Characterizing nerve growth factor–p75 interactions and small molecule inhibition using surface plasmon resonance spectroscopy

Nerve growth factor (NGF) is critical for the proliferation, differentiation, and survival of neurons through its binding to the p75^NTR and TrkA receptors. Dysregulation of NGF has been implicated in several pathologies, including neurodegeneration (i.e., Parkinson's and Alzheimer's diseases) and both inflammatory and neuropathic pain states. Therefore, small molecule inhibitors that block NGF–receptor interactions have significant therapeutic potential. Small molecule antagonists ALE-0540, PD90780, Ro 08-2750, and PQC 083 have all been reported to inhibit NGF from binding the TrkA receptor. Interestingly, the characterization of the ability of these molecules to block NGF–p75^NTR interactions has not been performed. In addition, the inhibitory action of these molecules has never been evaluated using surface plasmon resonance (SPR) spectroscopy, which has been proven to be highly useful in drug discovery applications. In the current study, we used SPR biosensors to characterize the binding of NGF to the p75^NTR receptor in addition to characterizing the inhibitory potential of the known NGF antagonists. The results of this study provide the first evaluation of the ability of these compounds to block NGF binding to p75^NTR receptor. In addition, only PD90780 was effective at inhibiting the interaction of NGF with p75^NTR, suggesting receptor selectivity between known NGF inhibitors.     

Haloperidol and atypical antipsychotics share a same action of decreasing P75(NTR) mRNA levels in PC12 cells

P75(NTR) is a common neurotrophin receptor which binds all neurotrophins with similar affinities and has been shown to be capable of mediating programmed cell death. In this study, we investigated effects of the antipsychotic drugs (APDs) haloperidol, clozapine, quetiapine, and risperidone on p75(NTR) mRNA levels in PC12 cells. Haloperidol is a prototype of typical APDs, and the other three drugs are atypical APDs, which are effective in reducing negative symptoms and cognitive deficits of schizophrenia, cause less side effects, and are more tolerable compared to haloperidol. PC12 cells were cultured with various concentrations of haloperidol, clozapine, quetiapine, or risperidone, in their media. After culture for 48h, the cell viabilities and p75(NTR) mRNA levels were measured. It was shown that both haloperidol and the atypical APDs used in this study deceased p75(NTR) mRNA levels in PC12 cells in a dose dependent manner, while not affecting cell viabilities. In further experiments, doses that produced significant/greatest effects were chosen and provided in the culture media for various periods. Decreases in p75(NTR) mRNA levels were observed in cultures treated for 12h with quetiapine, 24h with clozapine or risperidone, or for 48h with haloperidol. These results suggest that both haloperidol and atypical APDs have the same action of decreasing p75(NTR) mRNA levels in PC12 cells. Although the underlying molecular mechanism of this action remains to be elucidated, this finding is particularly relevant given the neurodevelopmental deficits associated with schizophrenia and important roles of p75(NTR) in mediating cell death.     

The p75(NTR) tumor suppressor induces cell cycle arrest facilitating caspase mediated apoptosis in prostate tumor cells

The p75 neurotrophin receptor (p75(NTR)) is a death receptor which belongs to the tumor necrosis factor receptor super-family of membrane proteins. This study shows that p75(NTR) retarded cell cycle progression by induced accumulation of cells in G0/G1 and a reduction in the S phase of the cell cycle. The rescue of tumor cells from cell cycle progression by a death domain deleted (DeltaDD) dominant-negative antagonist of p75(NTR) showed that the death domain transduced anti-proliferative activity in a ligand-independent manner. Conversely, addition of NGF ligand rescued retardation of cell cycle progression with commensurate changes in components of the cyclin/cdk holoenzyme complex. In the absence of ligand, p75(NTR)-dependent cell cycle arrest facilitated an increase in apoptotic nuclear fragmentation of the prostate cancer cells. Apoptosis of p75(NTR) expressing cells occurred via the intrinsic mitochondrial pathway leading to a sequential caspase-9 and -7 cascade. Since the death domain deleted dominant-negative antagonist of p75(NTR) rescued intrinsic caspase associated apoptosis in PC-3 cells, this shows p75(NTR) was integral to ligand independent induction of apoptosis. Moreover, the ability of ligand to ameliorate the p75(NTR)-dependent intrinsic apoptotic cascade indicates that NGF functioned as a survival factor for p75(NTR) expressing prostate cancer cells.     

p53 is important for the anti-proliferative effect of ibuprofen in colon carcinoma cells

S-ibuprofen which inhibits the cyclooxygenase-1/-2 and R-ibuprofen which shows no COX-inhibition at therapeutic concentrations have anti-carcinogenic effects in human colon cancer cells; however, the molecular mechanisms for these effects are still unknown. Using HCT-116 colon carcinoma cell lines, expressing either the wild-type form of p53 (HCT-116 p53^wt) or being p(HCT-116 p53^−/−), we demonstrated that both induction of a cell cycle block and apoptosis after S- and R-ibuprofen treatment is in part dependent on p53. Also in the in vivo nude mice model HCT-116 p53^−/− xenografts were less sensitive for S- and R-ibuprofen treatment than HCT-116 p53^wt cells. Furthermore, results indicate that induction of apoptosis in HCT-116 p53^wt cells after ibuprofen treatment is in part dependent on a signalling pathway including the neutrophin receptor p75^NTR, p53 and Bax.     

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75^NTR), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75^NTR and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75^NTR and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75^NTR/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75^NTR or TrkA. Interestingly, immunoreactivity to anti-p75^NTR antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75^NTR, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75^NTR is turned on.     

Rho activation patterns after spinal cord injury and the role of activated Rho in apoptosis in the central nervous system

Growth inhibitory proteins in the central nervous system (CNS) block axon growth and regeneration by signaling to Rho, an intracellular GTPase. It is not known how CNS trauma affects the expression and activation of RhoA. Here we detect GTP-bound RhoA in spinal cord homogenates and report that spinal cord injury (SCI) in both rats and mice activates RhoA over 10-fold in the absence of changes in RhoA expression. In situ Rho-GTP detection revealed that both neurons and glial cells showed Rho activation at SCI lesion sites. Application of a Rho antagonist (C3-05) reversed Rho activation and reduced the number of TUNEL-labeled cells by approximately 50% in both injured mouse and rat, showing a role for activated Rho in cell death after CNS injury. Next, we examined the role of the p75 neurotrophin receptor (p75NTR) in Rho signaling. After SCI, an up-regulation of p75NTR was detected by Western blot and observed in both neurons and glia. Treatment with C3-05 blocked the increase in p75NTR expression. Experiments with p75NTR-null mutant mice showed that immediate Rho activation after SCI is p75NTR dependent. Our results indicate that blocking overactivation of Rho after SCI protects cells from p75NTR-dependent apoptosis.     

The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells

The densely ciliated granule cell layer of the adult murine hippocampal dentate gyrus is one of two sites of adult neurogenesis. The granule cells have already been proven to localize their SSTR3 (somatostatin receptor 3) receptors to their so-called primary cilia. Here we show for the first time that 70-90% of these cells in 7-18 months-old wild-type and 3×Tg-AD (Alzheimer disease transgenic) mice also load p75^NTR receptors into the structures containing SSTR3, i.e., their primary cilia. On the other hand, p75^NTR’s TrkA co-receptors were not localized to cilia but conventionally distributed throughout the cell surface. Significantly fewer cells (20-40%) in the hippocampal CA1 and CA3 regions and cerebral cortex have p75^NTR containing cilia. While we don’t know what the impact of the cilial localization of p75^NTR on dentate gyral adult neurogenesis and memory encoding might be, the cilia’s amyloid β-activatable p75^NTR receptors could be damaging or lethal to the hippocampal functioning of amyloid β-accumulating Alzheimer brain.     

Identification of the dipeptidyl aminopeptidase responsible for N-terminal clipping of recombinant Exendin-4 precursor expressed in Pichia pastoris

Exendin-4 is a naturally occurring 39 amino acid peptide that is useful for the control of Type 2 diabetes. Recombinant Exendin-4, with an extra glycine at the carboxy-terminus (Exdgly), was expressed in the methylotropic yeast Pichia pastoris. A high proportion of the Exdgly molecules secreted into medium were found to be clipped, lacking the first two amino acids (His–Gly) from the N-terminus. Disruption of the P. pastoris homolog of the Saccharomyces cerevisiae dipeptidyl aminopeptidase (STE13) gene in Pichia genome resulted in a clone that expressed N-terminally intact Exdgly. Elimination of N-terminal clipping enhanced the yield and simplified the purification of Exdgly from P. pastoris culture supernatant.     

Functional inhibition of the p75 receptor using a small interfering RNA

The neurotrophin receptor p75(NTR) mediates a wide variety of biological effects. Consistent with the function in controlling the survival and neurite formation, p75(NTR) is expressed during the developmental stages of the nervous system. Importantly, p75(NTR) is re-expressed in various pathological conditions and is suggested to contribute to the inhibition of neuronal regeneration and the death of the neurons. Here we develop a tool to knock down the expression of p75(NTR) by employing a small interfering RNA (siRNA). The siRNA for p75(NTR) effectively reduces the expression of endogenous p75(NTR) both in Schwann cells and dorsal root ganglion neurons in vitro. NGF-induced cell death in Schwann cells and the neurite retraction in DRG neurons induced by myelin-associated glycoprotein are attenuated by the siRNA. Inhibition of p75(NTR) in specific pathological conditions by the siRNA may provide a potential therapeutic agent.     

Modulation of Fas-induced apoptosis by p75 neurotrophin receptor in a human neuroblastoma cell line

Fas and p75 neurotrophin receptors (p75^NTR) are death receptors that alone induce apoptosis of SH-SY5Y neuroblastoma cell line respectively by Fas ligand or brain-derived neurotrophic factor (BDNF, a p75^NTR ligand). We report on the modulation of Fas-mediated apoptosis by concomitant p75^NTR activation. The exposure to both ligands suppressed the apoptotic effect. A co-localisation of Fas and p75^NTR receptors was evidenced by co-capping and immunoprecipitation assays. Moreover, a caspase-8 inhibitor suppressed the protective effect of the concomitant BDNF and Fas ligand stimulation, suggesting that p75^NTR and Fas receptors could share common signalling pathways.     

Effect of p75NTR on the regulation of naturally occurring cell death and retinal ganglion cell number in the mouse eye

Neurotrophins induce neural cell survival and differentiation during retinal development and regeneration through the high-affinity tyrosine kinase (Trk) receptors. On the other hand, nerve growth factor (NGF) binding to the low-affinity neurotrophin receptor p75 (p75(NTR)) might induce programmed cell death (PCD) in the early phase of retinal development. In the present study, we examined the retinal cell types that experience p75(NTR)-induced PCD and identify them to be postmitotic retinal ganglion cells (RGCs). However, retinal morphology, RGC number, and BrdU-positive cell number in p75(NTR) knockout (KO) mouse were normal after embryonic day 15 (E15). In chick retina, migratory RGCs express p75(NTR), whereas layered RGCs express the high-affinity NGF receptor TrkA, which may switch the pro-apoptotic signaling of p75(NTR) into a neurotrophic one. In contrast to the chick model, migratory RGCs express TrkA, while stratified RGCs express p75(NTR) in mouse retina. However, RGC number in TrkA KO mouse was also normal at birth. We next examined the expression of transforming growth factor beta (TGFbeta) receptor, which modulates chick RGC number in combination with p75(NTR), but was absent in mouse RGCs. p75(NTR) and TrkA seem to be involved in the regulation of mouse RGC number in the early phase of retinal development, but the number may be later adjusted by other molecules. These results suggest the different mechanism of RGC number control between mouse and chick retina.     

P75NTR在绒山羊毛囊生长周期中的表达

实验旨在研究绒山羊（Capra hircus）毛囊生长相关基因的表达规律,为绒山羊分子育种提供参考。本实验采用RTPCR、组织免疫荧光、Western blot等方法,研究神经营养素受体P75NTR在辽宁绒山羊皮肤组织中的表达和分布情况。结果表明：在毛囊生长周期的三个时期,均监测到P75NTR mRNA及蛋白的存在,P75NTR的荧光信号在退行期要强于其他两个时期,且在退行期毛囊外根鞘细胞中检测到P75NTR的高表达。以上结果表明,P75NTRmRNA及蛋白的表达与毛囊生长周期变化有一定的相关性,P75NTR受体在绒山羊的毛囊周期性变化中发挥着重要的功能。     

Capsaicin inhibits intestinal Cl- secretion and promotes Na+ absorption by blocking TRPV4 channels in healthy and colitic mice

Although capsaicin has been studied extensively as an activator of the transient receptor potential vanilloid cation channel subtype 1 (TRPV1) channels in sensory neurons, little is known about its TRPV1-independent actions in gastrointestinal health and disease. Here, we aimed to investigate the pharmacological actions of capsaicin as a food additive and medication on intestinal ion transporters in mouse models of ulcerative colitis (UC). The short-circuit current (I_sc) of the intestine from WT, TRPV1-, and TRPV4-KO mice were measured in Ussing chambers, and Ca²⁺ imaging was performed on small intestinal epithelial cells. We also performed Western blots, immunohistochemistry, and immunofluorescence on intestinal epithelial cells and on intestinal tissues following UC induction with dextran sodium sulfate. We found that capsaicin did not affect basal intestinal I_sc but significantly inhibited carbachol- and caffeine-induced intestinal I_sc in WT mice. Capsaicin similarly inhibited the intestinal I_sc in TRPV1 KO mice, but this inhibition was absent in TRPV4 KO mice. We also determined that Ca²⁺ influx via TRPV4 was required for cholinergic signaling–mediated intestinal anion secretion, which was inhibited by capsaicin. Moreover, the glucose-induced jejunal I_scvia Na⁺/glucose cotransporter was suppressed by TRPV4 activation, which could be relieved by capsaicin. Capsaicin also stimulated ouabain- and amiloride-sensitive colonic I_sc. Finally, we found that dietary capsaicin ameliorated the UC phenotype, suppressed hyperaction of TRPV4 channels, and rescued the reduced ouabain- and amiloride-sensitive I_sc. We therefore conclude that capsaicin inhibits intestinal Cl^- secretion and promotes Na⁺ absorption predominantly by blocking TRPV4 channels to exert its beneficial anti-colitic action.     

Pro-domain in precursor nerve growth factor mediates cell death

Nerve growth factor (NGF) is synthesized as a precursor, proNGF that undergoes post-translational processing to generate the biologically active mature NGF. While the neurotrophic function of NGF is well established, the activity of the proNGF precursor is still unclear. In this study, we have cloned the pro-domain of the precursor NGF molecule and have elucidated its function. We have used both mature and the furin resistant pro((R/G))NGF as controls in our experiments. Both pro((R/G))NGF and mature NGF (NGF) exhibited neurotrophic activity on PC12 cells while the pro-domain itself promoted cell death. The pro-domain, has been found to mediate apoptosis possibly by promoting the formation of a signaling complex comprising of endogenous p75(NTR) receptor, Bim/Bcl2 group of proteins and JNK and MEK1/2 signaling pathways.     

Exendin-4 inhibits structural remodeling and improves Ca2+ homeostasis in rats with heart failure via the GLP-1 receptor through the eNOS/cGMP/PKG pathway

The glucagon-like peptide-1 receptor (GLP-1R) agonist exendin-4 is a long-acting analog of GLP-1, which stimulates insulin secretion and is clinically used in the treatment of type 2 diabetes. Previous studies have demonstrated that GLP-1 agonists and analogs serve as cardioprotective factors in various conditions. Disturbances in calcium cycling are characteristic of heart failure (HF); therefore, the aim of this study was to investigate the effect of exendin-4 (a GLP-1 mimetic) on the regulation of calcium handling and to identify the underlying mechanisms in an HF rat model after myocardial infarction (MI). Rats underwent surgical ligation of the left anterior descending coronary artery or sham surgery prior to infusion with vehicle, exendin-4, or exendin-4 and exendin9-39 for 4 weeks. Exendin-4 treatment decreased MI size, suppressed chamber dilation, myocyte hypertrophy, and fibrosis and improved in vivo heart function in the rats subjected to MI. Exendin-4 resulted in an increase in circulating GLP-1 and GLP-1R in ventricular tissues. Additionally, exendin-4 activated the eNOS/cGMP/PKG signaling pathway and inhibited the Ca²⁺/calmodulin-dependent kinase II (CaMKII) pathways. Myocytes isolated from exendin-4-treated hearts displayed higher Ca²⁺ transients, higher sarcoplasmic reticulum Ca²⁺ content, and higher l-type Ca²⁺ current densities than MI hearts. Exendin-4 treatment restored the protein expression of sarcoplasmic reticulum Ca²⁺ uptake ATPase (SERCA2a), phosphorylated phospholamban (PLB) and Cav1.2 and decreased the levels of phosphorylated ryanodine receptor (RyR). Moreover, the favorable effects of exendin-4 were significantly inhibited by exendin9-39 (a GLP-1 receptor antagonist). Exendin-4 treatment of an HF rat model after MI inhibited cardiac and cardiomyocytes progressive remodeling. In addition, Ca²⁺ handling and its molecular modulation were also improved by exendin-4 treatment. The beneficial effects of exendin-4 on cardiac remodeling may be mediated through activation of the eNOS/cGMP/PKG pathway.     

Expression profiles of p53 and p66shc during oxidative stress-induced senescence in fetal bovine fibroblasts

Somatic cells undergo a permanent cell cycle arrest, called cellular senescence, after a limited number of cell divisions in vitro. Both the tumor suppressor protein p53 and the stress-response protein p66(shc) are suggested to regulate the molecular events associated with senescence. This study was undertaken to investigate the effect of different oxygen tensions and oxidative stress on cell longevity and to establish the role of p53 and p66(shc) in cells undergoing senescence. As a model of cellular senescence, primary fetal bovine fibroblasts were cultured in either 20% O(2) or 5% O(2) atmospheres until senescence was reached. Fibroblasts cultured under 20% O(2) tension underwent senescence after 30 population doublings (PD), whereas fibroblasts cultured under 5% O(2) tension did not exhibit signs of senescence. Oxidative stress, as measured by protein carbonyl content, was significantly elevated in senescent cells compared to their younger counterparts and to fibroblasts cultured under 5% O(2) at the same PD. p53 mRNA gradually decreased in 20% O(2) cultured fibroblasts until senescence was reached, whereas p53 protein levels were significantly increased as well as p53 phosphorylation on serine 20, suggesting that p53 might be stabilized by posttranslational modifications during senescence. Senescence was also associated with high levels of p66(shc) mRNA and protein levels, while the levels remained low and stable in dividing fibroblasts under 5% O(2) atmosphere. Taken together, our results show an effect of oxidative stress on the replicative life span of fetal bovine fibroblasts as well as an involvement of p53, serine 20-p53 phosphorylation and p66(shc) in senescence.