, cough, edema and diabetes. However, its function and mode of action within nervous system remain largely unclear. In the present study, we have attempted to determine the effects of Cortex Mori Radicis Extract (CMRE) on neuronal differentiation. Here, we reported that CMRE induces the neurite outgrowth in pheochromocytoma PC12 cells and primary cortical neuron. Following the generation of neurite outgrowth, extracellular Ca2+ influx was inhibited and intracellular Ca2+ decreased. In addition, CMRE induced the extracellular signal-regulated kinase 1/2 (ERK1/2) activation and also stimulated the Rap1-GTP expression, which is closely linked to neuritogenesis. Moreover, the neurite outgrowth induced by CMRE was antagonized to a marked degree by suppressing activation of p-ERK1/2 with the specific ERK1/2 inhibitor (PD98059), suggesting the involvement of Rap1-GTP and ERK1/2 in CMRE-induced neurite outgrowth. Taken together, these results demonstrate that CMRE induces neurite outgrowth of PC12 cells through Rap1-ERK signaling pathway via inhibiting Ca2+ influx, and provide a novel insight into the manner in which CMRE participates in neuritogenesis.
PMCID: PMC4483855  PMID: 26131075
Cortex Mori Radicis Extract; PC12 cells; ERK signaling pathway; Rap1; Ca2+ influx
24.  The role of cAMP in nerve growth factor-promoted neurite outgrowth in PC12 cells 
The Journal of Cell Biology  1986;102(3):821-829.
Nerve growth factor (NGF)-mediated neurite outgrowth in rat pheochromocytoma PC12 cells has been described to be synergistically potentiated by the simultaneous addition of dibutyryl cAMP. To elucidate further the role of cAMP in NGF-induced neurite outgrowth we have used the adenylate cyclase activator forskolin, cAMP, and a set of chemically modified cAMP analogues, including the adenosine cyclic 3',5'-phosphorothioates (cAMPS) (Rp)-cAMPS and (Sp)-cAMPS. These diastereomers have differential effects on the activation of cAMP- dependent protein kinases, i.e., (Sp)-cAMPS behaves as a cAMP agonist and (Rp)-cAMPS behaves as a cAMP antagonist. Our data show that the establishment of a neuritic network, as observed from PC12 cells treated with NGF alone, could not be induced by either forskolin, cAMP, or cAMP analogues alone. The presence of NGF in combination with forskolin or cAMP or its agonistic analogues potentiated the initiation of neurite outgrowth from PC12 cells. The (Sp)-cAMPS-induced stimulation of NGF-mediated process formation was successfully blocked by the (Rp)-cAMPS diastereomer. On the other hand, NGF-stimulated neurite outgrowth was not inhibited by the presence of the cAMP antagonist (Rp)-cAMPS. We conclude that the morphological differentiation of PC12 cells stimulated by NGF does not require cAMP as a second messenger. The constant increase of intracellular cAMP, caused by either forskolin or cAMP and the analogues, in combination with NGF, not only rapidly stimulated early neurite outgrowth but also exerted a maintaining effect on the neuronal network established by NGF.
PMCID: PMC2114106  PMID: 3005337
25.  Hepatoprotective effect of silymarin 
World Journal of Hepatology  2014;6(3):144-149.
The use of medicinal plants in treating illnesses has been reported since ancestral times. In the case of hepatic diseases, several species such as Silybum marianum, Phyllanthus niruri, and Panus giganteus (Berk.) have been shown to ameliorate hepatic lesions. Silymarin is a natural compound derived from the species Silybum marianum, which is commonly known as Milk thistle. This plant contains at least seven flavoligands and the flavonoid taxifolin. The hepatoprotective and antioxidant activity of silymarin is caused by its ability to inhibit the free radicals that are produced from the metabolism of toxic substances such as ethanol, acetaminophen, and carbon tetrachloride. The generation of free radicals is known to damage cellular membranes and cause lipoperoxidation. Silymarin enhances hepatic glutathione and may contribute to the antioxidant defense of the liver. It has also been shown that silymarin increases protein synthesis in hepatocytes by stimulating RNA polymerase I activity. A previous study on humans reported that silymarin treatment caused a slight increase in the survival of patients with cirrhotic alcoholism compared with untreated controls.
PMCID: PMC3959115  PMID: 24672644
Silybum marianum; Hepatoprotector; Lipoperoxidation; Silymarin

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