Inhibition of the electrostatic interaction between β-amyloid peptide and membranes prevents β-amyloid-induced toxicity

Abstract

The accumulation of ␤-amyloid peptides (A␤) into senile plaques is one of the hallmarks of Alzheimer disease. Aggregated A␤ is toxic to cells in culture and this has been considered to be the cause of neurodegeneration that occurs in the Alzheimer disease brain. The discovery of compounds that prevent A␤ toxicity may lead to a better understanding of the processes involved and ultimately to possible therapeutic drugs. Low nanomolar concentrations of A␤1-42 and the toxic fragment A␤25-35 have been demonstrated to render cells more sensitive to subsequent insults as manifested by an increased sensitivity to formazan crystals following MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide) reduction. Formation of the toxic ␤-sheet conformation by A␤ peptides is increased by negatively charged membranes. Here we demonstrate that phloretin and exifone, dipolar compounds that decrease the effective negative charge of membranes, prevent association of A␤1-40 and A␤25-35 to negatively charged lipid vesicles and A␤ induced cell toxicity. These results suggest that A␤ toxicity is mediated through a nonspecific physicochemical interaction with cell membranes.␤-amyloid, the major constituent of senile plaques in Alzheimer disease patients (1) has been proposed to be the cause of the neurodegeneration that occurs in Alzheimer disease brains. A␤1-42, A␤1-40, and certain fragments, notably A␤25-35, are directly toxic to neuronal cell cultures at high micromolar concentrations (2-5). The observed cell death has been correlated with an effect of amyloid peptides on the membrane integrity as determined by lipid peroxidation (2). Furthermore, it has recently been shown that low nanomolar concentrations of A␤ peptides increase the susceptibility of the plasma membrane to additional insults (6).Substantial evidence has been provided suggesting that a crucial step for the formation of toxic A␤ is the transition of random coil to ␤-sheet conformation that is necessary for fibril aggregation. Those fibrils have been demonstrated to cause cell death (7-9). On the other hand studies with lipid vesicles demonstrated that formation of ␤-sheet structures is enhanced in the presence of negatively charged lipid vesicles (10-12). Decreasing the negative charge of a membrane may, therefore, result in a decrease in membrane association of A␤ peptides. Such a decrease in the negative charge of lipid membranes by a decrease in the membrane dipole potential has been demonstrated for phloretin, a lipophilic dipolar substance shown to decrease the membrane dipole potential (13-15).Here we demonstrate that phloretin and a structural analogue, exifone, not only reduce the association of toxic A␤ peptides with the membrane but also prevent A␤ toxicity to neuron-like PC12 cells. These results suggest that a physicochemical interaction of A␤ peptides with negatively charged membranes might be responsible for the toxic effect of A␤ to neuronal cells. MATERIALS AND METHODSMaterials. Rat PC12 pheochromocytoma cells were a gift from E. Sho...

Distribution and Fluidizing Action of Soluble and Aggregated Amyloid β-Peptide in Rat Synaptic Plasma Membranes

“…A␤ peptides have been shown to modify various cellular functions, leading to increased neuronal vulnerability. It has been recently reported that the effects of A␤ on neuronal viability were not mediated by specific receptor interactions (12) but potentially by changes in the structure and dynamics of membrane lipid constituents (17). Therefore, the purpose of the present experiments was to examine the effects of both soluble and aggregated A␤ on membrane structure using a combination of x-ray diffraction and fluorescence spectroscopy approaches.…”

“…Ethanol, for example, also increases membrane fluidity, enhancing Ca 2ϩ flux into neurons (46). The combined physico-chemical effects of A␤ on bulk and protein annular fluidity may contribute to mechanisms of neurotoxicity (17). A␤-induced membrane structure changes may also lead to further increases in A␤ production by increasing the access of certain proteases to abnormal, membrane-associated cleavage sites on amyloid precursor protein following disruptions in the organization of the lipid bilayer.…”

“…Indeed, previous membrane equilibrium binding experiments have demonstrated that the A␤ [25][26][27][28][29][30][31][32][33][34][35] fragment is highly lipophilic (K P Ͼ 10 2 ); the peptide intercalates deep into the membrane bilayer hydrocarbon core, as determined by small angle x-ray diffraction approaches (15). In addition, aggregated A␤ has strong electrostatic interactions with the surface of model membranes that appear to mediate its neurotoxicity (17). It was also reported that soluble A␤ has pronounced effects on the molecular anisotropy properties of rat SPM samples, consistent with peptide-induced changes in the conformation of membrane lipid constituents (14).…”

Amino acid substitutions [K16A] and [K28A] distinctly affect amyloid β-protein oligomerization

“…[K16A] substitution induces formation of more compact oligomers than the [K28A] subcharged residues are likely to interact with a cellular membrane because they can participate in both effective electrostatic interactions with negatively charged phospholipid head groups and effective hydrophobic interactions with lipid hydrocarbon groups [35][36][37][38][39]. Substitutions of positively charged residues R5, K16, and K28 with alanine were reported to significantly reduce Aβ-induced toxicity to human embryonic kidney (HEK293) cells [40].…”

Disruption of Calcium Homeostasis in Alzheimer’s Disease: Role of Channel Formation by β Amyloid Protein

“…Figure 5 exhibits the structures of these compounds. Phloretin, a plant-derived flavonoid, decreases the membrane potential and inhibits the electrostatic interaction between AβP and membrane lipids (Hertel et al, 1997). Cholesterol decreases membrane fluidity and inhibits channel formation by peptides such as α-toxin, gramicidine, amylin, and AβP.…”