p75NTR Expression Research Articles

Expression pattern of p75 neurotrophin receptor protein in human scalp skin and hair follicles: Hair cycle-dependent expression

The p75 neurotrophin receptor (p75NTR) is a death factor (apoptosis-promoting protein) that belongs to the tumor necrosis factor receptor superfamily of membrane proteins. In the murine hair follicle (HF) model, p75NTR plays a critical role during HF morphogenesis, functioning as a receptor that negatively controls HF development. p75NTR signaling is involved in the control of keratinocyte apoptosis during catagen. To date, knowledge about the expression pattern of p75NTR protein in human scalp skin and HFs is limited. In this investigation we hypothesized that p75NTR protein is expressed in human scalp skin and its expression in HFs fluctuates with the transitions from anagen --> catagen --> telogen stages. To test this hypothesis, the immunoreactivity of p75NTR protein was examined in human scalp skin by immunofluorescent and immunoalkaline phosphatase methods. A total of 50 normal-appearing human scalp skin biopsy specimens were examined (healthy women age 53-57 years). In each case, 50 HFs were analyzed (35, 10, and 5 follicles in anagen, catagen, and telogen, respectively). We found variations in p75NTR protein expression with HF cycling. p75NTR expression was negligible in early, mid, and mature anagen and weak during late anagen. p75NTR expression was moderate during anagen-catagen transition. It was strong in both catagen and telogen HF. Also, p75NTR protein expression was strong in the stratum corneum (epidermis), dermal fibroblasts, blood vessels, nerve endings, adipocytes, and both sebaceous and sweat glands. Our knowledge about other proteins (prosurvival and pro-apoptotic molecules) interacting with p75 is incomplete. Our investigation reports, for the first time, the expression patterns of p75NTR in human scalp skin and HFs. p75NTR protein expression exhibited significant hair cycle-dependent fluctuation, suggesting a possible role in human HF biology.

Enhanced neuronal loss under perinatal hypothyroidism involves impaired neurotrophic signaling and increased proteolysis of p75 NTR

Recognition of the molecular events that lead to enhanced cell death is vital to understand the developmental cerebellar defects under hypothyroidism. Though neurotrophins promote the survival and development of neurons in the cerebellum, but the mechanism of their insufficiency mediated cell loss under hypothyroidism is unknown. Here in developmental hypothyroid rat model we report that hypothyroidism induced neuronal loss involve down regulation of neurotrophic survival signaling and increased truncation of the receptor p75 NTR. Results showed that perinatal hypothyroidism besides repressing the expression of BDNF also impairs the maturation of NGF which results in decreased activation of ERK, CREB, NF-kappaB and AKT. Furthermore hypothyroidism caused an enhanced expression and proteolysis of p75 NTR. The increased proteolysis of p75 NTR in vivo and its association with death of granule neurons brings forward hitherto a p75 NTR dependence signaling which along with compromised survival signaling could provide a neurotrophic basis of understanding the cause of enhanced cell death in developing cerebellum under hypothyroidism.

Expression of p75NTR in fetal brain and medulloblastomas: evidence of a precursor cell marker and its persistence in neoplasia

p75 neurotrophin receptor (p75NTR) is a member of the tumor necrosis factor superfamily, and plays a significant role in nervous system development. p75NTR has a dual (proliferative/apoptotic) role in neurogenesis and binds pro-neurotrophins with high affinity. Recent work suggests p75NTR is overexpressed in the developing cerebellum and in nodular/desmoplastic medulloblastomas. We analyzed p75NTR expression in various parts of the fetal and adult human central nervous system, and in 75 patients with medulloblastomas. The expression of p75NTR in the fetal brain was seen solely within the external granular layer with weaker expression in the Purkinje layer, which most likely represents Purkinje cell staining. The staining was present in gestational weeks 20-40, while no staining was identified elsewhere in the fetal brain or within the adult cerebellum. p75NTR positive cells were also positive with the proliferation marker ki-67, but were negative for ret, reelin, CD133, CD34, and cleaved caspase 3. Nine of 75 medulloblastomas (12%) were also showed positive immunostaining for p75NTR. The staining was seen in four classic, two desmoplastic, and three anaplastic medulloblastomas. The persistence of p75NTR in a small group of medulloblastomas raises the possibility that in such tumors, the receptor could be a potential therapeutic target.

Proteomic assessment of sympathetic ganglia from adult mice that possess null mutations of ExonIII or ExonIV in the p75 neurotrophin receptor gene

Neurotrophins, such as nerve growth factor (NGF), are capable of binding to the transmembrane p75 neurotrophin receptor (p75NTR), which regulates a variety of cellular responses including apoptosis and axonal elongation. While the development of mutant mouse strains that lack functional p75NTR expression has provided further insight into the importance of this neurotrophin receptor, there remains a paucity of information concerning how the loss of p75NTR expression may alter neural phenotypes. To address this issue, we assessed the proteome of the cervical sympathetic ganglia from two mutant lines of mice, which were compared to the ganglionic proteome of age-matched wild type mice. The ganglionic proteome of mice possessing two mutant alleles of either exonIII or exonIV for the p75NTR gene displayed detectable alterations in levels of Lamin A, tyrosine hydroxylase, and Annexin V, as compared to ganglionic proteome of wild type mice. Decreased expression of the basic isoform of tyrosine hydroxylase may be linked to perturbed NGF signaling in the absence of p75NTR in mutant mice. Stereological measurement showed significant increases in the number of sympathetic neurons in both lines of p75NTR-deficient mice, relative to wild type mice. This enhanced survival of sympathetic neurons coincides with shifts toward the more basic isoforms of Annexin V in mutant mice. This study, in addition to providing the first comparative proteomic assessment of sympathetic ganglia, sheds new light onto the phenotypic changes that occur as a consequence of a loss of p75NTR expression in adult mice.

Abstract A33: Superior efficacy of dietary 3,3’diindolylmethane (DIM) amongst several indoles to inhibit prostate cancer growth via induction of the p75NTR tumor suppressor protein

Abstract A33 A comparison of the indoles etodolac, indomethacin, 5-methylindole-3-acetic acid, indole-3-carbinol, 3,3’diindolylmethane (DIM) and the related ketorolac molecule showed superior efficacy of DIM, a dietary indole, to induce expression of the p75NTR tumor suppressor and inhibit survival of the PC-3 prostate cancer cell line. Each indole that induced p75NTR expression exhibited the same rank-order for inhibition of prostate cell survival. Comparison of the activity of DIM, the most potent of the indoles, between several cell lines, showed that DIM induced p75NTR at low concentrations in prostate cells (PC-3 , DU-145), followed by bladder (T24), with breast (MCF-7) and fibroblast cells (3T3) being the least responsive to DIM. DIM induction of p75NTR levels in the various cell lines occurred in the same rank-order as inhibition of cell survival. Since the consumption of cruciferous vegetables has been reported to reduce the risk of prostate cancer through the release of indole-3-carbinol (I3C) which is subsequently metabolized in the acidic environment of the stomach to DIM, the superior activity of DIM to induce expression of the p75NTR tumor suppressor protein and inhibit cell survival suggests a mechanism of action for the anticancer activity of DIM in the prevention of prostate cancer. In order to demonstrate a cause and effect between DIM and p75NTR inhibition of survival, we used a dominant negative of p75NTR to rescue DIM induced loss of cell survival. In order to investigate the mechanism of action by which p75NTR transduces a biochemical signaling cascade leading to cell death of prostate cancer cells we utilized knockdown with p38 MAPK siRNA to rescue DIM induction of p75NTR thereby implicating p38 MAPK in DIM induction of cell death. Moreover, DIM treatment induced phosphorylation of p38 MAPK within one minute, suggesting the target of DIM is proximal to p38 MAPK. Hence, these results suggest that the anti-cancer activity of cruciferous vegetables may be mediated in part by DIM and that the mechanism of action occurs, in part, by activation of the p38 MAPK pathway leading to expression of the p75NTR death receptor with subsequent apoptosis of prostate cancer cells. Citation Information: Cancer Prev Res 2008;1(7 Suppl):A33.

Carprofen induction of p75NTR-dependent apoptosis via the p38 mitogen-activated protein kinase pathway in prostate cancer cells

The p75 neurotrophin receptor (p75(NTR)) functions as a tumor suppressor in prostate epithelial cells, where its expression declines with progression to malignant cancer. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in several prostate cancer cell lines leading to p75(NTR)-mediated decreased survival. Using the 2-phenyl propionic acid moiety of these profens as a pharmacophore, we screened an in silico database of 30 million compounds and identified carprofen as having an order of magnitude greater activity for induction of p75(NTR) levels and inhibition of cell survival. Prostate (PC-3 and DU-145) and bladder (T24) cancer cells were more sensitive to carprofen induction of p75(NTR)-associated loss of survival than breast (MCF-7) and fibroblast (3T3) cells. Transfection of prostate cell lines with a dominant-negative form of p75(NTR) before carprofen treatment partially rescued cell survival, showing a cause-and-effect relationship between carprofen induction of p75(NTR) levels and inhibition of survival. Carprofen induced apoptotic nuclear fragmentation in prostate but not in MCF-7 and 3T3 cells. Furthermore, small interfering RNA knockdown of the p38 mitogen-activated protein kinase (MAPK) protein prevented induction of p75(NTR) by carprofen in both prostate cell lines. Carprofen treatment induced phosphorylation of p38 MAPK as early as within 1 min. Expression of a dominant-negative form of MK2, the kinase downstream of p38 MAPK frequently associated with signaling cascades leading to apoptosis, prevented carprofen induction of the p75(NTR) protein. Collectively, we identify carprofen as a highly potent profen capable of inducing p75(NTR)-dependent apoptosis via the p38 MAPK pathway in prostate cancer cells.

Role of p75NTR in female rat urinary bladder with cyclophosphamide-induced cystitis

Previous studies demonstrated changes in urinary bladder neurotrophin content and upregulation of neurotrophin receptors, TrkA and the p75 neurotrophin receptor (p75(NTR)), in micturition reflex pathways after cyclophosphamide (CYP)-induced cystitis. p75(NTR) can bind nerve growth factor (NGF) and modulate NGF-TrkA binding and signaling. We examined p75(NTR) expression and the role of p75(NTR) in the micturition reflex in control and CYP-treated rats. p75(NTR) Immunoreactivity was present throughout the urinary bladder. CYP-induced cystitis (4 h, 48 h, chronic) increased (P < or = 0.05) p75(NTR) expression in whole urinary bladder as shown by Western blotting. The role of p75(NTR) in bladder function in control and CYP-treated rats was determined using conscious cystometry and immunoneutralization or PD90780, a compound known to specifically block NGF binding to p75(NTR). An anti-p75(NTR) monoclonal antibody or PD90780 was infused intravesically and cystometric parameters were evaluated. Both methods of p75(NTR) blockade significantly (P < or = 0.05) decreased the intercontraction interval and void volume in control and CYP-treated rats. Intravesical infusion of PD90780 also significantly (P < or = 0.001) increased intravesical pressure and increased the number of nonvoiding contractions during the filling phase. Control intravesical infusions of isotype-matched IgG and vehicle were without effect. Intravesical instillation of PD90780 significantly (P < or = 0.01) reduced the volume threshold to elicit a micturition contraction in control rats (no inflammation) and CYP-treated in a closed urinary bladder system. These studies demonstrate 1) ubiquitous p75(NTR) expression in urinary bladder and increased expression with CYP-induced cystitis and 2) p75(NTR) blockade at the level of the urinary bladder produces bladder hyperreflexia in control and CYP-treated rats. The overall activity of the urinary bladder reflects the balance of NGF-p75(NTR) and NGF-TrkA signaling.

Pan‐neurotrophin receptor p75NTR expression is strongly induced in lesional atopic mast cells

Neurotrophins such as nerve growth factor or brain-derived neurotrophic factor influence neuronal proliferation and differentiation via the low-affinity pan-neurotrophin receptor p75NTR that may play a pivotal role in linking the immune with the nervous system. Because the precise regulation of p75NTR gene transcription in mast cells under states of allergic inflammation has not been investigated in detail so far, the present studies assessed the gene regulation and expression of this receptor. Transcriptional expression of p75NTR in human skin was studied in isolated cutaneous cells by means of RT-PCR. In situ lesional mast cell p75NTR expression was analysed by immunohistochemistry. The p75NTR mRNA expression was found in isolated human skin mast cells and keratinocytes. Lower mRNA levels were present in fibroblasts and melanocytes but no transcripts were found in endothelial cells. The p75NTR protein expression was found in situ in lesional and non-lesional mast cells. A significantly increased expression of p75NTR protein was found in atopic dermatitis lesional mast cells when compared with control mast cell expression (P<0.05). The demonstration of an increased level of p75NTR gene transcription in lesional mast cells points to an induction of low-affinity neurotrophin receptor sensitivity of mast cells under states of allergic inflammation. Topically administered neurotrophin receptor-modulating compounds may act as anti-inflammatory mediators in cutaneous allergic inflammation.

Nerve growth factor receptor (p75NTR) and pattern of invasion predict poor prognosis in oral squamous cell carcinoma

To evaluate the expression of p75 neurotrophin receptor (p75(NTR)) in oral squamous cell carcinoma (OSCC). The results were related to tumour node metastasis (TNM) stage, World Health Organization (WHO) grade, invasive front grading (IFG) and prognosis. Immunohistochemically, the expression of p75(NTR) was assessed in 53 T1-T2 OSCCs. Clinical data were recorded prospectively. The end-point was disease-free survival. All tumours expressed p75(NTR), and this expression, both in central/superficial tumour areas and at the invasive front, was associated with poor prognosis (P = 0.03 and P = 0.02) (log rank test). Tumours with marked cellular dissociation (IFG parameter) had more recurrences than tumours with collective tumour cell invasion (P = 0.03). In tumours showing both p75(NTR) at the invasive front and marked tumour cell dissociation, the average risk of recurrence was increased about 17 times (Cox regression analysis) compared with tumours with low p75(NTR) expression and collective invasion. Traditional prognostic systems were of no prognostic significance. p75(NTR) was expressed in all OSCCs. p75(NTR) expression and the pattern of invasion were significantly associated with a poor prognosis in OSCCs, and both were better prognostic factors than traditional prognostic parameters. The combination of p75(NTR) expression and the pattern of invasion strongly increased precision in the identification of tumours with poor disease-free survival.

Identification and kainic acid-induced up-regulation of low-affinity p75 neurotrophin receptor (p75NTR) in the nigral dopamine neurons of adult rats

Parkinson's disease is a common and severe debilitating neurological disease that results from massive and progressive degenerative death of dopamine neurons in the substantia nigra, but the mechanisms of neuronal degeneration and disease progression remains largely obscure. We are interested in possible implications of low-affinity p75 neurotrophin receptor (p75NTR), which may mediate neuronal apoptosis in the central nervous system, in triggering cell death of the nigral dopamine neurons. The RT-PCR and immunohistochemistry were carried out to detect if p75NTR is expressed in these nigral neurons and up-regulated by kainic acid (KA) insult in adult rats. It revealed p75NTR-positive immunoreactivity in the substantia nigra, and co-localization of p75NTR and tyrosine hydroxylase (TH) was found in a large number of substantia nigra neurons beside confirmation of p75NTR in the choline acetyltransferase (ChAT)-positive forebrain neurons. Cell count data further indicated that about 47–100% of TH-positive nigral neurons and 98–100% of ChAT-positive forebrain neurons express p75NTR. More interestingly, significant increasing in both p75NTR mRNA and p75NTR-positive neurons occurred rapidly following KA insult in the substantia nigra of animal model. The present study has provided first evidence on p75NTR expression and KA-inducing p75NTR up-regulation in substantia nigra neurons in rodent animals. Taken together with previous data on p75NTR functions in neuronal apoptosis, this study also suggests that p75NTR may play important roles in neuronal cell survival or excitotoxic degeneration of dopamine neurons in the substantia nigra in pathogenesis of Parkinson's disease in human beings.

Gene expression alterations of neurotrophins, their receptors and prohormone convertases in a rat model of spinal cord contusion

We have used a semi-quantitative RT-PCR approach to investigate the alterations in the expression of the main regulators of neuronal survival and death, neurotrophins (NTs), NT receptors, and prohormone convertases (PC), in a rat model of spinal cord contusion. Our results revealed that the expression of the members of NT family (Nerve-Growth Factor (NGF), Brain-Derived Neurotrophic Factor (BDNF), and Neurotrophin-3 (NT-3)) is significantly declined in the injured spinal cord, as early as 6h after the induction of the contusion. The expression was recovered afterward to that of the control levels. Furthermore, the expression of all NTs high-affinity Trk receptors decreased severely after the contusion. While the expression of TrkA and TrkC were completely shut down after 6 and 12h after injury respectively, the expression of TrkB receptor declined at 12h after injury and remained at this low level thereafter. In contrast to the pattern of Trk receptor expression, p75NTR receptor showed a significant upregulation after contusion. The expression of PC members functioning in the constitutive secretory pathway, i.e. furin, PACE4 and PC7, increased after damage, while the expression of PC members acting in regulated secretory pathway, PC1 and PC2, reduced after spinal cord injury. All together, the down-regulation of NTs, their designated Trk receptors and PC1/PC2 enzymes along with an upregulation of p75NTR promote neuronal death after injury. Our results suggest that either overexpression of NTs, Trk receptors and PC1/PC2 or interfering with the expression of p75NTR in host and/or grafted cells before transplantation could increase the success of the transplantation.

P75 NTR activation of NF-κB is involved in PrP106-126-induced apoptosis in mouse neuroblastoma cells

Neuronal death is a pathological hallmark of prion diseases. Synthetic prion peptide PrP106-126 can convert PrP C into protease-resistant aggregates, which can cause neurotoxicity in vivo and in vitro. Various cell surface proteins can participate in the infection process of prions. p75 NTR can interact with PrP106-126 and has a neurotoxic effect on neurons. However, for p75 NTR lacking intrinsic catalytic activity domain in cytoplasm, p75 NTR -associated signaling molecular and the signaling events in cytoplasm in p75 NTR-mediated apoptosis responding to PrP106-126 remain still unknown. Thus p75 NTR -associated NF-κB signaling pathway was investigated in this study. Herein PrP106-126-induced apoptosis in mouse neuroblastoma cell line N2a, PrP106-126 significantly up-regulated p75 NTR expression on mRNA and protein levels. For the first time we found that PrP106-126 induced activation of NF-κB by Western blot assay, and blocking the interaction of p75 NTR with PrP106-126 by p75 NTR polyclonal antibody sc-6189 or pretreatment with inhibitor NF-κB SN50 reduced the activation of NF-κB and attenuated the apoptotic effect by PrP106-126. This study offers a possible interpretation that NF-κB signaling pathway was activated by the interaction of PrP106-126 with p75 NTR, and NF-κB activity showed the pro-apoptotic effect in PrP106-126-induced apoptosis in N2a cells. Involvement of NF-κB signaling pathway in p75 NTR-mediated apoptosis may partially account for the PrP106-126-induced neurotoxicity in N2a cells.

The inhibitory effect of p75 neurotrophin receptor on growth of human hepatocellular carcinoma cells

p75 neurotrophin receptor (p75NTR), a member of the TNF receptor superfamily, is a focus for study at present. Up to now, its role and functions in hepatocellular carcinoma were not fully elucidated. In this study, we investigated the expression of p75NTR in hepatocellular carcinoma and the impact of its alteration on tumor growth. We found that the expression of p75NTR was decreased significantly in 158 cases of hepatocellular carcinoma tissues as compared with their adjacent noncancerous counterparts, and its expression was also significantly decreased in various human hepatocellular carcinoma cell lines. Down-regulating p75NTR by specific siRNA promoted the growth of normal liver cell lines, whereas up-regulating p75NTR inhibited the growth of hepatocellular carcinoma cell lines in vitro and caused dramatic attenuation of tumor growth in vivo by induction of cell cycle arrest. Furthermore, we found that up-regulating p75NTR could down-regulate the expression of cyclin A, cyclin D1, cyclin E, cdk2, p-Rb and PCNA, but up-regulate the expression of Rb. Conversely, the results were inverse when p75NTR was down-regulated by specific siRNA. Therefore, we provided the evidence that p75NTR was a potential tumor suppressor and might be used as a therapeutic target for hepatocellular carcinoma.

Neurotrophin-induced upregulation of p75NTR via a protein kinase C-delta-dependent mechanism

Neurotrophins exert their biological effects via p75NTR and Trk receptors. Functional interplay between these two receptors has been widely explored with respect to p75NTR enhancing the activation and signalling of Trk, but few studies address the bidirectional aspects. We have previously demonstrated that the expression of p75NTR can be differentially modulated by different Trk receptor mutations. Here we investigate the mechanism of Nerve Growth Factor (NGF)-induced upregulation of p75NTR expression. We utilize pharmacological inhibition to investigate the role of various TrkA-associated signalling intermediates in this regulatory cascade. Notably, the inhibition of phospholipase C-γ (PLC-γ) using U73122, prevented the NGF-induced upregulation of p75NTR protein and mRNA. The inhibition of protein kinase C-δ (PKC-δ) activation by rottlerin, a selective PKC-δ inhibitor, and by small interfering RNA (siRNA) directed against PKC-δ also inhibited this NGF-induced upregulation. Finally, we also show that in cerebellar granule neurons, BDNF acting via TrkB increases p75NTR expression in a PKC-δ dependent manner. These results indicate the importance of Trk-dependent PLC-γ and PKC-δ activation for downstream regulation of p75NTR protein expression in response to neurotrophin stimulation, a process that has implications to the survival and growth of the developing nervous system.

Peroxynitrite Mediates Retinal Neurodegeneration by Inhibiting Nerve Growth Factor Survival Signaling in Experimental and Human Diabetes

Recently we have shown that diabetes-induced retinal neurodegeneration positively correlates with oxidative stress and peroxynitrite. Studies also show that peroxynitrite impairs nerve growth factor (NGF) survival signaling in sensory neurons. However, the causal role of peroxynitrite and the impact of tyrosine nitration on diabetes-induced retinal neurodegeneration and NGF survival signaling have not been elucidated. Expression of NGF and its receptors was examined in retinas from human and streptozotocin-induced diabetic rats and retinal ganglion cells (RGCs). Diabetic animals were treated with FeTPPS (15 mg x kg(-1) x day(-1) ip), which catalytically decomposes peroxynitrite to nitrate. After 4 weeks of diabetes, retinal cell death was determined by TUNEL assay. Lipid peroxidation and nitrotyrosine were determined using MDA assay, immunofluorescence, and Slot-Blot analysis. Expression of NGF and its receptors was determined by enzyme-linked immunosorbent assay (ELISA), real-time PCR, immunoprecipitation, and Western blot analyses. Analyses of retinal neuronal death and NGF showed ninefold and twofold increases, respectively, in diabetic retinas compared with controls. Diabetes also induced increases in lipid peroxidation, nitrotyrosine, and the pro-apoptotic p75(NTR) receptor in human and rat retinas. These effects were associated with tyrosine nitration of the pro-survival TrkA receptor, resulting in diminished phosphorylation of TrkA and its downstream target, Akt. Furthermore, peroxynitrite induced neuronal death, TrkA nitration, and activation of p38 mitogen-activated protein kinase (MAPK) in RGCs, even in the presence of exogenous NGF. FeTPPS prevented tyrosine nitration, restored NGF survival signal, and prevented neuronal death in vitro and in vivo. Together, these data suggest that diabetes-induced peroxynitrite impairs NGF neuronal survival by nitrating TrkA receptor and enhancing p75(NTR) expression.

Chronic hypobaric hypoxia induced apoptosis in CA1 region of hippocampus: A possible role of NMDAR mediated p75 NTR upregulation

Hypobaric hypoxia has been implicated with neural degeneration and memory loss. Though there has been considerable knowledge on the role of the p75 NTR in triggering apoptosis, the occurrence of a similar mechanism in hypoxic stress still remains to be explored. We, in the present study, have tried to explore the role of p75 NTR in mediating apoptosis in hypobaric hypoxia. Male Sprague Dawley rats were exposed to an altitude of 7,620 m for different durations. To study the contribution of apoptosis to hypobaric hypoxia induced cell death in the hippocampus, rat brains were examined for the occurrence of apoptosis by determining the number of cells showing DNA breaks using terminal deoxynucleotidyl transferase-mediated UTP nick end labeling (TUNEL) assay and chromatin condensation using Hoechst staining along with estimation of caspase activity and expression of active Caspase 3. Expression of p75 NTR was studied to determine its possible role in triggering apoptosis in hypobaric hypoxia. Exposure to hypobaric hypoxia was found to progressively increase the number of TUNEL positive and Hoechst positive cells along with increase in caspase activity, thus suggesting apoptotic mode of cell death. p75 NTR was found to be upregulated on prolonged exposure to hypobaric hypoxia corresponding to the increase in the number of apoptotic cells. Further, reduced expression of p75 NTR expression by antisense nucleotide administration significantly decreased apoptosis in the CA1 region of hippocampus. Blocking of NMDA receptors by MK801 interestingly decreased p75 NTR expression and the number of TUNEL positive cells as compared to hypoxic animals. These findings suggest the regulation of p75 NTR by NMDA receptors and its role in inducing apoptosis in hypoxia.

Effects of estrogen on basal forebrain cholinergic neurons and spatial learning

Estrogen receptors are expressed in several areas of the brain associated with cognition, including the basal forebrain cholinergic nuclei, and numerous reports have described improvements in memory in response to estrogen supplementation. The relationship between estrogen's effects on the basal cholinergic system and improvements in cognitive function, however, are obscure. We therefore undertook a study to determine the effects of estrogen on several parameters of the cholinergic system in ovariectomized rats and measured the concomitant effects on performance in the Barnes maze, a test of spatial memory. Six weeks of estradiol treatment caused an increase in choline acetyltransferase activity throughout the projection fields of the basal forebrain, including the hippocampal formation (14%), olfactory bulb (30%), and cerebral cortex (35%). Estrogen treatment also caused an increase in cell soma size of cholinergic neurons in the horizontal diagonal limb of the band of Broca and in the basal nucleus of Meynert. There was no change in the number of neurons positive for p75(NTR), nor in the level of p75(NTR) expression per neuron. Barnes maze performance was markedly improved after estradiol treatment, reinforcing the view that estrogen has beneficial cognitive effects, particularly on spatial memory. The beneficial cognitive effect was likely mediated in part by stimulation of the basal forebrain cholinergic system, especially in its neocortical projection, but was not associated with changes in the level of p75(NTR) expression.

P75NTR and Sortilin Increase After Facial Nerve Injury

After axotomy, Schwann cells (SCs), required for successful nerve regeneration, undergo a number of cellular changes including dedifferentiation, proliferation, expression of molecules that support axon growth, and apoptosis. This study investigated the role of p75, sortilin, and proneurotrophins in SC survival after facial nerve (FN) axotomy. Preliminary animal study. With use of FN SCs, expression of p75 and its coreceptor sortilin were quantified by immunofluorescence on days 12, 22, and 52 after axotomy in vivo and by Western blot in vitro. Contralateral FNs served as a control. SC apoptosis was detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL). To verify a causative role for p75 in FN SC death, cultured FN SCs were treated with pro-nerve growth factor (NGF), and apoptosis was determined by TUNEL. Expression of p75 and sortilin increased in FN SCs distal (P < .05) to the axotomy compared with the contralateral controls for all time points. SC apoptosis also significantly increased in the distal segment compared with the contralateral and proximal portions (P < .05). ProNGF, a p75 ligand, increased apoptosis and p75 expression in primary FN SC cultures. FN axotomy increases p75 and sortilin expression in SCs, which correlates with increased apoptosis. These findings suggest roles for p75 and sortilin in SC loss after FN injury. Sortilin is a novel target in promoting FN healing after injury.

The p38 MAPK Pathway Mediates Aryl Propionic Acid–Induced Messenger RNA Stability of p75NTR in Prostate Cancer Cells

The p75(NTR) acts as a tumor suppressor in the prostate, but its expression is lost as prostate cancer progresses and is minimal in established prostate cancer cell lines such as PC-3, DU-145, and LNCaP. Previously, we showed that treatment with R-flurbiprofen or ibuprofen induced p75(NTR) expression in PC-3 and DU-145 cells leading to p75(NTR)-mediated decreased survival. Here, we investigate the mechanism by which these drugs induce p75(NTR) expression. We show that the observed increase in p75(NTR) protein due to R-flurbiprofen and ibuprofen treatment was accompanied by an increase in p75(NTR) mRNA, and this increase in mRNA was the result of increased mRNA stability and not by an up-regulation of transcription. In addition, we show that treatment with R-flurbiprofen or ibuprofen led to sustained activation of the p38 mitogen-activated protein kinase (MAPK) pathway. Furthermore, inhibition of the p38 MAPK pathway with the p38 MAPK-specific inhibitor SB202190 or by small interfering RNA (siRNA) knockdown of p38 MAPK protein prevented induction of p75(NTR) by R-flurbiprofen and ibuprofen. We also observed that siRNA knockdown of MAPK-activated protein kinase (MK)-2 and MK3, the kinases downstream of p38 MAPK that are responsible for the mRNA stabilizing effects of the p38 MAPK pathway, also prevented an induction of p75(NTR) by R-flurbiprofen and ibuprofen. Finally, we identify the RNA stabilizing protein HuR and the posttranscriptional regulator eukaryotic translation initiation factor 4E as two possible mechanisms by which the p38 MAPK pathway may increase p75(NTR) expression. Collectively, the data suggest that R-flurbiprofen and ibuprofen induce p75(NTR) expression by increased mRNA stability that is mediated through the p38 MAPK pathway.

Expression of Acid-Sensing Ion Channel 3 (ASIC3) in Nucleus Pulposus Cells of the Intervertebral Disc Is Regulated by p75NTR and ERK Signaling

Although a recent study has shown that skeletal tissues express ASICs, their function is unknown. We show that intervertebral disc cells express ASIC3; moreover, expression is uniquely regulated and needed for survival in a low pH and hypoeromsotic medium. These findings suggest that ASIC3 may adapt disc cells to their hydrodynamically stressed microenvironment. The nucleus pulposus is an avascular, hydrated tissue that permits the intervertebral disc to resist compressive loads to the spine. Because the tissue is hyperosmotic and avascular, the pH of the nucleus pulposus is low. To determine the mechanisms by which the disc cells accommodate to the low pH and hypertonicity, the expression and regulation of the acid sensing ion channel (ASIC)3 was examined. Expression of ASICs in cells of the intervertebral disc was analyzed. To study its regulation, we cloned the 2.8-kb rat ASIC3 promoter and performed luciferase reporter assays. The effect of pharmacological inhibition of ASICs on disc cell survival was studied by measuring MTT and caspase-3 activities. ASIC3 was expressed in discal tissues and cultured disc cells in vitro. Because studies of neuronal cells have shown that ASIC3 expression and promoter activity is induced by nerve growth factor (NGF), we examined the effect of NGF on nucleus pulposus cells. Surprisingly, ASIC3 promoter activity did not increase after NGF treatment. The absence of induction was linked to nonexpression of tropomyosin-related kinase A (TrkA), a high-affinity NGF receptor, although a modest expression of p75NTR was seen. When treated with p75NTR antibody or transfected with dominant negative-p75NTR plasmid, there was significant suppression of ASIC3 basal promoter activity. To further explore the downstream mechanism of control of ASIC3 basal promoter activity, we blocked p75NTR and measured phospho extracellular matrix regulated kinase (pERK) levels. We found that DN-p75NTR suppressed NGF mediated transient ERK activation. Moreover, inhibition of ERK activity by dominant negative-mitogen activated protein kinase kinase (DN-MEK) resulted in a dose-dependent suppression of ASIC3 basal promoter activity, whereas overexpression of constitutively active MEK1 caused an increase in ASIC3 promoter activity. Finally, to gain insight in the functional importance of ASIC3, we suppressed ASIC activity in nucleus pulposus cells. Noteworthy, under both hyperosmotic and acidic conditions, ASIC3 served to promote cell survival and lower the activity of the pro-apoptosis protein, caspase-3. Results of this study indicate that NGF serves to maintain the basal expression of ASIC3 through p75NTR and ERK signaling in discal cells. We suggest that ASIC3 is needed for adaptation of the nucleus pulposus and annulus fibrosus cells to the acidic and hyperosmotic microenvironment of the intervertebral disc.