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 (p75NTR) 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 p75NTR 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 p75NTR depends on nerve growth factor and promotes cell survival. However, the structural mechanism and functional significance of TRADD recruitment in neuronal p75NTR signaling remain poorly understood. Here we report an NMR structure of the p75NTR-DD and TRADD-DD complex and reveal the mechanism of specific recognition of the TRADD-DD by the p75NTR-DD mainly through electrostatic interactions. Furthermore, we identified spatiotemporal overlap of p75NTR and TRADD expression in developing cerebellar granule neurons (CGNs) at early postnatal stages and discover the physiological relevance of the interaction between TRADD and p75NTR 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 p75NTR through DD interactions creates a membrane-proximal platform, which can be efficiently regulated by various neurotrophic factors through extracellular domains of p75NTR, to propagate downstream signaling in developing neurons.

Oxygen-dependent cleavage of the p75 neurotrophin receptor triggers stabilization of HIF-1α.

Homeostatic control of oxygen availability allows cells to survive oxygen deprivation. Although the transcription factor hypoxia-inducible factor 1α (HIF-1α) is the main regulator of the hypoxic response, the upstream mechanisms required for its stabilization remain elusive. Here, we show that p75 neurotrophin receptor (p75(NTR)) undergoes hypoxia-induced γ-secretase-dependent cleavage to provide a positive feed-forward mechanism required for oxygen-dependent HIF-1α stabilization. The intracellular domain of p75(NTR) directly interacts with the evolutionarily conserved zinc finger domains of the E3 RING ubiquitin ligase Siah2 (seven in absentia homolog 2), which regulates HIF-1α degradation. p75(NTR) stabilizes Siah2 by decreasing its auto-ubiquitination. Genetic loss of p75(NTR) dramatically decreases Siah2 abundance, HIF-1α stabilization, and induction of HIF-1α target genes in hypoxia. p75(NTR-/-) mice show reduced HIF-1α stabilization, vascular endothelial growth factor (VEGF) expression, and neoangiogenesis after retinal hypoxia. Thus, hypoxia-induced intramembrane proteolysis of p75(NTR) constitutes an apical oxygen-dependent mechanism to control the magnitude of the hypoxic response.

Generation of rationally-designed nerve growth factor (NGF) variants with receptor specificity.

Nerve growth factor (NGF) is the prototypic member of the neurotrophin family and binds two receptors, TrkA and the 75 kDa neurotrophin receptor (p75NTR), through which diverse and sometimes opposing effects are mediated. Using the FoldX protein design algorithm, we generated eight NGF variants with different point mutations predicted to have altered binding to TrkA or p75NTR. Of these, the I31R NGF variant exhibited specific binding to p75NTR. The generation of this NGF variant with selective affinity for p75NTR can be used to enhance understanding of neurotrophin receptor imbalance in diseases and identifies a key targetable residue for the development of small molecules to disrupt binding of NGF to TrkA with potential uses in chronic pain.

Death Domain Signaling by Disulfide-Linked Dimers of the p75 Neurotrophin Receptor Mediates Neuronal Death in the CNS.

UNLABELLED: The p75 neurotrophin receptor (p75(NTR)) mediates neuronal death in response to neural insults by activating a caspase apoptotic pathway. The oligomeric state and activation mechanism that enable p75(NTR) to mediate these effects have recently been called into question. Here, we have investigated mutant mice lacking the p75(NTR) death domain (DD) or a highly conserved transmembrane (TM) cysteine residue (Cys(259)) implicated in receptor dimerization and activation. Neuronal death induced by proneurotrophins or epileptic seizures was assessed and compared with responses in p75(NTR) knock-out mice and wild-type animals. Proneurotrophins induced apoptosis of cultured hippocampal and cortical neurons from wild-type mice, but mutant neurons lacking p75(NTR), only the p75(NTR) DD, or just Cys(259) were all equally resistant to proneurotrophin-induced neuronal death. Homo-FRET anisotropy experiments demonstrated that both NGF and proNGF induce conformational changes in p75(NTR) that are dependent on the TM cysteine. In vivo, neuronal death induced by pilocarpine-mediated seizures was significantly reduced in the hippocampus and somatosensory, piriform, and entorhinal cortices of all three strains of p75(NTR) mutant mice. Interestingly, the levels of protection observed in mice lacking the DD or only Cys(259) were identical to those of p75(NTR) knock-out mice even though the Cys(259) mutant differed from the wild-type receptor in only one amino acid residue. We conclude that, both in vitro and in vivo, neuronal death induced by p75(NTR) requires the DD and TM Cys(259), supporting the physiological relevance of DD signaling by disulfide-linked dimers of p75(NTR) in the CNS. SIGNIFICANCE STATEMENT: A detailed understanding of the physiological significance of distinct structural determinants in the p75 neurotrophin receptor (p75(NTR)) is crucial for the identification of suitable drug targets in this receptor. We have tested the relevance of the p75(NTR) death domain (DD) and the highly conserved transmembrane residue Cys(259) for the ability of p75(NTR) to induce apoptosis in neurons of the CNS using gene-targeted mutant mice. The physiological importance of these determinants had been contested in some recent in vitro studies. Our results indicate a requirement for DD signaling by disulfide-linked dimers of p75(NTR) for neuronal death induced by proneurotrophins and epileptic seizures. These new mouse models will be useful for clarifying different aspects of p75(NTR) physiology.

Heterodimerization of p45-p75 modulates p75 signaling: structural basis and mechanism of action.

The p75 neurotrophin receptor, a member of the tumor necrosis factor receptor superfamily, is required as a co-receptor for the Nogo receptor (NgR) to mediate the activity of myelin-associated inhibitors such as Nogo, MAG, and OMgp. p45/NRH2/PLAIDD is a p75 homologue and contains a death domain (DD). Here we report that p45 markedly interferes with the function of p75 as a co-receptor for NgR. P45 forms heterodimers with p75 and thereby blocks RhoA activation and inhibition of neurite outgrowth induced by myelin-associated inhibitors. p45 binds p75 through both its transmembrane (TM) domain and DD. To understand the underlying mechanisms, we have determined the three-dimensional NMR solution structure of the intracellular domain of p45 and characterized its interaction with p75. We have identified the residues involved in such interaction by NMR and co-immunoprecipitation. The DD of p45 binds the DD of p75 by electrostatic interactions. In addition, previous reports suggested that Cys257 in the p75 TM domain is required for signaling. We found that the interaction of the cysteine 58 of p45 with the cysteine 257 of p75 within the TM domain is necessary for p45-p75 heterodimerization. These results suggest a mechanism involving both the TM domain and the DD of p45 to regulate p75-mediated signaling.

Nerve Growth Factor Signaling from Membrane Microdomains to the Nucleus: Differential Regulation by Caveolins.

Membrane microdomains or "lipid rafts" have emerged as essential functional modules of the cell, critical for the regulation of growth factor receptor-mediated responses. Herein we describe the dichotomy between caveolin-1 and caveolin-2, structural and regulatory components of microdomains, in modulating proliferation and differentiation. Caveolin-2 potentiates while caveolin-1 inhibits nerve growth factor (NGF) signaling and subsequent cell differentiation. Caveolin-2 does not appear to impair NGF receptor trafficking but elicits prolonged and stronger activation of MAPK (mitogen-activated protein kinase), Rsk2 (ribosomal protein S6 kinase 2), and CREB (cAMP response element binding protein). In contrast, caveolin-1 does not alter initiation of the NGF signaling pathway activation; rather, it acts, at least in part, by sequestering the cognate receptors, TrkA and p75NTR, at the plasma membrane, together with the phosphorylated form of the downstream effector Rsk2, which ultimately prevents CREB phosphorylation. The non-phosphorylatable caveolin-1 serine 80 mutant (S80V), no longer inhibits TrkA trafficking or subsequent CREB phosphorylation. MC192, a monoclonal antibody towards p75NTR that does not block NGF binding, prevents exit of both NGF receptors (TrkA and p75NTR) from lipid rafts. The results presented herein underline the role of caveolin and receptor signaling complex interplay in the context of neuronal development and tumorigenesis.

Detection of p75NTR Trimers: Implications for Receptor Stoichiometry and Activation.

The p75 neurotrophin receptor (p75(NTR)) is a multifunctional receptor that participates in many critical processes in the nervous system, ranging from apoptosis to synaptic plasticity and morphological events. It is a member of the tumor necrosis factor receptor (TNFR) superfamily, whose members undergo trimeric oligomerization. Interestingly, p75(NTR) interacts with dimeric ligands (i.e., proneurotrophins or mature neurotrophins), but several of the intracellular adaptors that mediate p75(NTR) signaling are trimeric (i.e., TNFR-associated factor 6 or TRAF6). Consequently, the active receptor signaling unit remains uncertain. To identify the functional receptor complex, we evaluated its oligomerization in vitro and in mice brain tissues using a combination of biochemical techniques. We found that the most abundant homotypic arrangement for p75(NTR) is a trimer and that monomers and trimers coexist at the cell surface. Interestingly, trimers are not required for ligand-independent or ligand-dependent p75(NTR) activation in a growth cone retraction functional assay. However, monomers are capable of inducing acute morphological effects in neurons. We propose that p75(NTR) activation is regulated by its oligomerization status and its levels of expression. These results indicate that the oligomeric state of p75(NTR) confers differential responses and offers an explanation for the diverse and contradictory actions of this receptor in the nervous system. SIGNIFICANCE STATEMENT: The p75 neurotrophin receptor (p75(NTR)) regulates a wide range of cellular functions, including apoptosis, neuronal processes remodeling, and synaptic plasticity. The goal of our work was to inquire whether oligomers of the receptor are required for function. Here we report that p75(NTR) predominantly assembles as a trimer, similar to other tumor necrosis factor receptors. Interestingly, monomers and trimers coexist at the cell surface, but trimers are not required for p75(NTR) activation in a functional assay. However, monomers are capable of inducing acute morphological effects in neurons. Identification of the oligomerization state of p75(NTR) begins to provide insights to the mechanisms of signal initiation of this noncatalytic receptor, as well as to develop therapeutic interventions to diminish its activity.

p75NTR ectodomain is a physiological neuroprotective molecule against amyloid-beta toxicity in the brain of Alzheimer's disease.

In Alzheimer's disease (AD), neurodegenerative signals such as amyloid-beta (Aß) and the precursors of neurotrophins, outbalance neurotrophic signals, causing synaptic dysfunction and neurodegeneration. The neurotrophin receptor p75 (p75NTR) is a receptor of Aß and mediates Aß-induced neurodegenerative signals. The shedding of its ectodomain from the cell surface is physiologically regulated; however, the function of the diffusible p75NTR ectodomain (p75ECD) after shedding remains largely not known. Here, we show that p75ECD levels in cerebrospinal fluid and in the brains of Alzheimer's patients and amyloid-beta precursor protein (APP)/PS1 transgenic mice were significantly reduced, due to inhibition of the sheddase-tumor necrosis factor-alpha-converting enzyme by Aß. Restoration of p75ECD to the normal level by brain delivery of the gene encoding human p75ECD before or after Aß deposition in the brain of APP/PS1 mice reversed the behavioral deficits and AD-type pathologies, such as Aß deposit, apoptotic events, neuroinflammation, Tau phosphorylation and loss of dendritic spine, neuronal structures and synaptic proteins. Furthermore, p75ECD can also reduce amyloidogenesis by suppressing ß-secretase expression and activities. Our data demonstrate that p75ECD is a physiologically neuroprotective molecule against Aß toxicity and would be a novel therapeutic target and biomarker for AD.

NMR Dynamics of Transmembrane and Intracellular Domains of p75NTR in Lipid-Protein Nanodiscs.

P75NTR is a type I integral membrane protein that plays a key role in neurotrophin signaling. However, structural data for the receptor in various functional states are sparse and controversial. In this work, we studied the spatial structure and mobility of the transmembrane and intracellular parts of p75NTR, incorporated into lipid-protein nanodiscs of various sizes and compositions, by solution NMR spectroscopy. Our data reveal a high level of flexibility and disorder in the juxtamembrane chopper domain of p75NTR, which results in the motions of the receptor death domain being uncoupled from the motions of the transmembrane helix. Moreover, none of the intracellular domains of p75NTR demonstrated a propensity to interact with the membrane or to self-associate under the experimental conditions. The obtained data are discussed in the context of the receptor activation mechanism.

Silica Nanopills for Targeted Anticancer Drug Delivery.

Multifunctional SiO2 microtubes for targeted drug delivery are produced with precise control over shape and size by combining lithography and electrochemical etching. The hollow core is loaded with a lipophilic anticancer drug generating nanopills and an antibody is conjugated to the external surface for cancer cell targeting. Results demonstrate selective killing of neuroblastoma cells that express the cognate receptor.

Modulation of lipid kinase PI4KIIα activity and lipid raft association of presenilin 1 underlies γ-secretase inhibition by ginsenoside (20S)-Rg3.

Amyloid ß-peptide (Aß) pathology is an invariant feature of Alzheimer disease, preceding any detectable clinical symptoms by more than a decade. To this end, we seek to identify agents that can reduce Aß levels in the brain via novel mechanisms. We found that (20S)-Rg3, a triterpene natural compound known as ginsenoside, reduced Aß levels in cultured primary neurons and in the brains of a mouse model of Alzheimer disease. The (20S)-Rg3 treatment induced a decrease in the association of presenilin 1 (PS1) fragments with lipid rafts where catalytic components of the γ-secretase complex are enriched. The Aß-lowering activity of (20S)-Rg3 directly correlated with increased activity of phosphatidylinositol 4-kinase IIα (PI4KIIα), a lipid kinase that mediates the rate-limiting step in phosphatidylinositol 4,5-bisphosphate synthesis. PI4KIIα overexpression recapitulated the effects of (20S)-Rg3, whereas reduced expression of PI4KIIα abolished the Aß-reducing activity of (20S)-Rg3 in neurons. Our results substantiate an important role for PI4KIIα and phosphoinositide modulation in γ-secretase activity and Aß biogenesis.

Acute administration of the small-molecule p75(NTR) ligand does not prevent hippocampal neuron loss or development of spontaneous seizures after pilocarpine-induced status epilepticus.

Neurotrophins, such as brain-derived neurotrophic factor (BDNF), are initially expressed in a precursor form (e.g., pro-BDNF) and cleaved to form mature BDNF (mBDNF). After pilocarpine-induced status epilepticus (SE), increases in neurotrophins regulate a wide variety of cell-signaling pathways, including prosurvival and cell-death machinery in a receptor-specific manner. Pro-BDNF preferentially binds to the p75 neurotrophin receptor (p75(NTR) ), whereas mBDNF is the major ligand of the tropomyosin-related kinase receptor. To elucidate a potential role for p75(NTR) in acute stages of epileptogenesis, rats were injected prior to and at onset of SE with LM11A-31, a small-molecule ligand that binds to p75(NTR) to promote survival signaling and inhibit neuronal cell death. Modulation of early p75(NTR) signaling and its effects on electrographic SE, SE-induced neurodegeneration, and subsequent spontaneous seizures were examined after LM11A-31 administration. Despite an established neuroprotective effect of LM11A-31 in several animal models of neurodegenerative disorders (e.g., Alzheimer's disease, traumatic brain injury, and spinal cord injury), high-dose LM11A-31 administration prior to and at onset of SE did not reduce the intensity of electrographic SE, prevent SE-induced neuronal cell injury, or inhibit the progression of epileptogenesis. Further studies are required to understand the role of p75(NTR) activation during epileptogenesis and in seizure-induced cell injury in the hippocampus, among other potential cellular pathologies contributing to the onset of spontaneous seizures. Additional studies utilizing more prolonged treatment with LM11A-31 are required to reach a definite conclusion on its potential neuroprotective role in epilepsy.

Efficacious anticancer drug delivery mediated by a pH-sensitive self-assembly of a conserved tripeptide derived from tyrosine kinase NGF receptor.

We present herein a short tripeptide sequence (Lys-Phe-Gly or KFG) that is situated in the juxtamembrane region of the tyrosine kinase nerve growth factor (Trk NGF) receptors. KFG self-assembles in water and shows a reversible and concentration-dependent switching of nanostructures from nanospheres (vesicles) to nanotubes, as evidenced by dynamic light scattering, transmission electron microscopy, and atomic force microscopy. The morphology change was associated with a transition in the secondary structure. The tripeptide vesicles have inner aqueous compartments and are stable at pH 7.4 but rupture rapidly at pH≈6. The pH-sensitive response of the vesicles was exploited for the delivery of a chemotherapeutic anticancer drug, doxorubicin, which resulted in enhanced cytotoxicity for both drug-sensitive and drug-resistant cells. Efficient intracellular release of the drug was confirmed by fluorescence-activated cell sorting analysis, fluorescence microscopy, and confocal microscopy.

A novel ETV6-NTRK3 gene fusion in congenital fibrosarcoma.

Congenital (or infantile) fibrosarcoma (CFS) is a malignant tumour of fibroblasts that occurs in patients aged two years or younger. CFS is unique among human sarcomas in that it has an excellent prognosis and very low metastatic rate. CFS is histologically identical to adult-type fibrosarcoma (ATFS); however, ATFS is an aggressive malignancy of adults and older children that has a poor prognosis. We report a novel recurrent t(12;15)(p13;q25) rearrangement in CFS that may underlie the distinctive biological properties of this tumour. By cloning the chromosome breakpoints, we show that the rearrangement fuses the ETV6 (also known as TEL) gene from 12p13 with the 15q25 NTRK3 neurotrophin-3 receptor gene (also known as TRKC). Analysis of mRNA revealed the expression of ETV6-NTRK3 chimaeric transcripts in all three CFS tumours analysed. These were not detected in ATFS or infantile fibromatosis (IFB), a histologically similar but benign fibroblastic proliferation occurring in the same age-group as CFS. ETV6-NTRK3 fusion transcripts encode the helix-loop-helix (HLH) protein dimerization domain of ETV6 fused to the protein tyrosine kinase (PTK) domain of NTRK3. Our studies indicate that a chimaeric PTK is expressed in CFS and this may contribute to oncogenesis by dysregulation of NTRK3 signal transduction pathways. Moreover, ETV6-NTRK3 gene fusions provide a potential diagnostic marker for CFS.

Molecular dissection of the myelin-associated glycoprotein receptor complex reveals cell type-specific mechanisms for neurite outgrowth inhibition.

Neuronal Nogo66 receptor-1 (NgR1) binds the myelin inhibitors NogoA, OMgp, and myelin-associated glycoprotein (MAG) and has been proposed to function as the ligand-binding component of a receptor complex that also includes Lingo-1, p75(NTR), or TROY. In this study, we use Vibrio cholerae neuraminidase (VCN) and mouse genetics to probe the molecular composition of the MAG receptor complex in postnatal retinal ganglion cells (RGCs). We find that VCN treatment is not sufficient to release MAG inhibition of RGCs; however, it does attenuate MAG inhibition of cerebellar granule neurons. Furthermore, the loss of p75(NTR) is not sufficient to release MAG inhibition of RGCs, but p75(NTR-/-) dorsal root ganglion neurons show enhanced growth on MAG compared to wild-type controls. Interestingly, TROY is not a functional substitute for p75(NTR) in RGCs. Finally, NgR1(-/-) RGCs are strongly inhibited by MAG. In the presence of VCN, however, NgR1(-/-) RGCs exhibit enhanced neurite growth. Collectively, our experiments reveal distinct and cell type-specific mechanisms for MAG-elicited growth inhibition.

Identification of a linear epitope in sortilin that partakes in pro-neurotrophin binding.

Sortilin acts as a cell surface receptor for pro-neurotrophins (pro-NT) that upon complex formation with the p75 neurotrophin receptor (p75(NTR)) is able to signal neuronal cell death. Here we screened a sortilin peptide library comprising 16-mer overlapping sequences for binding of the pro-domains of nerve growth factor and brain-derived neurotrophic factor. We find that a linear surface-exposed sequence, (163)RIFRSSDFAKNF(174), constitutes an important pro-NT binding epitope in sortilin. Systematic mutational analysis revealed residues Arg(163), Phe(165), Arg(166), and Phe(170) to be critical for the interaction. Expression of a sortilin mutant in which these four amino acids were substituted by alanines disrupted pro-NT binding without affecting receptor heterodimerization with p75(NTR) or binding of ligands that selectively engages the centrally located tunnel in the beta-propeller of sortilin. We furthermore demonstrate that a peptide comprising the ligand-binding epitope can prevent pro-NT-induced apoptosis in RN22 schwannoma cells.

Evolution of GITRL immune function: murine GITRL exhibits unique structural and biochemical properties within the TNF superfamily.

Glucocorticoid-induced TNF receptor ligand (GITRL), a recently identified member of the TNF superfamily, binds to its receptor, GITR, on both effector and regulatory T cells and generates positive costimulatory signals implicated in a wide range of T cell functions. In contrast to all previously characterized homotrimeric TNF family members, the mouse GITRL crystal structure reveals a previously unrecognized dimeric assembly that is stabilized via a unique "domain-swapping" interaction. Consistent with its crystal structure, mouse GITRL exists as a stable dimer in solution. Structure-guided mutagenesis studies confirmed the determinants responsible for dimerization and support a previously unrecognized receptor-recognition surface for mouse GITRL that has not been observed for any other TNF family members. Taken together, the unique structural and biochemical behavior of mouse GITRL, along with the unusual domain organization of murine GITR, support a previously unrecognized mechanism for signaling within the TNF superfamily.

Structural basis for ligand-mediated mouse GITR activation.

Glucocorticoid-induced TNF receptor ligand (GITRL) is a member of the TNF super family (TNFSF). GITRL plays an important role in controlling regulatory T cells. The crystal structure of the mouse GITRL (mGITRL) was determined to 1.8-A resolution. Contrary to the current paradigm that all ligands in the TNFSF are trimeric, mGITRL associates as dimer through a unique C terminus tethering arm. Analytical ultracentrifuge studies revealed that in solution, the recombinant mGITRL exists as monomers at low concentrations and as dimers at high concentrations. Biochemical studies confirmed that the mGITRL dimer is biologically active. Removal of the three terminal residues in the C terminus resulted in enhanced receptor-mediated NF-kappaB activation than by the wild-type receptor complex. However, deletion of the tethering C-terminus arm led to reduced activity. Our studies suggest that the mGITRL may undergo a dynamic population shift among different oligomeric forms via C terminus-mediated conformational changes. We hypothesize that specific oligomeric forms of GITRL may be used as a means to differentially control GITR receptor signaling in diverse cells.