Hericium erinaceus (Bull.: Fr) Pers. cultivated under tropical conditions: isolation of hericenones and demonstration of NGF-mediated neurite outgrowth in PC12 cells via MEK/ERK and PI3K-Akt signaling pathways.

Hericium erinaceus (Bull.: Fr.) Pers. is an edible and medicinal mushroom used traditionally to improve memory. In this study, we investigated the neuritogenic effects of hericenones isolated from H. erinaceus and the mechanisms of action involved. H. erinaceus was cultivated and the secondary metabolites were elucidated by high performance liquid chromatography (HPLC), liquid chromatography-mass spectrometry (LC-MS), and nuclear magnetic resonance (NMR). The secondary metabolites were tested for neurite outgrowth activity (if any). Rat pheochromocytoma (PC12) cells were employed and the nerve growth factor (NGF) level was also determined. The signaling pathways involved in the mushroom-induced neuritogenesis were investigated using several pharmacological inhibitors. Hericenones B-E (1-4), erinacerin A (5) and isohericerin (6) were isolated from the basidiocarps of H. erinaceus. The hericenones did not promote neurite outgrowth but when induced with a low concentration of NGF (5 ng mL(-1)), the neuritogenic activity was comparable to that of the positive control (50 ng mL(-1) of NGF). Hericenone E was able to stimulate NGF secretion which was two-fold higher than that of the positive control. The neuritogenesis process was partially blocked by the tyrosine kinase receptor (Trk) inhibitor, K252a, suggesting that the neuritogenic effect was not solely due to NGF. Hericenone E also increased the phosphorylation of extracellular-signal regulated kinases (ERKs) and protein kinase B (Akt). Taken together, this study suggests that hericenone E potentiated NGF-induced neuritogenesis in PC12 cells via the MEK/ERK and PI3K/Akt pathways.

Diabetes impairs an interleukin-1ß-dependent pathway that enhances neurite outgrowth through JAK/STAT3 modulation of mitochondrial bioenergetics in adult sensory neurons.

BACKGROUND: A luminex-based screen of cytokine expression in dorsal root ganglia (DRG) and nerve of type 1 diabetic rodents revealed interleukin-1 (IL-1α) and IL-1ß to be significantly depressed. We, therefore, tested the hypothesis that impaired IL-1α and IL-1ß expression in DRG may contribute to aberrant axon regeneration and plasticity seen in diabetic sensory neuropathy. In addition, we determined if these cytokines could optimize mitochondrial bioenergetics since mitochondrial dysfunction is a key etiological factor in diabetic neuropathy. RESULTS: Cytokines IL-1α and IL-1ß were reduced 2-fold (p<0.05) in DRG and/or nerve of 2 and 5 month streptozotocin (STZ)-diabetic rats. IL-2 and IL-10 were unchanged. IL-1α and IL-1ß induced similar 2 to 3-fold increases in neurite outgrowth in cultures derived from control or diabetic rats (p<0.05). STAT3 phosphorylation on Tyr705 or Ser727 was depressed in DRG from STZ-diabetic mice and treatment of cultures derived from STZ-diabetic rats with IL-1ß for 30 min raised phosphorylation of STAT3 on Tyr705 and Ser727 by 1.5 to 2-fold (p<0.05). shRNA-based or AG490 inhibition of STAT3 activity or shRNA blockade of endogenous IL-1ß expression completely blocked neurite outgrowth. Cultured neurons derived from STZ-diabetic mice were treated for 24 hr with IL-1ß and maximal oxygen consumption rate and spare respiratory capacity, both key measures of bioenergetic fidelity that were depressed in diabetic compared with control neurons, were enhanced 2-fold. This effect was blocked by AG490. CONCLUSIONS: Endogenous synthesis of IL-1ß is diminished in nerve tissue in type 1 diabetes and we propose this defect triggers reduced STAT3 signaling and mitochondrial function leading to sup-optimal axonal regeneration and plasticity.

Neuritogenic effect of standardized extract of Centella asiatica ECa233 on human neuroblastoma cells.

BACKGROUND: In order to gain insight into neuroprotective effects of ECa 233, a standardized extract of Centella asiatica, previously demonstrated in animal models of memory impairment induced by transient global ischemia or intracerebroventricular injection of ß-amyloid, the effect of ECa 233 on neurite outgrowth of human IMR-32 neuroblastoma cell line was investigated. METHODS: Cells were seeded and incubated with various concentrations of ECa 233. Morphometric analysis was carried out by a measurement of the longest neurite growth of cells at 24 and 48 h. Contributing signaling pathways possibly involved were subsequently elucidated by western blot analysis. RESULTS: While ECa 233 had only limited effects on cell viability, it significantly enhanced neurite outgrowth of IMR-32 cells at the concentrations of 1-100 µg/ml. Western blot analysis revealed that ECa 233 significantly upregulated the level of activated ERK1/2 and Akt of the treated cells suggesting their involvement in the neuritogenic effect observed, which was subsequently verified by the finding that an addition of their respective inhibitors could reverse the effect of ECa 233 on these cells. CONCLUSIONS: The present study clearly demonstrated neurite outgrowth promoting activity of ECa 233. ERK1/2 and Akt signaling pathways seemed to account for the neurotrophic effect observed. In conjunction with in vivo neuroprotective effect of ECa 233 previously reported, the results obtained support further development of ECa 233 for clinical use in neuronal injury or neurodegenerative diseases.

Curcuminoids promote neurite outgrowth in PC12 cells through MAPK/ERK- and PKC-dependent pathways.

Curcuminoids, the predominant polyphenolic compounds in the rhizome of Curcuma longa Linn., consist of curcumin, demethoxycurcumin (DMC), and bisdemethoxycurcumin (BDMC). They exhibit multiple desirable characteristics for a neuroprotective agent including antioxidant, anti-inflammatory, and antiamyloid activities. In this work, we report the first investigation of the neurotrophic action and mechanism of curcuminoids in PC12 cells, which respond to nerve growth factor (NGF) and therefore serve as a model system for primary neuronal cells. The percentages of neurite-bearing cells for those treated with 20 µM curcumin, DMC, and BDMC for 72 h reached 21.6 ± 2.0%, 16.3 ± 2.4%, and 19.9 ± 2.5%, respectively, and were significantly higher than that of the negative control (2.0 ± 0.3%, p < 0.05). In parallel, increased expression of the neuronal differentiation markers, growth-associated protein-43 (GAP-43), and neurofilament-L (NF-L) was found in curcuminoid-treated cells. All three curcuminoids (20 µM) activated extracellular signal-regulated protein kinase 1/2 (ERK1/2) and protein kinase C (PKC) signalings, and inhibition of these kinases with the respective pharmacological inhibitors effectively attenuated curcuminoid-induced neurite outgrowth. Furthermore, our results show that both curcumin and DMC, but not BDMC, induced phosphorylation of cAMP response element-binding protein (CREB) and CRE-reporter gene activity significantly (p < 0.05). These inductions were markedly attenuated by the addition of MEK/ERK or PKC inhibitor; as a consequence, ERK- and PKC-dependent pathways may be involved in curcuminoid-mediated neuritogenesis in PC12 cells. Moreover, activation of CREB coupling with CRE-dependent gene transcription may play a vital role for curcumin- or DMC-induced PC12 differentiation.

Development of a cell transducible RhoA inhibitor TAT-C3 transferase and its encapsulation in biocompatible microspheres to promote survival and enhance regeneration of severed neurons.

PURPOSE: Neurons in post-traumatized mammalian central nervous system show only limited degree of regeneration, which can be attributed to the presence of neurite outgrowth inhibitors in damaged myelin and glial scar, and to the apoptosis of severed central neurons and glial cells during secondary Wallerian degeneration. RhoA GTPase has been implicated as the common denominator in these counter-regeneration events, which shows significant and persistent up-regulation for weeks in injured spinal cord and cerebral infarct after stroke. While the exoenzyme C3 transferase is a potent RhoA inhibitor, its extremely low efficiency of cell entry and degradation in vivo has restricted the therapeutic value. This study aims to circumvent these problems by developing a membrane-permeating form of C3 transferase and a biopolymer-based microsphere depot system for sustainable controlled release of the protein. MATERIALS AND METHODS: A membrane-permeating form of C3 transferase was developed by fusing a Tat (trans-activating transcription factor) transduction domain of human immunodeficiency virus to its amino terminal using standard molecular cloning techniques. After confirming efficient cell entry into epithelial and neuroblastoma cells, the resulting recombinant protein TAT-C3 was encapsulated in biocompatible polymer poly(D,L -lactide-co-glycolide) in the form of microspheres by a water-in-oil-in-water (W/O/W) emulsion method. By blending capped and uncapped form of the polymer at different ratios, TAT-C3 protein release profile was modified to suit the expression pattern of endogenous RhoA during CNS injuries. Bioactivity of TAT-C3 released from microspheres was assessed by RhoA ribosylation assay. RESULTS: In contrast to wild-type C3 transferase, the modified TAT-C3 protein was found to efficiently enter NIH3T3 and N1E-115 neuroblastoma cells as early as 6 hours of incubation. The fusion of TAT sequence to C3 transferase imposed no appreciable effects on its biological activity in promoting neurite outgrowth through RhoA inhibition. Characterization of TAT-C3 encapsulation in various blends of capped/uncapped PLGA polymer revealed the 30:70 formulation to be optimal in attaining a mild initial burst release of 25%, followed by a subsequent average daily release of 2.3% of encapsulated protein over one month, matching the change in RhoA level in severed brain and spinal cord. Importantly, TAT-C3 released from the microspheres remained active up to the first three weeks of incubation. CONCLUSION: Enhanced cell entry of TAT-C3 circumvents the need to administer high dose of the protein to site of injury. The encapsulation of TAT-C3 in different blends of capped/uncapped PLGA microspheres allows adjustment of protein release profile to suit the pattern of RhoA expression in injured CNS.

Oestrogen synthesis in the hippocampus: role in axon outgrowth.

Ovarian oestrogens have been postulated to be neuroprotective. It has also been shown that considerable amounts of oestrogens are synthesised in hippocampal neurones. In the present study, we focused on a potential role of hippocampus-derived oestradiol compared to gonad-derived oestradiol on axon outgrowth of hippocampal neurones. To address the role of hippocampus-derived oestradiol, we inhibited oestrogen synthesis by treatment of neonatal hippocampal cell cultures with letrozole, a specific aromatase inhibitor. As an alternative, we used siRNA against steroidogenic acute regulatory protein (StAR). Axon outgrowth and GAP-43 expression were significantly down-regulated in response to letrozole and in siRNA-StAR transfected cells. The effects after inhibition of oestrogen synthesis in response to letrozole and in siRNA-StAR transfected cells were reversed by oestrogen supplementation. No difference was found between ovariectomised animals, cycling animals at pro-oestrus and ovariectomised and subsequently oestradiol-treated animals. However, high pharmacological doses of oestradiol promoted axon outgrowth, which was possible to abolish by the oestrogen receptor antagonist ICI 182,780. Our results show that oestradiol-induced neurite outgrowth is very likely mediated by genomic oestrogen receptors and requires higher doses of oestradiol than physiological serum concentrations derived from the gonads.

Establishment and application of a high throughput model for Rho kinase inhibitors screening based on fluorescence polarization.

Rho kinase (ROCK) inhibitors are effective candidates for neural or cardiovascular disorders. High throughput model for screening ROCK inhibitors is a basic foundation to pick up ROCK inhibitors from thousands of compounds for drug developing. The high throughput model was established based on purified recombinant rat ROCK catalytic domain (rROCK-CD) from Escherichia coli (E. coli). There are two steps of reaction in the model: incubation of 5.0 microl recombinant rROCK-CD (2.0 microg/ml), 5.0 microl different compounds, 5.0 microl fluorescent S6-peptide (200 nM), and 5.0 microl ATP (10 microM) at 37 degrees C for 60 min was made the first reaction, and the second reaction was made by incubating them with additional 60 microl binding reagent at ambient temperature for 30 min. The phosphorylated S6 peptide can bind to a binding reagent, and the fluorescence varies from low polarization to high according to the amount of the phosphorylated peptide. IC50 was calculated based on polarization variation. Compound, which IC50 was less than 10 microM, was recognized as a lead compound which taken bioactivity evaluation in PC12 by observing neurite outgrowth. The Z'-factor of the model is 0.81 (above 0.5). The model screened five lead compounds from 3294, which promoted neurite outgrowth to different extent. The results suggested that the model is suitable for high throughput screening (HTS), and the five lead compounds are worth of further investigation.

Participation of protein kinase C alpha isoform and extracellular signal-regulated kinase in neurite outgrowth of GT1 hypothalamic neurons.

In the present study, we investigated the selective role of protein kinase C (PKC) isoforms on neurite outgrowth of the GT1 hypothalamic neurons using several PKC isoform-selective inhibitors and transfection-based expression of enhanced green fluorescence protein (EGFP)-fused PKC isoforms. 12-O-Tetradecanoylphorbol-13-acetate (TPA) induced neurite outgrowth and growth cone formation, effects that were blocked by GF 109203X (a PKC inhibitor), safingolTM(a PKCalpha-selective inhibitor), but not by rottlerinTM (a PKCdelta-selective inhibitor), indicating that PKCalpha may be selectively involved in neurite outgrowth and cytoskeletal changes of filamentous actin and beta-tubulin. To define the differential localization of PKC isoforms, EGFP-tagged PKCalpha, PKCgamma, and PKCdelta were transfected into GT1 neuronal cells. TPA treatment induced relocalization of PKCalpha-EGFP to growth cones and cell-cell adhesion sites, PKCgamma-EGFP to the nucleus, and PKCdelta-EGFP to the membrane ruffle, respectively. An EGFP chimera of the catalytic domain of PKCalpha (PKCalpha-Cat-EGFP), the expression of which was inducible by doxycycline, was employed to directly ascertain the effect of PKCalpha enzymatic activity on neurite outgrowth of GT1 cells. Transient transfection of PKCalpha-Cat-EGFP alone increased the neurite-outgrowth and doxycycline treatment further augmented the number of neurite-containing cells. We also examined the involvement of the extracellular signal-regulated kinase (ERK) MAP kinase in TPA-induced neurite outgrowth. TPA treatment increased phosphorylated ERK MAP kinase, but not p38 MAP kinase. Specific inhibition of PKCalpha with safingol blocked the phosphorylation of ERK induced by TPA. More importantly, both neurite outgrowth and phosphorylation of ERK by TPA were blocked by PD 098059, a specific inhibitor of MEK (MAP kinase/ERK kinase-1), but not by SB203580, a specific inhibitor of p38 MAP kinase. These results demonstrate that PKCalpha isoform-specific activation is involved in neurite outgrowth of GT1 hypothalamic neuronal cells via ERK, but not the p38 MAP kinase signal pathway.

Investigation of the Ca(2+)-independent form of Ca(2+)/calmodulin-dependent protein kinase II in neurite outgrowth.

Neuronal Ca(2+)/calmodulin-dependent protein kinase II (CaM kinase II) plays important roles in the control of nerve functions in response to intracellular Ca(2+) (for reviews [Annu. Rev. Physiol. 57 (1995) 417-445; Trends Neurosci. 17 (1994) 406-412]). Brief Ca(2+) signals activate CaM kinase II, and stimulate an autophosphorylation of Thr-286 which allows the kinase to maintain its activated state even after the Ca(2+) concentration has returned to basal levels [J. Biol. Chem. 264 (1989) 16759-16763; Neuron 3 (1989) 59-70; J. Biochem. 109 (1991) 137-143]. Autophosphorylation of CaM kinase II occurs in situ, but it occurs relatively quickly, within just a few minutes [Endocrinology 134 (1994) 2245-2250; J. Biol. Chem. 268 (1993) 7863-7867; J. Biol. Chem. 265 (1990) 18055-18058]. In the present study, we investigated the involvement of the autophosphorylated/Ca(2+)-independent form of CaM kinase II in neurite outgrowth. When neuroblastoma Neruo2a (Nb2a) cells expressing the alpha isoform of CaM kinase II (Nb2a/alpha cells) were stimulated by plating, they formed neurites. The autophosphorylation of Thr-286 and appearance of Ca(2+)-independent activity preceded the neurite formation. The effect of mutating of the kinase autophosphorylation site replacing Thr-286 with Ala (alpha T286A kinase) or Asp (alpha T286D kinase) was examined. alpha T286A kinase was not converted to a Ca(2+)-independent form, and alpha T286D kinase had Ca(2+)-independent activity significantly as an autophosphorylated kinase. Cells expressing alpha T286D kinase had much longer neurites than Nb2a/alpha cells, whereas cells with alpha T286A kinase did not form neurites. These results indicated that the Ca(2+)-independent form of CaM kinase II autophosphorylated at Thr-286 is involved in neurite outgrowth.

Docosahexaenoic acid decreases phospholipase A2 activity in the neurites/nerve growth cones of PC12 cells.

Docosahexaenoic acid (DHA) accumulates in nerve growth cones (NGC) during perinatal development and it is neuroprotective in ischemia. Because the phospholipases A2 (PLA2) are present in NGC and these enzymes function in both ischemia and long-term potentiation, the relationship between DHA and PLA2 was investigated in the NGC of nerve growth factor-differentiated PC12 cells. When PC12 cells were incubated with [3H]DHA, it primarily esterified in ethanolamine glycerolipids and concentrated initially in cell bodies with similar levels present in the neurite/nerve growth cone (N/NGC) fraction after 4 days. PLA2 activity in the N/NGC fraction was investigated using [14C]arachidonic acid-labeled phosphatidylinositol ([14C-AA]PI) as substrate. Heat denaturation and pharmacological inhibition showed that much of the PLA2 activity was calcium-independent and secretory rather than cytosolic. Supplementing the media with as little as 33 nM DHA significantly reduced PLA2 activity in the N/NGC fraction.

Inhibition of protein tyrosine kinases impairs axon extension in the embryonic optic tract.

The role of protein tyrosine kinase (PTK) activity in the development of the retinal projection was examined in vivo by applying inhibitors of cytoplasmic PTKs, herbimycin A and lavendustin A, to intact brain preparations of Xenopus embryos. The inhibitors were present during the period when retinal ganglion cell axons first navigate through the optic tract to reach their target, the optic tectum. A majority of inhibitor-treated retinal axons stalled at the beginning of the optic tract, leading to an 80% reduction in projection length at the highest doses. All inhibitor-treated axons that did extend into the optic tract exhibited normal pathfinding behavior. Tyrosine kinase assays of inhibitor-treated brains demonstrated that at doses at which retinal axon extension was severely impaired, PTK activity, including that of src family proteins, was reduced by 50-60%. Consistent with the in vivo findings, PTK inhibitors reduced neurite outgrowth from cultured retinal neurons by 70-80%. This contrasts with the strong enhancement of outgrowth induced by the same inhibitors in cultured chick ciliary ganglion neurons and suggests that the mediation of outgrowth by PTK activity may vary in different neuronal types. Inhibitor-treated growth cones cultured on laminin were larger than normal, suggesting that tyrosine phosphorylation can modulate growth cone-substrate adhesive interactions. Our results in vivo and in vitro provide complementary evidence that retinal axon outgrowth is inhibited by pharmacological blockers of PTK activity and indicate that inhibitor-sensitive PTKs normally play a role in promoting retinal neurite extension.