Background: The beta-amyloid peptide (Aβ) involved in Alzheimer’s disease (AD) has been described to associate/aggregate on the cell surface disrupting the membrane through pore formation and breakage. However, molecular determinants involved for this interaction (e.g., some physicochemical properties of the cell membrane) are largely unknown. Since cholesterol is an important molecule for membrane structure and fluidity, we examined the effect of varying cholesterol content with the association and membrane perforation by Aβ in cultured hippocampal neurons.Methods: To decrease or increase the levels of cholesterol in the membrane we used methyl-β-cyclodextrin (MβCD) and MβCD/cholesterol, respectively. We analyzed if membrane fluidity was affected using generalized polarization (GP) imaging and the fluorescent dye di-4-ANEPPDHQ. Additionally membrane association and perforation was assessed using immunocytochemistry and electrophysiological techniques, respectively.Results: The results showed that cholesterol removal decreased the macroscopic association of Aβ to neuronal membranes (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.05) and induced a facilitation of the membrane perforation by Aβ with respect to control cells (half-time for maximal charge transferred: control = 7.2 vs. MβCD = 4.4). Under this condition, we found an increase in membrane fluidity (46 ± 3.3% decrease in GP value, p < 0.001). On the contrary, increasing cholesterol levels incremented membrane rigidity (38 ± 2.7% increase in GP value, p < 0.001) and enhanced the association and clustering of Aβ (fluorescent-puncta/20 μm: control = 18 ± 2 vs. MβCD = 10 ± 1, p < 0.01), but inhibited membrane disruption.Conclusion: Our results strongly support the significance of plasma membrane organization in the toxic effects of Aβ in hippocampal neurons, since fluidity can regulate distribution and insertion of the Aβ peptide in the neuronal membrane.

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Ethanol increased the frequency of miniature glycinergic currents [miniature inhibitory postsynaptic currents (mIPSCs)] in cultured spinal neurons. This effect was dependent on intracellular calcium augmentation, since preincubation with BAPTA (an intracellular calcium chelator) or thapsigargin [a sarco(endo)plasmic reticulum Ca(2+)-ATPase (SERCA) pump inhibitor] significantly attenuated this effect. Similarly, U73122 (a phospholipase C inhibitor) or 2-aminoethoxydiphenyl borate [2-APB, an inositol 1,4,5-trisphosphate (IP₃) receptor (IP3R) inhibitor] reduced this effect. Block of ethanol action was also achieved after preincubation with Rp-cAMPS, inhibitor of the adenylate cyclase (AC)/PKA signaling pathway. These data suggest that there is a convergence at the level of IP₃R that accounts for presynaptic ethanol effects. At the postsynaptic level, ethanol increased the decay time constant of mIPSCs in a group of neurons (30 ± 10% above control, n = 13/26 cells). On the other hand, the currents activated by exogenously applied glycine were consistently potentiated (55 ± 10% above control, n = 11/12 cells), which suggests that ethanol modulates synaptic and nonsynaptic glycine receptors (GlyRs) in a different fashion. Supporting the role of G protein modulation on ethanol responses, we found that a nonhydrolyzable GTP analog [guanosine 5'-O-(3-thiotriphosphate) (GTPγS)] increased the decay time constant in ∼50% of the neurons (28 ± 12%, n = 11/19 cells) but potentiated the glycine-activated Cl(-) current in most of the neurons examined (83 ± 29%, n = 7/9 cells). In addition, confocal microscopy showed that α1-containing GlyRs colocalized with Gβ and Piccolo (a presynaptic cytomatrix protein) in ∼40% of synaptic receptor clusters, suggesting that colocalization of Gβγ and GlyRs might account for the difference in ethanol sensitivity at the postsynaptic level.

The ability of beta-amyloid peptide (Aβ) to disrupt the plasma membrane through formation of pores and membrane breakage has been previously described. However, the molecular determinants for these effects are largely unknown. In this study, we examined if the association and subsequent membrane perforation induced by Aβ was dependent on GM1 levels. Pretreatment of hippocampal neurons with D-PDMP decreased GM1 and Aβ clustering at the membrane (Aβ fluorescent-punctas/20μm, control=16.2±1.1 vs. D-PDMP=6.4±0.4, p<0.001). Interestingly, membrane perforation with Aβ occurred with a slower time course when the GM1 content was diminished (time to establish perforated configuration (TEPC) (min): control=7.8±2 vs. low GM1=12.1±0.5, p<0.01), suggesting that the presence of GM1 in the membrane can modulate the distribution and the membrane perforation by Aβ. On the other hand, increasing GM1 facilitated the membrane perforation (TEPC: control=7.8±2 vs. GM1=6.2±1min, p<0.05). Additionally, using Cholera Toxin Subunit-B (CTB) to block the interaction of Aβ with GM1 attenuated membrane perforation significantly. Furthermore, pretreatment with CTB decreased the membrane association of Aβ (fluorescent-punctas/20μm, Aβ: control=14.8±2.5 vs. CTB=8±1.4, p<0.05), suggesting that GM1 also plays a role in both association of Aβ with the membrane and in perforation. In addition, blockade of the Aβ association with CTB inhibited synaptotoxicity. Taken together, our results strongly suggest that membrane lipid composition can affect the ability of Aβ to associate and subsequently perforate the plasma membrane thereby modulating its neurotoxicity in hippocampal neurons.

It is well accepted that cortical and hippocampal synaptic densities are reduced in Alzheimer's disease (AD). These alterations in neuronal networking occur at the very onset of AD and may lead to the neuronal loss displayed in later stages of the disease, which is characterized by severe cognitive and behavioral impairments. Many studies suggest that amyloid-β (Aβ) oligomers are responsible for synaptic disconnections and neuronal death. The effects of Aβ in different brain regions are pleotropic, thus suggesting a common mechanism for toxicity. One potential site for this mechanism of toxicity is the neuronal membrane. It is recognized that Aβ can associate to the plasma membrane and induce the formation of pores after the interaction with lipids like GM1 and cholesterol, and proteins such as APP and NMDA receptors. After this early event, the membrane increases its permeability allowing the influx of small ions and larger molecules. Thus, one of the main toxic consequences of Aβ oligomer interaction with neurons is an increase in intracellular Ca(2+) concentration that causes alterations in ionic homeostasis. It has been proposed that Aβ perforates the membrane similarly to pore-forming toxins producing a series of effects that include synaptic failure and cell death. These actions of Aβ appear to be potentiated by neuroinflammation, which results in a series of effects that, when prolonged, will affect membrane integrity, pore formation and cellular homeostasis. Here, we will review the most recent data on Aβ actions at the membrane level and how its relationship with neuroinflammation could further potentiate brain impairment in AD. The notion of having drugs acting with dual inhibitory actions, inhibition of membrane damage and inflammation, could serve as a starting conceptual point for the development of new therapies for the disease.