Interactions between beta-neuregulin and neurotrophins in motor neuron apoptosis

Neuregulins play a major role in the formation and stabilization of neuromuscular junctions, and are produced by both motor neurons and muscle. Although the effects and mechanism of neuregulins on skeletal muscle (e.g. regulation of acetylcholine receptor expression) have been studied extensively, the effects of neuregulins on motor neurons remain unknown. We report that neuregulin-1beta (NRGbeta1) inhibited apoptosis of rat motor neurons for up to 7 days in culture by a phosphatidylinositol 3 kinase-dependent pathway and synergistically enhanced motor neuron survival promoted by glial-derived neurotrophic factor (GDNF). However, binding of neurotrophins, including brain-derived neurotrophic factor (BDNF) and nerve growth factor (NGF), to the p75 neurotrophin receptor (p75NTR) abolished the neuregulin anti-apoptotic effect on motor neurons. Inhibitors of the c-Jun N-terminal kinase (JNK) mitogen-activated protein kinase prevented motor neuron death caused by co-incubation of NRGbeta1 and BDNF or NGF, as well as by trophic factor deprivation. Motor neuron apoptosis resulting from both trophic factor deprivation and exposure to NRGbeta1 plus neurotrophins required the induction of neuronal nitric oxide synthase and peroxynitrite formation. Because motor neurons express both p75NTR and neuregulin erbB receptors during the period of embryonic programmed cell death, motor neuron survival may be the result of complex interactions between trophic and death factors, which may be the same molecules acting in different combinations.     

Opposing Roles of pka and epac in the cAMP-Dependent Regulation of Schwann Cell Proliferation and Differentiation

In Schwann cells (SCs), cyclic adenosine monophosphate (cAMP) not only induces differentiation into a myelinating SC-related phenotype, but also synergistically enhances the mitogenic action of growth factors such as neuregulin. To better understand the molecular mechanism by which cAMP exerts these apparently contradictory functions, we investigated the role of the two main effectors of cAMP, protein kinase A (PKA) and the exchange protein activated by cAMP (EPAC), on the proliferation and differentiation of both isolated and axon-related SCs. For these studies, a variety of PKA and EPAC agonists and antagonists were used, including pathway-selective analogs of cAMP and pharmacological inhibitors. Our studies indicated that the activity of PKA rather than EPAC was required for the adjuvant effect of cAMP on S-phase entry, whereas the activity of EPAC rather than PKA was required for SC differentiation and myelin formation. Even though selective EPAC activation had an overall anti-proliferative effect in SCs, it failed to drive the expression of Krox-20, a master regulator of myelination, and that of myelin-specific proteins and lipids, suggesting that EPAC activation was insufficient to drive a full differentiating response. Interestingly, inhibition of EPAC activity resulted in a drastic impairment of SC differentiation and myelin formation but not Krox-20 expression, which indicates an independent mechanism of Krox-20 regulation in response to cAMP. In conclusion, our data supports the idea that the outcome of cAMP signaling in SCs depends on the particular set of effectors activated. Whereas the mitogenic action of cAMP relies exclusively on PKA activity, the differentiating action of cAMP requires a PKA-independent (non-canonical) cAMP-specific pathway that is partially transduced by EPAC.     

Differential regulation of Shc adaptor proteins in skeletal muscle,spinal cord and forebrain of aged rats with sensorimotor impairment

The Shc family of proteins participates in mitogenic and survival signalling through binding to receptor tyrosine kinases. We report here on the expression of Shc in forebrain, spinal cord and hind limb muscles from 30-month-old rats with different degrees of sensorimotor impairment. ShcA (mRNA and protein) is up-regulated in skeletal muscles and spinal cord of aged rats, and this change relates to biological age, i.e. degree of behavioural incapacitation, rather than to chronological age. Western blot and RT-PCR revealed that the increase in ShcA selectively affected the p46 isoform in the spinal cord, whereas in muscle tissue a robust increase of p66(ShcA) was also evident. Furthermore, in parallel with the up-regulation of ShcA, an increase of p75(NTR) mRNA in the aged animals was observed. ShcB mRNA showed a tendency for down-regulation in both spinal cord and skeletal muscles, whereas the expression of ShcC was unaltered. Our data show that the regulation of Shc mRNAs in senescence is region as well as isoform specific. The regulatory changes may reflect changes in mitogenic/survival signalling induced by age-related cell and tissue damage. The coup-regulation of p66(ShcA) and p75(NTR) is interesting since both molecules have been associated with apoptosis.     

Krüppel-Like Factor 6 Rendered Rat Schwann Cell More Sensitive to Apoptosis via Upregulating FAS Expression

Krüppel-like factor 6 (KLF6) is a tumor suppressor gene and play a role in the regulation of cell proliferation and apoptosis. After the peripheral nerve injury (PNI), the microenvironment created by surrounding Schwann cells (SCs) is a critical determinant of its regenerative potential. In this study, we examined the effects of KLF6 on SCs responses during PNI. Both KLF6 mRNA and protein expression levels were upregulated in the injured sciatic nerve, and immunofluorescence results showed that many KLF6-positive cells simultaneously expressed the SC markers S-100 and p75NTR. The apoptosis inducers TNFα and cisplatin upregulated KLF6 expression in primary cultured SCs and the SC line RSC96. Although KLF6 overexpression exacerbated cisplatin- and TNFα-induced apoptosis, expression levels of the apoptosis regulators Bcl2 and Bax were not significantly affected in either KLF6-overexpressing or KLF6-depleted RSC96 cells. Realtime PCR arrays and qRT-PCR demonstrated that KLF6 overexpression upregulated four pro-apoptotic genes, FAS, TNF, TNFSF12, and PYCARD, and inhibited expression of the anti-apoptotic IL10 gene expression. Further analysis revealed that FAS protein expression was positively correlated with KLF6 expression in SCs. These data suggest that KLF6 upregulation may render SCs more vulnerable to apoptosis after injury via upregulating FAS expression.     

Neuregulin-1 increases the proliferation of neuronal progenitors from embryonic neural stem cells

Neuregulins are a family of proteins expressed in the developing brain and in brain regions that continue to undergo neurogenesis in adult animals. We investigated the effects of neuregulins on embryonic neural stem cells (NSCs) isolated from E11 mouse telencephalon. Treatment of basic fibroblast growth factor (bFGF)-expanded neurosphere cultures with the EGF-like domain of neuregulin1-beta1 (NRG-1(177-244)) resulted in a 4-fold increase of bromodeoxyuridine (BrDU)-labeled cells, suggesting that NRG-1 stimulated proliferation. The majority of the BrdU-positive cells co-labeled with an antibody against MAP2, indicating that the proliferating cells were neuronal. No BrDU labeling was seen in GFAP- or O4-positive cells. In NRG-1-treated cultures, many of the MAP2-positive cells co-labeled with an anti-nestin antibody, suggesting that these cells are neuron-restricted progenitors (NRPs). Few MAP2/nestin-positive cells were seen in control cultures. The increase in the number of neuronal cells in NRG-1-treated cultures was due to increased proliferation of MAP2-positive cells rather than the regulation of cell survival or fate determination. These results suggest that neuregulins are mitogenic to NRPs, thus endogenous neuregulins may play important roles during CNS neurogenesis.     

Let-7a-5p regulated by lncRNA-MEG3 promotes functional differentiation to Schwann cells from adipose derived stem cells via directly inhibiting RBPJ-mediating Notch pathway

Schwann cells (SCs) have important roles in supporting and repairing peripheral neurons, and thus have great potential for nerve injury treatment. Adipose tissue-derived stem cells (ADSCs) can be reliably induced to differentiate into SCs. However, the underlying molecular mechanisms are unclear. We explored the roles of MEG3/let-7a-5p/RBPJ axis in the differentiation into SCs from ADSCs. Primary ADSCs were induced to differentiate into SCs by appropriate reagents. ELISA, immunostaining, Western blotting, and qRT-PCR were employed to examine levels of SC-markers such as S100, GFAP, SOX10, p75NTR, GAP43, MPZ, β-NGF, BDNF, and NCAM and let-7 family, MEG3, RBPJ, and Notch signaling related proteins. Dual luciferase assay and RNA immunoprecipitation were performed to validate interactions of let-7a-5p/RBPJ mRNA and MEG3/let-7a-5p. Cultured ADSCs could be induced to differentiate into functional SCs. Let-7a-5p and let-7d-5p were elevated during the differentiation while MEG3 and RBPJ/Notch-signaling were suppressed. Let-7a-5p mimics promoted ADSC differentiation into SCs and up-regulated the levels of SC-related markers including S100, GFAP, SOX10, p75NTR, GAP43, MPZ, β-NGF, and NCAM, while RBPJ or MEG3 overexpression retarded the differentiation and reduced those levels. Let-7a-5p directly targeted RBPJ and MEG3 disinhibited Notch-RBPJ signaling via sponging let-7a-5p. RBPJ overexpression reversed the acceleration of let-7a-5p mimics on SC differentiation while let-7a-5p mimics blocked MEG3-mediated suppression on SC differentiation. Let-7a-5p sponged by MEG3 promotes differentiation of ADSCs into SCs via suppressing Notch signaling by targeting RBPJ. These findings shed light on mechanisms underlying the differentiation of ADSCs to SCs and provide avenues to accelerate the process.

     

NRAGE,a p75 neurotrophin receptor-interacting protein,induces caspase activation and cell death through a JNK-dependent mitochondrial pathway

The p75 neurotrophin receptor (p75NTR) mediates signaling events leading to activation of the JNK pathway and cell death in a variety of cell types. We recently identified NRAGE, a protein that directly interacts with the p75NTR cytosolic region and facilitates p75NTR-mediated cell death. For the present study, we developed an inducible recombinant NRAGE adenovirus to dissect the mechanism of NRAGE-mediated apoptosis. Induced NRAGE expression resulted in robust activation of the JNK pathway that was not inhibited by the pharmacological mixed lineage kinase (MLK) inhibitor CEP1347. NRAGE induced cytosolic accumulation of cytochrome c, activation of Caspases-3, -9 and -7, and caspase-dependent cell death. Blocking JNK and c-Jun action by overexpression of the JNK-binding domain of JIP1 or dominant-negative c-Jun ablated NRAGE-mediated caspase activation and NRAGE-induced cell death. These findings identify NRAGE as a p75NTR interactor capable of inducing caspase activation and cell death through a JNK-dependent mitochondrial apoptotic pathway.     

SMAD-Independent Down-Regulation of Caveolin-1 by TGF-β: Effects on Proliferation and Survival of Myofibroblasts

Transforming growth factor-β (TGF-β) mediates growth-inhibitory effects on most target cells via activation of the canonical SMAD signaling pathway. This growth-inhibitory activity may be coupled with cellular differentiation. Our studies demonstrate that TGF-β1 inhibits proliferation of primary, non-transformed human lung fibroblasts in association with the induction of myofibroblast differentiation. Differentiated myofibroblasts maintain the capacity to proliferate in response to exogenous mitogenic stimuli and are resistant to serum deprivation-induced apoptosis. These proliferative and anti-apoptotic properties of myofibroblasts are related, in part, to the down-regulation of caveolin-1 (Cav-1) by TGF-β1. Cav-1 down-regulation is mediated by early activation of p38 MAPK and does not require SMAD signaling. In contrast, myofibroblast differentiation is dependent on activation of the SMAD pathway, but not on p38 MAPK. Thus, combinatorial signaling by TGF-β1 of myofibroblast differentiation and down-regulation of Cav-1 by SMAD and p38 MAPK pathways, respectively, confer proliferative and apoptosis-resistant properties to myofibroblasts. Selective targeting of this SMAD-independent, p38-MAPK/Cav-1-dependent pathway is likely to be effective in the treatment of pathological conditions characterized by TGF-β signaling and myofibroblast activation.     

The p75 neurotrophin receptor activates Akt (protein kinase B) through a phosphatidylinositol 3-kinase-dependent pathway

The Akt kinase plays a crucial role in supporting Trk-dependent cell survival, whereas the p75 neurotrophin receptor (p75NTR) facilitates cellular apoptosis. The precise mechanism that p75NTR uses to promote cell death is not certain, but one possibility is that p75NTR-dependent ceramide accumulation inhibits phosphatidylinositol 3-kinase-mediated Akt activation. To test this hypothesis, we developed a system for examining p75NTR-dependent apoptosis and determined the effect of p75NTR on Akt activation. Surprisingly, p75NTR increased, rather than decreased, Akt phosphorylation in a variety of cell types, including human Niemann-Pick fibroblasts, which lack acidic sphingomyelinase activity. The p75NTR expression level required to elicit Akt phosphorylation was much lower than that required to activate the JNK pathway or to mediate apoptosis. We show that p75NTR-dependent Akt phosphorylation was independent of TrkA signaling, required active phosphatidylinositol 3-kinase, and was associated with increased tyrosine phosphorylation of p85 and Shc and with reduced cytosolic tyrosine phosphatase activity. Finally, we show that p75NTR expression increased survival in cells exposed to staurosporine or subjected to serum withdrawal. These findings indicate that p75NTR facilitates cell survival through novel signaling cascades that result in Akt activation.     

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.     

Molecular characterization of neurohybrid cell death induced by Alzheimer's amyloid-beta peptides via p75NTR/PLAIDD

One of the most important pathological features of Alzheimer's disease (AD) is extracellular senile plaques, whose major component is amyloid-beta peptides (Abeta). Abeta binds to the extracellular domain of p75NTR (p75 neurotrophin receptor) and induces neuronal cell death. We investigated the molecular mechanism of Abeta-induced neurotoxicity in detail from the standpoint of interaction between p75NTR and its recently identified relative, PLAIDD (p75-like apoptosis-inducing death domain). Using F11 neuronal hybrid cells, we demonstrate that there are two distinct pathways for Abeta-induced toxicity mediated by p75NTR. One pathway that has been previously elucidated, is mediated by p75NTR, Go, JNK, NADPH oxidase and caspase3-related caspases. We found that PLAIDD and Gi proteins, heterotrimeric G proteins, are involved in the alternative Abeta-induced neurotoxicity mediated by p75NTR. The alternative pathway triggered by Abeta is thus mediated by p75NTR, PLAIDD, Gi, JNK, NADPH oxidase and caspase3-related caspases. In addition, we found that HN, ADNF, IGF-I, or bFGF inhibits both pathways of Abeta-induced neurotoxicity mediated by p75NTR.     

Activation of neutral-sphingomyelinase,MAPKs, and p75 NTR-mediating caffeic acid phenethyl ester–induced apoptosis in C6 glioma cells

Background

Caffeic acid phenethyl ester (CAPE), a component of propolis, is reported to possess anti-inflammatory, anti-bacterial, anti-viral, and anti-tumor activities. Previously, our laboratory demonstrated the in vitro and in vivo bioactivity of CAPE and addressed the role of p53 and the p38 mitogen-activated protein kinase (MAPK) pathway in regulating CAPE-induced apoptosis in C6 glioma cells.

Results

C6 cancer cell lines were exposed to doses of CAPE; DNA fragmentation and MAPKs and NGF/P75NTR levels were then determined. SMase activity and ceramide content measurement as well as western blotting analyses were performed to clarify molecular changes. The present study showed that CAPE activated neutral sphingomyelinase (N-SMase), which led to the ceramide-mediated activation of MAPKs, including extracellular signal-regulated kinase (ERK), Jun N-terminus kinase (JNK), and p38 MAPK. In addition, CAPE increased the expression of nerve growth factor (NGF) and p75 neurotrophin receptor (p75NTR). The addition of an N-SMase inhibitor, GW4869, established that NGF/p75NTR was the downstream target of N-SMase/ceramide. Pretreatment with MAPK inhibitors demonstrated that MEK/ERK and JNK acted upstream and downstream, respectively, of NGF/p75NTR. Additionally, CAPE-induced caspase 3 activation and poly [ADP-ribose] polymerase cleavage were reduced by pretreatment with MAPK inhibitors, a p75NTR peptide antagonist, or GW4869.

Conclusions

Taken together, N-SMase activation played a pivotal role in CAPE-induced apoptosis by activation of the p38 MAPK pathway and NGF/p75NTR may explain a new role of CAPE induced apoptosis in C6 glioma.     

NRAGE, a p75NTR adaptor protein, is required for developmental apoptosis in vivo

NRAGE (also known as Maged1, Dlxin) is a member of the MAGE gene family that may play a role in the neuronal apoptosis that is regulated by the p75 neurotrophin receptor (p75NTR). To test this hypothesis in vivo, we generated NRAGE knockout mice and found that NRAGE deletion caused a defect in developmental apoptosis of sympathetic neurons of the superior cervical ganglia, similar to that observed in p75NTR knockout mice. Primary sympathetic neurons derived from NRAGE knockout mice were resistant to apoptosis induced by brain-derived neurotrophic factor (BDNF), a pro-apoptotic p75NTR ligand, and NRAGE-deficient sympathetic neurons show attenuated BDNF-dependent JNK activation. Hair follicle catagen is an apoptosis-like process that is dependent on p75NTR signaling; we show that NRAGE and p75NTR show regulated co-expression in the hair follicle and that identical defects in hair follicle catagen are present in NRAGE and p75NTR knockout mice. Interestingly, NRAGE knockout mice have severe defects in motoneuron apoptosis that are not observed in p75NTR knockout animals, raising the possibility that NRAGE may facilitate apoptosis induced by receptors other than p75NTR. Together, these studies demonstrate that NRAGE plays an important role in apoptotic-signaling in vivo.     

p75 neurotrophin receptor inhibits invasion and metastasis of gastric cancer

The p75 neurotrophin receptor (p75NTR) is a focus for study at present. However, its function in gastric cancer was not elucidated. Here, we investigated its relation with metastasis of gastric cancer. By immunohistochemistry, we found that the positive rate of p75NTR expression in metastatic gastric cancer was 15.09% (16 of 106), which was lower compared with nonmetastatic gastric cancer (64.15%; 68 of 106). The average staining score in nonmetastatic gastric cancer was significantly higher than in metastatic gastric cancer (1.21 +/- 0.35 versus 0.23 +/- 0.18; P<0.01). p75NTR protein level was also lowly expressed in the highly liver-metastatic gastric cancer cell line XGC9811-L compared with other gastric cancer cell lines by Western blotting. It could also significantly inhibit the in vitro adhesive, invasive, and migratory and in vivo metastatic abilities of gastric cancer cell lines SGC7901 and MKN45 by reducing urokinase-type plasminogen activator (uPA) and matrix metalloproteinase (MMP)-9 proteins and by increasing tissue inhibitor of matrix metalloproteinase (TIMP)-1 protein. Further studies showed that p75NTR could suppress the nuclear factor-kappaB (NF-kappaB) signal. SN50, a specific inhibitor of NF-kappaB, which could inhibit in vitro invasive and migratory abilities of gastric cancer cells, reduced expression of uPA and MMP9 proteins and increased expression of TIMP1 protein. Taken together, p75NTR had the function of inhibiting the invasive and metastatic abilities of gastric cancer cells, which was mediated, at least partially, by down-regulation of uPA and MMP9 proteins and up-regulation of TIMP1 protein via the NF-kappaB signal transduction pathway. Our studies suggested that p75NTR may be used as a new potential therapeutic target in metastatic gastric cancer.