Cysteine-Rich Repeat Domains 2 and 4 are Amyloid-β Binding Domains of Neurotrophin Receptor p75NTR and Potential Targets to Block Amyloid-β Neurotoxicity

Abstract

The p75 neurotrophin receptor (p75NTR) is an amyloid-β (Aβ) receptor that both mediates Aβ neurotoxicity and regulates Aβ production and deposition, thus playing an important role in the pathogenesis of Alzheimer’s disease (AD). The extracellular domain of p75NTR (p75ECD), consisting of four cysteine-rich repeat domains (CRDs), was recently reported to be an endogenous anti-Aβ scavenger to block p75NTR-mediated neuronal death and neurite degeneration signaling of Aβ and pro-neurotrophins. Identification of the specific Aβ binding domains of p75NTR is crucial for illuminating their interactions and the etiology of AD. CRDs of p75ECD were obtained by expression of recombinant plasmids or direct synthesis. Aβ aggregation inhibiting test and immunoprecipitation assay were applied to locate the specific binding domains of Aβ to p75ECD. The Aβ neurotoxicity antagonistic effects of different CRDs were examined by cytotoxicity experiments including neurite outgrowth assay, propidium iodide (PI) staining, and MTT assay. In the Aβ aggregation inhibiting test, the fluorescence intensity in the CRD2 and CRD4 treatment groups was significantly lower than that in the CRD1 and CRD3 treatment groups. Immunoprecipitation assay and western blot confirmed that Aβ could bind to CRD2 and CRD4. Besides, CRD2 and CRD4 antagonized Aβ neurotoxicity suggested by longer neurite length, less PI labelled cells, and higher cell viability than the control group. Our results indicate that CRD2 and CRD4 are Aβ binding domains of p75NTR and capable of antagonizing Aβ neurotoxicity, and therefore are potential therapeutic targets to block the interaction of Aβ and p75NTR in the pathogenesis of AD.

Keywords

Alzheimer’s disease amyloid-β cysteine-rich repeat domains p75 neurotrophin receptor

INTRODUCTION

Alzheimer’s disease (AD) is the most common form of dementia that affects aging population worldwide. Pathological hallmarks of AD include extracellular senile plaques consisting of amyloid-β (Aβ) deposition, and intraneuronal hyperphosphorylated tau-positive neurofibrillary tangles. Although the molecular pathological mechanism underlying AD remains unclear, mounting evidence has pointed to the central role of Aβ in the etiology of the disease [1].

The p75 neurotrophin receptor (p75NTR) is the receptor for nerve growth factor (NGF) and other neurotrophins including brain-derived neurotrophic factor (BDNF), neurotrophin-3 (NT3) and neurotrophin-4/5 (NT4/5). Under physiological conditions, the binding of neurotrophins to p75NTR and tropomyosin-related kinase (Trks) can trigger several signal transduction pathways, which regulate the neurite outgrowth, survival, differentiation, and development of neuronal cells [2, 3]. The p75NTR has a wide tissue and cellular distribution in a wide range of species and its distribution varies during the different stages of the life. It is well known that cholinergic neurons in the basal forebrain which are severely affected in AD, especially in the nucleus basalis of Meynert, express p75NTR throughout adult life [4]. While neurons in other brain regions normally express little or no p75NTR. In the normal metabolic process, p75NTR is cleaved by α-secretase, mainly tumor necrosis factor α-converting enzyme (TACE) [5, 6], subsequently extracellular domain of p75NTR (p75ECD) is released. Structurally, p75ECD consists of four cysteine-rich repeat domains (from CRD1 to CRD4) accounting for ligands binding, and a stalk domain that links these CRDs to the transmembrane domain. Aside from neurotrophins, Aβ was also found to bind to p75NTR [7] and induces Aβ production and deposition, neuronal death, neurite degeneration, tau phosphorylation, and cell cycle re-entry in the pathogenesis of AD [8, 9].

Our previous studies have shown that the expression of p75NTR was upregulated during aging and further activated by Aβ in the AD brain. Aβ/p75NTR signaling also increases the production and accumulation of Aβ, which in turn activates the expression of p75NTR and aggravates Aβ-induced neurotoxicity via p75NTR, thus forms a positive feedback loop between Aβ and p75NTR, accelerating the development of AD [10, 11]. Since the expression of p75NTR is upregulated while the level of p75ECD is dramatically decreased in the AD brain, the shedding of p75ECD is considered to be impaired in AD [12, 13]. The restoration of p75ECD levels by intracranial and intramuscular administration of AAV-p75ECD can alleviate AD pathologies and improve cognition function of the amyloid-β protein precursor (AβPP)/PS1 transgenic mice [13, 14]. However, the binding domains of p75ECD to Aβ is not known. Locating the binding domain of Aβ to p75ECD is helpful to better understand their interactions, and to specifically block Aβ neurotoxicity through p75NTR with minimal effects on its physiological function. Therefore, we conducted the present study to identify the specific Aβ binding domains of p75NTR.

MATERIALS AND METHODS

Plasmids construction

The plasmids encoding different cysteine-rich domains (CRDs) or p75ECD fused with Fc fragment of human IgG were constructed by Western Biotechnology (China), including p75-CRD1-2-Fc, p75-CRD2-3-Fc, p75-CRD3-4-Fc, and p75-ECD-Fc. The plasmids were transformed to DH5α competent cells for amplification and then to BL21 for expression. The plasmids encoding different CRDs or p75ECD fused with yellow fluorescent protein (YFP), or YFP only were constructed by GeneArt (Germany), including p75-CRD1-YFP, p75-CRD2-YFP, p75-CRD3-YFP, p75-CRD4-YFP, p75-ECD-YFP, and pe-YFP. The integrity of all cloned constructs was determined by DNA sequencing in both directions. The plasmids were transformed to DH5α competent cells for amplification and then transfected to HEK293T cells for expression.

Transfection of cell lines

HEK293T cells (1.0×10⁶) were seeded into six-well plates with 80% confluence and cultured in DMEM supplemented with 10% FBS and 2 mM glutamine for 12 h. Plasmid DNA constructs were transfected into cells using Lipofectamine2000 (Invitrogen) according to the manufacturer’s recommendation. Transfection with YFP constructs served to monitor transfection efficiency, and the percentage of fluorescent cells was calculated using microscopy and subjected to normalization.

Aβ aggregation inhibiting test

To test the inhibitory effect of different domains within p75ECD on Aβ aggregation, 1μM Aβ₄₂ monomer was first incubated with PBS, p75-CRD1-2-Fc, p75-CRD2-3-Fc, p75-CRD3-4-Fc, p75-ECD-Fc, human IgG or different synthetic CRDs (including CRD1, CRD2, CRD3, and CRD4, produced by GL Biochem, China), respectively with a mole ratio of 1:2 in DMEM at 37°C for 3 d. For the Thioflavin T (ThT) fluorescence assay, the resultants were then incubated with 10μM ThT solution in dark. Fluorescence emission of ThT is shifted when it binds to β-sheet aggregate structures of amyloid fibrils. Fluorescence intensity was monitored at an excitation wavelength of 450 nm and an emission wavelength of 482 nm by a spectrometer (Synergy H4; BioTek). Each experiment was performed in triplicate.

Immunoprecipitation (IP) assay

All of the HEK293T cells were collected by RIPA buffer, which included 50 mM Tris (pH 7.4), 150 mM NaCl, 1% Triton X-100, 1% sodium deoxycholate, 0.1% SDS, 1 mM sodium orthovanadate, and 0.5 ug/ml leupeptin. After boiled, the extracts of cell lysates were stored at – 80°C until use. The extracts of cell lysates (600μg) were firstly incubated with 1μg Aβ₄₂ at 4°C for 2 h. The solutions were then incubated at 4°C for 2 h with goat anti-GFP antibody (Chemicon), and finally incubated at 4°C for 2 h with protein G beads (Sigma) for immunoprecipitation. The beads were washed five times and boiled in loading buffer for western blot. For negative control, goat IgG was used in the same way for validation.

Western blot

The protein concentration of the co-incubates solutions mentioned above was determined using BCA protein assay kit (Thermo Scientific). Proteins (30μg) were analyzed by 4–20% Mini-PROTEAN TEX Gels (Biored) and transferred to nitrocellulose membrane (Hybond ECL; GE Healthcare). Corresponding primary antibodies (1:1000), 6E10 (anti-Aβ antibody, Chemicon) or goat anti-GFP antibody (Chemicon), were incubated with blots at 4°C overnight. HRP-conjugated secondary antibodies (1:1000, Sigma) were used for detection. Imaging was performed using protein imaging analysis system (Biored).

Neurite outgrowth assay

To investigate the antagonistic effects of different CRDs against Aβ toxicity in vitro, human neuroblastoma cell line SH-SY5Y was applied. SH-SY5Y cells were cultured for 7 d in a medium with 1% FBS and 10μM all-trans-retinoic acid (RA) (Sigma), followed by co-incubation with Aβ oligomers (1μM) and CRDs (1μM) for 72 h. The cells were then stained by MAP-2 antibody (Sigma) and visualized by Alexa Fluor fluorescent dyes. The cell images were taken under microscopy and quantified by ImageJ. The lengths of the five longest neurites per view field were measured, and data from six view fields per group were analyzed. The average length of neurites of each group was used for statistical analysis.

Propidium iodide staining

SH-SY5Y cells were co-incubated with Aβ oligomers (10μM) and CRDs (10μM) for 72 h, then stained in live with propidium iodide (PI). Briefly, cells were treated with PI diluted in the HEPES buffer (2μg/mL) for 15 min and then thoroughly washed with HEPES buffer. Cells were fixed with 2% (wt/vol) parafomaldehyde in PBS for 20 min and counterstained with DAPI (1:1000) for 5 min. Cell imaging was performed with B50 Fluorescence microscope.

MTT assay

SH-SY5Y cells were co-incubated with Aβ oligomers (10μM) and CRDs (10μM) for 72 h. The neurotoxicity of the co-incubation samples was measured using an MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide) viability assay as previously described [15]. Briefly, SH-SY5Y cells were seeded at a density of 10⁵ cells/mL in a 96-well plate in DMEM containing 10% FBS at 50μL per well. The co-incubates of Aβ (10μM) and CRD1, CRD2, CRD3, CRD4, or PBS were added to the wells. After 20 h of incubation, 10μL of MTT (Sigma-Aldrich, USA, 5 mg/mL in PBS) was added to each well, and the samples were incubated for 4 h. A solubilization solution (10% SDS in 0.01M hydrochloric acid) was added to dissolve the insoluble purple formazan product to produce a colored solution. Each assay was performed in triplicate. The optical density (OD) was read at 600 nm on a multi-well scanning spectrophotometer (BIO-RAD Model 2550 EIA Reader).

Statistical analysis

Statistical comparisons among groups were tested using Tukey’s test or one-way analysis of variance. p < 0.05 were considered significant. All analyses were performed using SPSS 13.0 (SPSS Inc., USA).

RESULTS

The schematic diagram of domain structure of p75NTR and amino acid sequences of different CRDs

The relatively locations of four different CRDs, transmembrane domain and intracellular domain of p75NTR were shown in Fig. 1. The amino acid sequences of different CRDs were shown in four rectangles at the bottom. The amino acid sequences of CRD1-2 applied in the primary ThT fluorescence assay consist of amino acid sequences of CRD1, CRD2 and their junction region sequence “ep”, and the amino acid sequences of CRD2-3 and CRD3-4 followed the same pattern.

Fig.1

The schematic diagram of domain structure of p75NTR and amino acid sequences of different CRDs. The four light gray ovals represent different CRDs of p75NTR, and their amino acid sequences were shown in the four rectangles at the bottom. The ladder-shaped figure, dark gray rectangle and black rectangle represent cell membrane, chopper domain and death domain of p75NTR respectively.

The inhibitory effect of CRDs on Aβ aggregation by ThT fluorescence assay

The primary ThT fluorescence assay showed that the fluorescence intensity generated by Aβ fibrils in CRD1-2, CRD2-3 and CRD3-4 treatment groups was similar to the p75ECD treatment groups (p > 0.05), yet was significantly lower than that in IgG treatment group (p < 0.05) (Fig. 2a), which indicated the inhibitory effects of CRD1-2, CRD2-3, CRD3-4 on Aβ aggregation were comparable to that of p75ECD.

Fig.2

The inhibitory effect of CRD1-2, CRD2-3, CRD3-4 or different CRDs on Aβ aggregation by ThT fluorescence assay. a) CRD1-2, CRD2-3, CRD3-4, and ECD represent the fusion protein of human IgG-Fc and CRD1-2, CRD2-3, CRD3-4 or extracellular domain of p75NTR respectively. The p75ECD-Fc and IgG treatment group was set as the positive and negative control, and the last group without any treatment was set as the blank control. b) CRD1, CRD2, CRD3, and CRD4 represent direct synthetic CRDs of p75NTR. The PBS treatment group was set as the negative control, and the last group without any treatment was set as the blank control. The intensity of each group was normalized to that of the negative control group. **indicates compared with negative control group, p < 0.01; ***indicates compared with negative control group, p < 0.001.

The inhibitory effects of four separate CRDs on Aβ aggregation were further tested. The results of ThT fluorescence assay showed that the fluorescence intensity generated by Aβ fibrils in CRD2 and CRD4 treatment groups was significantly lower than that in CRD1 and CRD3 treatment groups (p < 0.05) (Fig. 2b). While the fluorescence intensity in CRD1 and CRD3 treatment groups was not significantly different from that in PBS group (p > 0.05). The results suggested the binding domains of p75NTR and Aβ are located in CRD2 and CRD4.

The interaction of Aβ and different CRDs by IP assay

IP assay and Western blot were applied to confirm the interaction between four different CRDs and Aβ. Firstly, the binding of Aβ to four different CRDs or p75ECD was detected by 6E10 (an Aβ antibody). As shown in Fig. 3, lane a to lane f represented samples from co-incubates of Aβ and p75-CRD1-YFP, p75-CRD2-YFP, p75-CRD3-YFP, p75-CRD4-YFP, p75-ECD-YFP or pe-YFP respectively. Compared with the negative control group, in which goat IgG cannot pull down Aβ-p75-CRDs-YFP complex (Fig. 3b), the 40kD band indicating Aβ oligomers were only seen in lane b, d and e in Fig. 3a, suggesting that Aβ can only bind to CRD2 and CRD4, as well as p75ECD. Aβ could be detected in the IP supernatants (Fig. 3c, d).

Fig.3

The interaction between Aβ and different CRDs. Western blot images of IP resultants pulled down by goat anti-GFP antibody (a) or goat IgG (b), and of IP supernatants pulled down by goat anti-GFP antibody (c) or goat IgG (d) detected by 6E10. a) The 40kD Aβ oligomers indicated by the white triangle were seen in lane b (CRD2), d (CRD4) and e (p75ECD). b) Goat IgG, as the negative control, did not bind to Aβ. c, d) Aβ was present in the IP supernatants. Lane a-d, p75-CRD1-YFP, p75-CRD2-YFP, p75-CRD3-YFP, p75-CRD4-YFP; lane e, p75-ECD-YFP; lane f, pe-YFP. P75-CRDs-YFP, fusion protein of CRDs and YFP; ECD-YFP, fusion protein of p75NTR extracellular domain and YFP; pe-YFP, YFP expressed by the control plasmid.

We subsequently detected the binding of different CRDs and Aβ by the goat anti-GFP antibody. As shown in Fig. 4, lane a to lane f represented samples from co-incubates of Aβ and p75-CRD1-YFP, p75-CRD2-YFP, p75-CRD3-YFP, p75-CRD4-YFP, p75-ECD-YFP or pe-YFP respectively. Compared with the negative control group (Fig. 4b), fusion protein of p75-CRD1-YFP (31kD), p75-CRD2-YFP (32kD), p75-CRD3-YFP (31kD), p75-CRD4-YFP (32kD), p75-ECD-YFP (55kD) and peYFP (27kD) could all be detected in corresponding lanes. The fusion proteins of p75-CRDs-YFP, p75-ECD-YFP and peYFP could also be detected in the IP supernatants (Fig. 4c, d). Based on the results of IP assay, Aβ indeed bind to CRD2 and CRD4 of p75ECD.

Fig.4

The interaction between different CRDs and Aβ. Western blot images of IP resultants pulled down by goat anti-GFP antibody (a) or goat IgG (b), and of IP supernatants pulled down by goat anti-GFP antibody (c) or goat IgG (d) detected by goat anti-GFP antibody. a) p75-CRDs-YFP (indicated by asterisks), p75-ECD-YFP, or YFP were seen in lane a-d, lane e or lane f respectively. b) Goat IgG, as negative control, did not bind to fusion protein of CRDs and YFP. c,d) the fusion proteins of p75-CRDs-YFP were present in the IP supernatants. Lane a-d, p75-CRD1-YFP, p75-CRD2-YFP, p75-CRD3-YFP, p75-CRD4-YFP; lane e, p75-ECD-YFP; lane f, pe-YFP. P75-CRDs-YFP, fusion protein of CRDs and YFP; ECD-YFP, fusion protein of p75NTR extracellular domain and YFP; pe-YFP, YFP expressed by the control plasmid.

CRD2 and CRD4 antagonize Aβ neurotoxicity

The antagonistic effects of different CRDs against Aβ neurotoxicity were further tested. The neurite lengths of SH-SY5Y cells incubated with CRD2 and CRD4 were significantly longer than the PBS group (p < 0.001), yet was slightly shorter than the control group without Aβ treatment (p > 0.05) (Fig. 5a, c). The neurite length of the CRD1 and CRD3 treatment group was not significantly different from the PBS group (p > 0.05). Besides, the percentage of dead neurons, as indicated by PI labelled cells, was significantly lower in the CRD2 and CRD4 treatment groups than that in the PBS group (p < 0.001) (Fig. 5b, d). Similarly, in CRD2 and CRD4 treatment groups, the cell viability was significantly higher than that in the PBS group (p < 0.001) (Fig. 5e). No significant difference was found in the cell viability or the percentage of PI labelled neurons among the CRD1, CRD3 treatment groups and the PBS group. These results suggested that CRD2 and CRD4 are capable of antagonizing Aβ neurotoxicity in vitro.

Fig.5

CRD2 and CRD4 antagonize Aβ neurotoxicity in vitro. a) Representative images of MAP-2 staining of SH-SY5Y cells treated with Aβ (1μM) and CRDs (1μM), PBS or cell culture medium. Scale bar, 100μm. b) Representative images of PI staining of SH-SY5Y cells treated with Aβ (10μM) and CRDs (10μM), PBS or cell culture medium. The cells were counterstained with DAPI. Scale bar, 100μm. c) Quantification of neurite length of SH-SY5Y cells (mean±SEM, one-way ANOVA, Tukey’s test, ***p < 0.001). d) Quantification of percentages of PI labelled cells (mean±SEM, one-way ANOVA, Tukey’s test, ***p < 0.001). e) Quantitative analysis of cell viability in different groups. SH-SY5Y cells were treated with Aβ (10μM) plus different CRDs (10μM), PBS or cell culture medium (8 repeated wells, mean±SEM, one-way ANOVA, Tukey’s test, ***p < 0.001). The neurite length and cell viability of each group was normalized to that of the PBS treatment group.

DISCUSSION

In the present study, we firstly identified that CRD2 and CRD4 are Aβ binding domains of p75ECD with a biochemical method. Besides, CRD2 and CRD4 antagonize Aβ neurotoxicity suggested by longer neurite length, less PI labelled cells and higher cell viability than the control group.

This finding is consistent with a previous study which suggests that NGF and Aβ binding sites within p75NTR might be distinct [16, 17], and is also supported by the finding that one cysteine amino acid (Cys79) from CRD2 may participate in hydrogen bond with Aβ₄₂ and plays a major role in Aβ binding from a computational study [18]. However, this computational study showed that besides CRD2, Aβ₄₂ specifically recognizes CRD1 and forms a “cap” like structure at the N-terminal of receptor [18]. P75NTR is a member of tumor necrosis factor receptor (TNFR) superfamily, and most TNFRs with more than two CRDs are preclustered as dimers, known as preligand assembly, at the cell surface. This process is mainly mediated by a preligand assembly domain (PLAD) located in the CRD1, and CRD1 is considered to be not directly in contact with ligands [19, 20]. This might partly explain the discrepancy between our findings and those from the computational study which only applies a single monomer p75NTR extracellular domain structure for molecular docking and simulation.

Our previous study has demonstrated that full length p75NTR mediates Aβ-induced upregulation of β-site amyloid precursor protein-cleaving enzyme 1 (BACE1) [13] and the vicious cycle of amyloidogenesis by regulating the interaction and endocytosis of BACE1 and AβPP in response to the binding of Aβ and proNGF [11]. In addition, p75NTR also regulates the phosphorylation of AβPP and BACE1, which is an essential step for amyloidogenesis, in response to Aβ and proneurotrophins [11]. In contrast, the cleaved fragment of p75NTR, p75ECD, can protect neurons from Aβ cascade in the pathogenesis of AD on multiple fronts, including suppression of Aβ overproduction, aggregation and deposition, and Aβ-induced neurotoxicity and tau phosphorylation [13, 14]. Therefore, p75ECD is considered as an endogenous scavenger of Aβ and pro-neurotrophins that blocks neuronal death and neurite degeneration signaling through p75NTR. The shedding of p75ECD becomes a critical event of switching the neurotoxic signaling to the neuroprotective one. The therapeutic effect of p75ECD against AD should be further studied, particularly given the failure of current clinical trials on Aβ immunotherapy, secretase or tau aggregation inhibitors, and the urgent need for novel treatments for AD [21, 22].

Meanwhile, the physiological function of full length p75NTR should not be ignored. The distinct signaling pathways mediated by p75NTR mostly depend on different ligands (e.g., neurotrophins, pro-neurotrophins, Aβ) and co-receptors (e.g., Trk receptors, sortilin, NogoR, and Lingo-1) [3, 23]. It is reported that the junction regions between CRD1 and CRD2, and CRD3 and CRD4 are responsible for the binding of NGF to p75NTR [17]. The junction regions between CRD1 and CRD2, CRD3 and CRD4, and the C-terminal loop of p75NTR create three binding sites for NT-3 [24]. Based on our results along with previous findings, CRD2 and CRD4 are the main regions that interact with Aβ and can be candidates for drug development as a scavenger for Aβ. Whether CRD2 and CRD4 interfere with the signaling of mature neurotrophins requires further investigations.

Previous studies discovered a cyclic peptide (CATDIKGAEC) containing the KGA motif that is present in the toxic part of Aβ and closely resembles the binding site of NGF for p75NTR can rescue neurons from Aβ₄₀-induced neurotoxicity signaling in vitro, and significantly suppress the Aβ related inflammation in vivo, which may due to its competitive motive to antagonize Aβ toxicity [25, 26]. Thus we propose a parallel idea that the synthetic CRD2 and CRD4 may have similar effects as this cyclic peptide or p75ECD, since they can also bind with Aβ and may serve as competitive inhibitors. As shown in our preliminary results, CRD2 and CRD4 indeed attenuate Aβ neurotoxicity on neurite growth and cell viability in vitro by blocking Aβ-induced neurotoxic signaling via p75NTR, due to their scavenger activity against Aβ.

In conclusion, the present study indicates that CRD2 and CRD4 are Aβ binding domains of p75NTR, and capable of antagonizing Aβ neurotoxicity. These findings will help us to better understand the interaction between Aβ and p75NTR, and to develop therapeutics to target Aβ without interfering physiological function of p75NTR.

Footnotes

ACKNOWLEDGMENTS

This study was funded by the National Natural Science Foundation of China (grant No. 81401055) and National Health and Medical Research Council (NHMRC) of Australia (grants No. APP1021408, APP1021409).

Authors’ disclosures available online ().

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