Potentiated Neurite Outgrowth Research Articles

Bacopa monnieri protects neuronal cell line and Caenorhabditis elegans models of Alzheimer’s disease through sigma-1 receptor antagonist sensitive and antioxidant pathways

BACKGROUND: Due to better health care and improved nutritional status of the world’s population, many people live into old age. This has resulted in more diseases related to aging, such as neurodegenerative diseases. Bacopa monnieri (BM) is a medicinal herb found in Southeast Asia and is a popular memory-enhancing supplement. OBJECTIVE: This study aimed to investigate how BM may provide protection in neurodegenerative disease, and whether the sigma-1 receptor is involved. METHODS: PC-12 cells were differentiated with the addition of nerve growth factor. The potentiation by BM of PC-12 neurite growth was measured by counting the number of differentiated cells and by measuring their length. Differentiated PC-12 cells were also subjected to amyloid-β (Aβ) toxicity in the presence and absence of BM. The cell survival (MTT and cell counting) and neurite lengths were then measured as indicators of cellular health. Total protein was extracted from control and treated cells and expression of various signalling pathway molecules was assessed via western blotting. We also assessed the effects of BM on the lifespans of various mutant strains plus wild-type C. elegans. RESULTS: We show that BM can protect against Aβ toxicity in PC-12 cells. Furthermore, BM can potentiate neurite outgrowth in PC-12, in a sigma-1 receptor antagonist sensitive fashion, and Neuro2A cell lines. BM induced a reduction in pAKT expression and upregulated BDNF expression in PC-12 cells. BM was also able to increase the lifespan and health-span of Aβ expressing C. elegans mutants via the DAF-16 mediated pathway. BM reduced oxidative stress in wild-type C. elegans exposed to UV-A with pre-exposure and post-exposure treatments. CONCLUSIONS: This all further identifies BM as a potential agent to treat neurodegenerative diseases, by modulating different pathways.

Insulin stimulates atypical protein kinase C-mediated phosphorylation of the neuronal adaptor FE65 to potentiate neurite outgrowth by activating ARF6-Rac1 signaling.

Neurite outgrowth is a fundamental process in neurons that produces extensions and, consequently, neural connectivity. Neurite damage and atrophy are observed in various brain injuries and disorders. Understanding the intrinsic pathways of neurite outgrowth is essential for developing strategies to stimulate neurite regeneration. Insulin is a pivotal hormone in the regulation of glucose homeostasis. There is increasing evidence for the neurotrophic functions of insulin, including the induction of neurite outgrowth. However, the associated mechanism remains elusive. Here, we demonstrate that insulin potentiates neurite outgrowth mediated by the small GTPases ADP-ribosylation factor 6 (ARF6) and Ras-related C3 botulinum toxin substrate 1 (Rac1) through the neuronal adaptor FE65. Moreover, insulin enhances atypical protein kinase Cι/λ (PKCι/λ) activation and FE65 phosphorylation at serine 459 (S459) in neurons and mouse brains. In vitro and cellular assays show that PKCι/λ phosphorylated FE65 at S459. Consistently, insulin potentiates FE65 S459 phosphorylation only in the presence of PKCι/λ. Phosphomimetic studies show that an FE65 S459E mutant potently activates ARF6, Rac1, and neurite outgrowth. Notably, this phosphomimetic mutation enhances the FE65-ARF6 interaction, a process that promotes ARF6-Rac1-mediated neurite outgrowth. Likewise, insulin treatment and PKCι/λ overexpression potentiate the FE65-ARF6 interaction. Conversely, PKCι/λ knockdown suppresses the stimulatory effect of FE65 on ARF6-Rac1-mediated neurite outgrowth. The effect of insulin on neurite outgrowth is also markedly attenuated in PKCι/λ knockdown neurons, in the presence and absence of FE65. Our findings reveal a novel mechanism linking insulin with ARF6-Rac1-dependent neurite extension through the PKCι/λ-mediated phosphorylation of FE65.

Botanical Drug Puerarin Promotes Neuronal Survival and Neurite Outgrowth against MPTP/MPP+-Induced Toxicity via Progesterone Receptor Signaling.

Background Progesterone receptor (PR) modulates neuroprotective and regenerative responses in Parkinson's disease and related neurological diseases. Objectives The present study was designed to determine whether botanical drug puerarin could exhibit neuroprotective and neurorestorative activities via PR signaling. Methods The neuroprotective and neurotrophic activities of puerarin were investigated in MPTP-lesioned mice and MPP+-challenged primary rat midbrain neurons. Rotarod performance test and tail suspension test were used to assess motor functions. Tyrosine hydroxylase (TH) and PR were determined by immunostaining, Western blotting, and luciferase reporter assays. Neurite outgrowth was assessed by fluorescence staining and immunostaining. Results Puerarin effectively ameliorated the MPTP-induced motor abnormalities in MPTP-lesioned mice and protected primary rat midbrain neurons against MPP+-induced toxicity via PR signaling although progesterone exhibited the neuroprotection. PR antagonist mifepristone (RU486) diminished the neuroprotection of puerarin in MPTP-lesioned mice and MPP+-induced primary rat midbrain neurons. Moreover, puerarin promoted the differentiation of primary rat midbrain neurons and potentiated NGF to induce neuritogenesis in PC12 cells. RU486 and PR-siRNA could inhibit the effect of puerarin. Puerarin and progesterone could enhance the PR promoter. Conclusion Puerarin attenuated MPTP- and MPP+-induced toxicity and potentiated neurite outgrowth via PR. These results suggested that puerarin may become an alternative hormone for suppressing MPTP- and MPP+-induced toxicity in neurodegenerative diseases.

Dipentylammonium Binds to the Sigma-1 Receptor and Protects Against Glutamate Toxicity, Attenuates Dopamine Toxicity and Potentiates Neurite Outgrowth in Various Cultured Cell Lines.

Alzheimer's disease is a neurodegenerative disease that affects 44 million people worldwide, costing the world $605 billion to care for those affected not taking into account the physical and psychological costs for those who care for Alzheimer's patients. Dipentylammonium is a simple amine, which is structurally similar to a number of other identified sigma-1 receptor ligands with high affinities such as (2R-trans)-2butyl-5-heptylpyrrolidine, stearylamine and dodecylamine. This study investigates whether dipentylammonium is able to provide neuroprotective effects similar to those of sigma-1 receptor agonists such as PRE-084. Here we identify dipentylammonium as a sigma-1 receptor ligand with nanomolar affinity. We have found that micromolar concentrations of dipentylammonium protect from glutamate toxicity and prevent NFκB activation in HT-22 cells. Micromolar concentrations of dipentylammonium also protect stably expressing amyloid precursor protein Swedish mutant (APP/Swe) Neuro2A cells from toxicity induced by 150μM dopamine, suggesting that dipentylammonium may be useful for the treatment of Parkinsonian symptoms in Alzheimer's patients which are often associated with a more rapid deterioration of cognitive and physical ability. Finally, we found that low micromolar concentrations of dipentylammonium could out preform known sigma-1 receptor agonist PRE-084 in potentiating neurite outgrowth in Neuro2A cells, further suggesting that dipentylammonium has a potential use in the treatment of neurodegenerative diseases and could be acting through the sigma-1 receptor.

Chemical Constituents from Hericium erinaceus Promote Neuronal Survival and Potentiate Neurite Outgrowth via the TrkA/Erk1/2 Pathway.

Hericium erinaceus is a culinary-medicinal mushroom used traditionally in Eastern Asia to improve memory. In this work, we investigated the neuroprotective and neuritogenic effects of the secondary metabolites isolated from the MeOH extract of cultured mycelium of H. erinaceus and the primary mechanisms involved. One new dihydropyridine compound (6) and one new natural product (2) together with five known compounds (1,3–5,7) were obtained and their structures were elucidated by spectroscopic analysis, including 2D NMR and HRMS. The cell-based screening for bioactivity showed that 4-chloro-3,5-dimethoxybenzoic methyl ester (1) and a cyathane diterpenoid, erincine A (3), not only potentiated NGF-induced neurite outgrowth but also protected neuronally-differentiated cells against deprivation of NGF in PC12 pheochromocytoma cells. Additionally, compound 3 induced neuritogenesis in primary rat cortex neurons. Furthermore, our results revealed that TrkA-mediated and Erk1/2-dependant pathways could be involved in 1 and 3-promoted NGF-induced neurite outgrowth in PC12 cells.

Microtopographical cues promote peripheral nerve regeneration via transient mTORC2 activation.

Peripheral nerve injury is common and functionally devastating. Despite microsurgical repair, healing is slow and incomplete, with lasting functional deficit. There is a clear need to translate bioengineering approaches and increase our knowledge of the molecular processes controlling nerve regeneration to improve the rate and success of healing. Topographical cues are powerful determinants of neurite outgrowth and represent a highly translatable engineering strategy. Here we demonstrate, for the first time, that microtopography potentiates neurite outgrowth via the mTOR pathway, with the mTORC2 subtype being of particular importance. These results give further evidence for the incorporation of microtopographical cues into peripheral nerve regeneration conduits and indicate that mTORC2 may be a suitable therapeutic target to potentiate nerve regeneration.

Medicinal Chemistry of σ1 Receptor Ligands: Pharmacophore Models, Synthesis, Structure Affinity Relationships, and Pharmacological Applications.

In the first part of this chapter, we summarize the various pharmacophore models for σ1 receptor ligands. Common to all of them is a basic amine flanked by two hydrophobic regions, representing the pharmacophoric elements. The development of computer-based models like the 3D homology model is described as well as the first crystal structure of the σ1 receptor. The second part focuses on the synthesis and biological properties of different σ1 receptor ligands, identified as 1-9. Monocyclic piperazines 1 and bicyclic piperazines 2 and 3 were developed as cytotoxic compounds, thus the IC50 values of cell growth and survival inhibition studies are given for all derivatives. The mechanism of cell survival inhibition, induction of time-dependent apoptosis, of compound ent-2a is discussed. Experimentally determined σ1 affinity shows good correlation with the results from molecular dynamics simulations based on a 3D homology model. Spirocyclic compounds 4 and 5 represent well-established σ1 receptor ligands. The homologous fluoroalkyl derivatives 4 have favorable pharmacological properties for use as fluorinated PET tracers. The (S)-configured fluoroethyl substituted compound (S)-4b is under investigation as PET tracer for imaging of σ1 receptors in the brain of patients affected by major depression. 1,3-Dioxanes 6c and 6d display a very potent σ1 antagonist profile and the racemic 1,3-dioxane 6c has high anti-allodynic activity at low doses. The arylpropenylamines 7 are very potent σ1 receptor ligands with high σ1/σ2 selectivity. The top compound 7g acts as an agonist as defined by its ability to potentiate neurite outgrowth at low concentrations. Among the morpholinoethoxypyrazoles 8, 8c (known as S1RA) reveals the most promising pharmacokinetic and physicochemical properties. Due to its good safety profile, 8c is currently being investigated in a phase II clinical trial for the treatment of neuropathic pain. The most potent ligand 9e of 3,4-dihydro-2(1H)-quinolones 9 shows promising anti-nociceptive activity in the formalin test.

Pulsed electromagnetic fields potentiate neurite outgrowth in the dopaminergic MN9D cell line

Pulsed electromagnetic fields (PEMF) exert biological effects and are in clinical use to facilitate bone repair and wound healing. Research has demonstrated that PEMF can induce signaling molecules and growth factors, molecules that play important roles in neuronal differentiation. Here, we tested the effects of a low-amplitude, nonthermal, pulsed radiofrequency signal on morphological neuronal differentiation in MN9D, a dopaminergic cell line. Cells were plated in medium with 10% fetal calf serum. After 1 day, medium was replaced with serum-containing medium, serum-free medium, or medium supplemented with dibutyryl cyclic adenosine monophosphate (Bt2 cAMP), a cAMP analog known to induce neurite outgrowth. Cultures were divided into groups and treated with PEMF signals for either 30 min per day or continuously for 15 min every hour for 3 days. Both serum withdrawal and Bt2 cAMP significantly increased neurite length. PEMF treatment similarly increased neurite length under both serum-free and serum-supplemented conditions, although to a lesser degree in the presence of serum, when continuous treatments had greater effects. PEMF signals also increased cell body width, indicating neuronal maturation, and decreased protein content, suggesting that this treatment was antimitotic, an effect reversed by the inhibitor of cAMP formation dideoxyadenosine. Bt2 cAMP and PEMF effects were not additive, suggesting that neurite elongation was achieved through a common pathway. PEMF signals increased cAMP levels from 3 to 5 hr after treatment, supporting this mechanism of action. Although neuritogenesis is considered a developmental process, it may also represent the plasticity required to form and maintain synaptic connections throughout life.

Interaction of new antidepressants with sigma-1 receptor chaperones and their potentiation of neurite outgrowth in PC12 cells

The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [(3)H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect.

Interleukin-17A Increases Neurite Outgrowth from Adult Postganglionic Sympathetic Neurons

Inflammation can profoundly alter the structure and function of the nervous system. Interleukin (IL)-17 has been implicated in the pathogenesis of several inflammatory diseases associated with nervous system plasticity. However, the effects of IL-17 on the nervous system remain unexplored. Cell and explant culture techniques, immunohistochemistry, electrophysiology, and Ca2+ imaging were used to examine the impact of IL-17 on adult mouse sympathetic neurons. Receptors for IL-17 were present on postganglionic neurons from superior mesenteric ganglia (SMG). Supernatant from activated splenic T lymphocytes, which was abundant in IL-17, dramatically enhanced axonal length of SMG neurons. Importantly, IL-17-neutralizing antiserum abrogated the neurotrophic effect of splenocyte supernatant, and incubation of SMG neurons in IL-17 (1 ng/ml) significantly potentiated neurite outgrowth. The neurotrophic effect of IL-17 was accompanied by inhibition of voltage-dependent Ca2+ influx and was recapitulated by incubation of neurons in a blocker of N-type Ca2+ channels (ω-conotoxin GVIA; 30 nM). IL-17-induced neurite outgrowth in vitro appeared to be independent of glia, as treatment with a glial toxin (AraC; 5 μM) did not affect the outgrowth response to IL-17. Moreover, application of the cytokine to distal axons devoid of glial processes enhanced neurite extension. An inhibitor of the NF-κB pathway (SC-514; 20 μM) blocked the effects of IL-17. These data represent the first evidence that IL-17 can act on sympathetic somata and distal neurites to enhance neurite outgrowth, and identify a novel potential role for IL-17 in the neuroanatomical plasticity that accompanies inflammation.

Total Synthesis of (±)-4,5-Bis-epi-Neovibsanin A and B: A Neurite Outgrowth Comparison Study

(+/-)-4,5-Bis-epi-neovibsanin A and B were synthesized in 12 steps. The acid-catalyzed, one-pot, five-step cascade reaction was central toward the formation of the tricyclic core. The two diastereomers of natural neovibsanin A and B acted as desirable derivatives for structure-activity relationship studies to probe neurotrophic activity. Both (+/-)-4,5-bis-epi-neovibsanin A and B strongly potentiate neurite outgrowth in NGF-stimulated PC12 cells. Furthermore, the tricyclic core appears to be largely responsible for promoting a biological response.

Liquiritin potentiate neurite outgrowth induced by nerve growth factor in PC12 cells

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.

Donepezil Potentiates Nerve Growth Factor-Induced Neurite Outgrowth in PC12 Cells

Donepezil is a potent and selective acetylcholinesterase inhibitor developed for the treatment of Alzheimer’s disease. To elucidate whether donepezil causes neuronal differentiation, we examined its effect on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. Donepezil (10 µM) significantly potentiated the neurite outgrowth evoked by low (1 ng/ml) and high (50 ng/ml) concentrations of NGF. The effect of donepezil (1 – 10 µM) was concentration-dependent. The enhancement of neurite outgrowth caused by donepezil was not blocked by the acetylcholine receptor (AChR) antagonists mecamylamine and scopolamine. Furthermore, the AChR agonists nicotine and carbachol did not affect the neurite outgrowth induced by NGF. Donepezil (10 µM) also significantly potentiated neurite outgrowth evoked by dibutyryl cyclic AMP. Moreover, donepezil potentiated the NGF-induced phosphorylation of extracellular signal-regulated kinase (ERK). These results suggest that donepezil potentiated neuronal differentiation by enhancing the activation of ERK.

Potentiation of neurite outgrowth and reduction of apoptosis by immunosuppressive agents: implications for neuronal injury and transplantation

Immunosuppressive agents are believed to play a role in recovery from spinal cord injury, but the underlying mechanisms by which neuronal function is improved by these agents are poorly understood. In this study, the authors evaluate the effect of immunosuppressive medications on neurite outgrowth and cell survival after a pharmacologically induced injury. Differentiated human neuroblastoma SH-SY5Y cells were injured using the calcium agonist thapsigargin. After cellular injury, neurite outgrowth in the presence or absence of immunosuppressive agents was measured. Apoptosis was quantified with the aid of a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay. Neurite outgrowth was severely restricted following thapsigargin injury. Outgrowth was potentiated, however, by the addition of concentrations of 1 and 10 mM cyclosporin A in a dose-dependent fashion. Similarly, addition of 10 nM FK506 increased the percentage of neurites in the 20- to 40-micron range. A low dose (1 mM) of dexamethasone did not have a significant effect on neurite outgrowth, but a higher dose (10 mM) increased the percentage of neurites in the 10- to 45-micron range. These agents also lessened the degree of thapsigargin-induced apoptosis. Immunosuppressive agents such as cyclosporin A, FK506, and dexamethasone can potentiate neurite outgrowth and protect against apoptotic cell death in a human postmitotic neuronal cell line. Such effects may have implications for lessening neuronal injury after neurotrauma, stroke, or neurodegeneration.

C.14.03 The long-term - maximising potential for rehabilitation

The sigma-1 receptor chaperone located in the endoplasmic reticulum (ER) may be implicated in the mechanistic action of some antidepressants. The present study was undertaken to examine whether new antidepressant drugs interact with the sigma-1 receptor chaperone. First, we examined the effects of selective serotonin reuptake inhibitors (SSRIs) (fluvoxamine, paroxetine, sertraline, citalopram and escitalopram), serotonin and noradrenaline reuptake inhibitors (SNRIs) (duloxetine, venlafaxine, milnacipran), and mirtazapine, a noradrenaline and specific serotonergic antidepressant (NaSSA), on [3H](+)-pentazocine binding to rat brain membranes. Then, we examined the effects of these drugs on nerve growth factor (NGF)-induced neurite outgrowth in PC12 cells. The order of potency for drugs at the sigma-1 receptor chaperone was as follows: fluvoxamine>sertraline>fluoxetine>escitalopram>citalopram>paroxetine>duoxetine. Venlafaxine, milnacipran, and mirtazapine showed very weak affinity for this chaperone. Furthermore, fluvoxamine, fluoxetine, escitalopram, and mirtazapine significantly potentiated NGF-induced neurite outgrowth in cell assays, and the effects of all these drugs, excluding mirtazapine, were antagonized by NE-100, a selective antagonist of the sigma-1 receptor chaperone. Moreover, the effects of fluvoxamine and fluoxetine on neurite outgrowth were also antagonized by sertraline, indicating that sertraline may be an antagonist at the sigma-1 receptor chaperone. The effect of mirtazapine on neurite outgrowth was antagonized by the selective 5-hydroxytryptamine1A receptor antagonist WAY-100635. These findings suggest that activation at the sigma-1 receptor chaperone may be involved in the action of some SSRIs, such as fluvoxamine, fluoxetine and escitalopram. In contrast, mirtazapine independently potentiated neurite outgrowth in PC12 cells, indicating that this beneficial effect may mediate its pharmacological effect.

Nicotinic Acid Adenine Dinucleotide Phosphate Potentiates Neurite Outgrowth

Ca(2+) regulates a spectrum of cellular processes including many aspects of neuronal function. Ca(2+)-sensitive events such as neurite extension and axonal guidance are driven by Ca(2+) signals that are precisely organized in both time and space. These complex cues result from both Ca(2+) influx across the plasma membrane and the mobilization of intracellular Ca(2+) stores. In the present study, using rat cortical neurons, we have examined the effects of the novel intracellular Ca(2+)-mobilizing messenger nicotinic acid adenine dinucleotide phosphate (NAADP) on neurite length and cytosolic Ca(2+) levels. We show that NAADP potentiates neurite extension in response to serum and nerve growth factor and stimulates increases in cytosolic Ca(2+) from bafilomycin-sensitive Ca(2+) stores. Simultaneous blockade of inositol trisphosphate and ryanodine receptors abolished the effects of NAADP on neurite length and reduced the magnitude of NAADP-mediated Ca(2+) signals. This is the first report demonstrating functional NAADP receptors in a mammalian neuron. Interplay between NAADP receptors and more established intracellular Ca(2+) channels may therefore play important signaling roles in the nervous system.

Neurotrophin-dependent Tyrosine Phosphorylation of Ras Guanine-releasing Factor 1 and Associated Neurite Outgrowth Is Dependent on the HIKE Domain of TrkA

Ras guanine-releasing factor 1 (RasGrf1), a guanine nucleotide exchange factor for members of the Ras and Rho family of GTPases, is highly expressed in the brain. It is regulated by two separate mechanisms, calcium regulation through interaction with its calcium/calmodulin-binding IQ domain and serine and tyrosine phosphorylation. RasGrf1 is activated downstream of G-protein-coupled receptors and the non-receptor tyrosine kinases, Src and Ack1. Previously, we demonstrated a novel interaction between the intracellular domain of the nerve growth factor-regulated TrkA receptor tyrosine kinase and an N-terminal fragment of RasGrf1. We now show that RasGrf1 is phosphorylated and interacts with TrkA, -B, and -C in co-transfection studies. This interaction and phosphorylation of RasGrf1 is dependent on the HIKE domain of TrkA (a region shown to interact with pleckstrin homology domains) but not on any of the phosphotyrosine residues that act as docking sites for intracellular signaling molecules such as Shc and FRS-2. The PH1 domain alone of RasGrf1 is sufficient for phosphorylation by the TrkA receptor. A potential role for Trk activation of RasGrf1 is suggested through transfection studies in PC12 cells in which RasGrf1 significantly increases neurite outgrowth at low doses of neurotrophin stimulation. Notably, this neurite outgrowth is dependent on an intact HIKE domain, as nnr5-S10 cells expressing a TrkA HIKE domain mutant do not exhibit potentiated neurite outgrowth in the presence of RasGrf1. These studies identify RasGrf1 as a novel target of neurotrophin activation and suggest an additional pathway whereby neurotrophin-stimulated neurite outgrowth may be regulated.

Isolation of human NURF: a regulator of Engrailed gene expression.

The modification of chromatin structure is an important regulatory mechanism for developmental gene expression. Differential expression of the mammalian ISWI genes, SNF2H and SNF2L, has suggested that they possess distinct developmental roles. Here we describe the purification and characterization of the first human SNF2L-containing complex. The subunit composition suggests that it represents the human ortholog of the Drosophila nucleosome-remodeling factor (NURF) complex. Human NURF (hNURF) is enriched in brain, and we demonstrate that it regulates human Engrailed, a homeodomain protein that regulates neuronal development in the mid-hindbrain. Furthermore, we show that hNURF potentiates neurite outgrowth in cell culture. Taken together, our data suggess a role for an ISWI complex in neuronal growth.

FK506 potentiates NGF‐induced neurite outgrowth via the Ras/Raf/MAP kinase pathway

Nerve growth factor (NGF) and other members of the neurotrophin family are critical for the survival and differentiation of neurons within the peripheral and central nervous systems. Neurophilin ligands, including FK506, potentiate NGF-induced neurite outgrowth in several experimental models, although the mechanism of this potentiation is unclear. Therefore, we tested which signaling pathways were involved in FK506-potentiated neurite outgrowth in SH-SY5Y neuroblastoma cells using specific pharmacological inhibitors of various signaling molecules. Inhibitors of Ras (lovastatin), Raf (GW5074), or MAP kinase (PD98059 and U0126) blocked FK506 activity, as did inhibitors of phospholipase C (U73122) and phosphatidylinositol 3' kinase (LY294002). Protein kinase C inhibitors (Go6983 and Ro31-8220) slightly but significantly inhibited neurite outgrowth, whereas inhibitors of p38 MAPK (SB203580) or c-Jun N-terminal kinase (SP600125) had no effect. These data suggest that FK506 potentiates neurite outgrowth through the Ras/Raf/MAP kinase signaling pathway downstream of phospholipase C and phosphatidylinositol 3' kinase.

Induction of neurites by the regulatory domains of PKCδ and ε is counteracted by PKC catalytic activity and by the RhoA pathway

We have shown that protein kinase C (PKC) ε, independently of its kinase activity, via its regulatory domain (RD), induces neurites in neuroblastoma cells. This study was designed to evaluate whether the same effect is obtained in nonmalignant neural cells and to dissect mechanisms mediating the effect. Overexpression of PKCε resulted in neurite induction in two immortalised neural cell lines (HiB5 and RN33B). Phorbol ester potentiated neurite outgrowth from PKCε-overexpressing cells and led to neurite induction in cells overexpressing PKCδ. The effects were potentiated by blocking the PKC catalytic activity with GF109203X. Furthermore, kinase-inactive PKCδ induced more neurites than the wild-type isoform. The isolated regulatory domains of novel PKC isoforms also induced neurites. Experiments with PKCδ-overexpressing HiB5 cells demonstrated that phorbol ester, even in the presence of a PKC inhibitor, led to a decrease in stress fibres, indicating an inactivation of RhoA. Active RhoA blocked PKC-induced neurite outgrowth, and inhibition of the RhoA effector ROCK led to neurite outgrowth. This demonstrates that neurite induction by the regulatory domain of PKCδ can be counteracted by PKCδ kinase activity, that PKC-induced neurite outgrowth is accompanied by stress fibre dismantling indicating an inactivation of RhoA, and that the RhoA pathway suppresses PKC-mediated neurite outgrowth.