Depolarization promotes survival of ciliary ganglion neurons by BDNF-dependent and -independent mechanisms

Membrane activity upregulates brain derived neurotrophic factor (BDNF) expression to coordinately support neuronal survival in many systems. In parasympathetic ciliary ganglion (CG) neurons, activity mimicked by KCl depolarization provides nearly full trophic support. While BDNF has been considered unable to influence CG neuronal survival, we now document its expression during CG development and show that low concentrations do support survival via high-affinity TrkB receptors. Furthermore, a contribution of BDNF to activity-induced trophic support was demonstrated by showing that KCl depolarization increased BDNF mRNA and protein in, and release of BDNF from, CG neuron cultures. Application of anti-BDNF blocking antibody or mitogen activated protein kinase (MAPK) kinase inhibitor, attenuated depolarization-supported survival, implicating canonical BDNF/TrkB signaling. Ca2+-Calmodulin kinase II (CaMKII) was also required since its inhibition combined with anti-BDNF or MAPK kinase inhibitor abolished or greatly reduced the trophic effects of depolarization. Membrane activity may thus support CG neuronal survival both by stimulating release of BDNF that binds high-affinity TrkB receptors to activate MAPK and by recruiting CaMKII. This mechanism could have relevance late in development in vivo as ganglionic transmission and the effectiveness of BDNF over other growth factors both increase.     

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.     

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.     

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.     

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.     

Characteristics of taurine release in slices from adult and developing mouse brain stem

Summary. Taurine has been thought to function as a regulator of neuronal activity, neuromodulator and osmoregulator. Moreover, it is essential for the development and survival of neural cells and protects them under cell-damaging conditions. Taurine is also involved in many vital functions regulated by the brain stem, including cardiovascular control and arterial blood pressure. The release of taurine has been studied both in vivo and in vitro in higher brain areas, whereas the mechanisms of release have not been systematically characterized in the brain stem. The properties of release of preloaded [³H]taurine were now characterized in slices prepared from the mouse brain stem from developing (7-day-old) and young adult (3-month-old) mice, using a superfusion system. In general, taurine release was found to be similar to that in other brain areas, consisting of both Ca²⁺-dependent and Ca²⁺-independent components. Moreover, the release was mediated by Na⁺-, Cl⁻-dependent transporters operating outwards, as both Na⁺-free and Cl⁻ -free conditions greatly enhanced it. Cl⁻ channel antagonists and a Cl⁻ transport inhibitor reduced the release at both ages, indicating that a part of the release occurs through ion channels. Protein kinases appeared not to be involved in taurine release in the brain stem, since substances affecting the activity of protein kinase C or tyrosine kinase had no significant effects. The release was modulated by cAMP second messenger systems and phospholipases at both ages. Furthermore, the metabotropic glutamate receptor agonists likewise suppressed the K⁺-stimulated release at both ages. In the immature brain stem, the ionotropic glutamate receptor agonists N-methyl-D-aspartate (NMDA) and 2-amino-3-hydroxy-5-methyl-4-isoxazolepropionate (AMPA) potentiated taurine release in a receptor-mediated manner. This could constitute an important mechanism against excitotoxicity, protecting the brain stem under cell-damaging conditions.     

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.     

Exendin-4 improves hepatocyte injury by decreasing proliferation through blocking NGF/TrkA in diabetic mice

The hepatocytes express nerve growth factor (NGF) and its high affinity receptor tyrosine kinase A (TrkA). However, the link between NGF/TrkA system and hepatocyte proliferation in diabetic animals and the effects of exendin-4, a glucagon like peptide-1 (GLP-1) receptor agonist, on this system are not known. BALB/c male mice were divided into four groups. The first group was given citrate buffer only, the second group was administered exendin-4 alone, the third group received streptozotocin (STZ), and the fourth group was given both STZ and exendin-4. Exendin-4 (3 μg/kg) was administered by subcutaneous injection daily for 30 days after the animals were rendered diabetic by administration of STZ (200 mg/kg). With treatment of exendin-4 to the diabetic mice the following results were noted (i) NGF, TrkA and proliferating cell nuclear antigen positive hepatocytes were decreased; (ii) p75 neurotrophin receptor and caspase-3 positive hepatocyte could not be detected; (iii) liver alanine transaminase and aspartate transaminase activities, lipid peroxidation, protein carbonyl and myeloperoxidase levels were decreased; (iv) liver catalase, superoxide dismutase, glutathione peroxidase activities and glutathione levels were increased. These data suggest that exendin-4 might exerts its anti-proliferative action through blocking NGF/TrkA system and stimulating oxidative defense system in liver of diabetic mice.     

Analysis of retrograde transport in motor neurons reveals common endocytic carriers for tetanus toxin and neurotrophin receptor p75NTR.

Axonal retrograde transport is essential for neuronal growth and survival. However, the nature and dynamics of the membrane compartments involved in this process are poorly characterized. To shed light on this pathway, we established an experimental system for the visualization and the quantitative study of retrograde transport in living motor neurons based on a fluorescent fragment of tetanus toxin (TeNT HC). Morphological and kinetic analysis of TeNT HC retrograde carriers reveals two major groups of organelles: round vesicles and fast tubular structures. TeNT HC carriers lack markers of the classical endocytic pathway and are not acidified during axonal transport. Importantly, TeNT HC and NGF share the same retrograde transport organelles, which are characterized by the presence of the neurotrophin receptor p75NTR. Our results provide the first direct visualization of retrograde transport in living motor neurons, and reveal a novel retrograde route that could be used both by physiological ligands (i.e., neurotrophins) and TeNT to enter the central nervous system.     

SORT1 Mutation Resulting in Sortilin Deficiency and p75NTR Upregulation in a Family With Essential Tremor

Essential tremor (ET) is the most prevalent movement disorder affecting millions of people in the United States. Although a positive family history is one of the most important risk factors for ET, the genetic causes of ET remain unknown. In this study, whole exome sequencing and subsequent approaches were performed in a family with an autosomal dominant form of early-onset ET. Functional analyses including mutagenesis, cell culture, gene expression, enzyme-linked immunosorbent, and apoptosis assays were also performed. A disease-segregating mutation (p.Gly171Ala), absent in normal population, was identified in the SORT1 gene. The p.Gly171Ala mutation was shown not only to impair the expression of its encoding protein sortilin but also the mRNA levels of its binding partner p75 neurotrophin receptor that is known to be implicated in brain injury, neuronal apoptosis, and neurotransmission.     

Establishing a cellular FRET-based fluorescence plate reader assay to monitor proNGF-induced cross-linking of sortilin and the neurotrophin receptor p75NTR

Whereas the proform of the nerve growth factor (proNGF) is crucial for eliminating superfluous cells during neuronal development it also promotes apoptosis following brain trauma and neuronal injury. The apoptotic signal is elicited upon formation of a trimeric receptor complex also containing the vps10p domain receptor sortilin and the neurotrophin receptor p75^NTR. However, proNGF-induced receptor complex formation has been difficult to directly assess other than by western blotting. We here describe a fluorescence resonance energy transfer (FRET) based fluorescence plate reader assay to monitor the interaction between fluorescently tagged sortilin and p75^NTR in live cells. The method is based on a standard fluorescent plate reader found in many biochemical laboratories and the results are evaluated using a microscopy-based quantified sensitized acceptor emission FRET approach making use of a pair of FRET standard constructs. As a result, the effect of proNGF on the interaction between sortilin and p75^NTR can be evaluated in live cells allowing for screening and selection of therapeutic compounds interfering with proNGF-induced 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.     

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.     

Myelin-associated glycoprotein-mediated signaling in central nervous system pathophysiology

The myelin-associated glycoprotein (MAG) is a type I membrane-spanning protein expressed exclusively in oligoden drocytes and Schwann cells. It has two generally known pathophysiological roles in the central nervous system (CNS): maintenance of myelin integrity and inhibition of CNS axonal regeneration. The subtle CNS phenotype resulting from genetic ablation of MAG expression has made mechanistic analysis of its functional role in these difficult. However, the past few years have brought some major revelations, particularly in terms of mechanisms of MAG signaling through the Nogo-66 receptor (NgR) complex. Although apparently converging through NgR, a readily noticeable fact is that the neuronal growth inhibitory effect of MAG differs from that of Nogo-66. This may result from the influence of coreceptors in the form of gangliosides or from MAG-specific neuronal receptors such as NgR2. MAG has several other neuronal binding partners, and some of these may modulate its interaction with the NgR complex or downstream signaling. This article discusses new findings in MAG-forward and-reverse signaling and its role in CNS pathophysiology.     

A novel in vivo method for isolating antibodies from a phage display library by neuronal retrograde transport selectively yields antibodies against p75NTR

The neurotrophin receptor p75^NTR is utilized by a variety of pathogens to gain entry into the central nervous system (CNS). We tested if this entry portal might be exploited using a phage display library to isolate internalizing antibodies that target the CNS in vivo. By applying a phage library that expressed human single chain variable fragment (scFv) antibodies on their surface to a transected sciatic nerve, we showed that (1) phage conjugated to anti-p75^NTR antibody or phage scFv library pre-panned against p75^NTR are internalized by neurons expressing p75^NTR; (2) subsequent retrograde axonal transport separates internalized phage from the applied phage; and, (3) internalized phage can be recovered from a proximal ligature made on a nerve. This approach resulted in 13-fold increase in the number of phage isolated from the injured nerve compared with the starting population, and isolation of 18 unique internalizing p75^NTR antibodies that were transported from the peripheral nerve into the spinal cord, through the blood-brain barrier. In addition, antibodies recognizing other potentially internalized antigens were identified through in vivo selection using a fully diverse library. Because p75^NTR expression is upregulated in motor neurons in response to injury and in disease, the p75^NTR antibodies may have substantial potential for cell-targeted drug/gene delivery. In addition, this novel selection method provides the potential to generate panels of antibodies that could be used to identify further internalization targets, which could aid drug delivery across the blood-brain barrier.     

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。     

Effect of FK506 and cyclosporine A on the expression of BDNF, tyrosine kinase B and p75 neurotrophin receptors in astrocytes exposed to simulated ischemia in vitro

We investigated whether the immunosuppressive drugs, FK506 and cyclosporine A, increase BDNF protein and/or mRNA expression in ischemic astrocytes and if an increase could be related to changes in the nuclear expression of p-CREB, p-Erk1/2 and p-Akt. The influence of these immunosuppressants on protein and mRNA levels of TrkB and p75^NTR receptors was also examined. On day 21, cultures of rat astrocytes were subjected to ischemic conditions simulated in vitro (combined oxygen glucose deprivation, OGD) for 8 h and exposed to FK506 (10-1000 nM) and cyclosporine A (0.25-10 μM). FK506 and cyclosporine A (at 1000 nM and 0.25 μM, respectively) stimulated the expression and release of BDNF in cultured rat cerebral cortical astrocytes exposed to OGD. The immunosuppressants at these doses simultaneously increased p-CREB and p-Erk1/2 expression in the nuclear fraction of astrocytes. The results RT-PCR and Western blot analysis provided further evidence of a modulating influence of the drugs on the expression of trkB and p75^NTR genes and their protein products in ischemic astrocytes.