NRH2 is a trafficking switch to regulate sortilin localization and permit proneurotrophin‐induced cell death

Proneurotrophins mediate neuronal apoptosis using a dual receptor complex of sortilin and p75^NTR. Although p75^NTR is highly expressed on the plasma membrane and accessible to proneurotrophin ligands, sortilin is primarily localized to intracellular membranes, limiting the formation of a cell surface co‐receptor complex. Here, we show that the mammalian p75^NTR homologue NRH2 critically regulates the expression of sortilin on the neuronal cell surface and promotes p75^NTR and sortilin receptor complex formation, rendering cells responsive to proneurotrophins. This is accomplished by interactions between the cytoplasmic domains of NRH2 and sortilin that impair lysosomal degradation of sortilin. In proneurotrophin‐responsive neurons, acute silencing of endogenous NRH2 significantly reduces cell surface‐expressed sortilin and abolishes proneurotrophin‐induced neuronal death. Thus, these data suggest that NRH2 acts as a trafficking switch to impair lysosomal‐dependant sortilin degradation and to redistribute sortilin to the cell surface, rendering p75^NTR‐expressing cells susceptible to proneurotrophin‐induced death.     

ProBDNF collapses neurite outgrowth of primary neurons by activating RhoA

Background

Neurons extend their dendrites and axons to build functional neural circuits, which are regulated by both positive and negative signals during development. Brain-derived neurotrophic factor (BDNF) is a positive regulator for neurite outgrowth and neuronal survival but the functions of its precursor (proBDNF) are less characterized.

Methodology/Principal Findings

Here we show that proBDNF collapses neurite outgrowth in murine dorsal root ganglion (DRG) neurons and cortical neurons by activating RhoA via the p75 neurotrophin receptor (p75NTR). We demonstrated that the receptor proteins for proBDNF, p75NTR and sortilin, were highly expressed in cultured DRG or cortical neurons. ProBDNF caused a dramatic neurite collapse in a dose-dependent manner and this effect was about 500 fold more potent than myelin-associated glycoprotein. Neutralization of endogenous proBDNF by using antibodies enhanced neurite outgrowth in vitro and in vivo, but this effect was lost in p75NTR^−/− mice. The neurite outgrowth of cortical neurons from p75NTR deficient (p75NTR^−/−) mice was insensitive to proBDNF. There was a time-dependent reduction of length and number of filopodia in response to proBDNF which was accompanied with a polarized RhoA activation in growth cones. Moreover, proBDNF treatment of cortical neurons resulted in a time-dependent activation of RhoA but not Cdc42 and the effect was absent in p75NTR^−/− neurons. Rho kinase (ROCK) and the collapsin response mediator protein-2 (CRMP-2) were also involved in the proBDNF action.

Conclusions

proBDNF has an opposing role in neurite outgrowth to that of mature BDNF. Our observations suggest that proBDNF collapses neurites outgrowth and filopodial growth cones by activating RhoA through the p75NTR signaling pathway.     

Motoneuron programmed cell death in response to proBDNF

Motoneurons (MN) as well as most neuronal populations undergo a temporally and spatially specific period of programmed cell death (PCD). Several factors have been considered to regulate the survival of MNs during this period, including availability of muscle-derived trophic support and activity. The possibility that target-derived factors may also negatively regulate MN survival has been considered, but not pursued. Neurotrophin precursors, through their interaction with p75(NTR) and sortilin receptors have been shown to induce cell death during development and following injury in the CNS. In this study, we find that muscle cells produce and secrete proBDNF. ProBDNF through its interaction with p75(NTR) and sortilin, promotes a caspase-dependent death of MNs in culture. We also provide data to suggest that proBDNF regulates MN PCD during development in vivo.     

Mapping of the Interaction Site between Sortilin and the p75 Neurotrophin Receptor Reveals a Regulatory Role for the Sortilin Intracellular Domain in p75 Neurotrophin Receptor Shedding and Apoptosis

Neurotrophins comprise a group of neuronal growth factors that are essential for the development and maintenance of the nervous system. However, the immature pro-neurotrophins promote apoptosis by engaging in a complex with sortilin and the p75 neurotrophin receptor (p75^NTR). To identify the interaction site between sortilin and p75^NTR, we analyzed binding between chimeric receptor constructs and truncated p75^NTR variants by co-immunoprecipitation experiments, surface plasmon resonance analysis, and FRET. We found that complex formation between sortilin and p75^NTR relies on contact points in the extracellular domains of the receptors. We also determined that the interaction critically depends on an extracellular juxtamembrane 23-amino acid sequence of p75^NTR. Functional studies further revealed an important regulatory function of the sortilin intracellular domain in p75^NTR-regulated intramembrane proteolysis and apoptosis. Thus, although the intracellular domain of sortilin does not contribute to p75^NTR binding, it does regulate the rates of p75^NTR cleavage, which is required to mediate pro-neurotrophin-stimulated cell death.     

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.     

Amyloid beta1–42 (Aβ42) up‐regulates the expression of sortilin via the p75NTR/RhoA signaling pathway

Sortilin, a Golgi sorting protein and a member of the VPS10P family, is the co‐receptor for proneurotrophins, regulates protein trafficking, targets proteins to lysosomes, and regulates low density lipoprotein metabolism. The aim of this study was to investigate the expression and regulation of sortilin in Alzheimer's disease (AD). A significantly increased level of sortilin was found in human AD brain and in the brains of 6‐month‐old swedish‐amyloid precursor protein/PS1dE9 transgenic mice. Aβ₄₂ enhanced the protein and mRNA expression levels of sortilin in a dose‐ and time‐dependent manner in SH‐SY5Y cells, but had no effect on sorLA. In addition, proBDNF also significantly increased the protein and mRNA expression of sortilin in these cells. The recombinant extracellular domain of p75^NTR (P75ECD‐FC), or the antibody against the extracellular domain of p75^NTR, blocked the up‐regulation of sortilin induced by Amyloid‐β protein (Aβ), suggesting that Aβ₄₂ increased the expression level of sortilin and mRNA in SH‐SY5Y via the p75^NTR receptor. Inhibition of ROCK, but not Jun N‐terminal kinase, suppressed constitutive and Aβ₄₂‐induced expression of sortilin. In conclusion, this study shows that sortilin expression is increased in the AD brain in human and mice and that Aβ₄₂ oligomer increases sortilin gene and protein expression through p75^NTR and RhoA signaling pathways, suggesting a potential physiological interaction of Aβ₄₂ and sortilin in Alzheimer's disease.

     

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.     

A Role for the p75 Neurotrophin Receptor in Axonal Degeneration and Apoptosis Induced by Oxidative Stress

The p75 neurotrophin receptor (p75^NTR) mediates the death of specific populations of neurons during the development of the nervous system or after cellular injury. The receptor has also been implicated as a contributor to neurodegeneration caused by numerous pathological conditions. Because many of these conditions are associated with increases in reactive oxygen species, we investigated whether p75^NTR has a role in neurodegeneration in response to oxidative stress. Here we demonstrate that p75^NTR signaling is activated by 4-hydroxynonenal (HNE), a lipid peroxidation product generated naturally during oxidative stress. Exposure of sympathetic neurons to HNE resulted in neurite degeneration and apoptosis. However, these effects were reduced markedly in neurons from p75^NTR−/− mice. The neurodegenerative effects of HNE were not associated with production of neurotrophins and were unaffected by pretreatment with a receptor-blocking antibody, suggesting that oxidative stress activates p75^NTR via a ligand-independent mechanism. Previous studies have established that proteolysis of p75^NTR by the metalloprotease TNFα-converting enzyme and γ-secretase is necessary for p75^NTR-mediated apoptotic signaling. Exposure of sympathetic neurons to HNE resulted in metalloprotease- and γ-secretase-dependent cleavage of p75^NTR. Pharmacological blockade of p75^NTR proteolysis protected sympathetic neurons from HNE-induced neurite degeneration and apoptosis, suggesting that cleavage of p75^NTR is necessary for oxidant-induced neurodegeneration. In vivo, p75^NTR−/− mice exhibited resistance to axonal degeneration associated with oxidative injury following administration of the neurotoxin 6-hydroxydopamine. Together, these data suggest a novel mechanism linking oxidative stress to ligand-independent cleavage of p75^NTR, resulting in axonal fragmentation and neuronal death.     

Modulation of Fas-induced apoptosis by p75 neurotrophin receptor in a human neuroblastoma cell line

Fas and p75 neurotrophin receptors (p75^NTR) are death receptors that alone induce apoptosis of SH-SY5Y neuroblastoma cell line respectively by Fas ligand or brain-derived neurotrophic factor (BDNF, a p75^NTR ligand). We report on the modulation of Fas-mediated apoptosis by concomitant p75^NTR activation. The exposure to both ligands suppressed the apoptotic effect. A co-localisation of Fas and p75^NTR receptors was evidenced by co-capping and immunoprecipitation assays. Moreover, a caspase-8 inhibitor suppressed the protective effect of the concomitant BDNF and Fas ligand stimulation, suggesting that p75^NTR and Fas receptors could share common signalling pathways.     

Expression and function of Xenopus laevis p75NTR suggest evolution of developmental regulatory mechanisms

Neurotrophins signal through two different classes of receptors, members of the trk family of receptor tyrosine kinases, and p75 neurotrophin receptor (p75^NTR), a member of the tumor necrosis factor receptor family. While neurotrophin binding to trks results in, among other things, increased cell survival, p75^NTR has enigmatically been implicated in promoting both survival and cell death. Which of these two signals p75^NTR imparts depends on the specific cellular context. Xenopus laevis is an excellent system in which to study p75^NTR function in vivo because of its amenability to experimental manipulation. We therefore cloned partial cDNAs of two p75^NTR genes from Xenopus, which we have termed p75^NTRa and p75^NTRb. We then cloned two different cDNAs, both of which encompass the full coding region of p75^NTRa. Early in development both p75^NTRa and p75^NTRb are expressed in developing cranial ganglia and presumptive spinal sensory neurons, similar to what is observed in other species. Later, p75^NTRa expression largely continues to parallel p75^NTR expression in other species. However, Xenopus p75^NTRa is additionally expressed in the neuroepithelium of the anterior telencephalon, all layers of the retina including the photoreceptor layer, and functioning axial skeletal muscle. Finally, misexpression of full length p75^NTR and each of two truncated mutants in developing retina reveal that p75^NTR probably signals for cell survival in this system. This result contrasts with the reported role of p75^NTR in developing retinae of other species, and the possible implications of this difference are discussed. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 79–98, 2001     

Mature and precursor brain-derived neurotrophic factor have individual roles in the mouse olfactory bulb

Background

Sensory deprivation induces dramatic morphological and neurochemical changes in the olfactory bulb (OB) that are largely restricted to glomerular and granule layer interneurons. Mitral cells, pyramidal-like neurons, are resistant to sensory-deprivation-induced changes and are associated with the precursor to brain-derived neurotrophic factor (proBDNF); here, we investigate its unknown function in the adult mouse OB.

Principal Findings

As determined using brain-slice electrophysiology in a whole-cell configuration, brain-derived neurotrophic factor (BDNF), but not proBDNF, increased mitral cell excitability. BDNF increased mitral cell action potential firing frequency and decreased interspike interval in response to current injection. In a separate set of experiments, intranasal delivery of neurotrophic factors to awake, adult mice was performed to induce sustained interneuron neurochemical changes. ProBDNF, but not BDNF, increased activated-caspase 3 and reduced tyrosine hydroxylase immunoreactivity in OB glomerular interneurons. In a parallel set of experiments, short-term sensory deprivation produced by unilateral naris occlusion generated an identical phenotype.

Conclusions

Our results indicate that only mature BDNF increases mitral cell excitability whereas proBDNF remains ineffective. Our demonstration that proBDNF activates an apoptotic marker in vivo is the first for any proneurotrophin and establishes a role for proBDNF in a model of neuronal plasticity.     

p75 NTR -Immunoreactivity in the subventricular zone of adult male rats: Expression by cycling cells

While the study of in vitro regulation of neural stem cell lineage from both embryonic and adult neurospheres is greatly advanced, much less is known about factors acting in situ for neural stem cell lineage in adult brain. We reported that neurotrophin low affinity receptor p75^NTR is present in the subventricular zone (SVZ) in adult male rats. We then characterized co-distribution of markers associated with precursor cells (nestin and PSA-NCAM) with growth factor receptors (p75^NTR, trkA, EGFr) and proliferation-associated antigens (Ki67 and BrDU-uptake) in adult male rat by immunocytochemistry and confocal laser scan microscopy. Distribution of p75^NTR-immunoreactivity (IR) was investigated using different mono- and polyclonal antisera. p75^NTR– is not co-distributed with glial fibrillary acid protein. It was found to be co-distributed with a small number of nestin-IR cells, whereas no coexistence with PSA-NCAM-IR was observed. Conversely, p75^NTR-IR was present in numerous dividing cells (Ki-67-positive) and co-distributed with EGFr. In order to verify the possible association between p75^NTR and cell death, we investigated co-distribution of p75^NTR-IR with nuclear condensation images as visualized by Hoechst 33258 staining. While few images indicating nuclear condensation were observed in the SVZ, no coexistence with p75^NTR was found. TrkA- and trkB-IR was not found in the SVZ. We also investigated p75^NTR immunostaining on post-natal day 1 and day 16, because of the dramatic reduction of proliferating cells in SVZ over this time-interval. p75^NTR-IR was not increased in the early post-natal phase. Thus, p75^NTR seems to be associated with cell cycle regulation in SVZ in adult rat brain.     

TRAF6 and p62 inhibit amyloid β-induced neuronal death through p75 neurotrophin receptor

Amyloid β (Aβ) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient’s brain. Aβ is known to bind p75 neurotrophin receptor (p75^NTR) and mediates Aβ-induced neuronal death. Recently, we showed that NGF leads to p75^NTR polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that Aβ stimulation impaired the p75^NTR polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75^NTR on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75^NTR polyubiquitination upon Aβ/NGF treatment. Aβ significantly reduced NF-κB activity by attenuating the interaction of p75^NTR with IKKβ. p75^NTR increased NF-κB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the Aβ-mediated inhibition of p75^NTR polyubiquitination and restored neuronal cell survival.     

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.     

TRAF6 and p62 inhibit amyloid β-induced neuronal death through p75 neurotrophin receptor

Amyloid β (Aβ) aggregates are the primary component of senile plaques in Alzheimer disease (AD) patient’s brain. Aβ is known to bind p75 neurotrophin receptor (p75^NTR) and mediates Aβ-induced neuronal death. Recently, we showed that NGF leads to p75^NTR polyubiquitination, which promotes neuronal cell survival. Here, we demonstrate that Aβ stimulation impaired the p75^NTR polyubiquitination. TRAF6 and p62 are required for polyubiquitination of p75^NTR on NGF stimulation. Interestingly, we found that overexpression of TRAF6/p62 restored p75^NTR polyubiquitination upon Aβ/NGF treatment. Aβ significantly reduced NF-κB activity by attenuating the interaction of p75^NTR with IKKβ. p75^NTR increased NF-κB activity by recruiting TRAF6/p62, which thereby mediated cell survival. These findings indicate that TRAF6/p62 abrogated the Aβ-mediated inhibition of p75^NTR polyubiquitination and restored neuronal cell survival.     

Inflammatory cytokines IL-1β and TNF-α regulate p75NTR expression in CNS neurons and astrocytes by distinct cell-type-specific signalling mechanisms

The p75^NTR (where NTR is neurotrophin receptor) can mediate many distinct cellular functions, including cell survival and apoptosis, axonal growth and cell proliferation, depending on the cellular context. This multifunctional receptor is widely expressed in the CNS (central nervous system) during development, but its expression is restricted in the adult brain. However, p75^NTR is induced by a variety of pathophysiological insults, including seizures, lesions and degenerative disease. We have demonstrated previously that p75^NTR is induced by seizures in neurons, where it induces apoptosis, and in astrocytes, where it may regulate proliferation. In the present study, we have investigated whether the inflammatory cytokines IL (interleukin)-1β and TNF-α (tumour necrosis factor-α), that are commonly elevated in these pathological conditions, mediate the regulation of p75^NTR in neurons and astrocytes. We have further analysed the signal transduction pathways by which these cytokines induce p75^NTR expression in the different cell types, specifically investigating the roles of the NF-κB (nuclear factor κB) and p38 MAPK (mitogen-activated protein kinase) pathways. We have demonstrated that both cytokines regulate p75^NTR expression; however, the mechanisms governing this regulation are cytokine- and cell-type specific. The distinct mechanisms of cytokine-mediated p75^NTR regulation that we demonstrate in the present study may facilitate therapeutic intervention in regulation of this receptor in a cell-selective manner.     

Structural Characterization of the Self-Association of the Death Domain of p75NTR

The neurotrophin receptor p75^NTR conveys multiple signals via its intracellular death domain. However, how the death domain is activated and interacts with downstream adaptors remains unclear. Here, we report two crystal structures of the p75^NTR death domain in the form of a non-covalent asymmetric dimer and a Cys379-Cys379 disulfide bond linked symmetric dimer, respectively. These two dimer arrangements have not previously been observed in other death domain-containing proteins. Further analysis shows that both the Cys379-Cys379 disulfide linked and non-covalent full-length p75^NTR dimers are present on the cell surface. These observations suggest that various oligomers may exist simultaneously on the cell surface, and that p75^NTR activation and signalling may be modulated by neurotrophins or other factors via inducing a shift of the equilibrium between different oligomeric states.     

p75NTR optimizes the osteogenic potential of human periodontal ligament stem cells by up‐regulating α1 integrin expression

Human periodontal ligament stem cells (hPDLSCs) are a promising source in regenerative medicine. Due to the complexity and heterogeneity of hPDLSCs, it is critical to isolate homogeneous hPDLSCs with high regenerative potential. In this study, p75 neurotrophin receptor (p75NTR) was used to isolate p75NTR⁺ and p75NTR^? hPDLSCs by fluorescence‐activated cell sorting. Differences in osteogenic differentiation among p75NTR⁺, p75NTR^? and unsorted hPDLSCs were observed. Differential gene expression profiles between p75NTR⁺ and p75NTR^? hPDLSCs were analysed by RNA sequencing. α1 Integrin (ITGA1) small interfering RNA and ITGA1‐overexpressing adenovirus were used to transfect p75NTR⁺ and p75NTR^? hPDLSCs. The results showed that p75NTR⁺ hPDLSCs demonstrated superior osteogenic capacity than p75NTR^? and unsorted hPDLSCs. Differentially expressed genes between p75NTR⁺ and p75NTR^? hPDLSCs were highly involved in the extracellular matrix‐receptor interaction signalling pathway, and p75NTR⁺ hPDLSCs expressed higher ITGA1 levels than p75NTR^? hPDLSCs. ITGA1 silencing inhibited the osteogenic differentiation of p75NTR⁺ hPDLSCs, while ITGA1 overexpression enhanced the osteogenic differentiation of p75NTR^? hPDLSCs . These findings indicate that p75NTR optimizes the osteogenic potential of hPDLSCs by up‐regulating ITGA1 expression, suggesting that p75NTR can be used as a novel cell surface marker to identify and purify hPDLSCs to promote their applications in regenerative medicine.     

Overexpression of p75NTR increases survival of breast cancer cells through p21waf1

The p75 neurotrophin receptor (p75^NTR) plays a critical role in various neuronal and non-neuronal cell types by regulating cell survival, differentiation and proliferation. To evaluate the influence of p75^NTR in breast cancer development, we have established and characterized breast cancer cells which stably overexpress p75^NTR. We showed that p75^NTR overexpression per se promoted cell survival to apoptogens with a concomitant slowdown of cell growth. The pro-survival effect is associated with an increased expression of the inhibitor of apoptosis protein-1 (c-IAP1), a decrease of TRAIL-induced cleavage of PARP, procaspase 9 and procaspase 3, and a decrease of cytochrome C release from the mitochondria. The anti-proliferative effect is due to a cell accumulation in G0/G1, associated with a decrease of Rb phosphorylation and an increase of p21^waf1. Interestingly, inhibition of p21^waf1 with siRNA not only restores proliferation but also abolishes the pro-survival effect of p75^NTR, indicating the key role of p21^waf1 in the biological functions of p75^NTR. Finally, using a SCID mice xenograft model, we showed that p75^NTR overexpression favors tumor growth and strongly increases tumor resistance to anti-tumoral treatment.Together, our findings suggest that p75^NTR overexpression in breast tumor cells could favor tumor survival and contribute to tumor resistance to drugs. This provides a rationale to consider p75^NTR as a potential target for the future design of innovative therapeutic strategies.