Alzheimer's amyloid-beta peptide inhibits sodium/calcium exchange measured in rat and human brain plasma membrane vesicles

doi:10.1016/S0306-4522(97)00053-5

Neuroscience

Volume 80, Issue 3, 28 July 1997, Pages 675-684

https://doi.org/10.1016/S0306-4522(97)00053-5 Get rights and content

Abstract

Na⁺/Ca²⁺ exchange activity was measured by monitoring vesicular Ca²⁺ content after incubation in buffers containing ⁴⁵Ca²⁺. When Na⁺-loaded vesicles were placed into Na⁺-free buffer, vesicular Ca²⁺ content increased rapidly and reached a plateau after two to three minutes. Only preaggregated amyloid-beta_1–40 (Aβ_1–40) and Aβ_25–35 reduced vesicular Ca²⁺ content. Both peptides produced a maximal reduction in Ca²⁺ content of approximately 50%. The peptides reduced Ca²⁺ content with similar potency and half maximal effects were seen at less than 10 μM for Aβ_25–35. Calcium-loaded vesicles mediate a rapid Ca²⁺/Ca²⁺ exchange, which also was inhibited by aggregated Aβ_25–35. Aggregated Aβ_25–35 did not affect the passive Ca²⁺ permeability of the vesicles. Aggregated Aβ_25–35 reduced Ca²⁺ content in plasma membrane vesicles isolated from normal and Alzheimer's disease frontal cortex with less potency but the same efficacy as seen in rat brain. Aggregated Aβ_25–35 did not produce nonspecific effects on vesicle morphology such as clumping or loss of intact vesicles. When placed in the buffer used to measure Ca²⁺ content, Congo Red at molar ratios of less than one blocked the inhibitory effect of preaggregated Aβ_25–35. When added in equimolar concentrations to freshly dissolved and unaggregated Aβ_25–35, Congo Red also was effective at blocking the inhibitory effect on Ca²⁺ content. In contrast, vitamin E (antioxidant) and N-tert-butyl-α-phenylnitrone (spin trapping agent) failed to block the inhibitory action of aggregated Aβ_25–35.

The exact mechanisms of Aβ-induced neurotoxicity in cell culture has yet to be solved. Accumulation of free radicals play a necessary role, but disruptions of Ca²⁺ homeostasis are also important. The data presented here are consistent with a proposed mechanism where aggregated Aβ peptides directly interact with hydrophobic surfaces of the exchanger protein and/or lipid bilayer and interfere with plasma membrane Ca²⁺ transport.

Section snippets

Preparation of rat and human plasma membrane vesicles

Frozen tissue sections from both normal and Alzheimer's disease brain frontal cortex were obtained from the National Neurological Research Bank (VAMA Wadsworth, Los Angeles, CA). Plasma membrane vesicles from both rat and human brain were prepared as described previously.[55]

Preparation of Aβ peptides

Three amyloid peptides were used in this study. Purified Aβ_1–40 (H₂N-DAEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVGGVV-OH) was obtained from Quality Controlled Biochemicals (Hopkinton, MA), while Aβ_25–35 (H₂N-GSNKGAIIGLM-OH) and Aβ

Aggregation of synthetic Aβ_25–35 in 10 mM HCl solution

Aβ_25–35 was used to study aggregation kinetics, while non-aggregating peptides Aβ_scrambled and substance P were used as controls. The peptides were dissolved in 10 mM HCl at a concentration around 3–10 mg/ml, then incubated at 37°C. The reason for using 10 mM HCl was to slow aggregation of Aβ_25–35 such that the effects of unaggregated peptide on Na⁺/Ca²⁺ exchange could be studied. Fig. 1 shows the influence of incubation time upon aggregation as measured by thioflavine T fluorescence (ThT)

Do aggregated Aβ peptides inhibit Ca²⁺ transport by the Na⁺/Ca²⁺ exchanger?

Inhibition of vesicular Ca²⁺ accumulation during Na⁺-dependent Ca²⁺ uptake and Ca²⁺/Ca²⁺ exchange, as shown in this study, would be expected if aggregated Aβ peptides inhibited Ca²⁺ transport by the exchanger. Perhaps the most attractive alternative explanation for the mechanism of aggregated Aβ peptide effects on vesicular Ca²⁺ content is an increase in membrane ion permeability. Two findings from the present study argue against increases in Ca²⁺ permeability as the underlying mechanism of the

Conclusions

The exact mechanisms of Aβ-induced neurotoxicity in cell culture has yet to be solved. Accumulation of free radicals play a necessary role, but disruptions of Ca²⁺ homeostasis are also important. In the present study it was shown that exposure to aggregated Aβ_25–35 and Aβ_1–40 produced a partial reduction in Na⁺-dependent Ca²⁺ accumulation by plasma membrane vesicles. These data are consistent with a proposed mechanism where aggregated Aβ peptides directly interact with hydrophobic surfaces of

Unlinked References

4, 5, 9, 24, 31, 43

Acknowledgements

This work was supported by a grant from the National Institute of Neurological Disorders and Stroke, No. NS300384 and the Alzheimer's Association.

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Several lines of evidence suggest that a drop in intracellular ATP levels can drive the Amyloid Precursor Protein processing towards the amyloidogenic pathway rather than the non-amyloidogenic one, leading to Aβ accumulation [122–124]. Different laboratories have already documented that the Na+/Ca2+ exchange activity is compromised and potentially promotes remodeling of the Ca2+ cycling in AD [125–128]. In particular, available findings converge on a reduction of both the expression and activity of NCX [126,128].
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An increase in extracellular Ca2+ concentration indeed strengthens the driving force for Ca2+ influx into neurons during synaptic transmission, and thus favors excitotoxicity. Regulation of Ca2+ concentrations relies on several astroglial elements including the Ca2+ homeostasis modulator 1 (CALHM1) and Na+/Ca2+ exchangers (NCX), which have been associated with AD (Aqdam et al., 2010; Castaldo et al., 2009; Wu et al., 1997) and proposed as valuable neuroprotective targets in neurodegenerative diseases (Gomez-Villafuertes et al., 2007; Molinaro et al., 2013). Besides extracellular Ca2+ concentration, activation of many astroglial mechanisms depends on intracellular Ca2+ signaling (Verkhratsky et al., 2012a,b).
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Signaling effect of amyloid-β<inf>42</inf> on the processing of AβPP
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The effects of amyloid-β are extremely complex. Current work in the field of Alzheimer disease is focusing on discerning the impact between the physiological signaling effects of soluble low molecular weight amyloid-β species and the more global cellular damage that could derive from highly concentrated and/or aggregated amyloid. Being able to dissect the specific signaling events, to understand how soluble amyloid-β induces its own production by up-regulating BACE1 expression, could lead to new tools to interrupt the distinctive feedback cycle with potential therapeutic consequences. Here we describe a positive loop that exists between the secretases that are responsible for the generation of the amyloid-β component of Alzheimer disease. According to our hypothesis, in familial Alzheimer disease, the primary overproduction of amyloid-β can induce BACE1 transcription and drive a further increase of amyloid-β precursor protein processing and resultant amyloid-β production. In sporadic Alzheimer disease, many factors, among them oxidative stress and inflammation, with consequent induction of presenilins and BACE1, would activate a loop and proceed with the generation of amyloid-β and its signaling role onto BACE1 transcription. This concept of a signaling effect by and feedback on the amyloid-β precursor protein will likely shed light on how amyloid-β generation, oxidative stress, and secretase functions are intimately related in sporadic Alzheimer disease.
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Alzheimer's amyloid-beta peptide inhibits sodium/calcium exchange measured in rat and human brain plasma membrane vesicles

Abstract

Section snippets

Preparation of rat and human plasma membrane vesicles

Preparation of Aβ peptides

Aggregation of synthetic Aβ25–35 in 10 mM HCl solution

Do aggregated Aβ peptides inhibit Ca2+ transport by the Na+/Ca2+ exchanger?

Conclusions

Unlinked References

Acknowledgements

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Biochem. biophys. Res. Commun.

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J. biol. Chem.

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Biochem. biophys. Res. Commun.

Brain Res.

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J. Pharmac. exp. Ther.

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

Proc. natn. Acad. Sci. U.S.A.

β-amyloid Ca2+-channel hypothesis for neuronal death in Alzheimer Disease

Molec. Cell. Biochem.

Alzheimer disease amyloid beta-protein forms calcium channels in bilayer membranes – blockade by tromethamine and aluminum

Proc. natn. Acad. Sci. U.S.A.

Aggregation of synthetic Aβ_25–35 in 10 mM HCl solution

Do aggregated Aβ peptides inhibit Ca²⁺ transport by the Na⁺/Ca²⁺ exchanger?

Inhibition of the Na⁺-Ca²⁺ exchanger enhances anoxia and glucopenia-induced H-3 aspartate release in hippocampal slices

β-amyloid Ca²⁺-channel hypothesis for neuronal death in Alzheimer Disease