A DR6/p75(NTR) complex is responsible for β-amyloid-induced cortical neuron death

Abstract

The p75 neurotrophin receptor (p75(NTR)) 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 p75(NTR) and is a component of the p75(NTR) signaling complex responsible for Aβ-induced cortical neuron death. Cortical neurons isolated from either DR6 or p75(NTR) 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 p75(NTR) 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/p75(NTR) complex to prevent Aβ cytotoxicity represents a new approach for the treatment of neurodegenerative disorders such as AD.

Figure 1

DR6 is expressed in cortical neurons and upregulated in AD. (a) Quantitative RT-PCR analysis of DR6 mRNA expression in AD. (b) Western blot analysis of DR6 expression from four AD and three age-matched normal brains. (c) Densitometry quantification of DR6 protein level from DR6 western blotting from 10 AD and 9 age-matched normal brains. (d) In-situ hybridization analysis of DR6 (red) and co-localization with neurons (βIII-tubulin, green) in normal and AD cortex (yellow=merge, blue=DAPI). Arrows indicate cells with nuclear condensation characteristic of apoptosis. Arrowheads indicate area of image enlargement (top right corner). Scale bar=25 μm. (e) Quantification of DR6⁺ neurons from d. (f) Immunocytochemical staining of DR6 from age-matched normal and AD brains; arrows indicate intensely stained DR6-positive cells. Scale bar=95 μm. (g) Time-lapse images showing the effect of full-length DR6 (DR6 FL) overexpression in cultured neurons 0–92 h after transfection (bottom panel) versus vector control-transfected neurons (top panel). Scale bar=20 μm. (h) Quantification of neuronal survival from g

Figure 2

DR6 and p75^NTR form a receptor complex. (a) AP-DR6 binding to HEK 293 cells expressing p75^NTR and not to untransfected control cells. The non-homogenous staining pattern reflects incomplete transfection. Scale bar=25 μm. (b) Quantitative assessment of a by ELISA. (c) DR6/p75^NTR co-immunoprecipitation in HEK 293 cells overexpressing Myc-tagged DR6 and p75^NTR. (d) DR6/p75^NTR co-immunoprecipitation in fetal human spinal cord lysates. (e) Immunocytochemical staining to co-localized the DR6 and p75^NTR expression in cultured neurons. Scale bar=25 μm. (f) Competition ELISA showing the binding of europium-labeled human p75^NTR to human DR6 ectodomain-coated plates in the presence and absence of blocker constructs

Figure 3

Cortical neurons isolated from DR6 and p75^NTR null mice are resistant to Aβ42-induced cell death. (a) Western blot analysis of cleaved casp3 levels in DR6^−/− cortical neurons after Aβ42 exposure. (b) Quantification of cleaved casp3 levels from a. (c) Quantification of cleaved casp3 levels by western blot analysis in p75^NTR−/− cortical neurons after Aβ42 exposure. (d) Quantification of cleaved casp3 levels by western blot analysis in DR6-DN-infected neurons after Aβ42 exposure

Figure 4

Anti-DR6 antibodies that block the interaction between DR6 and p75^NTR inhibit Aβ42-induced neuronal cell death. (a) Anti-DR6 antibody 5D10 blocked DR6 and p75^NTR complex formation from DR6/p75^NTR co-transfected HEK 293 cells. Anti-DR6 antibody 2A9, which does not bind to DR6 at the p75^NTR-binding site, did not block p75^NTR from co-immunoprecipitating with DR6. (b) Top: Schematic domain structure of human DR6 protein depicting four extracellular CRD 1-4, a transmembrane domain (TM), and a cytoplasmic death domain (DD). Bottom: ELISA mapping 5D10-binding epitope within cysteine-rich domains of DR6 protein. (c) 5D10 blocks AP-DR6 binding to HEK 293 cells expressing p75^NTR. Scale bar=25 μm. (d) Quantitative assessment of 5D10 blocking AP-DR6 binding to p75^NTR from c. (e) TUNEL staining quantification of percent apoptotic cells from anti-DR6 antibody-treated and control-treated cortical neurons following Aβ42 exposure. (f) Western blot analysis of cleaved casp3 levels in anti-DR6 antibody-treated and control-treated cortical neurons following Aβ42 exposure. (g) Quantification of cleaved casp3 levels from f

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 Aβ 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 Aβ, upregulation of DR6 and p75^NTR, and decreased expression of Trk