Mechanism of membrane depolarization caused by the Alzheimer Aβ1–42 peptide

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Abstract

We report a novel observation that the neurotoxic Alzheimer peptide Aβ1–42, when pre-incubated, causes a dramatic and lasting membrane depolarization in differentiated human hNT neuronal cells and in rodent PC12 cells in a concentration-dependent manner. This phenomenon involves activation of the metabotropic glutamate receptor, mGluR1. Aβ-induced membrane depolarization in PC12 cells is sensitive to mGluR1 antagonists and to pertussis and cholera toxins, indicating the involvement of particular G-proteins. The effect is different from the known ability of aggregated Aβ1–42 to cause a calcium influx. Since mGluR1 agonists mimic the Aβ effect, we deduce that in this cell system glutamate can control the membrane potential and thereby the excitability of its target neurons. We propose that Aβ-induced membrane depolarization described here leads in Alzheimer's disease to hyperexcitability of affected neurons and is a crucially important molecular mechanism for β-amyloid toxicity and cognitive dysfunction in the disease.

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Materials and methods

mGluR Antagonists and agonists were obtained from Tocris Cookson Ltd., pertussis toxin and cholera toxin were from Sigma, Inc. Stock solutions of Aβ1–42 were prepared as described [4]. Experimental samples were prepared by diluting the stock solution of Aβ1–42 to 10μM (unless otherwise noted) in Tyrode's/2Ca buffer (150 mM NaCl, 3 mM KCl, 10 mM HEPES, pH 7.4, 2 mM CaCl2, 10 mM d-glucose, pH 7.4). Seed-free Aβ1–42 was prepared as described by Fezoui et al. [14] for Aβ1–40. In our case the seed-free

Results and discussion

In differentiated hNT neuronal cells, we find that the pre-aggregated Alzheimer peptide Aβ(1–42, aggr.) causes a large membrane depolarization. The peptide had been allowed to aggregate in Tyrode's/2Ca buffer at 37 °C and pH 7.4 for 48 h. For example, when Aβ(1–42, aggr.) at 20μM is added to the cells in the presence of the slow-acting voltage-sensitive fluorescent dye DiBAC4(3), there is an immediate sharp increase in fluorescence (Fig. 1A), indicating cell membrane depolarization. The new

Acknowledgements

This work was supported by the John & Dorothy Wilson Fund and by the Kurt & Johanna Immerwahr Fund for Alzheimer Research at MIT. Part of the work (V.T.) was funded by the MIT Undergraduate Research Opportunities Program. We are grateful to Prof. Elazer Edelman, Harvard-MIT Health Sciences Division, for allowing us to use his Fluoroskan II fluorometer, and to Prof. Jonathan King, Biology, MIT, for the use of his Hitachi fluorimeter. We gratefully acknowledge the many helpful discussions with

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Abbreviations: AMPA, α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid; CNQX, 6-cyano-7-nitroquinoxaline-2,3-dione; D-AP5, 2-amino-5-phosphonovaleric acid; DiBAC4(3), bis-(1,3-dibutylbarbituric acid)trimethine oxonol; Fura-2 salt, ratiometric Ca2+ indicator–Na salt; hNT neurons, a human teratocarcinoma cell line differentiated with retinoic acid; NMDA, N-methyl-d-aspartate; NMDG+30, N-methyl-d-glucamine; Tyrode's/2Ca, Tyrode's solution with 2mM Ca2+.

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