Amyloid precursor protein (APP), the precursor of Ab, has been shown to function as a cell surface receptor that mediates neuronal cell death by anti-APP antibody. The c-Jun N-terminal kinase (JNK) can mediate various neurotoxic signals, including Ab neurotoxicity. However, the relationship of APP-mediated neurotoxicity to JNK is not clear, partly because APP cytotoxicity is Ab independent. Here we examined whether JNK is involved in APP-mediated neuronal cell death and found that: (i) neuronal cell death by antibody-bound APP was inhibited by dominant-negative JNK, JIP-1b and SP600125, the specific inhibitor of JNK, but not by SB203580 or PD98059; (ii) constitutively active (ca) JNK caused neuronal cell death and (iii) the pharmacological profile of caJNK-mediated cell death closely coincided with that of APP-mediated cell death. Pertussis toxin (PTX) suppressed APP-mediated cell death but not caJNK-induced cell death, which was suppressed by Humanin, a newly identified neuroprotective factor which inhibits APP-mediated cytotoxicity. In the presence of PTX, the PTX-resistant mutant of Ga o , but not that of Ga i , recovered the cytotoxic action of APP. These findings demonstrate that JNK is involved in APP-mediated neuronal cell death as a downstream signal transducer of G o .

Neuronal pathology of the brain with Alzheimer's disease (AD) is characterized by numerous depositions of amyloid-beta peptides (Abeta). Abeta binding to the 75-kDa neurotrophin receptor (p75NTR) causes neuronal cell death. Here we report that Abeta causes cell death in neuronal hybrid cells transfected with p75NTR, but not in nontransfected cells, and that p75NTR(L401K) cannot mediate Abeta neurotoxicity. We analyzed the cytotoxic pathway by transfecting pertussis toxin (PTX)-resistant G protein alpha subunits in the presence of PTX and identified that Galpha(o), but not Galpha(i), proteins are involved in p75NTR-mediated Abeta neurotoxicity. Further investigation suggested that Abeta neurotoxicity via p75NTR involved JNK, NADPH oxidase, and caspases-9/3 and was inhibited by activity-dependent neurotrophic factor, insulin-like growth factor-I, basic fibroblast growth factor, and Humanin, as observed in primary neuron cultures. Understanding the Abeta neurotoxic mechanism would contribute significantly to the development of anti-AD therapies.

The biological function of full-length amyloid-␤ protein precursor (A␤PP), the precursor of A␤, is not fully understood. Multiple laboratories have reported that antibody binding to cell surface A␤PP causes neuronal cell death. Here we examined whether induced dimerization of the cytoplasmic domain of A␤PP (A␤PP CD ) triggers neuronal cell death. In neurohybrid cells expressing fusion constructs of the epidermal growth factor (EGF) receptor with A␤PP CD (EGFR/A␤PP hybrids), EGF drastically enhanced neuronal cell death in a manner sensitive to acetyl-Laspartyl-L-glutamyl-L-valyl-L-aspartyl-aldehyde (Ac-DEVD-CHO; DEVD), GSH-ethyl ester (GEE), and pertussis toxin (PTX). Dominant-negative apoptosis signal-regulating kinase 1 (ASK1) blocked this neuronal cell death, but not ␣-synuclein-induced cell death. Constitutively active ASK1 (caASK1) caused DEVD/GEEsensitive cell death in a manner resistant to PTX and sensitive to Humanin, which also suppressed neuronal cell death by EGFR/ A␤PP hybrid. ASK1 formed a complex with A␤PP CD via JIP-1b, the c-Jun N-terminal kinase (JNK)-interacting protein. EGFR/ A␤PP hybrid-induced and caASK1-induced neuronal cell deaths were specifically blocked by SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one), a specific JNK inhibitor. Combined with our earlier study, these data indicate that dimerization of A␤PP CD triggers ASK1/JNK-mediated neuronal cell death. We also noticed a potential role of ASK1/JNK in sustaining the activity of this mechanism after initial activation by A␤PP, which allows for the achievement of cell death by short-term anti-A␤PP antibody treatment. Understanding the function of A␤PP CD and its downstream pathway should lead to effective anti-Alzheimer's disease therapeutics.

One of the most important pathological features of Alzheimer's disease (AD) is extracellular senile plaques, whose major component is amyloid-b peptides (Ab). Ab binds to the extracellular domain of p75NTR (p75 neurotrophin receptor) and induces neuronal cell death. We investigated the molecular mechanism of Ab-induced neurotoxicity in detail from the standpoint of interaction between p75NTR and its recently identified relative, PLAIDD (p75-like apoptosis-inducing death domain). Using F11 neuronal hybrid cells, we demonstrate that there are two distinct pathways for Abinduced toxicity mediated by p75NTR. One pathway that has been previously elucidated, is mediated by p75NTR, Go, JNK, NADPH oxidase and caspase3-related caspases. We found that PLAIDD and Gi proteins, heterotrimeric G proteins, are involved in the alternative Ab-induced neurotoxicity mediated by p75NTR. The alternative pathway triggered by Ab is thus mediated by p75NTR, PLAIDD, Gi, JNK, NADPH oxidase and caspase3-related caspases. In addition, we found that HN, ADNF, IGF-I, or bFGF inhibits both pathways of Ab-induced neurotoxicity mediated by p75NTR. Amyloid-b peptides (Ab) are the major constituent of the senile plaques in Alzheimer's disease (AD). Ab, the 39-43 amino acids peptide, is produced by the b-and c-secretasemediated cleavage of amyloid precursor protein (APP). Formation and accumulation of Ab have been inferred to contribute to the development of AD. In vitro, increased levels of Ab concentration cause cell death in primary cultured neurons as well as some neuronal line cells (Yankner et al. 1989;Loo et al. 1993;Gschwind and Huber 1995;Kaneko et al. 1995;Pike et al. 1997;Giovanni et al. 1999;Sudo et al. 2001). However, the precise mechanism underlying Ab neurotoxicity remains to be elucidated.One possible mechanism for Ab-induced neurotoxicity is that Ab binds to its putative receptor on the neuronal cell membrane and triggers the intracellular death signal. Multiple proteins have been so far claimed to be candidates for the Ab receptor. They include the receptors for the endoplasmic reticulum Ab-binding dehydrogenase (ERAB;Yan et al. 1997a), the advanced glycation end products (Yan et al. 1997b), the a7 nicotinic acetylcholine receptor (Wang Address correspondence and reprint requests to M. Matsuoka, Department of Pharmacology, KEIO University School of Medicine, Shinanomachi, Tokyo, Japan. E-mail: sakimatu@sc.itc.keio.ac.jpAbbreviations used: Ab, amyloid beta; AD, Alzheimer's disease; ADNF, activity-dependent neurotrophic factor; APO, apocynin; APP, amyloid precursor protein; BBP-1, b-amyloid binding protein-1; bFGF, basic fibroblast growth factor; DPI, diphenyleneiodonium chloride; ERAB, endoplasmic reticulum Ab-binding dehydogenase; FBS, fetal bovine serum; GEE, gluthione-ethyl-ester; HN, Humanin; HRP, horseradish peroxidase; JNK, c-Jun N-terminal kinase; NFkB, nuclear factorkB; NOS, nitric oxide synthase; (p75-like apoptosis-inducing death domain) p75NTR, 75 kDa neurotrophin receptor; PTX, pertussis toxin; ROS, reactive ox...