A DR6/p75NTR complex is responsible for β-amyloid-induced cortical neuron death

The p75 neurotrophin receptor (p75NTR) is a known mediator of β-amyloid (Aβ)-induced neurotoxicity implicated in Alzheimer's disease (AD). Here, we demonstrate that death receptor 6 (DR6) binds to p75NTR and is a component of the p75NTR signaling complex responsible for Aβ-induced cortical neuron death. Cortical neurons isolated from either DR6 or p75NTR null mice are resistant to Aβ-induced neurotoxicity. Blocking DR6 function in cortical neurons by anti-DR6 antibodies that block the binding of DR6 to p75NTR receptor complex or by a dominant negative DR6 construct lacking the cytoplasmic signaling death domain attenuates Aβ-induced caspase 3 activation and cell death. DR6 expression is upregulated in AD cortex and correlates with elevated neuronal death. Targeting the disruption of the DR6/p75NTR complex to prevent Aβ cytotoxicity represents a new approach for the treatment of neurodegenerative disorders such as AD.


Subject Category: Neuroscience
Alzheimer's disease (AD) is a neurodegenerative disorder characterized by progressive cell death and subsequent cognitive impairment including memory loss. An early and constant feature of AD is the degeneration of cholinergic neurons in the basal forebrain that innervate the hippocampus and neocortex. 1 Although the cause of disease is unclear, the deposition of b-amyloid (Ab) into amyloid plaques is the hallmark pathological feature of AD. Many of the new experimental therapies for AD attempt to prevent the formation, aggregation, and accumulation or the facilitation of Ab removal from preexisting deposits.
Tumor necrosis factor receptor (TNFR) superfamily members, which have roles in immunological and oncological diseases, 2,3 have recently emerged as drug targets for the treatment of neurodegenerative diseases such as Alzheimer's and central nervous system (CNS) demyelination diseases. [4][5][6] A subset of the TNFR superfamily, the death receptors, contains four highly conserved cysteine-rich regions in their ectodomain and a cytoplasmic death domain that upon receptor oligomerization activates diverse downstream targets, including caspases. 7,8 Eight death receptors have been identified and each regulates cell death in selected cell populations: 8,9 Fas (also known as death receptor 2, CD95, and APO-1), TNFR1 (tumor necrosis factor receptor 1), TRAMP (also known as death receptor 3), TRAILR1 (TNFrelated apoptosis-inducing ligand receptor 1), TRAILR2, DR6 (death receptor 6), ectodermal dysplasia receptor, and p75 NTR (p75 neurotrophin receptor). Fas-induced cell death has a critical immunomodulatory role in the killing of autoaggressive lymphocytes and pathogen-infected cells. 10 TRAILRs have a critical role in apoptosis of tumor cells. 2 In the CNS, p75 NTR has a well-established role in neuronal cell death and axon degeneration. p75 NTR forms a receptor complex with sortilin that binds pro-nerve growth factor to induce neuronal cell death. 6,11 p75 NTR also forms a tripartite complex with NogoR (Nogo receptor) and LINGO-1 (Leucine-rich repeat and Ig domain containing NogoR interacting protein 1) to inhibit axon outgrowth. 12 In addition, p75 NTR has been shown to bind Ab to induce cell death in hippocampal neurons in vitro and cholinergic basal forebrain neurons in vivo. 13,14 There is increasing evidence that DR6 has an important role in neuronal cell death. Nikolaev et al. 4 have reported that DR6 induces neuronal cell death and axon degeneration during CNS development by directly binding N-terminal Ab precursor protein in the absence of trophic factors through activation of the caspase 6 and casp6 signaling pathway. 4 DR6 also mediates oligodendrocyte cell death during development. 5 Here, we demonstrate that DR6 forms a receptor complex with p75 NTR to induce cortical neuron death. Anti-DR6 antibody that blocks the formation of the DR6/p75 NTR receptor complex significantly reduces Ab-induced neurotoxicity. DR6 and p75 NTR expression levels are upregulated in AD brains. Blocking DR6/p75 NTR receptor complex function represents a novel therapeutic target for the treatment of neurodegenerative diseases such as AD.

Results
DR6 expression levels are correlated with neuronal death. DR6 is expressed in cortical neurons. To determine whether DR6 contributes to AD pathology, mRNA and protein expression levels were evaluated using post-mortem brain samples of AD patients with age-matched normal controls. Quantitative real-time PCR (RT-PCR) and western blot results (Figures 1a-c) showed a 1.6-fold and a 2.5-fold increase in DR6 mRNA and protein levels, respectively, in AD brain samples compared with normal brain samples. Consistent with this finding, in-situ hybridization revealed a 2.5-fold increase in the number of DR6-positive (DR6 þ ) neurons in the cortex of AD brains compared with normal human brains (Figures 1d and e). Cells that displayed nuclear DNA condensation characteristic of apoptosis ( Figure 1d, arrows) showed increased DR6 staining (red) when compared with normal brain cells (Figure 1d), suggesting that upregulation of DR6 may contribute to neuronal cell death. Immunocytochemical staining using anti-DR6 antibody also demonstrated an increased number of DR6-positive neurons with more intense staining in the AD brains compared with age-matched normal brain tissue (Figure 1f).
To further confirm that DR6 expression level contributes to neuronal death, full-length DR6 (DR6 FL) was introduced into neocortical neurons by lentivirus infection. Time-lapse live images captured across 92 h revealed that ectopic expression of DR6 FL-induced neuronal death as evident by changes in cell morphology and a decrease in cell count (Figures 1g  and h). DR6 FL-infected neurons showed a twofold reduction in cell survival compared with control virus-infected neurons DR6 and p75 NTR form a receptor complex. The upregulation of DR6 expression in AD brain tissues suggests that DR6 may contribute to neurodegeneration. This result prompted us to look for a ligand or co-receptor, which participates with DR6 to induce cortical neuron death. As p75 NTR also contains a death domain and is also upregulated in AD cortical and hippocampal neurons, 15,16 we investigated whether DR6 binds to p75 NTR . First, we tested whether alkaline phosphatase-DR6 fusion protein (AP-DR6) could bind HEK 293 cells expressing p75 NTR . As shown in Figure 2a, AP-DR6 bound strongly to cells expressing p75 NTR compared with control non-transfected cells with an EC 50 of 90 nM ( Figure 2b). Second, to determine whether DR6 forms a receptor complex with p75 NTR , DR6 was immunoprecipitated from HEK293 cells co-transfected with p75 NTR and Myc-tagged DR6. The presence of p75 NTR in the immunoprecipitate was analyzed by anti-p75 western blot ( Figure 2c). In the input lanes, both p75 NTR and DR6 expressions were detected in transfected cells; however, a strong p75 NTR immunoreactive band was only detected in the DR6/p75 NTR co-transfected precipitate and no band was detected in the cells transfected with either DR6 or p75 NTR alone. Third, we determined if the DR6/p75 NTR receptor complex is present endogenously by subjecting human fetal spinal cord lysates to immunoprecipitation with an anti-DR6 antibody followed by western blot analysis to determine if p75 NTR co-precipitates with DR6. As shown in Figure 2d, DR6 and p75 NTR immunoreactive bands were detected. Fourth, an immunocytochemistry (ICC) study was performed to confirm the co-expression of DR6 and p75 NTR in aged cultured cortical neurons (Figure 2e), and greater than 85% of cultured neurons were DR6 and p75 NTR double positive.
The above four studies indicate that DR6 and p75 NTR form a receptor complex. We next determined which region of DR6 ectodomain binds to p75 NTR . Deletion constructs lacking different cysteine-rich domains (CRDs) of DR6 were generated and co-expressed with p75 NTR in HEK 293 cells. p75 NTR co-immunoprecipitated with DR6 FL protein and deletion mutants lacking CRD 1 and 2, but not DR6 protein lacking CRD 3 or CRD 4, or CRD 3-4 double deletion (data not shown), suggesting that p75 NTR binds to CRD 3-4 of the DR6 ectodomain. To verify this finding, we directly measured the binding of recombinant DR6 to p75 NTR in a competition ELISA ( Figure 2f). DR6 full ectodomain, CRD 1-4, and CRD 3-4 all bound to p75 NTR with an IC 50 of r30 nM.
Neurons from DR6 null mice are resistant to Ab-induced cell death. To determine the contribution of both DR6 and p75 NTR to Ab-induced neurotoxicity, cortical neurons isolated from DR6 and p75 NTR null mice (E16) were cultured for 4 days, and then treated with aggregated Ab42 for 24 h. Cell death was monitored by measuring the expression levels of cleaved caspase 3 (casp3) by western blot (Figure 3). When compared with control treated cells, Ab42 treatment induced a twofold increase in cleaved casp3 expression in wild-type neuron cultures, but had no effect on neurons isolated from DR6 null mice (Figures 3a and b). Consistent with the DR6 null neuron casp3 data, there was a twofold increase in casp3 activation in wild-type neurons treated with Ab42, with no differences observed in p75 NTR null cortical neurons ( Figure 3c). Thus, although we were able to confirm that p75 NTR is necessary for Ab-induced neurotoxicity in the p75 NTR À / À neurons, the lack of an effect of neurotoxicity in the DR6 À / À neurons implies that both p75 NTR and DR6 contribute to the Ab42-induced cytotoxicity, a role that has long been attributed to p75 NTR alone. 13,14 To further elucidate the contribution of DR6 to Ab42induced cytotoxicity, a dominant negative version of DR6 lacking the cytoplasmic death domain (DR6-DN) was expressed in cultured cortical neurons by lentivirus infection and then treated with aggregated Ab42. The cleaved casp3 levels were analyzed again by western blot (Figure 3d). Ab42 treatment significantly increased levels of cleaved casp3 in control-infected cells, with no changes seen in DR6-DNexpressing cell cultures when compared with control infected or the absence of Ab42 treatment. These results show that the DR6/p75 NTR complex responsible for Ab-induced cortical neuron death requires the DR6 death domain.
Antibodies that block the formation of the DR6/p75 NTR receptor complex inhibit Ab-induced neuronal death. To further confirm that the DR6/p75 NTR receptor complex is responsible for Ab-induced neurotoxicity, we first assessed if anti-DR6 monoclonal antibody 5D10 affected the ability of DR6 to bind to p75 NTR . As shown in Figure 4a, 5D10 blocks the binding of DR6 to p75 NTR as determined by anti-DR6 immunoprecipitation and then anti-p75 NTR western blot in DR6/p75 NTR co-transfected cells. Epitope mapping revealed that the 5D10 bound within DR6 CRD 3-4 ( Figure 4b). In contrast, 2A9, an antibody that binds to DR6 CRD 1-2, did    (Figure 4a). In a direct binding assay, 5D10 also blocked the binding of AP-DR6 to HEK 293 cells expressing p75 NTR (Figure 4c). The IC 50 for 5D10 was 9 nM (Figure 4d). Thus, 5D10 competes with p75 NTR by binding to the same region of DR6, thereby inhibiting the formation of the DR6/p75 NTR complex.
To test if 5D10 protects against Ab42-induced neurotoxicity, cultured cortical neurons were treated with 5D10 or control antibody following aggregated Ab42 exposure for 24 h. 5D10 treatment resulted in a twofold reduction in Ab42induced neuronal apoptosis by TUNEL staining (Figure 4e) and a significant decrease in casp3 activation by western blot (Figures 4f and g). Thus, 5D10 specifically binds to DR6 within the CRD 3-4 domains to block formation of the DR6/p75 NTR receptor complex, thereby preventing Ab42-induced cell death in cultured neocortical neurons.

Discussion
DR6 has emerged as an important regulator of neuronal cell death. Here, we show that DR6 is expressed in adult cortical neurons and is upregulated in the AD brain. In AD cortex, increased DR6 expression is co-localized with cells undergoing DNA condensation. To understand the linkage between DR6 overexpression and cell death, we performed an extensive series of mechanism of action studies. Most significantly, we discovered that DR6 forms a receptor complex with p75 NTR that mediates Ab-induced neurotoxicity in cortical neurons. Whereas the role of p75 NTR in Ab-induced cell death is well documented, our studies reveal that DR6 is essential for p75 NTR /Ab-induced neurotoxicity, as cortical neurons genetically deficient of either receptor were resistant to cell death induced by Ab42 exposure. Anti-DR6 antibody that blocks the formation of the DR6/p75 NTR receptor complex attenuated Ab-induced cell death. The ability of overexpression of DR6-DN to attenuate Ab-induced cell death suggests that the death domain of DR6 is responsible for the apoptotic signal transduction. The discovery that DR6 binds p75 NTR and is directly involved in Ab-induced cortical neuron death identifies a new therapeutic target for the treatment of AD. p75 NTR has a complex role in regulating neuronal survival and death that is dependent on its binding to ligands and co-receptors. 6,12,15-20 p75 NTR is a low affinity neurotrophin (NT) receptor that interacts with Trk (tropomyosin receptor kinase) to enhance the Trk response to NTs, thereby promoting cell survival. 21 As a cell death receptor, p75 NTR forms a receptor complex with sortilin to mediate proapoptotic signaling in response to pro-nerve growth factor binding. 11 As a co-receptor involved in axon degeneration, p75 NTR forms a tripartite complex with NogoR and LINGO-1 that results in the inhibition of axon growth in response to Nogo, myelin-associated glycoprotein, or oligodendrocyte myelin glycoprotein. 12 Several lines of evidence suggest that changes in the expression levels of p75 NTR , its ligands, or its co-receptors during disease may switch p75 NTR function from pro-survival to pro-death. Ab is increased in AD and has been shown to bind p75 NTR to induce neuronal death. 13,14 NTs, which also bind p75 NTR and protect neurons from Ab-induced neurotoxicity in vitro, 22 are decreased in AD. 23 Additionally, increased p75 NTR and decreased TrkA expression have been reported in AD. 15,16,24,25 Our data combined with the published studies demonstrate that DR6, p75 NTR , and Ab are all upregulated in AD brains, whereas NTs and TrkA are downregulated, suggesting a potential role for the DR6/ p75 NTR receptor complex in neuronal cell death in AD. Figure 5 shows a schematic model depicting the role of p75 NTR in regulating cortical neuron survival and death signaling. In the presence of NTs, p75 NTR binds NTs and Trk to promote neuronal survival. In the absence of NTs, p75 NTR binds Ab and DR6 to form a death domain oligomeric complex that activates casp3 to induce cortical neuron death. DR6 antagonists selectively block the pro-apoptotic function of the DR6/p75 NTR complex while preserving the pro-survival function of the p75 NTR /Trk complex. Pharmaceutical reagents capable of blocking the formation of the DR6/p75 NTR receptor complex, such as 5D10, could alleviate or reverse the progression of AD and other neurodegenerative diseases by promoting neuron survival.
In summary, we have discovered that DR6 and p75 NTR form a receptor complex that induces neuronal death upon binding of Ab42. Studies using cortical neurons isolated from p75 NTR and DR6 null mice reveal that both p75 NTR and DR6 contribute to Ab-induced neurotoxicity as loss of either death receptor prevented the cell death. Blocking the interaction between DR6 and p75 NTR represents a promising therapeutic target for AD.

Materials and methods
In-situ hybridization. Rat brain frozen sections were prepared and processed as described 26 and were probed with digoxigenin-labeled DR6 antisense probe (5 0 -TAATACGACTCACTATAGGGGCTGGTGGGTAAGTTGT GGT-3 0 ) and sense RNA probe (5 0 -ATTTAGGTGACACTATAGAACTCGCGG TACCTTCTCTGAC-3 0 ). Sections were stained using the TSA plus fluorescent anti-digoxigenin conjugated antibodies kit (Perkin Elmer, Waltham, MA, USA) Figure 5 Working model for DR6/p75 NTR receptor complex signaling in cortical neurons. In the presence of NTs, p75 NTR binds NTs and Trk to promote cortical neuron survival via the AKT (also known as Protein Kinase B) pathway. In the absence of NTs, p75 NTR binds Ab and DR6 to form a receptor complex, which activates the caspase 3 apoptotic signaling pathway via a cytoplasmic death domain oligomeric complex. In Alzheimer's disease, various factors might switch p75 NTR signaling from pro-survival to pro-death, including the increased level of Ab, upregulation of DR6 and p75 NTR , and decreased expression of Trk RT-PCR. Quantitative RT-PCR was performed using six snap-frozen brain tissue blocks from four different AD individuals (four frontal lobes, one temporal lobe, one basal ganglion and four unspecified regions collected from four individual donors, two females, two males; Cooperative Human Tissue Network, Philadelphia, PA, USA). Three normal matching brain blocks from three individuals (two females and one male) without neurological diseases were included as control. All mRNAs were extracted using the Absolutely RNA miniprep kit (Agilent Technologies, Inc, Santa Clara, CA, USA) following the manufacturer's instruction. cDNAs were generated using purified RNAs (High Capacity cDNA Archive Kit, Life Technologies, Grand Island, NY, USA) and served as the template for quantitative RT-PCR. TaqMan Gene Expression system (Mx3000P) was used to quantify the DR6 mRNA levels using premixed primer sets with MGB probes: DR6 (Mm00446361_m1, Life Technologies). b-Actin mRNA levels were quantified in parallel for normalization using primer/probe set Rn00667869_m1 (Life Technologies).
Cell culture. Rat neocortical neurons were prepared from E18 Sprague-Dawley rats (Charles River, Wilmington, MA, USA). Mouse cortical neurons were prepared from E16 DR6 null, p75 NTR null or WT (C57BL/6) mice. Briefly, cerebral cortices from the rat or mouse embryos were dissected out, minced, and incubated in 0.25% Trypsin-EDTA (Life Technologies) at 37 1C for 10 min. The cells were triturated after adding 10 mg/ml DNase I (Sigma, St. Louis, MO, USA) and 10% fetal bovine serum (Life Technologies) to stop the reaction. Cell pellets collected by centrifugation at 275 r.c.f. at room temperature for 5 min were mechanically dissociated by gently passing through a plastic pipette until no large fragments were visible. Tissue culture plates (Corning, Corning, NY, USA) were coated with 100 mg/ml Poly-D-lysine (Sigma) before cell seeding. Plating densities of 1 Â 10 6 /well on a 12-well plate for western blots and 2 Â 10 4 /well of 4-well chamber slides (LabTek, Fisher Scientific, Pittsburgh, PA, USA) for ICC and TUNEL assay were used. Cells were maintained in Neurobasal medium containing B27 supplement (Life Technologies) at 37 1C in humidified air with 5% CO 2 and allowed to mature 5 days before treatment.
Time-lapse analysis. Rat cortical neurons were subjected to time-lapse recording for 92 h after cells were infected with DR6 FL or control lentivirus. Neurons were cultured in neurobasal medium with B27 supplement on a poly-Llysine-coated 24-well tissue culture plate. The entire plate was placed onto a motorized stage (Zeiss, Thornwood, NY, USA) that was housed inside a sealed chamber (Zeiss) to maintain the culture at 37 1C in humidified air with 5% CO 2 during the entire recording. Six wide-field microscopic images of cells under each treatment were acquired through the transmitted light channel on a Zeiss Axiovert microscope equipped with a digital camera and the Axiovision software (Zeiss).
Ab preparation. Ab42 peptide was obtained from Life technologies and reconstituted according to the manufacturer's instruction. First, the peptide was dissolved in HPLC grade water at 6 mg/ml, then further diluted in Dulbecco's phosphate-buffered saline (PBS) to the final concentration of 1 mg/ml and incubated at 37 1C for 24 h for aggregate formation. A measure of 50 mg/ml of Ab42 was used for all the experiments. Reverse sequence peptide Ab(42-1) prepared in the same way was used as controls.
Europium ELISA. 96F MAXISORP black plates (NUNC 437111; Fisher Scientific) were coated with 50 ml/well of 10 mg/ml human DR6 (huDR6) ectodomain in PBS buffer (pH 7.2) and incubated at 37 1C for 1 h. After washing four times with PBS containing 0.05% Tween-20 buffer (PBST), the plates were blocked with 300 ml/well blocking buffer (Hank's buffered salt solution, pH 7.0, 25 mM HEPES, 1% bovine serum albumin, 0.1% ovalbumin, 0.1% non-fat dry milk, 0.001% NaN 3 ) for 1 h at room temperature. After washing with PBST, Europium-labeled human p75 NTR -Fc was added to the plates at a fixed concentration of 1 nM in Perkin Elmer assay buffer. Each sample was run in duplicate with and without competition using 0-400 nM of huDR6 CRD 3-4, huDR6 CRD 1-4, and huDR6 ectodomain. The huDR6 CRD 3-4 and huDR6 CRD 1-4 constructs consisted of residues 132-213 and 42-213 of the full-length human DR6 protein, respectively, and a C-terminal 12-His tag. The huDR6 ectodomain consisted of residues 1-349 of the full-length human DR6 protein. The plates were incubated at room temperature on a plate shaker for 1 h in the dark and washed eight times with PBST. 100 ml/well of enhancement solution (Perkin Elmer) was added and shaken at room temperature for 1 h in the dark. Signal was detected on Victor3 1420 Multilabel Counter fluorometer (Wallac, Perkin Elmer).
Epitope mapping ELISA. Cell lysates from HEK 293 cells transfected with individual DR6 constructs were processed as described above (immunoprecipitation), and incubated with anti-Myc antibody (Abcam, Cambridge, MA, USA)-coated ELISA plate at room temperature for 2 h. The plate was washed, then incubated with 5D10 (2 mg/ml) at room temperature for 2 h, followed by horseradish peroxidase-anti-mouse IgG (Jackson Immunoresearch, West Grove, PA, USA) at DR6/p75 NTR /Ab complex induces cortical neuron death Y Hu et al room temperature for 1 h. The signal was detected by 3,3 0 ,5,5 0 -Tetramethylbenzidine (Thermo Scientific, Waltham, MA, USA) and read at 450 nm.
Generation of DR6-deficient mice. DR6-deficient mice were generated at TaconicArtemis. Briefly, exons 2-3 of DR6 locus were replaced by an Flp-recombination-site-flanked, positive selection cassette. The targeting vector was generated using BAC clones from the C67BL/6J RPCI-23 BAC library and transfected into C57BL/6N Tac ES cell line. Homologous recombinant clones were isolated and used to generate chimeric animals by injection into C57BL/6 blastocysts. Replacing exons 2-3 by the positive selection cassette resulted in loss of expression of the DR6 gene by preventing transcription of exons 2-6. Heterozygous mice were interbred to generate DR6-deficient mice. The genotype of the DR6-locus was determined by PCR using tail DNA. p75 NTR null mice were purchased from Jackson Laboratory (Bar Harbor, ME, USA).
Quantitation of neuronal death. Cultured neurons (WT or DR6 null or p75 NTR null) were exposed to Ab42 for 24 h, with no addition or in the presence of anti-DR6 antibody (5D10, 10-30 mg/ml) or isotype control antibody (MOPC21). Stained neurons were visualized under a fluorescence microscope. Dead neurons were defined as those with shrunken cell bodies, retracted neurites, nuclear DNA condensation, and fragmentation. Apoptotic neuron was quantified with the TUNEL/DAPI system by dividing the number of nuclei exhibiting TUNEL or condensation/fragmentation by the total number of nuclei as identified by DAPI. At least 10 fields were counted for each experimental condition, and each study was repeated two to four times. Aged cortical neurons were within 5-10 divisions.
Statistical Methods. GraphPad Prism software (GraphPad Software, Inc., La Jolla, CA, USA) was used for statistical analysis. Data are shown as mean and standard error of the mean (S.E.M.). In all studies, comparison of mean values was conducted using unpaired Student's t-tests or one-way analysis of variance followed by Tukey post hoc test. Statistical significance was determined at the 5% level (Po0.05). For all figures, *Po0.05, **Po0.01, ***Po0.001, and ****Po0.0001.

Conflict of Interest
The authors declare no conflict of interest.