Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide Abdullah Md. Sheikh and Atsushi Nagai Department of Laboratory Medicine, Shimane University School of Medicine, Izumo, Japan Introduction Alzheimer’s disease (AD) is a neurodegenerative disorder that is manifested clinically as progressive dementia. Histopathologically, it is characterized by degenerative changes in the neurons, together with intra-neuronal deposition of hyperphosphorylated Tau and extracellular accumulation of peptides comprising 39–43 amino acids, called amyloid beta (Ab) peptides, which are generated by secretase-mediated cleavage of transmembrane amyloid precursor protein [1]. Bio- chemical analysis of the Ab peptides isolated from AD Keywords Alzheimer’s disease; amyloid beta peptide; fibril formation; lysophosphatidylcholine; phospholipid–Ab interaction Correspondence A. Nagai, Department of Laboratory Medicine, Shimane University Faculty of Medicine, 89-1 Enya-cho, Izumo 693-8501, Japan Tel ⁄ Fax: +81 853 20 2312 E-mail: anagai@med.shimane-u.ac.jp (Received 14 July 2010, revised 29 Novem- ber 2010, accepted 6 December 2010) doi:10.1111/j.1742-4658.2010.07984.x Phospholipids are known to influence fibril formation of amyloid beta (Ab) peptide. Here, we show that lysophosphatidylcholine (LPC), a polar phos- pholipid, enhances Ab(1-42) fibril formation, by decreasing the lag time and the critical peptide concentration required for fibril formation, and increasing the fibril elongation rate. Conversely, LPC did not have an enhancing effect on Ab(1-40) fibril formation, and appeared to be inhibi- tory. Tyrosine fluorescence spectroscopy showed that LPC altered the fluorescence spectra of A b(1-40) and Ab(1-42) in opposite ways. Further, 8-anilino-1-naphthalene sulfonic acid fluorescence spectroscopy showed that LPC significantly increased the hydrophobicity of Ab(1-42), but not of Ab(1-40). Tris-tricine gradient SDS ⁄ PAGE revealed that LPC increased the formation of higher-molecular-weight species of Ab(1-42), including trimers and tetramers. LPC had no such effect on Ab(1-40), and thus may specifi- cally influence the oligomerization and nucleation processes of Ab(1-42) in a manner dependent on its native structure. Dot-blot assays confirmed that LPC induced Ab(1-42) oligomer formation at an early time point. Thus our results indicate that LPC specifically enhances the formation of Ab(1- 42) fibrils, the main component of senile plaques in Alzheimer’s disease patients, and may be involved in Alzheimer’s disease pathology. Structured digital abstract l MINT-8077403: A beta (1-42) (uniprotkb:P05067) and A beta (1-42) (uniprotkb:P05067) bind ( MI:0407)byelectron microscopy (MI:0040) l MINT-8077463: A beta (1-42) (uniprotkb:P05067) and A beta (1-42) (uniprotkb:P05067) bind ( MI:0407)byfilter binding (MI:0049) l MINT-8077369, MINT-8077387: A beta (1-42) (uniprotkb:P05067) and A beta (1-42) (uni- protkb: P05067) bind (MI:0407)byfluorescence technology (MI:0051) l MINT-8077417, MINT-8077428, MINT-8077436, MINT-8077448: A beta (1-40) (uni- protkb: P05067) and A beta (1-40) (uniprotkb:P05067) bind (MI:0407)bycomigration in sds page ( MI:0808) Abbreviations Ab, amyloid beta peptide; AD, Alzheimer’s disease; LPC, lysophosphatidylcholine; ThT, thioflavin T. 634 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS brain indicated that an A b peptide consisting of 42 res- idues, Ab(1–42), is the principal species associated with senile plaques in AD, while an Ab peptide consisting of 40 residues, Ab(1–40), is more abundant in cerebro- vascular amyloid deposits and cerebrospinal fluid [2]. Genetic analysis of familial AD patients, as well as animal studies, showed that genetic alterations found in familial AD, such as amyloid precursor protein or pre-senilin mutations, increase the production and deposition of Ab in the brain [1,3–7]. This in turn initi- ates a cascade of events leading to AD-related neuro- toxicity and the appearance of AD plaques [8]. Moreover, the Ab is deposited mainly in fibrillary form, and the fibrils have been shown to be intimately associated with dystrophic neurons and activated glial cells [8]. Therefore, the Ab fibril formation process is considered to have a central role in AD pathology. Lysophosphatidylcholine (LPC) is a bioactive polar phospholipid that is produced by phospholipase A 2 -mediated hydrolysis of phosphatidylcholine [9]. Studies in our laboratory and others have established the neuroinflammatory and neurodegenerative proper- ties of LPC [10–12]. Neuroinflammatory processes have an essential role in AD pathology [13]. Phospho- lipase A 2 activity was reported to be specifically increased in astrocytes in the cortical area of AD patients where neurodegeneration was evident [14], indicating that lipid metabolism, including that of LPC, may be changed in the brain of AD patients. Indeed, the concentration of LPC is increased in the white matter of aged human brains exhibiting senile atrophy of the Alzheimer type; in addition, the LPC to phosphatidylcholine ratio is decreased in the cerebrospinal fluid of AD patients [15,16]. Moreover, LPC increases Ab-induced neuronal apoptosis [17]. Taken together, these reports suggest that LPC may have an important role in AD pathology. There have been many studies on the interaction of Ab and phospholipids in relation to AD pathology, including factors such as membrane disruption and neurotoxicity, conformational changes and Ab fibril formation process [18–21]. The Ab-interacting phos- pholipids are mainly acidic, including phosphatidic acid, phosphatidylserine, phosphatidylinositol, cardioli- pin and phosphatidylethanolamine [20]. Recently, it has been shown that neutral zwitterionic phospholipid, such as phosphatidylcholine, can also interact with Ab peptide and affect its conformation and fibril forma- tion [19]. However, the effects of LPC on Ab fibril for- mation have not been investigated in detail. In this study, we used an in vitro system to examine the mech- anisms by which LPC influences fibril formation of Ab(1-40) and Ab(1-42). Results Effects of LPC on Ab(1-42) fibril formation To investigate the effects of LPC on Ab(1-42) fibril formation, we incubated increasing concentrations (starting from 250 nm)ofAb(1-42) in a fibril-forming buffer with or without 20 lm LPC for 8 h. No fibrils were detectable at concentrations of Ab(1-42) of up to 10 lm, as revealed by thioflavin T (ThT) fluorescence assay. However, addition of LPC (20 lm) induced the fibril formation process at 5 lm Ab(1-42) (Fig. 1A). Subsequent fibril formation increased linearly with respect to Ab(1-42) concentration (r 2 > 0.94) in the presence or absence of LPC, although the slopes were significantly different (1.1 without LPC versus 1.89 with LPC, P < 0.001). To investigate the dose-depen- dent effect, we added increasing concentrations of LPC to 50 lm Ab(1-42), and fibril formation was allowed to proceed for 30 min at 37 °C. We observed a linear increase of ThT fluorescence with increasing LPC con- centration (r 2 = 0.98) (Fig. 1B). The effects of LPC on fibril formation became apparent at 5 lm (mean ThT fluorescence 5.7; arbitrary units), and reached a plateau at 120 lm LPC (mean ThT fluorescence 37.9). However, transmission electron microscopy showed that LPC did not change the overall morphology of Ab(1-42) fibrils (Fig. 1G). Previous reports have shown that peptide concen- tration affects the fibril formation process [22]. Our preliminary experiments also showed that the lag phase was decreased at a high Ab(1-42) concentra- tion, causing difficulties in the analysis of fibril formation kinetics (data not shown). Therefore, in order to investigate the effects of LPC on Ab(1-42) fibril formation kinetics, we choose a peptide concen- tration of 12.5 lm. At this concentration, Ab(1-42) fibril formation showed typical sigmoid kinetics, with a lag phase of between 4 and 8 h (Fig. 1C), and reached a plateau at 16 h. When 20 lm LPC was added, the lag phase became as short as 15 min, and the ThT fluorescence reached a plateau within 2 h (Fig. 1D). Next we investigated the effects of the vesicular form of LPC on the Ab(1-42) fibril formation process. For this purpose, an increasing concentration of LPC lipo- somes was added to 50 lm Ab(1-42), and fibril forma- tion was allowed to proceed for 30 min. As in the case of non-vesicular LPC, a linear increase of fibril forma- tion was observed with increasing LPC liposome concentration (r 2 < 0.93) (Fig. 1E). However, the rate of fibril formation was higher (slope 2.2 for LPC liposome versus slope 0.4 for non-vesicular LPC, A. M. Sheikh and A. Nagai LPC modulates Ab fibril formation FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS 635 P < 0.001). LPC liposomes affected the fibril forma- tion process from 5 lm LPC (mean ThT fluorescence 4.8), and the effect reached a plateau at 40 lm LPC (mean ThT fluorescence 84.1). Like non-vesicular LPC, LPC liposomes greatly decreased the lag phage to less than 30 min, and the fibril formation process reached a plateau within 2 h (Fig. 1F). Effects of LPC on Ab(1-40) fibril formation Next, we investigated the fibrillogenic properties of Ab(1-40). Significant fibril formation was observed at 20 lm Ab(1-40) after 8 h incubation, suggesting that the critical micelle concentrations (CMC) was between 10 and 20 lm (Fig. 2A) under these conditions. How- ever, LPC (20 lm) increased the CMC to between 20 and 50 lm (Fig. 2A). Similarly, a kinetic study showed that the lag phase of Ab(1-40) fibril formation at 50 lm was between 4 and 6 h (Fig. 2B). LPC (20 lm) increased the lag period to between 6 to 8 h, with a corresponding delay in reaching the plateau (Fig. 2B). Effects of LPC on the change of intrinsic tyrosine fluorescence during Ab fibril assembly Next we investigated the effect of LPC on tyrosine (Tyr) fluorescence of Ab during fibril assembly. Upon excitation at 277 nm, the emission maximum of Tyr fluorescence is approximately 304 nm [23]. Fibril formation of Ab peptides in the absence or presence of LPC did not produce any shift of the Tyr fluorescence maximum. Incubation of Ab(1-40) or Ab(1-42) in fibril-forming buffer caused a time-dependent decrease of Tyr fluorescence intensity (Fig. 3A–D), although the change was extremely small in the case of Ab(1-42). Incubation of Ab(1-40) for 2 h in the presence of LPC increased the Tyr fluorescence compared with 0 h incu- bated Ab(1-40) alone or Ab(1-40) plus LPC (Fig. 3A); 120 A 40 80 ThT fluorescence ThT fluorescenceThT fluorescence ThT fluorescence ThT fluorescenceThT fluorescence 0204060 0 Aβ β (1-42) µM: 8 12 C A β (1-42) 12.5 µM 0102030 0 4 Time (h): 40 60 80 100 A β (1-42) 50 µM 020406080100 0 20 LPC liposome (µM) a 40 B 20 30 A β (1-42) 50 µM 0 20406080100 0 10 D E G F LPC (µM): 8 12 16 0246810 0 4 Time (h): A β (1-42) 12.5 µM LPC lipo 20 µM 10 20 30 0102030 0 A β (1-42) 12.5 µM LPC lipo 20 µM Time (h): b Fig. 1. Effect of LPC on Ab (1-42) fibril formation. (A) Various concentrations of Ab(1-42) peptide were incubated in fibril- forming buffer with no LPC (open circles) or with 20 l M LPC (closed circles) at 37 °C for 8 h. (B) Ab(1-42) (50 l M) was incubated with increasing concentrations of LPC for 30 min at 37 °C. (C,D) Ab(1-42) (12.5 l M) was allowed to form fibrils in the absence (C) or presence (D) of 20 l M LPC for the indicated times. (E) Dose-dependent effect of LPC liposomes on Ab(1-42) fibril formation. LPC liposomes were prepared as described in Experimental procedures. Various concentra- tions of LPC liposomes were added to 50 l M Ab(1-42) peptide, and fibril formation was allowed to proceed for 30 min, with monitoring by ThT fluorescence measure- ment. (F) Fibril formation kinetics of Ab(1-42) in the presence of LPC liposomes. Ab(1-42) (12.5 l M) was allowed to form fibrils in the presence of 20 l M LPC liposomes for the indicated times. For (A–F), fibril formation was monitored by the ThT fluorescence assay as described in Experimental procedures, and expressed in arbitrary ThT fluorescence units. (G) Ab(1-42) (50 l M) was allowed to form fibrils for 24 h in the absence (a) or presence (b) of 20 l M LPC, and fibril morphology was investigated by electron microscopy. LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai 636 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS thereafter a time-dependent decrease of the fluores- cence was observed (Fig. 3B). Addition of LPC decreased the Tyr fluorescence of Ab(1-42) after 2 h of incubation compared to that of Ab(1-42) alone (Fig. 3C,D), but thereafter the fluorescence remained unchanged up to 24 h (Fig. 3D). Surface hydrophobicity of Ab aggregates The fluorescent dye 8-anilino-1-naphthalene sulfonic acid (ANS), which is widely used in protein-folding studies, was used to investigate the structural features of Ab aggregates. When ANS binds to solvent-exposed hydrophobic regions on protein surfaces, an increase in the fluorescence intensity and a blue shift of the emission maximum are observed [24]. We found that incubation of Ab peptides in fibril-forming buffer caused an increase of fluorescence with a blue shift (Fig. 3E–H). When Ab(1-42) was incubated in the presence of LPC, ANS fluorescence was significantly increased compared to Ab(1-42) alone (Fig. 3G,H), suggesting an increase of hydrophobicity. In the case of Ab(1-40), LPC did not cause an increase of ANS fluorescence (Fig. 3E,F). SDS ⁄ PAGE analysis of Ab peptide during fibril assembly Next we investigated the Ab species that were gener- ated during fibril formation, and the effects of LPC on them. A b(1-40) or Ab(1-42) (50 lm) were allowed to form fibrils in the presence of 0 or 20 lm LPC for 0, 1, 4, 8 and 24 h, and the products were separated by SDS ⁄ PAGE using a 10–20% gradient tris-tricine gel system. Both Ab(1-40) and Ab(1-42) produced dimeric and tetrameric species in fibril-forming buffer, although they mostly remained in monomeric form (Fig. 4A,B). When Ab(1-42) was further incubated in fibril-forming buffer, the amount of monomeric species decreased time-dependently, and an initial increase in tri- and tetrameric species was observed, followed by a time-dependent reduction (Fig. 4B). Addition of LPC affected the mono-, tri- and tetrameric species of Ab(1- 42), decreasing the amount of monomer, and increas- ing the amounts of tri- and tetrameric species, com- pared to the peptide alone at the same time point (Fig. 4B). However, no significant effect of either LPC or incubation time was apparent with regard to dimeric species of Ab(1-40) or Ab(1-42) (Fig. 4A, B). Conversely, LPC did not have any significant effect on the concentration of Ab(1-40) monomer (Fig. 4A). Dot-blot immunoassay of Ab oligomer Next we examined the oligomeric species formed during fibril formation of A b(1-42) peptide, using an oligomer-specific antibody [25]. Our dot-blot immuno- assay showed that, in the case of Ab(1-42) alone, oligomer was detectable after 8 h incubation in fibril- forming buffer (Fig. 4C). However, when LPC was added, oligomer formation was enhanced and became detectable as early as 1 h after the start of incubation (Fig. 4C). Effects of LPC on the rate of Ab fibril elongation To further examine the effect of LPC on Ab fibril for- mation, we investigated whether LPC influenced the elongation phase. To eliminate the nucleation process (lag phase) and focus on Ab elongation, we monitored fibril formation for Ab(1-40) and Ab(1-42) in the pres- ence of pre-formed sonicated fibrils. In a preliminary experiment, pre-formed sonicated fibrils were incu- bated in fibril-forming buffer for up to 48 h, and no increase in ThT fluorescence was observed during that 12 15 18 A B 0 3 6 9 * * 0.25 0.5 1 5 10 20 50 A β (1-40) µM: 25 30 35 ThT fluorescence ThT fluorescence 5 10 15 20 A β (1-40) 50 µM Time (h): 0 5 10 15 20 25 30 0 Fig. 2. Effect of LPC on Ab(1-40) fibril formation. (A) Various concentrations of Ab(1-40) peptide were incubated in fibril-forming buffer with 20 l M LPC (closed squares), or without LPC (open squares) at 37 °C for 8 h. (B) Fibril formation kinetics of Ab(1-40). Ab(1-40) (50 l M) was incubated in fibril-forming buffer at 37 °C for the indicated times in the absence (open circles) or in the presence of 20 l M LPC (closed circles). For (A) and (B), the fibril formation was monitored by ThT fluorescence assay as described in Experi- mental procedures, and expressed in arbitrary ThT fluorescence units. *P < 0.001 versus Ab(1-40) alone at the same time point. A. M. Sheikh and A. Nagai LPC modulates Ab fibril formation FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS 637 time (data not shown). Addition of pre-formed soni- cated fibrils effectively eliminated the lag phase, and a linear increase in fibril formation was observed (r 2 > 0.8) ( Fig . 5). In the case of Ab(1-42), addition of LPC significantly increased the rate of elongation (slope 0.89 versus 0.2, P < 0.001) (Fig. 5B). However, LPC had no effect on the rate of elongation of Ab(1- 40) (slope 0.8 versus 0.75, P = 0.74) (Fig. 5A). Discussion Our key observations are that LPC increased fibrillo- genesis of Ab(1-42) by decreasing both the lag phase and the critical peptide concentration required for fibril formation. This fibril formation-enhancing char- acteristic of LPC is specific for Ab(1-42). In the case of Ab(1-40), LPC (20 lm) actually increased the lag period and critical peptide concentration for fibril formation, suggesting that it may have an inhibitory effect on Ab(1-40) fibrillogenesis. This differential effect of LPC on Ab(1-40) and Ab(1-42) fibrillogenesis was also supported by the findings that the phospho- lipid differentially regulates the micro-environment during fibril formation of these two peptides. However, as we did not investigate Ab(1-40) fibrillogenesis in the presence of higher concentrations of non-vesicular and vesicular LPC, a fibril formation-enhancing effect at higher concentration cannot be ruled out. Lipids are known to influence several fibrillogenic processes. For example, negatively charged phospho- lipids, such as lysophosphatidic acid and lysophosphat- idylglycerol, increase fibrillogenesis of b 2 -microglobulin [26]. Ab has affinity for negatively charged lipids, such as phosphatidylinositol and ganglioside, and peptides bound to negatively charged lipid membranes can self- associate into b-sheets [20,27,28]. However, a recent study showed that zwitterionic phospholipid vesicles, such as phosphatidylcholine liposomes, can also inter- act with Ab(1-40), possibly through the phosphocho- line head group, and a-helix or b-sheet formation is promoted depending on the salt concentration, lipid:peptide ratio and temperature [19]. Although we used LPC in both vesicular and non-vesicular form, non-vesicular LPC showed a dose-dependent effect on Ab fibril formation, starting at a low LPC:peptide A B 35 Aβ β 1-40 30 35 A β 1-40 25 20 25 15 2 h 15 5 C 10 D 20 25 A β 1-42 30 A β 1-42 10 15 20 5 Tyrosine fluorescence Tyrosine fluorescence Tyrosine fluorescence Tyrosine fluorescence 2 h 10 280 300 320 340 0 h 024824 h 024824 0 E A β 1-40 30 30 A β 1-40 F GH 20 20 10 10 0 24 h 0 40 60 A β 1-42 A β 1-42 A β – 0 h A β + LPC – 0 h A β – 2 h, 24 h A β + LPC – 2 h, 24 h 20 ANS fluorescence ANS fluorescence ANS fluorescence ANS fluorescence 2 h A β 1-42 400 500 600 0 40 60 20 0 024824 h 024824 h Wavelength (nm) 400 500 600 Wavelength (nm) Wavelength (nm) 280 300 320 340 Wavelength (nm) A β 1-42 + LPC Fig. 3. Effect of LPC on Ab peptide fibril-forming micro-environ- ment. Ab peptide (50 l M) was incubated in fibril-forming buffer in the absence or presence of 20 l M LPC for the indicated times. Ab fibril samples (20 lL) were added to glycine buffer (pH 8.5, 50 m M final concentration) to make a total volume of 200 lL. Tyrosine (Tyr) fluorescence was analyzed using a spectrofluorimeter with excitation at 277 nm and emission in the range of 280–350 nm as described in Experimental procedures. (A,C) Normalized Tyr fluores- cence spectra of Ab(1-40) (A) or Ab(1-42) (C) alone or in the pres- ence of LPC, incubated for 0 or 2 h. (B,D) Time-dependent changes in the Tyr fluorescence maxima for Ab(1-40) (B) and Ab(1-42) (D). (E,F) ANS emission spectra (E) and time-dependent change of the fluorescence maximum (F), of Ab(1-40) are shown. (G,H) ANS emission spectra (G) and time-dependent fluorescence maximum (H) of Ab(1-42) are shown. For ANS fluorescence analysis, 20 lLof Ab fibril samples and ANS (10 l M final concentration) were added to glycine buffer (pH 8.5, 50 m M final concentration) to make a total volume of 200 lL, and ANS fluorescence was analyzed using a spectrofluorimeter with excitation at 360 nm and emission in the range of 400–600 nm as described in Experimental procedures. *P < 0.05 versus Ab peptide alone at the same time point. LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai 638 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS ratio (1 : 2) and low LPC concentration (as low as 5 lm). However, vesicular LPC showed greater ability to enhance fibril formation than non-vesicular LPC, suggesting that the polar phosphate head group of LPC may play a critical role in interaction with Ab(1- 42) during fibril formation. The intrinsic Tyr fluorescence of Ab peptide is not highly sensitive to the local micro-environment, displaying modest decreases in fluorescence intensity during fibril formation [23]. On the other hand, the quantum yield of ANS fluorescence is greatly increased after binding to hydrophobic patches during the fibril formation process of Ab peptide, suggesting that it is an excellent probe to monitor the fibril formation micro-environment [29]. Our intrinsic Tyr fluorescence experiments demonstrated that LPC modulates the fluorescence of Ab(1-40) and Ab(1-42) in an opposite manner, albeit modestly. Also, ANS experiments showed that LPC exposes the hydrophobic patches of Ab(1-42) peptide only. The change in Tyr and ANS fluorescence induced by LPC is indicative of potential LPC–peptide interaction and differential changes in the micro-environment during the Ab(1-40) and Ab(1- 42) fibril formation processes [29,30]. The observations that LPC almost abolished the lag phase and decreased the critical concentration of Ab(1- 42) aggregation suggest that LPC may act as seeds in the fibril formation process. However, as LPC had no 40 60 A B Aβ β 1-40 20 0 40 50 A β 1-42 20 30 ThT fluorescence ThT fluorescence 0 1020304050 0 10 Time (min) 0 1020304050 Time (min) Fig. 5. Effect of LPC on the rate of Ab(1-40) and Ab(1-42) fibril elongation. (A,B) Ab(1-40) (A) or Ab(1-42) (B) (11 lg, 50 l M) were allowed to form fibrils in the presence of 0.2 lg of pre-formed sonicated fibrils in the absence (open circles) or presence (closed circles) of 20 l M LPC, for the indicated times. Fibril formation was monitored by ThT fluorescence assay, and expressed in arbitrary fluorescence units. Aβ β 1-40 198.5 kDa A B C 116.2 84.8 53.9 37.4 29 19.8 6.8 A β (50 µM): LPC (20 µ M): A β (50 µM): LPC (20 µ M): 0 h 1 h A β 1-42 A β 1-40 198.5 kDa 116.2 84.8 53.9 37.4 29 19.8 6.8 0 h 1 h LPC (–) (+) ++ +++ +++++ ++–– +++––– 4 h 8 h 24 h ++++++++++ ++–– +++––– 4 h8h 24 h 0 h 1 h 2 hh4 h 8h24 h Fig. 4. Effect of LPC on Ab peptide oligomerization. (A,B) Ab pep- tide (50 l M) was incubated in fibril-forming buffer in the absence or presence of 20 l M LPC for the indicated times. After fibril forma- tion, 2.5 lgofAb(1-40) (A) or Ab(1-42) (B) were separated by 10–20% gradient Tris ⁄ tricine SDS ⁄ PAGE, and bands were stained with Coomassie blue as described in Experimental procedures. (C) For dot-blot immunoassay, aliquots of 10 lLofAb fibrils were spotted on a poly(vinylidene difluoride) membrane, and Ab (1-42) oligomers were detected using an oligomer-specific antibody as described in Experimental procedures. A. M. Sheikh and A. Nagai LPC modulates Ab fibril formation FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS 639 such effect on Ab(1-40) fibril formation, it may not act as seeds, but rather may specifically influence the olig- omerization and nucleation processes of Ab peptides, depending on their native structure. This is consistent with our finding that LPC exclusively increased the tri- meric and tetrameric species of Ab(1-42) but not those of Ab(1-40). Indeed, Ab(1-42) oligomer was detectable as early as 1 h after the start of the fibril formation process, supporting the idea that LPC affects the oligo- merization process of Ab(1-42). Fibril formation of both Ab(1-40) and Ab(1-42) is nucleation-dependent, and both peptides have been shown to have surfactant properties due to the pres- ence of hydrophobic amino acids at the C-terminus, a region that is critical for nucleation and fibril forma- tion [31]. The presence of two more hydrophobic amino acids at the C-terminus causes Ab(1-42) to oligomerize much faster than A b(1-40) does [32], and it was proposed that this difference in fibril formation kinetics is due to conformational differences between the peptides [33]. These findings imply that hydropho- bicity is a determinant of Ab oligomerization and fibril formation processes. Our ANS experiments showed that LPC significantly increased the hydrophobicity of Ab(1-42) only, so this increased hydrophobicity may be critical for the enhanced nucleation and fibril formation of Ab(1-42). In conclusion, our findings show that LPC increases fibrillogenesis of Ab(1-42), the major component of Alzheimer’s disease plaque, and thus LPC may play a role in the pathology of Alzheimer’s disease. Experimental procedures Materials Lysophosphatidylcholine (LPC) was purchased from Avanti Polar Lipids (Alabaster, AL, USA) and dissolved in water. To prepare unilamellar LPC vesicles, 25 mg of lyophilized LPC was hydrated with 10 mL of water, followed by soni- cation at room temperature for 30 min in a bath sonicator. The Ab peptides Ab(1-40) and Ab(1-42) (Peptide Institute, Osaka, Japan) were each dissolved in 0.1% NH 3 at a con- centration of 250 lm, aliquoted immediately (in order to avoid the need for repeated freeze-thaw cycles), and stored at )70 °C, according to the manufacturer’s instructions. Chromatographic data provided by the manufacturer con- firmed the monomeric purity of the peptides. Thioflavin T (ThT) was obtained from Wako Pure Chemicals (Rich- mond, VA, USA), and deionized and filter sterile water was purchased from Sigma-Aldrich (St Louis, MO, USA). Pre- stained protein size markers were purchased from Bio-Rad (Hercules, CA, USA). Ab peptide fibril formation For fibril formation, a solution of synthetic Ab peptide in fibril formation buffer (50 mm phosphate buffer pH 7.5 and 100 mm NaCl) was prepared with or without LPC or LPC liposomes at the concentrations indicated. The reac- tion mixture was incubated at 37 °C without agitation for the indicated times, and then the fibril formation reaction was terminated by quickly freezing the samples. Assessment of Ab fibril formation on the basis of ThT fluorescence The presence of b-sheet structures and the kinetics of fibril formation were monitored by means of ThT fluorescence spectroscopy. Samples were diluted tenfold with glycine (pH 8.5, 50 mm final concentration) and ThT (5 lm final concen- tration). ThT fluorescence was measured using a fluorescence spectrophotometer (F2500 spectrofluorimeter, Hitachi, Tokyo, Japan), with excitation and emission wavelengths of 446 and 490 nm, respectively [34]. The normalized florescence intensity of fibrillary Ab was obtained by subtracting the flo- rescence intensity of buffer alone from that of the sample. Electron microscopy Electron microscopy was performed as described previously [35]. In brief, after Ab fibril formation, 10 lL of sample was applied to a carbon-coated Formvar grid (Nisshin EM, Tokyo, Japan) and incubated for 1 min. The droplet was then displaced with an equal volume of 0.5% v ⁄ v glutaral- dehyde solution and incubated for an additional 1 min. The grid was washed with a few drops of water and dried. Finally, the peptide was stained with 10 lLof2%w⁄ v ura- nyl acetate solution for 2 min. This solution was soaked off, and the grid was air-dried and examined under an elec- tron microscope (EM-002B, Topcon, Tokyo, Japan). Tyrosine fluorescence spectroscopy Tyrosine fluorescence of Ab peptide was measured using a Hitachi F2500 spectrofluorimeter, with excitation at 277 nm. Fluorescence emission was scanned in the range of 280–350 nm, at a scan rate of 300 nmÆmin )1 .Slitwidthsfor excitation and emission were 5 nm. The fluorescence emission spectrum of buffer only (background intensity) was subtracted from the emission spectrum of the samples. The emission max- imum data is presented as the mean of three independent experiments, and is expressed in arbitrary fluorescence units. ANS fluorescence spectroscopy The fluorescence intensity change of 8-anilino-1-naphtha- lene sulfonic acid (ANS) was used to evaluate the relative LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai 640 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS exposure levels of hydrophobic surfaces of Ab aggregates [30]. Fluorescence intensity measurements were obtained using a Hitachi F2500 spectrofluorimeter, with excitation at 360 nm. The emission spectra were read from 380 to 600 nm, at a scan rate of 300 nmÆmin )1 . Slit widths for excitation and emission were 5 nm. The data of emis- sion maximum is presented as the mean of three indepen- dent experiments, and is expressed in arbitrary fluorescence units. Gel electrophoresis and staining SDS ⁄ PAGE was performed using a 10–20% gradient tris- tricine gel system (Invitrogen, Carlsbad, CA, USA). After fibril formation, 2.5 lgofAb peptide was mixed with 2· SDS non-reducing sample buffer (Invitrogen) making a total volume of 20 lL, incubated at 85 °C for 2 min, and separated by electrophoresis. The gel was washed briefly with water, fixed in fixation buffer (40% methanol and 10% acetic acid) for 30 min, and stained with Coomassie Blue G250 (Biosafe Coomassie; Bio-Rad) for 1 h. The stained gel was washed with water overnight and scanned using a gel scanner (Bio-Rad). Dot-blot immunoassay After fibril formation, an aliquot (10 lL) of Ab(1-42) pep- tide was applied to poly(vinylidene difluoride) membrane using a manifold. Then the membrane was immunoblotted with an oligomer-specific antibody (A11, Invitrogen). This oligomer-specific antibody reacts specifically to a variety of soluble oligomeric protein ⁄ peptide aggregates regardless of their amino acid sequence, and does not react with either monomer species or insoluble fibrils; it reacts only with Ab oligomer species of at least octamer [25]. Immunoreactive oligomer was detected using horseradish peroxidase-conju- gated anti-rabbit IgG and an enhanced chemiluminescence kit (Amersham, Little Chalfont, UK), according to the manufacturer’s instructions. Elongation assay The elongation assay was performed as described previ- ously [34]. In brief, 50 lm Ab(1-40) or Ab(1-42) peptide monomer in fibril formation buffer was incubated at 37 °C for 48 h to prepare Ab fibrils. Then the whole reaction mixture was sonicated for 10 min. 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Biochem Biophys Res Commum 273, 1003–1007. 33 Shen L, Ji HF & Zhang HY (2008) Why is the C-termi- nus of Ab(1-42) more unfolded than that of Ab(1-40)? Clues from hydrophobic interaction J Phys Chem B 112, 3164–3167. 34 Naiki H & Nakakuki K (1996) First-order kinetic model of Alzheimer’s amyloid fibril extension in vitro. Lab Invest 74, 374–383. 35 Walsh DM, Lomakin A, Benedek GB & Condron MM (1997) Amyloid b-protein fibrillogenesis. Detection of a protofibrillar intermediate. J Biol Chem 272, 22364– 22372. LPC modulates Ab fibril formation A. M. Sheikh and A. Nagai 642 FEBS Journal 278 (2011) 634–642 ª 2011 The Authors Journal compilation ª 2011 FEBS . Lysophosphatidylcholine modulates fibril formation of amyloid beta peptide Abdullah Md. Sheikh and Atsushi Nagai Department of Laboratory. influence fibril formation of amyloid beta (Ab) peptide. Here, we show that lysophosphatidylcholine (LPC), a polar phos- pholipid, enhances Ab(1-42) fibril formation,