Cholesterol modulates amyloid beta peptide 1-42 channel formation in planar lipid membranes

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1 Cholesterol modulates amyloid beta peptide 1-42 channel formation in planar lipid membranes Meleleo D., Notarachille G., Gallucci E. and Micelli S. Dept. Farmaco-Biologico, Università degli Studi di Bari, via E. Orabona 4, Bari Italy Summary Cholesterol, an integral component of eukaryotic cell membranes, is assuming a pivotal role as a target in some neurodegenerative diseases such as Alzheimer, although its role remains elusive. Recent studies indicate that Ch can favour the conformational transition of many peptides in model membranes and vesicles. AβP1-42 is one of the pathological features of Alzheimer s disease and, together with AβP1-40, is among the major components of senile plaques. Notably, the AβP1-42 peptide is more neurotoxic than AβP1-40, owing to its much stronger aggregation propensity. In this work, we studied the role of cholesterol in the incorporation and channel formation of AβP1-42 in planar lipid membranes of different composition. Introduction Brain cholesterol is an essential component of cell membranes and is involved in many biological functions. There are also considerable amounts of cholesterol in neuronal plasmalemma and in lipid rafts. Cholesterol partitions between the raft and non-raft phase, having higher affinity to raft sphingolipids than to unsaturated phospholipids. Rafts are small platforms, composed of sphingolipids with cholesterol in the outer exoplasmic leaflet, connected to phospholipids and cholesterol in the inner cytoplasmic leaflet of the lipid bilayer. One interesting aspect of many proteins and peptides seems to be their affinity for the cholesterol molecule. In fact, cholesterol is assuming a key role as a target in some neurodegenerative diseases such as Alzheimer, although its role remains unclear. Some authors have found that membrane components, such as cholesterol and gangliosides, alter the affinity of AβP for phospholipid membranes. Cholesterol and gangliosides, once associated with phospholipid membranes, lead to an increase in β-sheet content and/or the rate of aggregation of AβP (1). On the other hand, recent studies have suggested that cholesterol promotes the transition from β-sheet to α-helix of many proteins and peptides (2). Furthermore the amount of cholesterol in the exoplasmic leaflet of the neuronal membranes seems to increase 2009 by MEDIMOND s.r.l. L311C

2 168 New Trends in Alzheimer and Parkinson Disorders with age (3). Notably, although AβP1-42 and AβP1-40 are the major components of senile plaques, the AβP1-42 peptide is more neurotoxic than AβP1-40, owing to its much stronger aggregation propensity (4,5). On the other hand, a new concept has been emerging in recent years: small intermediates, soluble oligomers, can be toxic causing synaptic dysfunction or forming channel-like pores within the membrane. Recent studies indicate that the peptide s ability to form ion channels depends on its conformational structure and on the peptide/lipid structure in the physiological environment. The aims of this study were to evaluate the influence of cholesterol (Ch) on AβP1-42 incorporation and channel formation in palmitoyl-oleoyl-phosphatidylcholine (POPC) planar lipid membranes (PLMs) and to monitor AβP1-42 channel current in PLMs made up of Oxidized Cholesterol (OxCh), a component of aging neuronal membranes. Materials and Methods Channel activities were recorded in a lipid bilayer membrane made up of POPC or POPC:Ch (70:30, w/w) in 1% of n-decane or oxidized cholesterol (OxCh) in n- decane (1:1,v:v) (Fluka). Bilayers were formed across a 300 µm hole diameter in a teflon partition separating two teflon chambers which held symmetrical 50 mm KCl solutions, ph = 7, temperature 23 ± 1 C. The salts used in the experiments were of analytical grade. AβP1-42 (Sigma) was added to the cis-side of the membrane, at a final concentration of M. The membrane current was monitored with an oscilloscope and recorded on a chart recorder. For a detailed description see references 6,7. Results In many different experiments on POPC/POPC:Ch PLMs, the addition of 5*10-8 M of AβP1-42 to the cis-side of the medium facing the membrane did not determine any conductance variation for a long period of time (24/6 h respectively), upon application of voltages as high as 120 mv. After 24/6 hours (lag time) respectively, PLM breakage and withdrawing it again, AβP1-42 channel activity appears as non-random discrete current fluctuations, compatible with channel-type openings and closures. The minimal potential at which channel activity can be observed was 100mV in both kinds of membranes. After the first channel formation, the applied voltage can be lowered as far as 80 mv and ± 60mV in POPC and POPC:Ch PLMs respectively. In POPC PLMs, no channel activity was observed at positive applied voltages below 80 mv and at negative applied voltages. Fig. 1A and 1B show some chart recordings of AβP1-42 channel formation in POPC and POPC:Ch (70:30, w/w) PLMs with associated histograms of the conductance fluctuations. These results seem to indicate that cholesterol (30% weight ratio) increases AβP1-42 affinity to POPC PLMs. We carried out experiments with OxCh PLMs because cholesterol and its oxidation products are present on the exoplasmic leaflet of aging neuronal membranes. In OxCh PLMs, AβP1-42 channel activity occurs spontaneously two hours after it 904

3 Prague, Czech Republic, March, Fig.1 Examples of chart recordings of AβP1-42 channel activity in PLMs made up of POPC (A), POPC:Ch (B) and OxCh (C) at an applied voltage of 100 mv with associated histograms of the conductance fluctuations. Probability of the conductance, P(Λ), is the number of observed steps within an interval of width ( Λ= 0.01/ 0.05 ns in POPC and POPC:Ch/OxCh PLMs respectively) divided by the total number of steps. The P(Λ) values are indicated as a percentage. is added to the medium facing the membrane. The minimal potential at which channel activity could be observed was 100mV. After the first channel formation, the applied voltage can be lowered as far as ±20 mv. Fig. 1C shows some chart recordings of AβP1-42 channel formation in OxCh PLMs with associated histograms of the conductance fluctuations. All single-channel events were used to construct a histogram of conductance distribution. The conductance fluctuations are not uniform in size but distributed over a certain range. Examples of conductance fluctuation histograms at an applied voltage of 100 mv are shown in Fig.1. Table 1 reports the average Λ values ( Λ) obtained for each distribution at different applied voltages in the three PLMs. The average conductance ( Λ) is determined by recording not less than 100 single-events and averaging over the distribution of conductance values (8). The results indicate that Λ values decrease as applied voltages increase for POPC:Ch and OxCh membranes. Besides, in OxCh PLMs, AβP1-42 Λ values are significantly higher at all applied voltages than those in POPC and POPC:Ch PLMs. The occurrence frequency values (number of events in 60 sec) are significantly higher in OxCh PLMs than in POPC and POPC:Ch PLMs at applied voltages of 80 and 100 mv. The distribution of open times has been found to follow a single-exponential or a two-exponential function (6). The mean lifetimes ± SE obtained in OxCh and 905

4 170 New Trends in Alzheimer and Parkinson Disorders Table 1 The average conductance ( Λ) at different applied voltages in PLMs POPC:Ch PLMs are τ 1 (sec)= 1.05/0.60 ± 0.34/0.33 respectively and τ 2 (sec)= 3.80/3.91 ± 0.28/0.54 respectively. Preliminary studies to identify the charge on the ion carrying the current indicate a poorly cation-selective channel in OxCh PLMs. More investigations are necessary to determine the ion selectivity of the AβP1-42 channel in POPC and POPC:Ch PLMs. Conclusion The results of this study show that: 1. AβP1-42 forms ion channels in POPC PLMs. The C-terminal alanine and isoleucine residues make the AβP1-42 molecule more hydrophobic than AβP1-40, forming stable oligomers (9). Therefore AβP1-42 could be prone to bind to POPC PLMs and to incorporate into the membrane. However, AβP1-42 forms channels in PC12 cells, should AβP1-42 form channel in nervous tissue of the organism then this could explain its greater toxicity respect to AβP AβP1-42 channel activity increases significantly with Ch concentrations of up to 30% in POPC PLMs. This finding indicates that the cholesterol molecule may be considered a target of AβP1-42. Recent studies show that after Aβ binds to raft 906

5 Prague, Czech Republic, March, membranes containing cholesterol, the peptide can be translocated to PC membranes to which monomeric soluble Aβ does not bind (10). 3. AβP1-42 easily forms ion channels in OxCh PLMs. It is possible to hypothesize that AβP1-42 could form ion channels in vivo owing to the presence of oxidized cholesterol products in aging neuronal plasmalemma. These results can contribute to clarify the function of cholesterol and rafts in the development of human diseases and suggest a possible mechanism for AβP1-42 toxicity in neuronal membranes. References [1] CHOO-SMITH L.P. ET AL. Acceleration of amyloid fibril formation by specific binding of Abeta-(1-40) peptide to ganglioside-containing membrane vesicles. J Biol Chem 272: ,1997. [2] JI S.R. ET AL. Cholesterol is an important factor affecting the membrane insertion of beta-amyloid peptide (A beta 1-40), which may potentially inhibit the fibril formation. J Biol Chem 277: ,2002. [3] IGBAVBOA U. ET AL. Increasing age alters transbilayer fluidity and cholesterol asymmetry in synaptic plasma membranes of mice. J Neurochem. 66: ,1996. [4] MOBLEY J. ET AL. Modelling amyloid beta-peptide insertion into lipid bilayers. Biophys J. 86: , [5] SATO T. ET AL. Inhibitors of amyloid toxicity based on β-sheet packing of Aβ40 and Aβ42. Biochemistry 45: , [6] GALLUCCI e. ET AL. Magainin 2 channel formation in planar lipid membranes: the role of lipid polar groups and ergosterol. Eur Biophys J 32:22-32,2003. [7] MICELLI S. ET AL. Effect of sterols on beta-amyloid peptide (AbetaP 1-40) channel formation and their properties in planar lipid membranes. Biophys J 86: ,2004. [8] LUDWIG O. ET.AL. Pore formation by the mithocondrial porin of rat brain in lipid bilayer membranes. Biochim Biophys Acta 860: ,1996. [9] HAASS C. ET AL. Soluble protein oligomers in neurodegeneration: lessons from the Alzheimer s amyloid β-peptide. Nat Rev Mol Cell Biol 8: , 2007 [10] KAKIO A. ET AL. Formation of a membrane-active form of amyloid beta-protein in raft-like model membranes. Biochem Biophys Res Commun. 303: ,