Modulation of Ca2+ channel currents in primary cultures of rat cortical neurones by amyloid β protein (1–40) is dependent on solubility status

doi:10.1016/S0006-8993(02)03547-3

Brain Research

Volume 956, Issue 2, 29 November 2002, Pages 254-261

https://doi.org/10.1016/S0006-8993(02)03547-3 Get rights and content

Abstract

The Alzheimer’s disease peptide amyloid β protein (Aβ) can exist in soluble and fibrillar, aggregated forms. Aβ in the aggregated form is thought to be pro-apoptotic, causing cell death when applied to cultured neurones by disrupting Ca²⁺ homeostasis. This process may involve changes in Ca²⁺ influx across the plasma membrane. The aim of this study was to quantify this effect by applying both the aggregated and unaggregated forms of Aβ to cultured rat cortical neurones. Unaggregated Aβ_1–40 (24-h pretreatment, 1 μM) stimulated an increase in voltage-dependent Ca²⁺ channel current activity, which was found to comprise of N- and P-type current. In the aggregated form, Aβ_1–40 pre-treatment reduced Ca²⁺ channel current density in cortical neurones via an action on N-type Ca²⁺ current. This failure of aggregated Aβ_1–40 to increase the Ca²⁺ channel current was confirmed on cerebellar granule neurone Ca²⁺ currents which normally undergo an increase in activity following soluble Aβ application. Using the MTT and TUNEL assays, aggregated Aβ_1–40 was found to promote apoptotic cell death in cortical neurones confirming that Aβ exhibited the expected biological activity. Unaggregated Aβ had no neurotoxic effect. These data indicate that the unaggregated, non-pathological form of Aβ_1–40, and not the aggregated form, cause changes in neuronal Ca²⁺ channel activity. This may reflect a normal functional role for amyloid peptides in the central nervous system.

Introduction

The deposition of amyloid β protein (Aβ), as insoluble fibrillar aggregates in senile plaques, is the dominant histological hallmark of Alzheimer’s disease (AD). It is widely believed that the cellular actions of Aβ are responsible for the neuronal cell loss observed in AD. The key to the cellular toxicity of Aβ appears to be in its aggregation state [18]. Aggregated Aβ has been shown to have clear neurotoxic effects when applied in high concentrations to a variety of cultured neurone systems [10], [13], [17], [23]. However, the unaggregated form has no clearly defined physiological or pathological function [24], [29]. This is surprising, since unaggregated Aβ_1–40 has been detected at submicromolar concentrations in the cerebrospinal fluid of healthy human subjects [22] and is secreted into growth media by cultured neurones [8]. Recent studies show amyloid peptides to be constantly anabolised and catabolised under normal conditions [11], [25], which supports the possibility of a physiological role for Aβ.

A growing body of evidence indicates that Aβ has modulatory effects on ion channel currents in central neurones. In some cases these changes have been related to the neurotoxic action of the peptide via a disruption of intracellular ion homeostasis. Specifically, several studies suggest that Aβ potentiates Ca²⁺ channel currents in vitro [4], [23], [26] and that this effect is associated with cell death, implying that the aggregated form of Aβ is responsible. However, we have found that the unaggregated form of Aβ_1–40 can increase Ca²⁺ channel activity in primary cultures of rat cerebellar granule neurones [20] and in rat cortical synaptosomes [14]. Furthermore, we found that unaggregated Aβ could also increase K⁺ currents in cerebellar granule neurones but that aggregated Aβ was without effect [21]. This raises the possibility that aggregated and unaggregated forms of Aβ can have differential effects on ion channel activity. In addition, it calls into question the idea that increased plasmalemmal Ca²⁺ channel activity is associated with Aβ-induced cell death. The aim of this study, therefore, was to determine whether it is the aggregated or unaggregated form of Aβ that gives rise to an increase in Ca²⁺ channel currents in central neurones. To this end, we have applied aggregated and unaggregated forms of Aβ to neurones cultured from the central nervous system in order to determine the effects of each on the voltage-gated Ca²⁺ channel currents in these cells.

Section snippets

Culturing of rat central neurones

All experiments were performed using primary cultures of rat cerebellar granule and cortical neurones. Cells were obtained by enzymatic and mechanical dissociation as previously described [9], [20]. Briefly, tissue was removed from 6–8-day-old rat pups (cerebellum) or 16–18-day fetal rats (cerebral cortex) and triturated following a 15-min trypsin (EC 4.4.21.4, 2.5 mg ml⁻¹ in phosphate buffered saline) digestion. Cells, plated at a density of 0.25×10⁶ cells per well on circular 10-mm diameter

Results

In order to determine the effect of unaggregated Aβ on the Ca²⁺ channel current in rat cortical neurones, cultures were preincubated with 1 μM unaggregated Aβ_1–40 for 24 h. This resulted in an increase in Ca²⁺ channel current when compared to controls (Fig. 1A,B). When cells were depolarised from a holding potential of −90 mV to a test potential of +10 mV, maximal inward Ca²⁺ channel current density was enhanced by 49% in the Aβ-treated neurones (control −95±10 pA/pF, n=39; Aβ_1–40 −141±12

Discussion

The aim of this study was to compare the effects of unaggregated and aggregated Aβ_1–40 on Ca²⁺ channel currents in rat central neurones. The results clearly demonstrated that unaggregated Aβ_1–40 stimulated an increase in voltage-dependent Ca²⁺ channel current activity, which was found to comprise of N- and P-type current. In the aggregated form, Aβ_1–40 pre-treatment reduced Ca²⁺ channel current density in cortical neurones and had no effect on cerebellar granule neurone Ca²⁺ currents. The

Acknowledgements

We thank Dr T. Rupniak of GlaxoSmithKline Research for Aβ_1–40 and Dr C Shukla for assistance with TUNEL assays. This research was funded by the Wellcome Trust and the MRC.

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Research reportModulation of Ca2+ channel currents in primary cultures of rat cortical neurones by amyloid β protein (1–40) is dependent on solubility status

Abstract

Introduction

Section snippets

Culturing of rat central neurones

Results

Discussion

Acknowledgements

Neuropharmacology

Brain Res.

Neurodegeneration

J. Biol. Chem.

J. Immunol. Methods

Neuropharmacology

Brain Res. Mol. Brain Res.

Giant multilevel cation channels formed by Alzheimer disease amyloid beta-protein [A beta P-(1–40)] in bilayer membranes

Proc. Natl. Acad. Sci. USA

Identification of microglial signal transduction pathways mediating a neurotoxic response to amyloidogenic fragments of beta-amyloid and prion proteins

J. Neurosci.

Alzheimer amyloid beta-peptides exhibit ionophore-like properties in human erythrocytes

Eur. J. Clin. Invest.

Effects of calcium channel antagonists on calcium entry and glutamate release from cultured rat cerebellar granule cells

J. Neurochem.

Amyloid beta peptides mediate hypoxic augmentation of Ca2+ channels

J. Neurochem.

Amyloid beta-peptide is produced by cultured cells during normal metabolism

Nature

Endothelin-1 inhibits voltage-sensitive Ca2+ channels in cultured rat cerebellar granule neurones via the ET-A receptor

Pflügers Arch.

Research report
Modulation of Ca²⁺ channel currents in primary cultures of rat cortical neurones by amyloid β protein (1–40) is dependent on solubility status

Amyloid beta peptides mediate hypoxic augmentation of Ca²⁺ channels

Endothelin-1 inhibits voltage-sensitive Ca²⁺ channels in cultured rat cerebellar granule neurones via the ET-A receptor