Cross-linking of NCAM receptors on neurons induces programmed cell death

doi:10.1016/S0006-8993(98)00309-6

Brain Research

Volume 796, Issues 1–2, 15 June 1998, Pages 20-26

https://doi.org/10.1016/S0006-8993(98)00309-6 Get rights and content

Abstract

Programmed cell death has been implicated in the loss of neurons that occurs in many neurodegenerative diseases. This has led to an increased interest in the types of stimuli that can initiate neurons to undergo programmed cell death. Previously, we have shown that cross-linking of membrane receptors with the lectin concanavalin A can trigger programmed cell death in neurons [D.H. Cribbs, V.M. Kreng, A.J. Anderson, C.W. Cotman, Cross-linking of Concanavalin A receptors on cortical neurons induces programmed cell death, Neuroscience 75 (1996) 173–185]. Concanavalin A, however, binds to many surface glycoproteins and therefore, it is important to determine whether certain specific receptors can initiate the program. We found that surface immobilized anti-neural cell adhesion molecules (NCAM) monoclonal antibodies provide a good substrate for adhesion and neurite outgrowth for cortical neurons. However, neurons treated directly with soluble anti-NCAM monoclonal antibodies show significant cell death after 24 h and exhibit the morphological and biochemical features indicative of apoptosis, including membrane blebbing, cell shrinkage, condensation of nuclear chromatin and internucleosomal DNA cleavage.

Introduction

Programmed cell death (PCD), is a physiological process leading to an orchestrated form of cell death, that plays an important role in growth and developmental regulation. More recently, neuronal loss by programmed cell death has been reported to occur in a number of neurodegenerative diseases such as Alzheimer's disease (AD) [29], Huntington's disease [23], Parkinson's disease and others 11, 30. However, little is known about the factors and conditions that trigger PCD leading to these neurodegenerative disorders. In vitro, PCD can be triggered by a variety of extrinsic and intrinsic signals.

Many extracellular agents, including Fas ligands [19], bacterial superantigens [7]and others bind to the surface of the cell and induce cell death. Fas ligand, a cell surface molecule, binds to its receptor thus initiating PCD of in the Fas-receptor bearing cells. Bacterial superantigens initiate the activation-induced PCD in T lymphocytes by binding to T-cell antigen receptor proteins on the cell surface, the resultant complex activates T cells and initiates PCD upon cross-linking 7, 12. This mechanism can be replicated/mimicked by divalent antibodies against the T-cell antigen receptor, but not with monovalent antibodies, indicating that the binding event is insufficient to activate PCD, and that cross-linking of receptors is required to initiate PCD. In addition, it has been shown that lectins bind and cross-link many glycosylated membrane receptors on cells, which can activate the receptors and thereby induce intracellular signaling cascades leading to alterations in cellular behavior, including cell death 13, 15. Recently, it has been reported that concanavalin A (Con A) induces programmed cell death in cultured neurons by cross-linking membrane receptors [6].

It has also been shown that β-amyloid peptide (Aβ), which accumulates in the senile plaques of Alzheimer's disease, activates programmed cell death in cultured neurons 9, 18. Since Aβ induces PCD only in a fibrillar β-sheet assembly structure 1, 4, 5and when bound to neuronal surfaces, these data suggest that neurons are very sensitive to receptor cross-linking.

In this investigation, we have examined the possibility that cross-linking of membrane receptors by the prominent cell surface adhesion molecule NCAM can initiate programmed cell death using several different anti-NCAM monoclonal antibodies.

Section snippets

Cell culture

Embryonic cultures of cortical neurons were prepared from Sprague–Dawley rats at gestational day 18 as described previously [20]. Briefly, cultures were plated at 2×10⁵ cells/cm² on poly-l-lysine-treated multiwell plates and maintained in Dulbecco's modified Eagle's medium (DMEM; GIBCO) supplemented with N₂ components 2, 3. The cortical neurons were kept in culture at 37°C with 5% CO₂ for 2 days prior to the addition of anti-NCAM monoclonal antibodies.

Cell death and viability

All antibodies were extensively dialyzed

Surface-bound NCAM antibodies promote neurite outgrowth and survival

Surface-immobilized NCAM antibodies 5A5 and 5B8 acted differently than soluble forms on neuronal growth and viability. Neurons plated on NCAM antibodies 5A5 and 5B8 adsorbed on tissue culture plastic promoted neurite outgrowth and adhesion after 24 h incubation (Fig. 1), whereas soluble forms of NCAM antibodies resulted in reduction of viability and adhesion. A similar behavior has been reported earlier 16, 17with concanavalin A immobilized on tissue culture plastic which promoted excellent

Discussion

All of the anti-NCAM monoclonal antibodies tested, OB11, HNK-1, 5A5 and 5B8 were found to induce cell death in cortical neurons. These antibodies induced neuronal cell death characterized by hallmarks of programmed cell death, including cytoskeletal disruption, membrane blebbing, cell shrinkage, nuclear condensation and DNA fragmentation. In addition, when bound to the culture surface, these anti-NCAM antibodies were found to attach and promote neurite outgrowth. Anti-NCAM antibodies 5A5 and

Acknowledgements

This work was supported in part by the National Institute of Aging (NIA), National Research Service Award (NRSA) AG 00096 (BYA), and by NIA Grants AG 13007 and AG 07919. The 5B8 and 5A5 monoclonal antibodies were obtained from the Developmental Studies Hybridoma Bank maintained by the Department of Biological Sciences, University of Iowa (Iowa City, IA) and the Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine (Baltimore, MD), under contract

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Research reportCross-linking of NCAM receptors on neurons induces programmed cell death

Abstract

Introduction

Section snippets

Cell culture

Cell death and viability

Surface-bound NCAM antibodies promote neurite outgrowth and survival

Discussion

Acknowledgements

Differential induction of immediate early gene proteins in cultured neurons by beta-amyloid (Aβ): association of c-Jun with Aβ-induced apoptosis

J. Neurochem.

Growth of a rat neuroblastoma cell line in serum-free supplemented medium

Proc. Natl. Acad. Sci. U.S.A.

Survival and growth of hippocampal neurons in defined medium at low density: advantages of a sandwich culture technique or low oxygen

Brain Res.

A potential role for apoptosis in neurodegeneration and Alzheimer's disease

Mol. Neurobiol.

Cross-linking of Concanavalin A receptors on cortical neurons induces programmed cell death

Neuroscience

Superantigens interact with MCH class II molecules outside of the antigen groove

Cell

Mechanisms and functions of cell death

Annu. Rev. Cell Biol.

Apoptosis mediated neurotoxicity induced by chronic application of β amyloid fragment 25–35

NeuroReport

Identification, purification, and characterization of a calcium-dependent endonuclease (NUC 18) from apoptotic rat thymocytes

J. Biol. Chem.