Apoptosis related protein-1 triggers melanoma cell death via interaction with the juxtamembrane region of p75 neurotrophin receptor

Although chemotherapeutic drugs could theoretically target all metastatic sites, current treatments do not provide complementary therapeutics. Therefore, the development of an alternative approach replacing the traditional therapy is urgently needed. To assess the killing efficiency of the functionally identified apoptosis-related protein (APR)-1 in melanoma cells, we established a system for the regulated expression of APR-1. The induction of APR-1 expression caused apoptosis of melanoma cells via the interaction with the juxtamembrane region of p75 neurotrophin receptor (p75NTR), and possible also via the competition with tumour necrosis factor receptor-associated factor-6 (TRAF6) and the catalytic receptor of neurotrophin (Trk) for the same p75NTR interacting site. The accumulation of APR-1 in melanoma cells may block the physical association of p75NRT with TRAF6 and/or Trk, leading to the disruption of both NF-κB and extracellular signal-regulated kinase (ERK) pathways. Also, accumulation of APR-1 protein enhanced the activity of both c-Jun-N-terminal kinase (JNK) and p38 pathways. However, the analysis of APR-1-modulated pathways demonstrated the involvement of apoptosis-regulating kinase 1-JNK/p38 pathway in the induction of Bax expression leading to both mitochondrial dysregulation [as demonstrated by the loss of mitochondrial membrane potential, the release of both cytochrome c and apoptosis-inducing factor into cytoplasm, and cleavage of caspase-9, caspase-3 and poly (ADP-ribose) polymerase (PARP)] and endoplasmic reticulum stress as demonstrated by the increase of intracellular Ca(2+) release. Thus, besides the analysis of its pro-apoptotic function, our data provide insight into the molecular mechanism of APR-1-induced apoptosis of melanoma cells.     

p75 neurotrophin receptor interacts with and promotes BACE1 localization in endosomes aggravating amyloidogenesis

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by a progressive deposition of amyloid beta (Aβ) and dysregulation of neurotrophic signaling, causing synaptic dysfunction, loss of memory, and cell death. The expression of p75 neurotrophin receptor is elevated in the brain of AD patients, suggesting its involvement in this disease. However, the exact mechanism of its action is not yet clear. Here, we show that p75 interacts with beta‐site amyloid precursor protein cleaving enzyme‐1 (BACE1), and this interaction is enhanced in the presence of Aβ. Our results suggest that the colocalization of BACE1 and amyloid precursor protein (APP) is increased in the presence of both Aβ and p75 in cortical neurons. In addition, the localization of APP and BACE1 in early endosomes is increased in the presence of Aβ and p75. An increased phosphorylation of APP‐Thr668 and BACE1‐Ser498 by c‐Jun N‐terminal kinase (JNK) in the presence of Aβ and p75 could be responsible for this localization. In conclusion, our study proposes a potential involvement in amyloidogenesis for p75, which may represent a future therapeutic target for AD.

Cover Image for this Issue: doi. 10.1111/jnc.14163 .

     

p75 Neurotrophin receptor mediates neurotrophin activation of NF‐kappa B and induction of iNOS expression in P19 neurons

P19 embryonic carcinoma cells can be differentiated into neurons that form synaptic connections and that produce a variety of neurotransmitters. Results of RT‐PCR indicate that P19 neurons express several neurotrophin receptors (p75^NTR, trkB, and trkC, but not trkA) but they do not express any of the four neurotrophins. Consistent with the presence of trkB but not trkA, BDNF causes rapid phosphorylation of MAP kinases ERK1 and ERK2, but NGF does not. Neurotrophins induce translocation of NF‐κB into the nucleus. All four neurotrophins induce activation of NF‐κB in a biphasic manner. This effect is apparently mediated by p75^NTR, because an inhibitor of trk receptors, K252a, does not inhibit activation of NF‐κB. Instead, K252a itself promotes activation of NF‐κB and this effect is additive with the effect of neurotrophins. Inhibition of reactive oxygen intermediates with PDTC completely abolishes basal activity of NF‐κB and strongly inhibits activation of NF‐κB by neurotrophins, indicating an important role of reactive oxygen intermediates in the pathway by which neurotrophins activate NF‐κB. NF‐κB is known to promote expression of the iNOS gene. We found that all four neurotrophins increased iNOS mRNA levels, resulting in increased accumulation of iNOS protein. In contrast, none of the neurotrophins stimulated nNOS mRNA or protein synthesis. PDTC abolishes constitutive and neurotrophin‐induced expression of iNOS mRNA and protein and abolishes constitutive expression of nNOS mRNA, suggesting that reactive oxygen intermediates promote expression of nNOS. © 2003 Wiley Periodicals, Inc. J Neurobiol 55: 191–203, 2003     

Constitutive phosphorylation of TrkC receptors in cultured cerebellar granule neurons might be responsible for the inability of NT-3 to increase neuronal survival and to activate p21 ras

The neurotrophins brain derived neurotrophic factor (BDNF) and neurotrophin-3 (NT-3) are both expressed in developing cerebellum in addition to their tyrosine kinase receptors, TrkB and TrkC. In contrast to BDNF, NT-3 has only a negligible or a transient survival activity on cultured cerebellar granule neurons. The granule neurons however, express both TrkC and Trk B receptors which suggests a basic difference in signaling between BDNF and NT-3 in these neurons. Here we have studied whether this difference can be attributed to the presence of alternative TrkC receptor variants on the granule neurons and which signaling pathway is specifically activated by BDNF but not by NT-3 in these neurons. Using RT-PCR it was shown that the cerebellar granule neurons express the full length TrkC receptor, in addition to variant receptors containing small inserts in the receptor tyrosine kinase domain. There was no dramatic change in the relative amounts of different TrkC receptors during development. However, we found the TrkC receptor constitutively phosphorylated even in the absence of added ligand suggesting an interaction of TrkC with endogenously produced NT-3. In addition, NT-3 was able to phosphorylate the BDNF receptor, TrkB but only at higher concentration (50 ng/ml). There were also distinct differences in the activation of intracellular molecules by BDNF and NT-3. Thus, p21 Ras and PLCγ were activated by BDNF but not by NT-3 whereas both BDNF and NT-3 increased calcium and c-fos mRNA in the granule neurons. These results show that differential activation of specific intracellular pathways such as that of p21 Ras determines the specific effects of BDNF and NT-3 on granule neuron survival. In addition, since calcium is increased by NT-3 in the cerebellar granule neurons, this neurotrophin might have some unknown important effects on these neurons. Special issue dedicated to Dr. Hans Thoenen.     

The p75 neurotrophin receptor: effects on neuron survival in vitro and interaction with death domain-containing adaptor proteins

The p75 neurotrophin receptor (p75NTR) is a death domain (DD) containing receptor of the TNF/FAS(APO-1) family. p75NTR has recently been shown to mediate apoptosis in certain types of neurons as well as in oligodendrocytes. The molecular mechanisms by which p75NTR stimulates apoptosis are still unknown. Here, we have tested whether overexpression of p75NTR could modulate survival of sympathetic neurons cultured in the presence or absence of NGF. Moreover, using the yeast two-hybrid system, we tested whether p75NTR intracellular domain was able to dimerize or interact with known DD-containing proteins including FADD, RIP, RAIDD and TRADD. We found that over-expression of p75NTR had no effect on the survival of sympathetic neurons cultured in the presence of NGF but instead delayed neuronal death following NGF deprivation. These results strongly support the finding that p75NTR is not involved in the apoptosis process induced by NGF deprivation in sympathetic neurons. We also foun d that the intracellular domain of p75NTR failed to associate either with itself or with other known DD-containing proteins. This suggests that the mechanisms by which p75NTR triggers apoptosis in certain cell types are different from those used by other receptors of the TNF/FAS family.     

The expression of p75 neurotrophin receptor protects against the neurotoxicity of soluble oligomers of beta-amyloid

In this paper, evidence is provided that p75 neurotrophin receptor (p75NTR) exerts an opposite role on the cytotoxic function of β-amyloid (Aβ) depending on the different state of the peptide, fibrillar or oligomeric soluble form. Previous work in our laboratory has shown that the expression of p75NTR is required for cell death in vitro by Aβ peptides in fibrillar form (G. Perini, V. Della-Bianca, V. Politi, G. Della Valle, I. Dal-Pra, F. Rossi, U. Armato. Role of p75 neurotrophin receptor in the neurotoxicity by beta-amyloid peptides and synergistic effect of inflammatory cytokines. J. Exp. Med. 195 (2002) 907-918). In the present study, performed by using the same cell clones and procedures as in previous paper, we show that: (a) soluble oligomers of Aβ(1-42) exert a cytotoxic activity independent of p75NTR, (b) the expression of p75NTR exerts a protective role against the toxic activity of soluble oligomers, (c) this role is due to an active function of the juxtamembrane sequence of the cytoplasmic region of p75NTR and (d) the protective function is mediated by phosphatidylinositide 3-kinase (PI3K) activity.     

Structural basis of NF-κB signaling by the p75 neurotrophin receptor interaction with adaptor protein TRADD through their respective death domains

The p75 neurotrophin receptor (p75^NTR) is a critical mediator of neuronal death and tissue remodeling and has been implicated in various neurodegenerative diseases and cancers. The death domain (DD) of p75^NTR is an intracellular signaling hub and has been shown to interact with diverse adaptor proteins. In breast cancer cells, binding of the adaptor protein TRADD to p75^NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75^NTR signaling remain poorly understood. Here we report an NMR structure of the p75^NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75^NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75^NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75^NTR in the regulation of canonical NF-κB signaling and cell survival in CGNs. Our results provide a new structural framework for understanding how the recruitment of TRADD to p75^NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75^NTR, to propagate downstream signaling in developing neurons.     

Insights into the interactions between HIV-1 integrase and human LEDGF/p75 by molecular dynamics simulation and free energy calculation

Human cellular protein LEDGF/p75 (lens epithelium-derived growth factor) is an important binding partner of human immunodeficiency virus type 1 (HIV-1) integrase (IN). Without LEDGF/p75, HIV-1 can not complete its life cycle. To study the detailed interactions between LEDGF/p75 and HIV-1 IN, and then obtain the hotspots at the binding interface, 13 ns molecular dynamics simulations were carried out here. One-hundred snapshots extracted from the last 4 ns trajectories were used for calculation of binding free energy and decomposition of the energy by residue. First, the structural changes and their dynamic interactions were investigated focused on the production stage. And then, the free energy was discussed. On the basis of the above results, it could be suggested that residues Gln168, Glu170, and Thr174 in chain A of IN, Thr125, and Trp131 in chain B of IN as well as Ile365, Asp366, Phe406, and Val408 in LEDGF/p75 were responsible for their binding. These results might be helpful for discovery and design of small molecules to interrupt the interaction between HIV-1 IN and LEDGF/p75.     

Temporally restricted death and the role of p75NTR as a survival receptor in the developing sensory nervous system

The peripheral somatosensory system overproduces neurons early in development followed by a period of cell death during final target innervation. The decision to survive or die in somatosensory neurons of the dorsal root ganglion (DRG) is mediated by target‐derived neurotrophic factors and their cognate receptors. Subsets of peripheral somatosensory neurons can be crudely defined by the neurotrophic receptors that they express: peptidergic nociceptors (TrkA+), nonpeptidergic nociceptors (Ret+), mechanoreceptors (Ret+ or TrkB+), and proprioceptors (TrkC+). A direct comparison of early developmental timing between these subsets has not been performed. Here we characterized the accumulation and death of TrkA, B, C, and Ret+ neurons in the DRG as a function of developmental time. We find that TrkB, TrkC, and Ret‐expressing neurons in the DRG complete developmental cell death prior to TrkA‐expressing neurons. Given the broadly defined roles of the neurotrophin receptor p75NTR in augmenting neurotrophic signaling in sensory neurons, we investigated its role in supporting the survival of these distinct subpopulations. We find that TrkA+, TrkB+, and TrkC+ sensory neuron subpopulations require p75NTR for survival, but proliferating progenitors do not. These data demonstrate how diverging sensory neurons undergo successive waves of cell death and how p75NTR represses the magnitude, but not developmental window of this culling. © 2018 Wiley Periodicals, Inc. Develop Neurobiol 78: 701–717, 2018     

Towards the PET radiotracer for p75 neurotrophin receptor: [11C]LM11A-24 shows biological activity in vitro,but unfavorable ex vivo and in vivo profile

Mature neurotrophins as well as their pro forms are critically involved in the regulation of neuronal functions. They are signaling through three distinct types of receptors: tropomyosin receptor kinase family (TrkA/B/C), p75 neurotrophin receptor (p75^NTR) and sortilin. Aberrant expression of p75^NTR in the CNS is implicated in a variety of neurodegenerative diseases, including Alzheimer’s disease. The goal of this work was to evaluate one of the very few reported p75^NTR small molecule ligands as a lead compound for development of novel PET radiotracers for in vivo p75^NTR imaging. Here we report that previously described ligand LM11A-24 shows significant inhibition of carbachol-induced persistent firing (PF) of entorhinal cortex (EC) pyramidal neurons in wild-type mice via selective interaction with p75^NTR. Based on this electrophysiological assay, the compound has very high potency with an EC₅₀ <10 nM. We optimized the radiosynthesis of [¹¹C]LM11A-24 as the first attempt to develop PET radioligand for in vivo imaging of p75^NTR. Despite some weak interaction with CNS tissues, the radiolabeled compound showed unfavorable in vivo profile presumably due to high hydrophilicity.     

Association between Presenilin-1 and TRAF6 modulates regulated intramembrane proteolysis of the p75NTR neurotrophin receptor

The p75 neurotrophin receptor (p75^NTR) is a member of the tumour necrosis factor superfamily, which relies on the recruitment of cytosolic protein partners including the tumour necrosis factor receptor-associated factor 6 (TRAF6) E3 ubiquitin ligase to produce cellular responses. Recently, p75^NTR was also shown to undergo presenilin-dependent, γ-secretase-mediated regulated intramembrane proteolysis. In this study, we report the characterization of a highly conserved TRAF6-binding site (PxExxAr/Ac) in presenilin-1 (PS1) that mediates nerve growth factor (NGF)-induced association between PS1 and TRAF6. We demonstrate that disruption of this interaction between PS1 and TRAF6 inhibits TRAF6 autoubiquitination and γ-secretase cleavage of p75^NTR. Additionally, we show that PS1-deficiency antagonizes NGF-induced I-κB degradation. Finally, we also show that p75^NTR is a substrate for TRAF6-mediated ubiquitination and that TRAF6 E3 ligase activity is required for regulated intramembrane proteolysis of p75^NTR. In summary, our data suggest that an NGF-induced association between PS1 and TRAF6 influences regulated intramembrane proteolysis of p75^NTR.     

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.     

Blockade of TrkB but not p75NTR activates a subpopulation of quiescent neural precursor cells and enhances neurogenesis in the adult mouse hippocampus

Brain‐derived neurotrophic factor (BDNF) signaling plays a major role in the regulation of hippocampal neurogenesis in the adult brain. While the majority of studies suggest that this is due to its effect on the survival and differentiation of newborn neurons, it remains unclear whether this signaling directly regulates neural precursor cell (NPC) activity and which of its two receptors, TrkB or the p75 neurotrophin receptor (p75^NTR) mediates this effect. Here, we examined both the RNA and protein expression of these receptors and found that TrkB but not p75^NTR receptors are expressed by hippocampal NPCs in the adult mouse brain. Using a clonal neurosphere assay, we demonstrate that pharmacological blockade of TrkB receptors directly activates a distinct subpopulation of NPCs. Moreover, we show that administration of ANA‐12, a TrkB‐selective antagonist, in vivo either by systemic intraperitoneal injection or by direct infusion within the hippocampus leads to an increase in the production of new neurons. In contrast, we found that NPC‐specific knockout of p75^NTR had no effect on the proliferation of NPCs and did not alter neurogenesis in the adult hippocampus. Collectively, these results demonstrate a novel role of TrkB receptors in directly regulating the activity of a subset of hippocampal NPCs and suggest that the transient blockade of these receptors could be used to enhance adult hippocampal neurogenesis.     

An adenosine A receptor agonist induces sleep by increasing GABA release in the tuberomammillary nucleus to inhibit histaminergic systems in rats

The adenosine A(2A) receptor (A(2A)R) has been demonstrated to play a crucial role in the regulation of the sleep process. However, the molecular mechanism of the A(2A)R-mediated sleep remains to be elucidated. Here we used electroencephalogram and electromyogram recordings coupled with in vivo microdialysis to investigate the effects of an A(2A)R agonist, CGS21680, on sleep and on the release of histamine and GABA in the brain. In freely moving rats, CGS21680 applied to the subarachnoid space underlying the rostral basal forebrain significantly promoted sleep and inhibited histamine release in the frontal cortex. The histamine release was negatively correlated with the amount of non-rapid eye movement sleep (r = - 0.652). In urethane-anesthetized rats, CGS21680 inhibited histamine release in both the frontal cortex and medial pre-optic area in a dose-dependent manner, and increased GABA release specifically in the histaminergic tuberomammillary nucleus but not in the frontal cortex. Moreover, the CGS21680-induced inhibition of histamine release was antagonized by perfusion of the tuberomammillary nucleus with a GABA(A) antagonist, picrotoxin. These results suggest that the A(2A)R agonist induced sleep by inhibiting the histaminergic system through increasing GABA release in the tuberomammillary nucleus.     

Strained contacts with the cell membrane may influence ligand affinity to G protein coupled receptors: a case of free fatty acid receptor 1 agonists

A set of 1,3,4-thiadiazole-2-carboxamides bearing a substituted biphenyl in the amide portion was synthesised and tested for agonistic activity towards free fatty acid receptor 1 (FFA1). The observed activity trends were impossible to rationalised based solely on the docking energy scores of Glide SP. On the contrary, when the phospholipid cell membrane bilayer was reconstructed around FFA1, it became apparent that inactive compounds displayed significant strained contacts with the membrane while for active compounds the strain was noticeably lower. These findings justify using the improved docking protocol for modelling GPCR-ligand interactions which uses the crystal structure of the receptor and a reconstructed portion of a cell membrane.     

Regulation of insulin resistance by targeting the insulin‐like growth factor 1 receptor with microRNA‐122‐5p in hepatic cells

Insulin resistance (IR) is a common etiology of type 2 diabetes (T2D) defined by a state of decreased reactivity to insulin in multiple organs, such as the liver. This study aims to investigate how microRNA‐122‐5p (miR‐122) regulates the hepatic IR in vitro. We first found that the miR‐122 level was upregulated in the liver of rats fed with a high‐fat diet and injected with streptozotocin (T2D rats), while the expression level of insulin‐like growth factor 1 receptor (IGF‐1R), a potential target of miR‐122, was downregulated in the diabetic liver. In vitro, glucosamine‐induced IR was introduced in HepG2 hepatic cells, and the levels of miR‐122 and IGF‐1R were further assessed. An increase of miR‐122 level and a decrease of IGF‐IR level were observed in IR hepatic cells, which was the same as that in the diabetic liver. Results of the luciferase reporter assay validated IGF‐1R as a direct target of miR‐122. Moreover, in IR HepG2 cells, antagonizing miR‐122 with its specific inhibitor enhanced glucose uptake and suppressed the expression of glucose 6‐phosphatase and phosphoenolpyruvate carboxykinase, two key enzymes in regulating gluconeogenesis. Such alterations induced by the miR‐122 inhibitor in IR hepatic cells were impaired when IGF‐1R was simultaneously knocked down. In addition, the PI3K/Akt pathway was deactivated in IR cells, and then reactivated with miR‐122 inhibitor transfection. In conclusion, our study demonstrates that miR‐122 is able to regulate IR in hepatic cells by targeting IGF‐1R.