An Alzheimer Disease-linked Rare Mutation Potentiates Netrin Receptor Uncoordinated-5C-induced Signaling That Merges with Amyloid β Precursor Protein Signaling

Abstract

A missense mutation (T835M) in the uncoordinated-5C (UNC5C) netrin receptor gene increases the risk of late-onset Alzheimer disease (AD) and also the vulnerability of neurons harboring the mutation to various insults. The molecular mechanisms underlying T835M-UNC5C-induced death remain to be elucidated. In this study, we show that overexpression of wild-type UNC5C causes low-grade death, which is intensified by an AD-linked mutation T835M. An AD-linked survival factor, calmodulin-like skin protein (CLSP), and a natural ligand of UNC5C, netrin1, inhibit this death. T835M-UNC5C-induced neuronal cell death is mediated by an intracellular death-signaling cascade, consisting of death-associated protein kinase 1/protein kinase D/apoptosis signal-regulating kinase 1 (ASK1)/JNK/NADPH oxidase/caspases, which merges at ASK1 with a death-signaling cascade, mediated by amyloid β precursor protein (APP). Notably, netrin1 also binds to APP and partially inhibits the death-signaling cascade, induced by APP. These results may provide new insight into the amyloid β-independent pathomechanism of AD.

Keywords: Alzheimer disease; UNC5C; amyloid beta-independent; amyloid precursor protein (APP); apoptosis; neurodegenerative disease; neuronal cell death; signal transduction.

FIGURE 1.

Overexpression of UNC5C induces neuronal cell death, intensified by the T835M mutation but inhibited by the binding of netrin1. A–C, F11 cells, transfected with the empty pHA vector (vector), pRK5-WT-UNC5C (WT-UNC5C), or pRK5-T835M-UNC5C (T835M-UNC5C), were co-incubated with or without 1.0 μm recombinant mouse netrin1 (mNetrin1) or bovine serum albumin (BSA) for 48 h. This co-incubation was started at 24 h after transfection. At 72 h after transfection, the cells were harvested for trypan blue exclusion assays (A) and WST-8 assays (B). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with the monoclonal antibody to HA (C). D–F, F11 cells, transfected with indicated amounts (μg/well) of the empty pHA vector (vector) or pRK5-T835M-UNC5C, were co-incubated with or without 1.0 μm recombinant mouse Netrin1 (mNetrin1) or BSA for 48 h. The co-incubation was started at 24 h after transfection. At 72 h after transfection, they were harvested for trypan blue exclusion assays (D) and WST-8 assays (E). The cell lysates were subject to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA (F). ***, p < 0.001; **, p < 0.01; n.s., not significant. WB, Western blot.

FIGURE 2.

CLSP inhibits T835M-UNC5C-induced death in F11 cells and SH-SY5Y cells. A–C, F11 cells, transfected with the empty pHA vector (vector) or pRK5-T835M-UNC5C, were co-incubated with or without 10 nm recombinant mouse CLSP1 (mCLSP1) or glutathione S-transferase (GST) as a negative control for 48 h. The co-incubation was started 24 h after transfection. At 72 h after transfection, the cells were harvested for trypan blue exclusion assays (A) and WST-8 assays (B). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA (C). D–F, SH-SY5Y cells, transfected with the empty pHA vector (vector) or pRK5-T835M-UNC5C, were co-incubated with or without 1.0 μm recombinant mouse netrin1 (mNetrin1), 10 nm recombinant human CLSP (hCLSP1), or 1.0 μm BSA as a negative control for 48 h. The co-incubation was started at 24 h after transfection. At 24 h after transfection, the cells were harvested for trypan blue exclusion assays (D) and calcein assays (E). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA (F). ***, p < 0.001; n.s., not significant; WB, Western blot.

FIGURE 3.

Death domain of UNC5C is essential for UNC5C toxicity. A–C, F11 cells, transfected with the empty pHA vector (vector), pRK5-T835M-UNC5C, or pRK5-T835M-UNC5CΔDD, were harvested at 72 h after transfection for trypan blue exclusion assays (A) and WST-8 assays (B). The cell lysates were then subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA (C). D, F11 cells were transfected with the pHA vector (vector), pHA-WT-APP (WT-APP) or pRK5-WT-UNC5C (WT-UNC5C), pRK5-WT-UNC5CΔDD (WT-UNC5CΔDD), the empty pRK5/Myc vector (Myc vector), or pRK5/Myc-DAPK1 (Myc DAPK1). At 24 h after transfection, the cells were harvested for the preparation of cell lysates. Two types of cell lysates were mixed and incubated at 4 °C overnight. The mixed cell lysates were then subjected to immunoprecipitation (IP) using a Myc antibody. Washed immunoprecipitates (IP with anti-Myc) and the cell lysates (Input) were then subjected to SDS-PAGE and immunoblot analysis with a Myc antibody or HA antibody. Overexpression of WT-APP was performed as a transfection control. ***, p < 0.001; n.s., not significant; WB, Western blot.

FIGURE 4.

T835M-UNC5C-induced cell death is mediated by DAPK and PKD. A–C, F11 cells, transfected with the empty pHA vector (vector) or pRK5-T835M-UNC5C, were co-incubated with or without 1 μm DAPK1 inhibitor or 1 μm PKD inhibitors (or DMSO as a negative control) for 48 h. This co-incubation was started at 24 h after transfection. At 72 h after transfection, the cells were harvested for trypan blue exclusion assays (A) and WST-8 assays (B). The cell lysates were then subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA (C). D and E, F11 cells, co-transfected with the empty pHA vector (vector) or pRK5-T835M-UNC5C together with the empty pRK5/Myc vector (vector 1) or pRK5/Myc-dominant-negative DAPK1 (dnDAPK1) or with the empty pcDNA3 vector (vector 2) or pcDNA3-kinase-dead PKD (kdPKD), were harvested at 72 h for trypan blue exclusion assays (D). The cell lysates were then subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA or a DAPK1 antibody mixed with PKD antibody (E). ***, p < 0.001; n.s., not significant; WB, Western blot.

FIGURE 5.

T835M-UNC5C-induced cell death is mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector (vector) or pRK5-T835M-UNC5C together with the pcDNA3 vector (vector), pcDNA3-dominant-negative ASK1 (dnASK1), or pcDNA3-dominant-negative JNK (dnJNK). At 72 h, the cells were harvested for trypan blue exclusion assays (A). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA (B). ***, p < 0.001; *, p < 0.05; n.s., not significant; WB, Western blot.

FIGURE 6.

Constitutively active PKD induces cell death, mediated by ASK1 and JNK. A and B, F11 cells were co-transfected with the empty pHA vector (vector) or pcDNA3-constitutively active PKD (caPKD) together with the pcDNA3 vector (vector2), pcDNA3-dominant-negative ASK1 (dnASK1), or pcDNA3-dominant-negative JNK (dnJNK). At 72 h, the cells were harvested for trypan blue exclusion assays (A). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a PKD antibody or a monoclonal antibody against HA (B). C, F11 cells were co-transfected with pcDNA3-FLAG-JNK1a1 and the empty pHA vector (vector), pRK5-T835M-UNC5C, or pHA-V642I-APP. At 48 h after transfection, the cells were harvested for the preparation of cell lysates. FLAG-JNK1a1 in the lysates was immunoprecipitated with a FLAG antibody (M2) and used for in vitro kinase assays with c-Jun-derived peptide as a substrate. Whole mixtures were subjected to SDS-PAGE and immunoblot analysis with an HA antibody, phospho-c-Jun antibody, phospho-JNK antibody, or FLAG antibody (M2). Similar results were obtained in three independent experiments. ***, p < 0.001; n.s., not significant; WB, Western blot.

FIGURE 7.

NADPH oxidase and caspases are involved in cell death, mediated by T835M-UNC5C. A–C, F11 cells, transfected with the empty pHA vector (vector) or pRK5-T835M-UNC5C were co-incubated with or without 100 μm NADPH oxidase inhibitor acetovanillone (or apocynin; APO) or 100 μm caspase-3 inhibitor Ac-DEVD-CHO (DEVD) for 48 h. This co-incubation was started 24 h after transfection. At 72 h after transfection, the cells were harvested for trypan blue exclusion assays (A) and WST-8 assays (B). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with a monoclonal antibody against HA (C). ***, p < 0.001; n.s., not significant; WB, Western blot.

FIGURE 8.

Neither DAPK1 nor PKD is involved in the cell death pathway mediated by APP. A–C, F11 cells, transfected with the empty pcDNA3.1/MycHis vector (vector) or pcDNA3.1/MycHis-V642I-APP (V642I-APP), were co-incubated with or without 1 μm DAPK1 inhibitor or 1 μm PKD inhibitors or DMSO for 48 h. The co-incubation was started at 24 h after transfection. At 72 h after transfection, the cells were harvested for trypan blue exclusion assays (A) and WST-8 assays (B). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with antibody against APP (C). ***, p < 0.001; **, p < 0.01; n.s., not significant; WB, Western blot.

FIGURE 9.

Netrin1 binds to APP and inhibits TGFβ2-induced neuronal cell death via APP. A, conditioned media, derived from the cells that were transfected with the empty pEF1/MycHis vector (vector) or pEF1/MycHis-mNetrin1 (mouse netrin1 C-terminally tagged with MycHis), were collected for pulldown with TALON beads (Beads) for the preparation of His₆-tagged mNetrin1. In parallel, F11 cells were transfected with the empty pHA vector (vector), pRK5-WT-UNC5C (WT-UNC5C-HA), or pHA-WT-APP (WT-APP-HA). 24 h after transfection, the cell lysates were prepared from the cells. The washed TALON beads capturing mNetrin1-MycHis (mNetrin1) or control (vector) were then mixed with one of these cell lysates. The whole mixtures were incubated at 4 °C overnight and then washed. The final beads (Pull-down) as well as the beads and the cell lysates (Input) were used for immunoblot analysis with a monoclonal antibody against HA or Myc. Similar results were obtained in two independent experiments. B and C, F11 cells, transfected with the empty pFLAG vector (vector) or pFLAG-WT-APP (WT-APP), were co-incubated with 1 μm mNetrin1 or BSA together with 20 nm TGFβ2 or PBS. This co-incubation was started 24 h after transfection. 72 h after transfection, they were harvested for trypan blue exclusion assays (B). The cell lysates were subjected to SDS-PAGE and immunoblot analysis with an antibody against APP (C). ***, p < 0.001; n.s., not significant; WB, Western blot.

FIGURE 10.

Schematic illustration of the proposed T835M-UNC5C- and V642I-APP-induced neuronal death-signaling pathways. The UNC5C-induced death-signaling pathway is mediated by DAPK1, PKD, ASK1, JNK, NADPH oxidase, and caspases (Figs. 1–8), and the APP-induced death-signaling pathway is mediated by G_o, Rac1/Cdc42, ASK1, MAPKK, JNK, NADPH oxidase, and caspases (9–13). The T835M mutation of UNC5C and a London-type mutation of APP, V642I, intensify each death signaling (Fig. 1) (9–13). CLSP binds to the heterotrimeric Humanin receptor and induces expression of SH3BP5, which inhibits the UNC5C- and APP-mediated death signaling by inhibiting JNK (Fig. 2) (21, 23, 24, 26). Netrin1 binds to UNC5C and inhibits the UNC5C-induced death signaling (Fig. 1). Netrin1 binds to APP and partially inhibits the APP-induced death signaling and reduces the production of Aβ (Fig. 9) (29).