Up-regulation of Bcl-xL in response to subtoxic β-amyloid: role in neuronal resistance against apoptotic and oxidative injury

doi:10.1016/S0306-4522(00)00458-9

Neuroscience

Volume 102, Issue 1, 2 January 2001, Pages 139-150

https://doi.org/10.1016/S0306-4522(00)00458-9 Get rights and content

Abstract

Neuron death in Alzheimer’s disease is believed to be triggered by an increased production of amyloidogenic β-amyloid peptides, involving both increased oxidative stress and activation of a conserved death program. Bcl-xL, an anti-apoptotic protein of the Bcl-2 family, is expressed at high levels in the adult nervous system. Exposure of neuronal cultures to subtoxic concentrations of β-amyloid peptide 1–40 (1–10 μM) or the fragment 25–35 (1–10 μM) up-regulated both bcl-xL mRNA and Bcl-xL protein levels, determined by reverse transcriptase–polymerase chain reaction and western blot analysis. Bcl-xL protein was also up-regulated during oxidative stress induced by exposure to hydrogen peroxide (3–100 μM) or ferric ions (1–10 μM). In contrast, apoptotic stimuli (exposure to staurosporine or serum withdrawal) actually decreased neuronal Bcl-xL expression. To investigate the role of Bcl-xL in cell death relevant to Alzheimer’s disease, we stably overexpressed Bcl-xL in human SH-SY5Y neuroblastoma cells. Cells overexpressing Bcl-xL were significantly protected from β-amyloid neurotoxicity and staurosporine-induced apoptosis compared to vector-transfected controls. In contrast, Bcl-xL overexpression only confered a mild protection against oxidative injury induced by hydrogen peroxide.

We conclude that up-regulation of Bcl-xL expression in response to subtoxic concentrations of β-amyloid is a stress response that increases the resistance of neurons to β-amyloid neurotoxicity primarily by inhibiting apoptotic processes.

Section snippets

Cell culture

Rat PC12 pheochromocytoma and human SH-SY5Y neuroblastoma cells were plated into tissue culture flasks (Falcon Becton Dickinson, Heidelberg, Germany) or 24-well plates (Nunc, Wiesbaden, Germany) and cultured in RPMI 1640 culture medium supplemented with 5% or 10% fetal calf serum, respectively, 100 U/ml penicillin and 100 μg/ml streptomycin (Life Technologies, Karlsruhe, Germany). The PC12 cell culture medium was additionally supplemented with 10% horse serum (Pansystems GmbH, Aidenbach,

Exposure to subtoxic Aβ up-regulates neuronal bcl-xL messenger RNA and Bcl-xL protein expression

Cultured rat PC12 pheochromocytoma cells were exposed to Aβ1-40, its toxic fragment Aβ25-35, or control peptide Aβ35-25. Treatment with 1–10 μM of Aβ1-40 or Aβ25-35 for 24 h did not induce a significant cell death determined by the uptake of membrane impermeable dyes Trypan-Blue or EthD-1 in the cultures (n=4 separate experiments; data not shown). After 6 h of treatment with peptides, total mRNA was extracted and subjected to RT–PCR. Exposure of PC12 cells to Aβ25-35 (1–10 μM) and Aβ1-40 (1–10 μM)

Discussion

The present study demonstrates that exposure of cultured neurons to subtoxic Aβ concentrations increased the expression of the anti-apoptotic protein, Bcl-xL (Fig. 1, Fig. 2, Fig. 3). Overexpression of Bcl-xL protected neurons against toxic concentrations of Aβ (Fig. 6). Bcl-xL overexpression also protected against apoptosis induced by staurosporine (Fig. 5), and to a lesser extent against oxidative neuronal injury induced by hydrogen peroxide (Fig. 7). It is therefore conceivable that the

Acknowledgements

We thank Elke Bauerbach, Gudrun Münstermann and Christiane Schettler for technical assistance, and Dr Lawrence H. Boise and Professor Craig B. Thompson for providing plasmid pSFFV-Bcl-xL and Bcl-x antibody. This study was supported by a grant from Alzheimer Forschung Initiative e.V. to J.H.M.P and by the IZKF Universität Münster.

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