Secreted Aβ Does Not Mediate Neurotoxicity by Antibody-Stimulated Amyloid Precursor Protein

doi:10.1006/bbrc.2001.4604

Biochemical and Biophysical Research Communications

Volume 282, Issue 2, 30 March 2001, Pages 548-556

https://doi.org/10.1006/bbrc.2001.4604 Get rights and content

Abstract

Antibodies against APP, a precursor of Aβ deposited in Alzheimer's disease brain, have been shown to cause neuronal death. Therefore, it is important to determine whether Aβ mediates antibody-induced neurotoxicity. When primary neurons were treated with anti-APP antibodies, Aβ40 and Aβ42 in the cultured media were undetectable by an assay capable of detecting 100 nM Aβ peptides. However, exogenously treated Aβ1-42 or Aβ1-43 required >3 μM to exert neurotoxicity, and 25 μM Aβ1-40 was not neurotoxic. Glutathione-ethyl-ester inhibited neuronal death by anti-APP antibody, but not death by Aβ1-42, whereas serum attenuated toxicity by Aβ1-42, but not by anti-APP antibody. Using immortalized neuronal cells, we specified the domain responsible for toxicity to be cytoplasmic His⁶⁵⁷-Lys⁶⁷⁶, but not the Aβ1-42 region, of APP. This indicates that neuronal cell death by anti-APP antibody is not mediated by secreted Aβ.

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A role of amyloid β (Aβ) peptide aggregation and deposition in Alzheimer's disease (AD) pathogenesis is widely accepted. Significantly, abnormalities induced by aggregated Aβ have been linked to synaptic and neuritic degeneration, consistent with the “dying-back” pattern of degeneration that characterizes neurons affected in AD. However, molecular mechanisms underlying the toxic effect of aggregated Aβ remain elusive. In the last 2 decades, a variety of aggregated Aβ species have been identified and their toxic properties demonstrated in diverse experimental systems. Concurrently, specific Aβ assemblies have been shown to interact and misregulate a growing number of molecular effectors with diverse physiological functions. Such pleiotropic effects of aggregated Aβ posit a mayor challenge for the identification of the most cardinal Aβ effectors relevant to AD pathology. In this review, we discuss recent experimental evidence implicating amyloid β precursor protein (APP) as a molecular target for toxic Aβ assemblies. Based on a significant body of pathologic observations and experimental evidence, we propose a novel pathologic feed-forward mechanism linking Aβ aggregation to abnormalities in APP processing and function, which in turn would trigger the progressive loss of neuronal connectivity observed early in AD.
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Furthermore, the proportion of APP cleaved by α-secretase is much higher than that cleaved by β-secretase in brain and can further increase severalfold (17) to the detriment of the major fraction of APP degraded intracellularly (74), Therefore, under conditions favorable for α-secretase cleavage (75), not only the ratio sAPPα/sAPPβ but also the total amount of sAPP is increased and may influence APP dimerization in the plasma membrane. As it had not been shown before that cell surface APP is already almost entirely predimerized, the known neurotoxic effect of certain antibodies (19–22, 24) could have been assumed to be exerted through the dimerization of cell surface APP. Rather, our novel data suggest that these antibodies act by retaining increased amounts of APP dimers at the cell surface and/or by inducing higher order APP aggregates.
The amyloid precursor protein (APP) is implied both in cell growth and differentiation and in neurodegenerative processes in Alzheimer disease. Regulated proteolysis of APP generates biologically active fragments such as the neuroprotective secreted ectodomain sAPPα and the neurotoxic β-amyloid peptide. Furthermore, it has been suggested that the intact transmembrane APP plays a signaling role, which might be important for both normal synaptic plasticity and neuronal dysfunction in dementia. To understand APP signaling, we tracked single molecules of APP using quantum dots and quantitated APP homodimerization using fluorescence lifetime imaging microscopy for the detection of Förster resonance energy transfer in living neuroblastoma cells. Using selective labeling with synthetic fluorophores, we show that the dimerization of APP is considerably higher at the plasma membrane than in intracellular membranes. Heparan sulfate significantly contributes to the almost complete dimerization of APP at the plasma membrane. Importantly, this technique for the first time structurally defines the initiation of APP signaling by binding of a relevant physiological extracellular ligand; our results indicate APP as receptor for neuroprotective sAPPα, as sAPPα binding disrupts APP dimers, and this disruption of APP dimers by sAPPα is necessary for the protection of neuroblastoma cells against starvation-induced cell death. Only cells expressing reversibly dimerized wild-type, but not covalently dimerized mutant APP are protected by sAPPα. These findings suggest a potentially beneficial effect of increasing sAPPα production or disrupting APP dimers for neuronal survival.
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Humanin (HN) inhibits neuronal cell death induced by various Alzheimer's disease (AD)-related insults. It has been proposed that HN binds to a putative receptor on the cell membrane and triggers a signal transduction cascade linked to neuroprotection. Recently, it was shown that HN binds to pertussis toxin (PTX)-sensitive G protein-coupled formylpeptide receptor-like-1 molecule (FPRL-1), reduces Aβ(1-42) aggregation and fibril formation, and suppresses the Aβ(1-42) toxicity on mononuclear phagocytic cells [Ying, G., Iribarren, P., Zhou, Y., Gong, W., Zhang, N., Yu, Z.X., Le, Y., Cui, Y., Wang, J.M., 2004. Humanin, a newly identified neuroprotective factor, uses the G protein-coupled formylpeptide receptor-like-1 as a functional receptor. Journal of Immunology 172 (11), 7078–7085.]. We here show that siRNA-mediated disruption of expression of the mouse counterpart of FPRL-1, FPR2, did not result in attenuation of HN-mediated rescue of neuronal cell death induced by AD-related insults. We simultaneously provide evidence that neuroprotection by HN in F11 cells is mediated by the STAT3 transcription factor as well as by certain tyrosine kinases. Altogether, we speculate that a receptor other than FPR2 exists that mediates HN neuroprotection in F11 neurohybrid cells
Antibody-bound β-amyloid precursor protein stimulates the production of tumor necrosis factor-α and monocyte chemoattractant protein-1 by cortical neurons
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Alzheimer's disease (AD) is an age-related neurodegenerative disorder characterized by the accumulation of extracellular depositions of fibrillar β-amyloid (Aβ), which is derived from the alternative processing of β-amyloid precursor protein (APP). Although APP is thought to function as a cell surface receptor, its mode of action still remains elusive. In this study, we found that the culture medium derived from cortical neurons treated with an anti-APP antibody triggers the death of naive neurons. Biochemical and immunocytochemical analyses revealed the presence, both in the conditioned medium and in neurons, of increased levels of tumor necrosis factor-α and monocyte chemoattractant protein-1. Furthermore, the expression of these proinflammatory mediators occurred through a c-Jun N-terminal protein kinase/c-Jun-dependent mechanism. Taken together, our findings provide evidence for a novel mechanism whereby neuronal APP in its full-length configuration induces neuronal death. Such a mechanism might be relevant to neuroinflammatory processes as those observed in AD.
Deposition of amyloid fibrils promotes cell-surface accumulation of amyloid β precursor protein
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Amyloid β protein (Aβ) deposition and neuronal degeneration are characteristic pathological features of Alzheimer's disease (AD). In vitro, Aβ fibrils (fAβ) induce neuronal degeneration reminiscent to AD, but the mechanism of neurotoxicity is unknown. Here we show that amyloid fibrils increase the level of cell-surface full-length amyloid β precursor protein (h-AβPP) and secreted AβPP (s-AβPP). Pulse-chase analysis indicated that fAβ selectively inhibited the turnover of cell-surface AβPP, without altering its intracellular levels. FAβ-induced AβPP accumulation was not abrogated by cycloheximide, suggesting that increased protein synthesis is not critically required. Aβ fibrils sequester s-AβPP from the culture medium and promote its accumulation at the cell surface, indicating that binding of Aβ fibrils mediates AβPP accumulation. A time course analysis of Aβ treatment showed that AβPP level is elevated before significant cell death can be detected, while other toxic insults do not augment AβPP level, suggesting that AβPP may be specifically involved in early stages of Aβ-induced neurodegeneration. Finally, Aβ fibrils promote clustering of h-AβPP in abnormal focal adhesion-like (FA-like) structures that mediate neuronal dystrophy, increasing its association with the cytoskeleton. These results indicate that the interaction of Aβ fibrils with AβPP is an early event in the mechanism of Aβ-induced neurodegeneration that may play a significant role in AD pathogenesis.
The Gtx Homeodomain Transcription Factor Exerts Neuroprotection Using Its Homeodomain
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Citation Excerpt :
In the pIND system, 1 μg of FAD gene (in pIND) with 1 μg of pEF4-hGtx (in pEF4/His) was transfected to F11/EcR cells; cells were then treated with EcD; and cell mortality was measured 72 h after the onset of transfection. The neurotoxicity by V642I-AβPP and NL-AβPP in pcDNA transfected by the 1.5-μg protocol was quantitatively comparable with neurotoxicity by these genes in pIND transfected by the pIND protocol (1 μg of pIND plasmid transfection and 40 μm EcD treatment) in the pIND system, as shown in multiple studies (13-15, 20-22, 24). In this pIND system, high induction of NL-AβPP causes DEVD-resistant non-apoptotic cell death different from DEVD-sensitive apoptotic cell death by its low induction, whereas low to high induction of V642IAβPP solely causes DEVD-sensitive cell death (13).
Certain cases of familial Alzheimer's disease are caused by mutants of amyloid-β precursor protein (AβPP), including V642I-AβPP, K595N/M596L-AβPP (NL-AβPP), A617G-AβPP, and L648P-AβPP. By using an unbiased functional screening with transfection and expression of a human brain cDNA library, we searched for genes that protect neuronal cells from toxicity by V642I-AβPP. One protective clone was identical to the human GTX, a neuronal homeobox gene. Human Gtx (hGtx) inhibited caspase inhibitor-sensitive neuronal cell death not only by V642I-AβPP but also by L648P-, NL-, A617G-AβPP, apolipoprotein E4, and Aβ. The region of hGtx responsible for this rescue function was specified to be its homeodomain (Lys¹⁴⁸-His²⁰⁷). The rescue function was shared by DLX4, a distal-less family gene with a homeodomain only 38.3% homologous to that of hGtx, suggesting that this function would be generally shared by homeodomains. The neuroprotective function of hGtx was attributable to hGtx-stimulated production and secretion of insulin-like growth factor-I. This study provides molecular clues to understand how neuronal cells developmentally regulate themselves against cell death as well as to develop reagents effective in curative therapeutics of Alzheimer's disease.

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¹: The first three authors contributed to this study equally.

²: To whom correspondence should be addressed. Fax: +81-3-5363-8482. E-mail: [email protected].

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Regular ArticleSecreted Aβ Does Not Mediate Neurotoxicity by Antibody-Stimulated Amyloid Precursor Protein

Abstract

J. Biol. Chem.

Mol. Cell. Neurosci.

Neuron

J. Biol. Chem.

J. Biol. Chem.

Brain Res.

Brain. Res. Bull.

J. Biol. Chem.

Clin. Neurol. Neurosurg.

The precursor of Alzheimer disease amyloid A4 protein resembles a cell-surface receptor

Nature

A monoclonal antibody to amyloid precursor protein induces neuronal apoptosis

J. Neurochem.

Alzheimer's disease: A central role for amyloid

J. Neuropathol. Exp. Neurol.

Alzheimer's disease: Etiologies, pathophysiology, cognitive reserve, and treatment opportunities

Neurology

Apoptosis is induced by β-amyloid in cultured central nervous system neurons

Proc. Natl. Acad. Sci. USA

Apoptotic cell death induced by β-amyloid 1-42 peptide is cell type dependent

J. Neurochem.

The Alzheimer's Aβ peptide induces neurodegeneration and apoptotic cell death in transgenic mice

Nat. Genet.

Expression in brain of amyloid precursor protein mutated in the α-secretase site causes disturbed behavior, neuronal degeneration and premature death in transgenic mice

EMBO J.

Aβ deposition is associated with neuropil changes, but not with overt neuronal loss in the human amyloid precursor protein V717F (PDAPP) transgenic mouse

J. Neurosci.

APPSw transgenic mice develop age-related Aβ deposits and neuropil abnormalities, but no neuronal loss in CA1

J. Neuropathol. Exp. Neurol.

Regular Article
Secreted Aβ Does Not Mediate Neurotoxicity by Antibody-Stimulated Amyloid Precursor Protein