Effect of liposome membranes on disaggregation of amyloid β fibrils by dopamine

Biochemical Engineering Journal 71 (2013) 118–126

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Regular article

Effect of liposome membranes on disaggregation of amyloid ␤ fibrils by dopamine Huong Thi Vu a , Toshinori Shimanouchi a , Daisuke Ishikawa a , Tadaharu Matsumoto a , Hisashi Yagi b , Yuji Goto b , Hiroshi Umakoshi a , Ryoichi Kuboi a,∗ a b

Graduate School of Engineering Science, Osaka University, 1-3 Machikaneyama, Toyonaka, Osaka 560-8531, Japan Institute for Protein Research, Osaka University, 3-2 Yamadaoka, Suita, Osaka 565-087, Japan

a r t i c l e

i n f o

Article history: Received 14 July 2012 Received in revised form 24 November 2012 Accepted 10 December 2012 Available online 20 December 2012 Keywords: Alzheimer’s disease Amyloid beta fibrils Catecholamines Liposome Disaggregation

a b s t r a c t The inhibition of fibril formation of amyloid ␤ (A␤) and the disaggregation of A␤ fibrils are the promising approaches for a medical treatment of Alzheimer’s disease (AD) therapy. In this study, we investigated the effects of liposomes on dopamine-induced disaggregation of A␤ fibrils by using the variety of liposomes. The used liposomes were normal liposomes, raft-forming liposomes, charged liposomes and oxidized liposomes. Those liposome could accelerate the disaggregation rate of fibrils. From the comparison of normal and charged liposomes, a certain contribution of dopamine via an electrostatic interaction to the disaggregation was confirmed. From raft-forming and oxidized liposomes, we revealed a significant contribution of bound water to liposomes, which could assist the formation of the quinine-form of dopamine by a removal of its proton. It is, therefore, concluded that the membrane surface of liposomes is considered to be an adequate environment for the dopamine-induced disaggregation of fibrils. © 2012 Elsevier B.V. All rights reserved.

1. Introduction Amyloid ␤ (A␤), a peptide of 39–43 amino acids, is the main constituent of amyloid plaques in the brains of patients of Alzheimer’s disease and is thought to be a causative protein of Alzheimer’s disease (AD) [1]. The soluble monomeric A␤ is found to be non-toxic although its physiological function remains unknown. The deposition of A␤ in the form of amyloid fibrils is considered to be linked to AD [2,3]. The aggregation of the soluble A␤ monomer into toxic oligomeric or fibrillar species is a crucial step in the pathology of the disease [4,5]. There is currently no way to cure AD or stop its progression. Some researchers are making encouraging advances in the clinical treatment of AD, including medications and nondrug approaches to improve the symptom management. Therefore, reducing the level of aggregated A␤ in the brain is one of the promising strategies for AD therapy [6]. The identification of molecules that could initiate the “reverse” reaction to the fibril formation may be a key step toward a better understanding of the pathology of inclusions and deposits in human diseases, which allows us to evaluate the role of fibrils in neurodegenerative processes and disease progression. If these molecules

∗ Corresponding author. Tel.: +81 06 6850 6287; fax: +81 06 6850 6286. E-mail addresses: [email protected], [email protected] (R. Kuboi). 1369-703X/$ – see front matter © 2012 Elsevier B.V. All rights reserved. http://dx.doi.org/10.1016/j.bej.2012.12.012

could exert neuroprotective effects, their discovery could open new avenues for therapeutic intervention. The formed amyloid fibrils were reported to be disaggregated by various compounds, such as dopamine (DA) and l-dopa [7], nordihydroguaiaretic acid, rifampicin, tannic acid, quercetin [8], and salvianolic acid B [9], Anti-A␤1–11 antibody [10]. Besides, some of those reagents could also inhibit the formation of amyloid fibrils [7–11]. The prevention of amyloid fibril formation and its disaggregation seem to be a promising strategy for the treatment of AD [12–16]. From in vivo experiments using dyes specific to amyloids, A␤ fibrils are commonly observed on cell membranes [17]. It has also been reported, from experiments in vitro, that the liposome membrane could recruit not only the fragmented or short peptides [18,19] but also A␤ fibrillar aggregates [20]. In neuronal cell system, a variety of catechol amine derivatives are secreted and some hydrophobic derivatives such as dopamine are subject to partition into the lipid membrane. Therefore, we expect that the disaggregation of A␤ fibrils on liposome membranes by catechol amine derivatives would occur, which might give a better understanding of the physiological process of AD and a construction of novel therapeutic approach of AD using liposomes. In this study, the disaggregation of fibrils prepared by A␤(1–40) and A␤(1–42) by catechol derivatives was investigated in the presence of the variety of liposomes. The kinetics on the disaggregation of fibrils was investigated with a direct observation using a total internal reflection fluorescence microscopy (TIRFM)

H.T. Vu et al. / Biochemical Engineering Journal 71 (2013) 118–126

combined with thioflavin T (ThT). The mechanism on disaggregation of fibrils was discussed on a basis of the effects on hydrogen bondings. The liposome as a model biomembrane is well-known to posses the water molecules bound to phospholipids weakening the nearby hydrogen bondings [21]. The effect of liposomes on the disaggregation of A␤ fibrils was then investigated to address a possible action of the above effect in the biological system. Based on these results, we discussed the role of catechol amines such as DA in the modulation of A␤ fibrils on the liposome membrane surface. 2. Experimental 2.1. Materials Amyloid-␤ proteins with 40 and 42 amino acid residues (A␤(1–40) and A␤(1–42), respectively) were purchased from the Peptide Institute (Osaka, Japan). Various zwitterionic phospholipids 1,2-dimirystoyl-sn-glycero-3-phosphocholine (DMPC), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine (POPC) were purchased from Avanti Polar lipids (Alabaster, AL, USA). Anionic 1-palmitoyl-2-oleoyl-phosphatidylgrycerol phospholipids, (POPG), 1,2-dimirystoyl-phosphatidylgrycerol (DMPG), 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphate (POPA), sphingomyelin (SM), and DOTAP were purchased from Sigma. Stearic acid (SA) was purchased from Sigma Aldrich (St. Louis, MO, USA). Thioflavin T (ThT) was obtained from Dojindo (Kumamoto, Japan). Dopamine hydrochloride (DA), l-tyrosine (Tyr), catechol (Cat), 3,4-dihydroxy-l-phenylalanine (l-dopa), norepinephrine (NE) and epinephrine (EP) were purchased from Sigma–Aldrich. Other reagents used here were purchased form Sigma Aldrich and Wako Pure Chemical (Osaka, Japan). 2.2. Liposome preparation The variety of liposomes, as shown in Table 1 was prepared according to the previous report [22]. The SAPC sample was oxidized by mixing with CuSO4 (2 mM) and H2 O2 (20 mM) during a 15 h stirring at room temperature, according to the previous method [23]. The fully oxidation of SAPC was confirmed by a dissipation of main peak for SAPC (m/z = 811) and an alternative increase in main peaks of peroxidized lipid (LOOH, m/z = 843), 4-hexanonenal (m/z = 156), with a mass spectroscopy. Besides, a quantity of LOOH was monitored for 15 h with a conventional method [24]. Afterwards, the oxidized lipid (SAPCox) was recovered to measure its concentration with a commercial kit. The lipid mixture was dissolved in chloroform and then dried onto the wall of a round-bottom flask in vacuum and was then left overnight to ensure the removal of all of the solvent. The dried thin lipid film was hydrated with adequate buffer solution to form multilamellar vesicles (MLVs). Large unilamellar vesicles (LUVs) were formed from MLVs with five cycles of freeze–thaw treatment. All the liposome was treated with a extrusion method to adjust their diameter to 100 nm. 2.3. Fibril preparation A␤(1–40) and A␤(1–42) peptide solutions were prepared from powder by dissolving on 0.2% NH3 solution to 200 ␮M as stock solution at 4 ◦ C. The stock solutions were stored at −80 ◦ C until its use. Just before the experiment, the A␤ stock solutions were thawed and then diluted 20-fold by Tris–HCl buffer (Tris–HCl buffer: 50 mM and 100 mM NaCl, pH 7.4) to a final concentration of 10 ␮M. The fibrils were prepared by incubating A␤ monomer solution at 37 ◦ C for at least 1 day.

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2.4. Aˇ seed preparation Seed stock solutions were prepared by the following procedure as previously reported [25]. In brief, the seed was prepared from 10 ␮M of fibrils prepared under the conditions as described above. Fibrils were disrupted into seeds by the sonication of fibrils with a probe sonicator in a 10-ml plastic tube on an ice-bath at 40 W with five-cycle sonication (each 1 min in sonication and 1 min in interval). The seed stocks were kept at 4 ◦ C and briefly vortex mixed before use. 2.5. Fibril disaggregation experiments Preformed A␤ fibrils were incubated with catechol derivatives at various concentrations at 37 ◦ C. The disaggregation of A␤ fibrils was monitored by ThT fluorescence measurement. The fibril disaggregation was monitored by measuring the ThT fluorescence intensity. The fluorescence measurements were carried out at 37 ◦ C using a JASSO PF-6500 fluorescence spectrometer with an excitation wavelength of 442 nm and an emission wavelength of 485 nm. The decrease in ThT fluorescence was fitted to a first orderexponential decay function: F(t) = Fi exp(−kd t)

(1)

where F(t) represents the ThT fluorescence at time t, Fi is the initial level of fluorescence, and kd [s−1 ] is the first order rate constant, which is defined as the apparent disaggregation rate constant of fibrils. 2.6. Dielectric dispersion analysis The dielectric dispersion analysis permits to monitor the dipole moment such as the water. An RF impedance analyzer (Agilent, 4219B; 1 MHz to 1 GHz) and a network analyzer (Agilent, N5230C; 500 MHz to 50 GHz) were used to monitor the bulk water and the water bound to liposomes. The liposome suspension (about 30 mM for lipids) was loaded to the electrode. We kept the sample for 30 min at constant temperature before starting measurement. Thereafter, the relative permittivity (ε ) and dielectric loss (ε ) for the liposome suspension were measured, as a function of frequency, at each temperature, according to the previous reports [26,27]. The frequency dependence of ε and ε (1 MHz to 50 GHz) were analyzed with the following Debye’s equation: ε − εh =

4  i=1

εi 1 + (f/fci )

(2)

2

 ε (f/f ) Gdc ci i = 2 2fC0 1 + (f/f ) 4

ε −

i=1

(3)

ci

where εh and Gdc means the limit of relative permittivity at higher frequency and direct current conductivity of liposome solutions. C0 is the cell constant to obtain by a calibration using water, methanol, and ethanol in the frequency range of 1 MHz to 1 GHz. Alternatively, a calibration was carefully done by using the distilled water in the range of 500 MHz to 50 GHz. It has been reported that the liposome suspension has four different characteristic relaxations: the lateral diffusion of ionic species (i = 1, first-step, several MHz); the mobility of lipid headgroup (i = 2, second-step, ∼50 MHz); the water bound to liposome membranes (i = 3; third-step, 200–500 MHz); the bulk water (i = 4, fourth-step, ∼20 GHz) [26,27]. Therefore, Eqs. (2) and (3) were assumed to be written by a summation of four relaxation terms. Relaxation time  was calculated from the observed characteristic

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Table 1 Entry of liposomes used (100 nm in diameter). Lipid

Lipid composition

Chargea [C/nm2 ]

Phase state (37 ◦ C)

Remarks

1 2 3 4 5 6 7 8

POPC/SM POPC/CH/SM POPC/SM/CH/GM1 POPC POPC/CH DMPC DMPC/SA (10:4) DMPC/SAPCox

70:30 33/33/33 30/30/30/10 – 70/30 – 10/4 60/40

Neutral Neutral Negative Neutral (0) Neutral Neutral Negative Negative

ld + so ld + lo Possibly ld + lo ld ld + lo ld ld + lo Possibly ld + lo

9

DMPG/SAPCox

60/40

Negative

Possibly ld + lo

10

POPA

–

Negative (−1.40)

ld

11

DOTAP

–

Positive (+1.40)

Not determined

12

POPC/DOTAP

70/30

Positive (+0.42)

Not determined

– Strong interaction of CH with amyloid fibrils [20] GM1, which is a ganglioside, interacts with A␤ [17] Unsaturated acyl chain (one C C) Strong interaction of CH with amyloid fibrils [20] Saturated acyl chain in DMPC Strong interaction of SA with amyloid fibrils [20] Interaction of this liposome with A␤ is the same as the DMPC/SA (no. 7) [41] Interaction of this liposome with A␤ to induce their spherulitic amyloid aggregates [23] PA interacted with monomeric A␤ to induce the fibrillogenesis [42] Monomeric A␤ interacted with DOTAP liposome at around pH 7 [43] –

Entry

a

The values in parenthesis are the charged density of liposomes calculated by assuming a unilamellarity, a spherical shape, a bilayer thickness of 3.7 nm [44], and a mean sectional area of headgroup of 0.72 nm2 [45]. Besides, POPA and DOTAP are fully ionized at pH 7.4 [46].

frequency fci :  i = (2fci )−1 (i = 3, 4). In general, the large  represents the low mobility of target materials.

2.7. FTIR measurement Samples for infrared spectroscopy analysis were prepared in 50-␮m thick cells with CaF2 windows. The infrared spectra were measured with an FT/IR-4100ST (Nihon Bunko Co., Ltd., Tokyo, Japan). The temperature of the sample was controlled by means of a block assembly equipped with a circulating water jacket and monitored by a thermosensor placed at the edge of the cell window. The resolution was 4 cm−1 . The subtraction of spectra in the buffer was carried out to remove the contribution from water bands. The accuracy of the frequency reading was better than ±0.4 cm−1 . 2.8. Transmission electron microscopy (TEM) The formation of A␤ fibrils and their disaggregation were monitored by transmission electron microscopy (TEM). TEM images were obtained by the following procedure as previously reported [28]. In brief, a 5 ␮l aliquot of diluted solution was placed on a copper grid (400-mesh) covered with a carbon-coated collodion film for 1 min and the excess sample solution was removed by blotting with filter paper. After the residual solution had dried up, the grid was negatively stained with a 2% (w/v) uranyl acetate solution. Again, the liquid on the grid was removed with filter paper and dried. EM images were acquired using a JEOL 100CX transmission microscope (JEOL, Tokyo, Japan) with an acceleration voltage of 80 kV.

2.9. Total internal reflection fluorescence microscope (TIRFM) TIRFM observations were performed by a previously reported procedure [15,16]. Briefly, the TIRFM system used to observe the aggregation of amyloid fibril formation was developed based on an inverted microscope (IX70, Olympus, Tokyo, Japan). A␤ stock solution was diluted with Tris buffer (pH 7.5), NaCl solution, ThT, and each liposome solution. The final solutions contained A␤ (1–40) (50 ␮M), Tris (50 mM), NaCl (100 mM), ThT (10 ␮M), and each liposome (250 or 0 ␮M). These samples were incubated at 37 ◦ C for more than 48 h. The ThT molecule was excited at 442 nm by a helium–cadmium laser (IK5552R-F, Kimmon, Tokyo, Japan). The laser power was 4–80 mW, and the observation period was 3–5 s. The fluorescence image was filtered with a bandpass filter

(D490/30, Omega Optical, Bratteboro, VT) and visualized using a digital steel camera (DP70, Olympus). 2.10. Statistical treatment of data All the experiment was performed by at least three times. A Student’s t-test was performed to show the significant difference between data. 3. Results and discussion 3.1. Effect of liposomes on disaggregation of Aˇ fibrils It has been reported that the disaggregation of fibrils was examined by a sonication, laser irradiation [15,16], and chemical reagent such as dopamine (DA) [7,11]. Meanwhile, the effect of biomembranes or their mimicking system on the disaggregation of fibrils has been still unclear. We investigated the influence of liposomes on the DA-induced disaggregation of A␤ fibrils. In the first series of experiments, we monitored the disaggregation of fibrils by the ThT fluorescence intensity because the thioflavin T (ThT) can specifically bind to fibrils [11]. We selected DMPC/stearic acid (SA) (10:4) liposome because it has been reported that SA favors to interact with A␤ fibrils [20]. Fig. 1(a) shows the time-course of ThT fluorescence intensity staining fibrils of A␤(1–40) in the presence and absence of liposomes. It was obvious that a reduction of ThT fluorescence intensity after addition of DA in the presence of DMPC/SA (10:4) was larger than that in the absence of liposome. We quantitatively evaluated the apparent disaggregation rate constant (kd [s−1 ]) from the reduction behavior of ThT fluorescence intensity. The kd values in the absence and presence of liposome were 5.1 × 10−5 s−1 and kd = 8.1 × 10−5 s−1 , respectively. Therefore, the addition of DMPC/SA liposomes appears to accelerate the disaggregation rate of fibrils. To examine the effect of liposomes on the disaggregation of fibrils by DA in more detailed, the kd value was evaluated in the presence of the variety of liposomes. We systematically prepared various liposomes such as normal liposomes (entry 4 and 6), raft-forming liposomes (1–3, 5, and 7), charged liposomes (entry 10–12) and oxidized liposomes (entry 8 and 9). It was found that the DA-induced disaggregation behavior of A␤ fibrils depended on the lipid composition of liposome as shown in Fig. 1(b) and (c). A small increase in disaggregation rate was observed in the presence of liposomes composing of single zwitterionic phospholipid (entry 4 and 6) and the liposomes with microdomain (entry 1–3, 5, and 7). The oxidized DMPC/SAPCox liposomes (entry 8)

H.T. Vu et al. / Biochemical Engineering Journal 71 (2013) 118–126

Relative ThT fluorescence intensity [-]

(a)

3.2. Direct observation of Aˇ fibril disaggregation in the presence of liposomes

Aβ (1-40): 10 μM Dopamine Lipid: 1 mM

1.2

DA :100 μM 0.8

(-) liposome

0.4

(+) liposome

0

0

6

12

Disaggregation time [h] 200

Aβ (1-40)

* p<0.01

*

150 *

6

-1

kd× 10 [s ]

(b)

100 *

*

*

*

*

*

50

(c)

200

* Aβ(1-42)

*

*

* p<0.01

*

150

6

-1

kd× 10 [s ]

*

*

100

*

*

*

*

*

*

*

none

50 1

2 3

4

5 6

7

8

121

*

9 10 11 12

Fig. 1. (a) Time-course of ThT fluorescence intensity in the absence and the presence of DMPC/SA (10:4) liposome. Solid curves were well-fitted curves with Eq. (1). Effect of liposome on apparent disaggregation rate constant for (b) A␤(1–40) and (c) A␤(1–42). Fibrils were disaggregated by 100 ␮M DA in the absence and presence of 1 mM liposomes as follows: (1) POPC/SM (7:3); (2) POPC/CH/SM (1:1:1); (3) POPC/SM/CH/GM1 (3:3:3:1); (4) POPC; (5) POPC/CH (7:3); (6) DMPC; (7) DMPC/SA (10:4); (8) DMPC/SAPCox (6:4); (9) DMPG/SAPCox (6:4); (10) POPA; (11) DOTAP; (12) POPC/DOTAP (7:3).

could evidently accelerate the disaggregation rate of fibrils by DA. Besides, the replacement of DMPC by DMPG resulted in the further increase in kd value (DMPG/SAPCox, entry 9). It is considered that the difference between entry 8 and 9 resulted from the effect of negatively charged lipid. The same is true for POPA liposome (entry 10). Inversely, the cationic liposomes DOTAP (entry 11) and DOTAP/POPC (entry 12) suppressed the DA-induced disaggregation of fibrils (kd ∼ (3.4 ± 0.2) × 10−5 s−1 ). The aforementioned results might be explained by the electrostatic interaction between positively charged DA and charged liposomes. Under our experimental conditions, it was confirmed that all the liposomes could not disaggregate A␤ fibrils (data not shown). It is, therefore, considered that the incremental change in kd value by the addition of liposomes did not result from a liposome-induced disaggregation as previously reported [29]. It is, therefore, concluded that the lipid composition contributed to accelerate the apparent disaggregation rate of fibrils of both A␤(1–40) and A␤(1–42).

To demonstrate the effect of liposomes on the disaggregation of A␤ fibrils by DA, the direct observation was performed with the total internal reflection fluorescence microscopy (TIRFM) and transmission electron microscopy (TEM). The TIRFM combining with ThT is a powerful tool to confirm the aggregate with an affinity to ThT. A 10 ␮M A␤(1–40) fibrils were first incubated with 100 ␮M DA at 37 ◦ C. The sample was observed at each 3 h with TIRFM. The time-course of TIRFM images showed the progressive loss of A␤(1–40) fibrils with passing time (Fig. 2(a1)–(a3)). The fibrils disappeared after 6 h of the incubation (Fig. 2(a3)), indicating the complete disaggregation of A␤ fibrils by DA. These results provide a strong evidence for the DA-induced disaggregation of A␤ fibrils. However, TIRFM cannot give mechanistic details on the microscopic structure of the spot with fluorescence intensity seen in Fig. 2(a3). The microscopic structure of fibrils disaggregated by DA was then visualized by the TEM observation (Fig. 2(b1)–(b3)). After 2 h later of the DA addition, many short fibrils with several hundreds nm in length were observed (Fig. 2(b2)). Furthermore, after 6 h later of the DA addition, not only the length but also the number of fibrils decreased significantly, indicating the disaggregation of A␤ fibrils by DA. Therefore, the spot with fluorescence intensity observed with TIRFM (Fig. 2(a3)) is considered to be the remaining fibrils with several tens nm in length. In the presence of DMPC/SA liposome, the disaggregation of fibrils could be observed significantly with TEM. In the beginning, some liposome along fibrils was observed (Fig. 2(c1)). After a 6 hincubation of fibrils in the presence of liposome and DA, almost all the fibrils were disaggregated (Fig. 2(c2)). Especially, fragmented fibrils were observed along liposomes (L1 and L2) as shown in Fig. 2(c3). This image directly suggested that the fragmentation occurred randomly rather than the decomposition of fibrils from their ends. Indeed, the number of fibrils at 3 h after addition of DA definitely increased as compared with that at 0 h. Likewise, the disaggregation of A␤(1–42) fibrils by dopamine was driven by their fragmentation. To discuss the accelerated effect of liposomes on the disaggregation of fibrils, we compared the length distribution of disaggregated fibrils in the absence and presence of liposome. Fig. 3(a)–(c) shows the microscopic structure of 10 ␮M A␤(1–40) fibrils incubated at 37 ◦ C for 6 h in the presence of 100 ␮M DA in the absence of liposomes, the presence of 1 mM DMPC/SA (10:4) and DMPC/SAPCox (6:4) liposome, respectively. The presence of liposomes obviously generated the fragmented fibrils with shorter length. The average length of fibrils were measured to be at around 80 nm for DA alone (Fig. 3(a2) and (a3)) and 40 nm for DA/liposome (Fig. 3(b2), (b3), (c2), and (c3)). As a comparison, the sonicated fibrils were also prepared (Fig. 3(d1) and (d2)). A considerable sonication could give the short fibrils with 100 nm in length (Fig. 3(d3)), consistent with previous findings [30]. From the direct observation with both TIFRM and TEM, it was concluded that the fragmentation of fibrils by DA resulted in the decrease in ThT fluorescence intensity as shown in Fig. 1(b) in the presence and absence of liposomes. Meanwhile, the DA-induced disaggregation of fibrils appears to be different from the mechanistic fragmentation by the sonication. 3.3. DA distribution to liposome membrane for DA-induced disaggregation of fibrils From Fig. 1(b) and (c), the electrostatic interaction between DA and liposomes appears to regulate the disaggregation rate of fibrils. As shown in Fig. 1(b) and (c), The order of kd value for charged liposomes was POPA > POPC > POPC/DOTAP > DOTAP.

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Fig. 2. Direct observation of disaggregation of A␤ fibrils by DA in the absence and the presence of DMPC/SA (10:4) liposome. (a) Disaggregation of fibrils of A␤(1–40) monitored by TIRFM (scale 10 ␮m). The preformed fibrils of A␤ were incubated in the presence of 100 ␮M DA at 37 ◦ C for (a1) 0 h, (a2) 3 h, and (a3) 6 h. (b) Disaggregation of fibrils of A␤(1–40) monitored by TEM (scale 200 nm). The preformed A␤ fibrils were incubated in the presence of 100 ␮M DA at 37 ◦ C for (b1) 0 h, (b2) 2 h, and (b3) 6 h. (c) Disaggregation of fibrils of A␤(1–40) monitored by TEM for (c1) 0 h, (c2) 6 h, and (c3) its magnification. (d) Disaggregation of A␤(1–42) fibrils monitored by (d1) TIRFM and (d2) TEM. Arrows indicates the fragmentation sites of fibrils. L1 and L2 indicate DMPC/SA (10:4) liposomes.

On the other hand, the order of calculated charge density was POPA (−1.4 C/nm2 ) > POPC (0 C/nm2 ) > POPC/DOTAP (+0.42 C/nm2 ) > DOTAP (+1.4 C/nm2 ). Both were correlated with each other, suggesting the DA favored to interact with liposome membranes in an electrostatic manner. And then, the concentration dependency of DA against the kd value was investigated in Fig. 4(a). The kd value for A␤(1–40) fibrils increased up to 120 × 10−6 s−1 at around 500 ␮M of DA. Assuming the simple electrostatic DA concentration effect, the concentration of DA by 2-folds in the case of POPA liposome (no. 10) would be required. Alternatively, the increase in kd value for liposome (no. 9) would require the concentration of DA by 5-folds or more. If all the oxidized SAPC lipids were the carboxylic derivatives with negatively charge, DMPG/SAPCox should have the same charge as POPA and could not compensate for the DA concentration to achieve the concentration by 5-folds. However, in our experiment, oxidized SAPC lipid mixture includes the variety of oxidants (data not shown). It is, therefore, considered that the simple electrostatic concentration of DA on the liposome membrane is not enough to explain

the effect of liposomes on the DA-induced disaggregation of fibrils and its mechanism in detailed. 3.4. Disaggregation of Aˇ fibrils by catechol derivatives in the presence of liposome Mechanistic details on the DA-induced disaggregation of fibrils are herein discussed from the molecular aspect of DA. DA and ldopa have been previously reported to disaggregate the fibrils of A␤(1–40) [7], suggesting that catechol derivatives might disaggregate fibrils. We investigated the disaggregation of fibrils by various catechol derivatives in the same manner as Fig. 1(a). The significant reduction of ThT fluorescence intensity could be observed with time. The resulting kd value for catechol (Cat), norepinephrine (NE) and epinephrine (EP), l-dopa, DA were summarized in Fig. 4(b). Those kd values were (23–84) × 10−6 s−1 in the case of A␤(1–40) fibrils. In contrast, no significant decrease in ThT fluorescence intensity was observed in the presence of 100 ␮M Tyr, indicating that Tyr had no effect on A␤ fibrils. These results suggest that the catechol

H.T. Vu et al. / Biochemical Engineering Journal 71 (2013) 118–126

123

Fig. 3. TEM images of fragmented A␤(1–40) fibrils and their length distribution. (a1–a3) Fibrils disaggregated by DA for 6 h at 37 ◦ C. (b1–b3) Fibrils disaggregated by DA in the presence of 1 mM DMPC/SA (10:4) for 6 h at 37 ◦ C. (c1–c3) Fibrils disaggregated by DA in the presence of 1 mM DMPC/SAPCox (6:4) for 6 h at 37 ◦ C. (d1–d3) Fibrils sonicated for 2 min. A␤ fibrils were prepared by incubating 10 ␮M of monomeric A␤(1–40) in 50 mM Tris–HCl buffer (100 mM NaCl, pH 7.4) at 37 ◦ C for 24 h.

group is necessary for the disaggregation both of A␤(1–40) and A␤(1–42) fibrils. It is, therefore, considered that the catechol ring was a key structure for inducing the disaggregation of fibrils. Furthermore, we examined the effect of DMPC/SA (10:4) liposomes on the disaggregation activity of these compounds. The accelerated rate of disaggregation of fibrils was observed in the case of DA and l-dopa. In contrast, other catechol derivatives (Cat, NE, and EP) showed the similar disaggregation rate to the case in the absence of liposomes. The same was true for A␤(1–42) fibrils. Catechol is in general oxidized to produce the quinone-form. Especially, DA easily converts to the quinone-form [31]. Li et al. [7] have reported that the quinine-form of DA was likely to disaggregate fibrils via a Schiff-base reaction with Lys-residue. Thus, it is supposed that the conversion of DA to quinone-form under the liposomal environment might contribute to the acceleration of disaggregation of fibrils. To confirm which molecule is a key for disaggregation of fibrils, we investigated the disaggregation of fibrils in the presence of DA pre-incubated for 6 h (aged DA: A-typed quinone) because DA considerably converted to DA-quinone with O2 − . The kd value for A-typed quinine of DA was higher than that of fresh DA (F-typed DA) as shown in Fig. 4(c). Since the life time of O2 − is too short to affect the disaggregation of fibrils, the increase in kd is considered to be mainly caused by A-typed quinone of DA. Alternatively, the A␤/Cu complex (A␤(1–42):Cu = 1:2) can also oxidize DA to form DA-quinone (O-typed quinone of DA) associated with the equi-molar H2 O2 [32]. The O-typed DA accelerated the disaggregation rate (Fig. 4(c)), although the addition of H2 O2 could not result

in the disaggregation of fibrils. This result indicated that quinineform of DA contributed to the disaggregation of fibrils. In the case of catechol derivatives other than DA, their O-typed quinone-forms also showed the higher kd value relative to those of F- and A-typed catechol derivatives (Fig. 4(c)). From these results, it is likely that quinone-form of catechol derivative was a key species for the disaggregation of fibrils. Meanwhile, the quinone-form could contribute to the kd value by at most +75%-incremental change, giving no plausible explanation on the incremental change of the kd value by using liposomes (up to +225% in Fig. 1(b)). In the following, we investigated the effect of liposome on the production of quinone-form of DA to clarify the mechanism on the DA-induced disaggregation of fibrils. 3.5. Possible mechanism of the enhancement of disaggregation of fibrils by liposome membranes If the quinone form of catechol derivatives is a key species for the disaggregation of fibrils, the liposome requires the removal of the proton from catechol derivatives to promote the production of quinone-forms. The previous study has revealed that the bound water of the liposome membrane could promote the removal of protons from cholesterol for its oxidation [39]. The bound water might be, therefore, effective for the disaggregation of fibrils. To clarify the role of the bound water to liposomes toward the disaggregation of fibrils, the D2 O solvent isotope effect was examined. The polarization of O D bond in D2 O needs more energy

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Fig. 4. (a) DA-concentration dependency of disaggregation rate constants for A␤(1–40) and A␤(1–42) fibrils. (b) Disaggregation rate constant for A␤(1–40) and A␤(1–42) fibrils in the absence and the presence of DMPC/SA (10/4) liposome. (c) The influence of quinine-form of catechol derivatives on the disaggregation of fibrils. F, fresh catechol; A, aged catechol; O, A␤/Cu2 -oxidized catechol. A␤ fibrils were prepared by incubating 10 ␮M A␤(1–40) monomer solution at 37 ◦ C for 24 h in Tris–HCl buffer containing 100 mM NaCl, pH 7.4. A␤(1–40) (10 ␮M) and DMPC/SA (10/4) liposome with 100 nm in diameter (1 mM) were used. All the experiments were performed at 37 ◦ C.

than that in O H bond in H2 O. If bound water attack the hydrogen bonds (HBs) of fibrils to disaggregate them, the disaggregation rate constant (kd,D2 O ) should be reduced in contrast to kd,H2 O . The resulting kd,D2 O /kd,H2 O values were 0.40 ± 0.03 for DMPC and 0.34 ± 0.05 for DMPC/SA (10/4, mol/mol), which were obviously smaller than that in bulk aqueous phase (0.42 ± 0.01). On the other hand, the kd,D2 O /kd,H2 O value for A␤(1–42) fibrils in the presence of DMPC/SA (10/4, mol/mol) was 0.52 ± 0.02 (Fig. 5(a)), which is larger than the case of A␤(1–40). This finding is consistent with the result in the absence of liposomes. In the case of DMPC/SAPCox, the reduction of the kd,D2 O /kd,H2 O value was larger than that of DMPC/SA in both cases of A␤(1–40) and A␤(1–42) fibrils. Those results suggested that the bound water present at DMPC/SAPCox rather than DMPC/SA easily attacked HBs of fibrils to disaggregate them. Alternatively, we discuss the influence of rigidity of fibrils against the disaggregation. In a scanning TEM experiment, the values of molecular weight per length value of A␤(1–40) and A␤(1–42) were estimated to be 30.3 kDa/nm [34] and 19 kDa/nm [35], respectively. A solid-state NMR experiment has revealed that the difference in conformation of fibrils between double layered structure with in register parallel ␤-sheet of A␤(1–40) [36] and in register parallel ␤-sheet of A␤(1–42) [35]. These findings suggests that A␤(1–40) fibrils indicated the solid-like nature as compared with A␤(1–42) fibrils. Such a rigid structure of A␤ fibrils results from a stabilization by interstrand HBs [37,38]. Therefore, A␤(1–40) fibrils should be affected by D2 O exchange in contrast to A␤(1–42) fibrils. In actual, the kd values for A␤(1–40) and A␤(1–42) were (5.1 ± 0.6) × 10−5 and (8.0 ± 0.7) × 10−5 s−1 , respectively. The effect of liposome against the disaggregation of A␤(1–42) fibrils was larger than that for A␤(1–40). This result supports the above consideration. We also evaluated the difference in the isotope effect of D2 O against the fibril disaggregation between A␤(1–40) and A␤(1–42)

fibrils. The kd,D2 O /kd,H2 O value for A␤(1–40) fibrils was evaluated to be 0.42 ± 0.01 whereas the kd,D2 O /kd,H2 O value 0.6 ± 0.05 for A␤(1–42) fibrils. In the presence of liposome, the similar results were obtained (Fig. 5(a)). It is, therefore, considered that weaker HBs in A␤(1–42) fibrils than in A␤(1–40) fibrils is unlikely to be affected by D2 O. In the experiment with a dielectric dispersion analysis, the difference between the bulk water and bound water could be also estimated in a basis of their relaxation time [39]. The relaxation time for the bulk water and bound water to liposome membranes were observed in the rage of 1–10 ps and 0.1–1 ns, respectively (Fig. 5(b)). The molecular mobility of bound waters is considered to be restricted by the phospholipids in contrast to the bulk water. According to Arrhenius’s law: (2)−1 = A exp(−E/RT) [39], the activation energy E was estimated to be ∼21 kJ/mol for bulk water, consistent with the previous literature [40]. As for the bound water, 31.8 ± 3.8, 32 ± 2.5, 32 ± 3.1 kJ/mol for DMPC, DMPC/SA, DMPC/SAPCox, respectively. The E value indicates the extent of polarization of O H bond in water molecule because the strong polarization along O H bond results in the increase in intermolecular interaction between water molecules [39]. The O H bond in bound water is strongly polarized by its binding to phospholipid. It is considered that the bound water with strong polarization along O H bond is advantageous to attack HBs within fibrils. Thus, the bound water is anticipated to be active water in contrast to bulk water although no definite discrepancy between DMPC/SA and DMPC/SAPCox was observed in the dielectric dispersion analysis. The bound water to DMPC (un)modified the stearic acid (SA) was then investigated since SA preferably binds to A␤ fibrils [20]. The liposome membrane includes the waters bound to the phosphoester group (PO2 − ) [21]. The peak shift of PO2 − antisymmetric stretching band observed at around 1230 cm−1 to lower frequency

H.T. Vu et al. / Biochemical Engineering Journal 71 (2013) 118–126

(a)

* p < 0.01 * *

0.6

*

*

DMPC/ SAPCox

DMPC/ SA

Aβ(1-40)

τdiel [psec]

10

ΔE = 31 kJ/mol ΔE = 32 kJ/mol ΔE = 32 kJ/mol

3

WL

DMPC DMPC/SA(10/4) DMPC/SAPCox(6/4) 2

(c) 0.01

(i) (ii)

0.005 (iii) (iv)

ΔE = 22 kJ/mol

1

(v)

Wb

0

DMPC/ SAPCox

Aβ(1-42)

Absorbance [a.u.]

(b)

10 /0 8/ 2 6/ 4

W lip ith o s ou om t e 10 10/0 10/2 /4

10 /0 8/ 2 6/ 4

0.4

DMPC/ SA

10

*

* *

W lip ith os ou om t e 10 10/0 10/2 /4

kd, D2O /kd, H2O [-]

*

10

125

20

40

60

80

o

Temperature [ C]

0 1300

1250

1200

1150 -1

Wavenumber [cm ]

Fig. 5. (a) Effect of liposome on the D2 O isotope effect of disaggregation rate for A␤(1–40) and A␤(1–42) fibrils. (b) Temperature dependency of relaxation time of bound water to liposomes and bulk water. Relaxation time was estimated by a dielectric dispersion analysis (see Section 2). DMPC, DMPC/SA (10/4), and DMPC/SAPCox (6/4) were used. (c) Infrared spectra of (i) DMPC, (ii) DMPC/SA (10/2), and (iii) DMPC/SA (10/4), (iv) DMPC/SAPCox (8/2) and DMPC/SAPCox (6/4) liposomes for the PO2 − double bond stretching region at 25 ◦ C. Arrows show the maximum frequencies of the antisymmetric stretching band (as ). Fibrils were disaggregated by 100 ␮M DA in the absence and presence of 1 mM DMPC/SA liposome. A␤ fibrils were prepared by incubating 10 ␮M A␤ monomer solution at 37 ◦ C for 24 h in Tris–HCl buffer containing 100 mM NaCl, pH 7.4. The data were obtained from at least three independent experiments.

corresponds well with the increase in the bound water [21]. Fig. 5(c) indicates that the peak for DMPC/SA and DMPC/SAPCox shifted to lower frequency with increasing the molar ratio of SA and SAPCox, respectively. Specifically, the shift of peak to lower frequency range was observed in the case of DMPC/SAPCox. This suggested that the DMPC/SAPCox could pool more bound water within liposome membranes than DMPC/SA. Therefore, the DMPC/SAPCox is expected to enhance the disaggregation of A␤ fibrils, in comparison with its disaggregation in the bulk phase and in the presence of DMPC/SA. Actually, the extent of bound water (max ) was corresponding to the kd,D2 O /kd,H2 O value, suggesting that the bound water can preferably contribute to the disaggregation of fibrils. As a possible consideration, there is a possibility that the bound water weakened the HB within fibrils associating with the liposome membrane to enhance the DA-dependent disaggregation of fibrils. The formation of quinone-form requires the removal of proton from DA. The bound water with polarized O H bond is advantageous for the proton removal, as reported previously [33]. Alternatively, the formation of DA-A␤ complex might induce the gap or distortion at random sites on fibrils and the resulting

hydration in the neighborhood (HB destabilization). Those mechanisms could affect the disaggregation process of fibrils on the liposome membranes. 4. Conclusion The effect of liposome on the dopamine-induced disaggregation of fibrils was examined in this study. Overall, liposomes could accelerate the disaggregation rate of fibrils of A␤(1–40) and A␤(1–42). The disaggregation of fibrils was likely to be driven by the reaction of dopamine with A␤s via the quinine-form of dopamine. Liposome could contribute to the supply of bound water advantageous for a proton removal to produce the quinine-form of dopamine, rather than to the concentration of dopamine into liposome membrane phase. It is, therefore, considered that the liposome was the environment advantageous for the dopamine-induced disaggregation of fibrils, unless the liposome has the positively charged surface. It is also likely that the results presented here help the development of strategies for the prevention and the treatment of protein deposition-related diseases such as Alzheimer’s disease and Parkinson’s disease.

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