Bicaudal‐D1 regulates the intracellular sorting and signalling of neurotrophin receptors

We have identified a new function for the dynein adaptor Bicaudal D homolog 1 (BICD1) by screening a siRNA library for genes affecting the dynamics of neurotrophin receptor‐containing endosomes in motor neurons (MNs). Depleting BICD1 increased the intracellular accumulation of brain‐derived neurotrophic factor (BDNF)‐activated TrkB and p75 neurotrophin receptor (p75^NTR) by disrupting the endosomal sorting, reducing lysosomal degradation and increasing the co‐localisation of these neurotrophin receptors with retromer‐associated sorting nexin 1. The resulting re‐routing of active receptors increased their recycling to the plasma membrane and altered the repertoire of signalling‐competent TrkB isoforms and p75^NTR available for ligand binding on the neuronal surface. This resulted in attenuated, but more sustained, AKT activation in response to BDNF stimulation. These data, together with our observation that Bicd1 expression is restricted to the developing nervous system when neurotrophin receptor expression peaks, indicate that BICD1 regulates neurotrophin signalling by modulating the endosomal sorting of internalised ligand‐activated receptors.     

p75 neurotrophin receptor is involved in proliferation of undifferentiated mouse embryonic stem cells

Neurotrophins and their receptors are known to play a role in the proliferation and survival of many different cell types of neuronal and non-neuronal lineages. In addition, there is much evidence in the literature showing that the p75 neurotrophin receptor (p75^NTR), alone or in association with members of the family of Trk receptors, is expressed in a wide variety of stem cells, although its role in such cells has not been completely elucidated. In the present work we have investigated the expression of p75^NTR and Trks in totipotent and pluripotent cells, the mouse pre-implantation embryo and embryonic stem and germ cells (ES and EG cells). p75^NTR and TrkA can be first detected in the blastocyst from which ES cell lines are derived. Mouse ES cells retain p75^NTR/TrkA expression. Nerve growth factor is the only neurotrophin able to stimulate ES cell growth in culture, without affecting the expression of stem cell markers, alkaline phosphatase, Oct4 and Nanog. Such proliferation effect was blocked by antagonizing either p75^NTR or TrkA. Interestingly, immunoreactivity to anti-p75^NTR antibodies is lost upon ES cell differentiation. The expression pattern of neurotrophin receptors in murine ES cells differs from human ES cells, that only express TrkB and C, and do not respond to NGF. In this paper we also show that, while primordial germ cells (PGC) do not express p75^NTR, when they are made to revert to an ES-like phenotype, becoming EG cells, expression of p75^NTR is turned on.     

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.     

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.     

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.     

Age‐related changes in hippocampal‐dependent synaptic plasticity and memory mediated by p75 neurotrophin receptor

The plasticity mechanisms in the nervous system that are important for learning and memory are greatly impacted during aging. Notably, hippocampal‐dependent long‐term plasticity and its associative plasticity, such as synaptic tagging and capture (STC), show considerable age‐related decline. The p75 neurotrophin receptor (p75^NTR) is a negative regulator of structural and functional plasticity in the brain and thus represents a potential candidate to mediate age‐related alterations. However, the mechanisms by which p75^NTR affects synaptic plasticity of aged neuronal networks and ultimately contribute to deficits in cognitive function have not been well characterized. Here, we report that mutant mice lacking the p75^NTR were resistant to age‐associated changes in long‐term plasticity, associative plasticity, and associative memory. Our study shows that p75^NTR is responsible for age‐dependent disruption of hippocampal homeostatic plasticity by modulating several signaling pathways, including BDNF, MAPK, Arc, and RhoA‐ROCK2‐LIMK1‐cofilin. p75^NTR may thus represent an important therapeutic target for limiting the age‐related memory and cognitive function deficits.     

Neuroprotection by NGF and BDNF Against Neurotoxin-Exerted Apoptotic Death in Neural Stem Cells Are Mediated Through Trk Receptors,Activating PI3-Kinase and MAPK Pathways

Neural stem cells (NSC) undergo apoptotic cell death during development of nervous system and in adult. However, little is known about the biochemical regulation of neuroprotection by neurotrophin in these cells. In this report, we demonstrate that Staurosporine (STS) and Etoposide (ETS) induced apoptotic cell death of NSC by a mechanism requiring Caspase 3 activation, poly (ADP-ribose) polymerase and Lamin A/C cleavage. Although C17.2 cells revealed higher mRNA level of p75 neurotrophin receptor (p75^NTR) compared with TrkA or TrkB receptor, neuroprotective effect of both nerve growth factor (NGF) and brain-derived growth factor (BDNF) mediated through the activation of tropomyosin receptor kinase (Trk) receptors. Moreover, both NGF and BDNF induced the activation of the phosphatidylinositide 3 kinase (PI3K)/Akt and the mitogen-activated protein kinase (MAPK) pathway. Inhibition of Trk receptor by K252a reduced PARP cleavage as well as cell viability, whereas inhibition of p75^NTR did not affect the effect of neurotrophin on neurotoxic insults. Thus our studies indicate that the protective effect of NGF and BDNF in NSC against apoptotic stimuli is mediated by the PI3K/Akt and MAPK signaling pathway via Trk receptors. An erratum to this article can be found at     

The p75 neurotrophin receptor regulates MC3T3-E1 osteoblastic differentiation

While the role of p75^NTR signaling in the regulation of nerve-related cell growth and survival has been well documented, its actions in osteoblasts are poorly understood. In this study, we examined the effects of p75^NTR on osteoblast proliferation and differentiation using the MC3T3-E1 pre-osteoblast cell line. Proliferation and osteogenic differentiation were significantly enhanced in p75^NTR-overexpressing MC3T3-E1 cells (p75GFP-E1). In addition, expression of osteoblast-specific osteocalcin (OCN), bone sialoprotein (BSP), and osterix mRNA, ALP activity, and mineralization capacity were dramatically enhanced in p75GFP-E1 cells, compared to wild MC3T3-E1 cells (GFP-E1). To determine the binding partner of p75^NTR in p75GFP-E1 cells during osteogenic differentiation, we examined the expression of trkA, trkB, and trkC that are known binding partners of p75^NTR, as well as NgR. Pharmacological inhibition of trk tyrosine kinase with the K252a inhibitor resulted in marked reduction in the level of ALPase under osteogenic conditions. The deletion of the GDI binding domain in the p75^NTR-GFP construct had no effect on mineralization. Taken together, our studies demonstrated that p75^NTR signaling through the trk tyrosine kinase pathway affects osteoblast functions by targeting osteoblast proliferation and differentiation.     

The p75 neurotrophin receptor is localized to primary cilia in adult murine hippocampal dentate gyrus granule cells

The densely ciliated granule cell layer of the adult murine hippocampal dentate gyrus is one of two sites of adult neurogenesis. The granule cells have already been proven to localize their SSTR3 (somatostatin receptor 3) receptors to their so-called primary cilia. Here we show for the first time that 70-90% of these cells in 7-18 months-old wild-type and 3×Tg-AD (Alzheimer disease transgenic) mice also load p75^NTR receptors into the structures containing SSTR3, i.e., their primary cilia. On the other hand, p75^NTR’s TrkA co-receptors were not localized to cilia but conventionally distributed throughout the cell surface. Significantly fewer cells (20-40%) in the hippocampal CA1 and CA3 regions and cerebral cortex have p75^NTR containing cilia. While we don’t know what the impact of the cilial localization of p75^NTR on dentate gyral adult neurogenesis and memory encoding might be, the cilia’s amyloid β-activatable p75^NTR receptors could be damaging or lethal to the hippocampal functioning of amyloid β-accumulating Alzheimer brain.     

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.     

Death domain of p75 neurotrophin receptor: a structural perspective on an intracellular signalling hub

The death domain (DD) is a globular protein motif with a signature feature of an all‐helical Greek‐key motif. It is a primary mediator of a variety of biological activities, including apoptosis, cell survival and cytoskeletal changes, which are related to many neurodegenerative diseases, neurotrauma, and cancers. DDs exist in a wide range of signalling proteins including p75 neurotrophin receptor (p75^NTR), a member of the tumour necrosis factor receptor superfamily. The specific signalling mediated by p75^NTR in a given cell depends on the type of ligand engaging the extracellular domain and the recruitment of cytosolic interactors to the intracellular domain, especially the DD, of the receptor. In solution, the p75^NTR‐DDs mainly form a symmetric non‐covalent homodimer. In response to extracellular signals, conformational changes in the p75^NTR extracellular domain (ECD) propagate to the p75^NTR‐DD through the disulfide‐bonded transmembrane domain (TMD) and destabilize the p75^NTR‐DD homodimer, leading to protomer separation and exposure of binding sites on the DD surface. In this review, we focus on recent advances in the study of the structural mechanism of p75^NTR‐DD signalling through recruitment of diverse intracellular interactors for the regulation and control of diverse functional outputs.     

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.     

Effect of FK506 and cyclosporine A on the expression of BDNF, tyrosine kinase B and p75 neurotrophin receptors in astrocytes exposed to simulated ischemia in vitro

We investigated whether the immunosuppressive drugs, FK506 and cyclosporine A, increase BDNF protein and/or mRNA expression in ischemic astrocytes and if an increase could be related to changes in the nuclear expression of p-CREB, p-Erk1/2 and p-Akt. The influence of these immunosuppressants on protein and mRNA levels of TrkB and p75^NTR receptors was also examined. On day 21, cultures of rat astrocytes were subjected to ischemic conditions simulated in vitro (combined oxygen glucose deprivation, OGD) for 8 h and exposed to FK506 (10-1000 nM) and cyclosporine A (0.25-10 μM). FK506 and cyclosporine A (at 1000 nM and 0.25 μM, respectively) stimulated the expression and release of BDNF in cultured rat cerebral cortical astrocytes exposed to OGD. The immunosuppressants at these doses simultaneously increased p-CREB and p-Erk1/2 expression in the nuclear fraction of astrocytes. The results RT-PCR and Western blot analysis provided further evidence of a modulating influence of the drugs on the expression of trkB and p75^NTR genes and their protein products in ischemic astrocytes.     

Chronic Nerve Growth Factor Exposure Increases Apoptosis in a Model of In Vitro Induced Conjunctival Myofibroblasts

In the conjunctiva, repeated or prolonged exposure to injury leads to tissue remodeling and fibrosis associated with dryness, lost of corneal transparency and defect of ocular function. At the site of injury, fibroblasts (FB) migrate and differentiate into myofibroblasts (myoFB), contributing to the healing process together with other cell types, cytokines and growth factors. While the physiological deletion of MyoFB is necessary to successfully end the healing process, myoFB prolonged survival characterizes the pathological process of fibrosis. The reason for myoFB persistence is poorly understood. Nerve Growth Factor (NGF), often increased in inflamed stromal conjunctiva, may represent an important molecule both in many inflammatory processes characterized by tissue remodeling and in promoting wound-healing and well-balanced repair in humans. NGF effects are mediated by the specific expression of the NGF neurotrophic tyrosine kinase receptor type 1 (trkA^NGFR) and/or the pan-neurotrophin glycoprotein receptor (p75^NTR). Therefore, a conjunctival myoFB model (TGFβ1-induced myoFB) was developed and characterized for cell viability/proliferation as well as αSMA, p75^NTR and trkA^NGFR expression. MyoFB were exposed to acute and chronic NGF treatment and examined for their p75^NTR/trkA^NGFR, αSMA/TGFβ1 expression, and apoptosis. Both NGF treatments significantly increased the expression of p75^NTR, associated with a deregulation of both αSMA/TGFβ1 genes. Acute and chronic NGF exposures induced apoptosis in p75^NTR expressing myoFB, an effect counteracted by the specific trkA^NGFR and/or p75^NTR inhibitors. Focused single p75^NTR and double trkA^NGFR/p75^NTR knocking-down experiments highlighted the role of p75^NTR in NGF-induced apoptosis. Our current data indicate that NGF is able to trigger in vitro myoFB apoptosis, mainly via p75^NTR. The trkA^NGFR/p75^NTR ratio in favor of p75^NTR characterizes this process. Due to the lack of effective pharmacological agents for balanced tissue repairs, these new findings suggest that NGF might be a suitable therapeutic tool in conditions with impaired tissue healing.     

HAP1 Is Required for Endocytosis and Signalling of BDNF and Its Receptors in Neurons

When BDNF binds to its receptors, TrkB and p75^NTR, the BDNF-receptor complex is endocytosed and trafficked to the cell body for downstream signal transduction, which plays a critical role in neuronal functions. Huntingtin-associated protein 1 (HAP1) is involved in trafficking of vesicles intracellularly and also interacts with several membrane proteins including TrkB. Although it has been known that HAP1 has functions in vesicular trafficking and receptor stabilisation, it is not yet established whether HAP1 has a role in BDNF and its receptor endocytosis. In the present study, we found that HAP1 is in an interacting complex with p75^NTR, TrkB and BDNF, especially newly endocytosed BDNF. BDNF and TrkB internalisation is abolished in HAP1 knock-out (KO) cortical neurons. TrkB downstream signalling pathways such as ERK, Akt and PLCγ-1 are also impaired in HAP1 KO cortical neurons upon BDNF stimulation. Proliferation of cerebellar granule cells is also impaired in cell culture and cerebellum of HAP1 KO mice. Our findings suggest that HAP1 may play a key role in BDNF and its receptor endocytosis and may promote neuronal survival and proliferation.     

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.