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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Excessive accumulation of amyloid β-protein (Aβ) is one of the primary mechanisms that leads to neuronal death with phosphorylated tau in the pathogenesis of Alzheimer's disease (AD). Protofibrils, one of the high-molecular-weight Aβ oligomers (HMW-Aβo), are implicated to be important targets of disease modifying therapy of AD.
We previously reported that phenolic compounds such as myricetin inhibit Aβ1-40, Aβ1-42, and α-synuclein aggregations, including their oligomerizations, which may exert protective effects against AD and Parkinson's disease. The purpose of this study was to clarify the detailed mechanism of the protective effect of myricetin against the neurotoxicity of HMW-Aβo in SH-SY5Y cells.
To assess the effect of myricetin on HMW-Aβo-induced oxidative stress, we systematically examined the level of membrane oxidative damage by measuring cell membrane lipid peroxidation, membrane fluidity, and cell membrane potential, and the mitochondrial oxidative damage was evaluated by mitochondrial permeability transition (MPT), mitochondrial reactive oxygen species (ROS), and manganese-superoxide dismutase (Mn-SOD), and adenosine triphosphate (ATP) assay in SH-SY5Y cells.
Myricetin has been found to increased cell viability by suppression of HMW-Aβo-induced membrane disruption in SH-SY5Y cells, as shown in reducing membrane phospholipid peroxidation and increasing membrane fluidity and membrane resistance. Myricetin has also been found to suppress HMW-Aβo-induced mitochondria dysfunction, as demonstrated in decreasing MPT, Mn-SOD, and ATP generation, raising mitochondrial membrane potential, and increasing mitochondrial-ROS generation.
These results suggest that myricetin preventing HMW-Aβo-induced neurotoxicity through multiple antioxidant functions may be developed as a disease-modifying agent against AD.
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We previously reported that EAAT3 (whose rat homologous is known as Excitatory Amino Acid Carrier 1- EAAC1 [26]) and NCX1 physically and functionally interact at both the plasma membrane and mitochondrial levels [23,24,31], and that in cardiac and neuronal models they orchestrate the entry of glutamate into the cytoplasm and then into the mitochondria, fueling ATP synthesis, in health and disease [13,22–25,34]. Although some abnormalities in expression/function of either EAATs or NCX have been described in AD patients [35–37], their contribution to the metabolic pathophysiology of AD has not been investigated yet. Based on these findings, in the present study we explored the hypothesis that glutamate might weaken the progression of the AD-like phenotype by boosting cell energy metabolism and assessed the role played by EAATs and NCX in this setting.
Increasing evidence suggests that metabolic dysfunctions are at the roots of neurodegenerative disorders such as Alzheimer’s disease (AD). In particular, defects in cerebral glucose metabolism, which have been often noted even before the occurrence of clinical symptoms and histopathological lesions, are now regarded as critical contributors to the pathogenesis of AD. Hence, the stimulation of energy metabolism, by enhancing the availability of specific metabolites, might be an alternative way to improve ATP synthesis and to positively affect AD progression. For instance, glutamate may serve as an intermediary metabolite for ATP synthesis through the tricarboxylic acid (TCA) cycle and the oxidative phosphorylation. We have recently shown that two transporters are critical for the anaplerotic use of glutamate: the Na⁺-dependent Excitatory Amino Acids Carrier 1 (EAAC1) and the Na⁺-Ca²⁺ exchanger 1 (NCX1). Therefore, in the present study, we established an AD-like phenotype by perturbing glucose metabolism in both primary rat cortical neurons and retinoic acid (RA)-differentiated SH-SY5Y cells, and we explored the potential of glutamate to halt cell damage by monitoring neurotoxicity, AD markers, ATP synthesis, cytosolic Ca²⁺ levels and EAAC1/NCX1 functional activities.
We found that glutamate significantly increased ATP production and cell survival, reduced the increase of AD biomarkers (amyloid β protein and the hyperphosphorylated form of tau protein), and recovered the increase of NCX reverse-mode activity. The RNA silencing of either EAAC1 or NCX1 caused the loss of the beneficial effects of glutamate, suggesting the requirement of a functional interplay between these transporters for glutamate-induced protection.
Remarkably, our results indicate, as proof‐of‐principle, that facilitating the use of alternative fuels, like glutamate, may be an effective approach to overcome deficits in glucose utilization and significantly slow down neuronal degenerative process in AD.
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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].
It is well established that mitochondria are the main source of ATP production within cells. However, mitochondria have other remarkable functions, serving as important modulators of cellular Ca²⁺ signaling, and it is now generally recognized that control over Ca²⁺ homeostasis is intrinsically interwoven with mitochondrial abilities to adjust and tune ATP production. In this review, we describe the mechanisms that mitochondria use to balance Ca²⁺ homeostasis maintenance and cell energy metabolism. In recent years, the knowledge on the molecular machinery mediating Ca²⁺ influx/efflux has been improved and, albeit still open to further investigations, several lines of evidence converge on the hypothesis that plasma membrane Na⁺/Ca²⁺ exchanger (NCX) isoforms are also expressed at the mitochondrial level, where they contribute to the Ca²⁺ and Na⁺ homeostasis maintenance. In particular, the connection between mitochondrial NCX activity and metabolic substrates utilization is further discussed here. We also briefly focus on the alterations of both mitochondrial Ca²⁺ handling and cellular bioenergetics in neurodegenerative diseases, such as Parkinson’s and Alzheimer’s disease.
Astrocytes as new targets to improve cognitive functions
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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).
Astrocytes are now viewed as key elements of brain wiring as well as neuronal communication. Indeed, they not only bridge the gap between metabolic supplies by blood vessels and neurons, but also allow fine control of neurotransmission by providing appropriate signaling molecules and insulation through a tight enwrapping of synapses. Recognition that astroglia is essential to neuronal communication is nevertheless fairly recent and the large body of evidence dissecting such role has focused on the synaptic level by identifying neuro- and gliotransmitters uptaken and released at synaptic or extrasynaptic sites. Yet, more integrated research deciphering the impact of astroglial functions on neuronal network activity have led to the reasonable assumption that the role of astrocytes in supervising synaptic activity translates in influencing neuronal processing and cognitive functions. Several investigations using recent genetic tools now support this notion by showing that inactivating or boosting astroglial function directly affects cognitive abilities. Accordingly, brain diseases resulting in impaired cognitive functions have seen their physiopathological mechanisms revisited in light of this primary protagonist of brain processing. We here provide a review of the current knowledge on the role of astrocytes in cognition and in several brain diseases including neurodegenerative disorders, psychiatric illnesses, as well as other conditions such as epilepsy. Potential astroglial therapeutic targets are also discussed.
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

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

J. Pharmac. exp. Ther.

Inhibition of Na+/Ca2+ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures

Brain Res.

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.

Vitamin E protects nerve cells from amyloid β protein toxicity

Biochem. biophys. Res. Commun.

Hydrogen peroxide mediates amyloid β protein toxicity

Cell

Fluorometric assay of proteins in the nanogram range

Archs Biochem. Biophys.

Assembly and aggregation properties of synthetic Alzheimer's A4/β amyloid peptide analogs

J. biol. Chem.

Beta-amyloid peptide free radical fragments initiate synaptosomal lipoperoxidation in a sequence specific fashion – implications to Alzheimer's disease

Biochem. biophys. Res. Commun.

Aging does not affect steady state expression of the Na+/Ca2+ exchanger in rat brain

Cell. molec. Neurobiol.

Analysis of Na+/Ca2+ exchange activity in human brain: the effect of normal aging

Neurobiol. Aging

Theoretical models of the ion structure of amyloid-β protein

Biophys. J.

Amyloid beta protein-induced irreversible current in rat cortical neurones

NeuroReport

Cloning of two isoforms of the rat brain Na+–Ca2+ exchanger gene and their functional expression in HeLa cells

Fedn Eur. biochem. Socs Lett.

Cytochalasins protect hippocampal neurons against amyloid beta-peptide toxicity: Evidence that actin depolymerization suppresses Ca2+ influx

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

Beta-amyloid nerotoxicity requires fibril formation and is inhibited by Congo red

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

Inhibition of Na⁺/Ca²⁺ exchange enhances delayed neuronal death elicited by glutamate in cerebellar granule cell cultures

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

Aging does not affect steady state expression of the Na⁺/Ca²⁺ exchanger in rat brain

Analysis of Na⁺/Ca²⁺ exchange activity in human brain: the effect of normal aging

Cloning of two isoforms of the rat brain Na⁺–Ca²⁺ exchanger gene and their functional expression in HeLa cells

Cytochalasins protect hippocampal neurons against amyloid beta-peptide toxicity: Evidence that actin depolymerization suppresses Ca²⁺ influx

Positively charged cyclic hexapeptides, novel blockers for the cardiac sarcolemma Na⁺-Ca²⁺ exchanger

Identification of a peptide inhibitor of the cardiac sarcolemmal Na⁺-Ca²⁺ exchanger