Induced expression of neuronal membrane attack complex and cell death by Alzheimer's β-amyloid peptide

doi:10.1016/S0006-8993(98)00346-1

Brain Research

Volume 796, Issues 1–2, 15 June 1998, Pages 187-197

https://doi.org/10.1016/S0006-8993(98)00346-1 Get rights and content

Abstract

β-amyloid peptide (Aβ) and complement-derived membrane attack complex (MAC) are co-localized in senile plaques of brains from Alzheimer's disease (AD) patients. But the relationship between Aβ and complement activation is unclear. We have used human neurotypic cells, differentiated SH-SY5Y, as a model system to examine regulation of neuronal MAC expression and cell death by Aβ. We demonstrated that mRNAs (C1q, C2, C3, C4, C5, C6, C7, C8 and C9) and proteins (C1q, C3 and C9) for the major components of the classical complement cascade are present in the SH-SY5Y neurotypic cells, indicating that neuronal cells can synthesize the necessary proteins required for MAC formation. Furthermore, immunocytochemical studies showed the Aβ-induced neuronal MAC expression on the SH-SY5Y cells after CD59 was removed by PIPLC or blocked by anti-CD59 antibody. Meanwhile, increased Aβ-induced neuronal cell death was observed following treatment with anti-CD59. Taken together, these results suggest that Aβ activates neuronal complement cascade to induce MAC, and a deficiency of endogenous complement regulatory proteins, e.g., CD59, may increase the vulnerability of neurons to complement-mediated cytotoxicity.

Introduction

Extracellular senile plaques and neuronal cell loss in the brain are characteristics of Alzheimer's disease (AD). A major component of senile plaques is β-amyloid peptide (Aβ), a group of 39–43 amino acid peptides derived from the amyloid precursor protein (APP) 15, 23. Aβ is detected in both normal and AD brains [26], indicating that Aβ alone may not be sufficient to cause AD. Immunoreactivity of various components of the complement cascade, including the membrane attack complex (MAC), colocalizes with Aβ in neuritic plaques 4, 5, 12, 16, 17, 34. In vitro, Aβ binds C1q to activate the complement cascade [24], and enhances complement-induced toxicity in neuronal culture [25]. These results suggest that Aβ-induced complement activation may contribute to the neuropathogenesis in AD. We recently reported that human complement is toxic to human neuronal cells only following removal of glycosylphosphatidylinositol (GPI)-anchored cell membrane proteins by phosphoinositide specific phospholipase C (PIPLC) [27]. One of the proteins removed by PIPLC is CD59, a membrane-bound glycoprotein capable of protecting against complement-mediated lysis by preventing the assembly of MAC 10, 21, 31. We hypothesized that cells might be more vulnerable to complement-mediated toxicity when CD59 is dysfunctional. Here we demonstrate that differentiated human neuronal cells, SH-SY5Y, can synthesize the major complement components and Aβ can induce MAC expression on these cells when CD59 was inactivated by either PIPLC or CD59 antibody treatment. These novel findings suggest that Aβ can activate the neuronal complement cascade to induce MAC formation and cell death when the endogenous complement inhibitor CD59 is compromised.

Section snippets

Cell cultures

Human neuroblastoma SH-SY5Y cells were cultured in a 1:1 ratio of MEM/F12 media, 15% heat-inactivated fetal calf serum (GIBCO, Grand Island, NY, USA) and 10 μM retinoic acid (RA, Sigma, USA). The cells were seeded at 25,000 cells/well in 24-well plates. The medium was replaced every 3 days. On day 7 after seeding, cells became neurotypic with multiple neurites. Treatments were given to the cells in serum-free media with 1:100 N₂ supplemented (GIBCO, Grand Island, NY, USA) 50% MEM and 50% F-12.

Cytotoxicity

We have used retinoic acid (RA)- differentiated human neuronal cell line (SH-SY5Y) as an in vitro human neuronal system 2, 11, 22. To test the hypothesis that cells become vulnerable to Aβ after removing endogenous CD59, we used 2 approaches to compromise CD59: (1) the antibody against human CD59 (anti-CD59) which specifically blocks CD59 [19]; (2) PI-PLC enzyme which removes CD59 from cell membrane [3]. A rat antibody against human CD59 (anti-CD59) was added 30 min prior to the addition of 30 μ

Discussion

Since Aβ deposits in senile plaques which contain complement proteins, it is tempting to speculate that Aβ deposit might play a role in complement activation. Glial cells synthesize complement proteins, e.g., astrocyte cultures produce C1 through C9 as well as CD59, and microglia produce C1 and C3 7, 8. Our in vitro studies are the first to provide the direct evidence that SH-SY5Y human neuronal cells can synthesize the major components (Fig. 4) and Aβ can induce neuronal MAC when CD59 is

Acknowledgements

We thank Dr. Mike Falduto for assisting us with our initial western blot experiment, Dr. Warren Wade, Richard Perner for providing Aβ(1–42) peptide and Dr. June Biedler of Memorial Sloan-Kettering Cancer Center, New York for providing an initial stock of SH-SY5Y cells. Dr. Chi-Ming Lee is currently at Institute for Dementia Research, Bayer, 400 Morgan Lane, West Haven, CT 06516.

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Research reportInduced expression of neuronal membrane attack complex and cell death by Alzheimer's β-amyloid peptide

Abstract

Introduction

Section snippets

Cell cultures

Cytotoxicity

Discussion

Acknowledgements

Biophys. J.

J. Biol. Chem.

Neurosci. Lett.

Brain Res.

Neurobiol. Aging

Neurosci. Lett.

Brain Res.

Brain Res.

Brain Res.

Morphology and growth, tumorigenicity and cytogenetics of human neuroblastoma cells in continuous culture

Cancer Res.

CD59, an Ly-6-like protein expressed in human lympoid cells, regulates the action of the complement membrane attack complex on homologous cells

J. Ex. Med.

Immunoglobulins and complement factors in senile plaques

Acta Neuropathol., Berlin

Cerebral amyloid plaques in Alzheimer's disease but not in Scrapie-affected mice are closely associated with a local inflammatory process

Virchows Archiv. B. Cell Path.

Expression of complement components of the alternative pathway by glioma cell lines

J. Immunol.

In vivo transfer of GPI-linked complement restriction factors from erythrocytes to the endothelium

Science

Research report
Induced expression of neuronal membrane attack complex and cell death by Alzheimer's β-amyloid peptide