Effect of Alanine Replacement of L17 and F19 on the Aggregation and Neurotoxicity of Arctic-Type Aβ40

Alzheimer’s disease is the most common form of neurodegenerative disease. Beta-amyloid peptides (Aβ) are responsible for neuronal death both in vitro and in vivo. Previously, L17 and F19 residues were identified as playing key roles in the stabilization of the Aβ40 conformation and in the reduction of its neurotoxicity. In this study, the effects of L17A/F19A mutations on the neurotoxicity of Aβ genetic mutant Arctic-type Aβ40(E22G) were tested. The results showed that compared to Aβ40(E22G), Aβ40(L17A/F19A/E22G) reduced the rate of conformation conversion, aggregation, and cytotoxicity, suggesting that L17 and F19 are critical residues responsible for conformational changes which may trigger the neurotoxic cascade of Aβ. Aβ40(L17A/F19A/E22G) also had decreased damage due to reactive oxygen species. The results are consistent with the discordant helix hypothesis, and confirm that residues 17–25 are in the discordant helix region. Compared to Aβ40(L17A/F19A), reduction in aggregation of Aβ40(L17A/F19A/E22G) was less significantly decreased. This observation provides an explanation based on the discordant helix hypothesis that the mutation of E22 to G22 of Aβ40(E22G) alters the propensity of the discordant helix. Arctic-type Aβ40(E22G) aggregates more severely than wild-type Aβ40, with a consequential increase in toxicity.


Introduction
Alzheimer's disease (AD) is the most common neurodegenerative disease in the elderly population [1][2][3]. There are two forms of AD, late onset sporadic (SAD) and early onset familial (FAD) [4]. SAD is predominantly diagnosed in people over 65 years of age, and less frequent FAD often occurs in patients under the age of 65 years [5,6]. FAD is clinically considered the most serious form of AD. Regardless of the form of AD, amyloid senile plaques (SPs) and neurofibrillary tangles (NFTs) are the two most important pathological hallmarks in the brains of AD patients [7]. In SPs, the main component is the b amyloid peptide (Ab), that is either 40 (Ab 40 ) or 42 (Ab 42 ) amino acids long, containing hydrophobic amino acid sequences at its C-termini. This peptide is the proteolytic product of the amyloid precursor protein (APP), which is sequentially cleaved first by b-secretase, then by csecretase [8,9].
Amyloid deposits of Ab peptides for both SAD and FAD, including oligomers, protofibrils, and fibrils have been demonstrated to be toxic to neural cells, and are the main causative agents leading to AD [10,11]. The neurotoxicity induced by Ab aggregates has been associated with formation of reactive oxygen species (ROS) and reactive nitrogen species (RNS) [12,13]. Furthermore, neurotoxicity is highly correlated with its structural and molecular states [14]. Fibrilligenesis of Ab is usually accompanied by a conformational conversion from either a-helix or random coil to b-sheet/strands during the aggregation process [15,16]. The conformation of Ab is well correlated with sequence and toxicity. Using different Ab fragments or truncated Ab peptides, several sequence regions, including residues 17-20, 30-35, and 41-42, have been shown to play roles in conformation and toxicity [17][18][19].
To reduce toxicity induced by Ab, it is necessary to prevent structural conversion and aggregation of Ab [15,16,30]. Several studies have suggested that residues 16-23 constitute a region of the discordant helix [30][31][32][33][34], and any factors which stabilize the conformation of this region can prevent the aggregation and reduce toxicity of Ab 40 [32,35]. Our previous studies found that 17-25 is the discordant helix region that can stabilize Ab structure and inhibit aggregation [36]. This identification provides the basis for determining key residues that can increase helical propensity. Previously, we identified L17 and F19 as key amino acids in the stabilization of the Ab 40 conformation. By constructing an Ab 40 (L17A/F19A) mutant, we further demonstrated that replacement of L17 and F19 with alanine can stabilize the Ab conformation, reduce Ab aggregation, and diminish Ab-induced neurotoxicity [37]. Most Ab peptides of FAD contain one point mutation of wild-type Ab [21][22][23][24][25][26]. Some of them, such as Arctictype Ab, can cause more severe cell death than wild-type Ab. Among these familial Ab mutants, the Arctic-type Ab has been shown to accelerate the development of clinical and pathological features indistinguishable from those of sporadic AD.
To further investigate the effects of L17A/F19A mutations on Ab properties, we used the same strategy as a previous study [37] and constructed an Ab 40 (L17A/F19A/E22G) mutant to charac-terize and compare the related properties with the genetic Arctic mutation of Ab 40 (E22G), which has been shown to be the most toxic FAD Ab mutant [23]. We wished to determine whether the L17A/F19A mutations can result in stable conformations and less neurotoxicity not only in wild-type Ab, but also in FAD. In vitro studies were used to show that the L17/F19 mutants decreased Ab aggregation and changed aggregation morphology. In vivo studies were used to verify neurotoxicity by using a cell viability assay. Our results show that compared to Ab 40 (E22G), Ab 40 (L17A/ F19A/E22G) can reduce aggregation and neurotoxicity. These results demonstrate that replacement of L17 and F19 with alanine residues decreases aggregation and neurotoxicity of Ab 40 (E22G), further suggesting that L17 and F19 are key residues in the stabilization of Ab. Our study also verifies the identification of critical residues responsible for conformational changes which may trigger the neurotoxic cascade of Ab 40 and its genetic mutations.

Ab Peptide Preparation
Production of recombinant Ab peptides used the cloning protocol as previously described [38]. cDNAs of Ab 40 were a kind gift from Professor Paul Greengard. Eschericha coli BL21(DE3) (Sigma, St. Louis, USA) was used for expression. All Ab peptides were purified on a reverse phase C 18 HPLC column (Waters, Milford, Massachusetts, USA) with a linear gradient from 0% to 100% acetonitrile. The molecular weight of the purified Ab peptides was verified by MALDI-TOF mass spectroscopy. They were freshly prepared in a 1 mM stock solution in 2,2,2trifluoroethanol (Sigma, St. Louis, USA).

Circular Dichroism (CD) Spectroscopy and Secondary Structure Analyses
Far-UV CD spectra were collected from 190-260 nm at 37uC using a synchrotron radiation circular dichroism (SRCD) spectropolarimeter at the 04B1 beam station of the national synchrotron radiation center in Taiwan. For SRCD measurements, a final peptide concentration of 60 mM in 20 mM phosphate buffer, pH 7.0, was used. All measurements were performed in CaF 2 cells with a path length of 0.1 mm. Each SRCD spectrum was reported as the average sum of three separate analyses. Secondary structure analysis was performed in an online web server Dichroweb [39,40] using the CDSSTR program.

Thioflavin-T Peptide Aggregation Assay
The peptide stock solution was dried under N 2 gas and resuspended in 20 mM phosphate buffer, pH 7.0, to a final concentration of 60 mM. Thioflavin T (Th-T) (Sigma, St. Louis, USA) dye (30 mM) was added to the freshly prepared peptide solution at a molar ratio of 1:2 with 0.01% NaN 3 at 37uC. Fluorescence measurements were performed using a microplate reader (FlexStation 3, DOWNINGTOWN, Pennsylvania, USA) every 30 minutes at 37.060.2uC. The excitation and emission wavelengths were 450 nm and 482 nm, respectively.
Aggregation kinetics was fitted using the following equation: Where Y is the fluorescence intensity, t is time, t o is the time to 50% of maximal fluorescence, and k app is apparent rate constant for the growth of fibrils [41]. Kinetic data obtained from spectroscopic measurements were fitted using the nonlinear curve fitting software Original 8.0 (OriginLab, Northampton, MA, USA). In the initial fitting stage, the Simplex method was used to calculate the initial input parameters to establish the parameter Effect of L17/F19 on Arctic-Type Ab 40 PLOS ONE | www.plosone.org region. These parameters were then used as constraints for further nonlinear curve fitting. A 0.95 confidence level target was set to constrain the quality of the curve fitting. The final fitting parameters were obtained when the value of x 2 was less than 0.05, and the parameters and errors for the parameters reached a convergent and steady state.

Western Blot Analysis of Ab Oligomers
Each Ab peptide was dissolved in phosphate buffer, pH 7.0, to a final concentration of 60 mM, and incubated for 0, 24, 48 and 72 hours at 37uC. Then the samples were separated by 4,20% gradient Tricine-SDS-PAGE and transferred onto a polyvinylidene difluoride (PVDF; PE, 0.22 mm) membrane for 2 hours. The PVDF membrane was blocked using 5% nonfat milk in phosphate-buffered saline (PBS) for 1 hour and probed with primary anti-mouse monoclonal antibody (6E10, Abcam, Cambridge, UK; 1:2000 dilution) overnight at 4uC. After probing with primary antibody, the PVDF membrane was washed three times with PBST and probed with anti-mouse secondary antibody (Sigma, Poole, UK; 1:6000 dilution). The labeled Ab peptides were detected using the western lighting chemiluminescence kit (GE, Pittsburgh, USA).

Fourier-transform Infrared Spectroscopy (FT-IR)
A FT-IR spectrometer (Jasco, FT-IR/4100) equipped with an accessory was used to study the conformation of Ab 40 during the aggregative process. One hundred ml of 200 mM Ab peptide solution was coated on a ZnSe crystal and kept dry overnight in a desiccator at room temperature. The spectra were recorded at a wavelength range of 1500-1800 cm 21 with 1 cm 21 intervals. The peak was identified from the first derivation of the IR spectrum in the amide I region, and the secondary structure was analyzed using Original 8.0 software.

Transmission Electron Microscope (TEM) Analysis
A TEM (JEM-2000 EXII, JEOL, Japan) with an accelerating voltage of 100 KeV was used to analyze the morphology of Ab peptides incubated at different time periods. Ten microliters of the Ab peptide samples used for the aggregation assay was placed onto a carbon-coated 200 mesh copper grid (Pelco, Ca, USA). Excess solution was wicked dry with tissue paper, and the sample was negatively stained with 5 ml of 2% uranyl acetate for 30 seconds. Excess solution was wicked dry, and the grid was allowed to air dry for further TEM analysis.

Cell Viability Assay
Human neuroblastoma SH-SY5Y cells [42] were cultured in DMEM/F12 (1:1) supplemented (Biochrom, Berlin, Germany) with 2 mM glutamine and 10% (v/v) heat-inactivated fetal bovine serum (Biowest, Nuaillé, France) at 37uC in a humidified atmosphere containing 5% CO 2 . Cell viability was measured using 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) (Sigma, Missouri, USA). All Ab peptides were prepared as a 1 mM stock solution in trifluoroethanol. Freshly prepared stock solution was then dried under N 2 gas and resuspended in PBS buffer, pH 7.0, to a final peptide concentration of 500 mM. The resulting solution was incubated at 25uC for 24 hours to obtain the Ab oligomers. This peptide solution was further diluted to 30 mM for the viability assay.
In a 96-well microtiter plate, 1610 4 cells were placed in each well and incubated in the absence or presence of Ab peptides in a total volume of 100 ml for 48 hours and 72 hours at 37uC in a humidified atmosphere containing 5% CO 2 before the viability assay. Ten microliters of MTT solution was added to each well and further incubated for another 4 hours at 37uC [12]. The absorbance was measured at a wavelength of 570 nm using a microplate reader (FlexStation 3).

Measurement of Intracellular ROS
ROS were determined using a 29,79-dichlorofluorescein diacetate (DCFH-DA)(Sigma, St. Louis, USA) assay in a flow cytometer (Beckman Coulter, Brea, CA, USA). Cells (1610 5 ) were incubated with 30 mM of Ab peptides for 48 hours. Before conducting flow cytometry, the cells were treated with 10 mM of DCFH-DA for 30 minutes at 37uC, followed by washing with PBS [12,13]. Data analyses were performed using program Kaluza software, version 1.2.

Statistical Analysis
Results were analyzed by the Student's t-Test using Original 8.0 software. Data are expressed as mean 6 standard deviation. A p value #0.05 was considered statistically significant.

Structural Stability of Ab 40 and Ab 40 Mutations
The aggregation of Ab is accompanied by a conformational conversion from either helix or random coil to b-sheet. Therefore, the conformational state of Ab plays a critical role in its aggregation ability and toxicity. We first applied CD spectroscopy to investigate the structural state of wild-type and Arctic-type Ab 40 . Figure 1 (A) and (C) shows the CD spectra of wild-type Ab 40 and Arctic-type Ab 40 (E22G). From the CD spectra, it can be seen that both Ab 40 and Arctic-type Ab 40 (E22G) peptides were converted from random coil to b-sheet after 48 hours. The bsheet conformation first appeared at 4 hours for Ab 40 (E22G), while for Ab 40 , the b-sheet structure was first observed later at 48 hours. Further analysis of b-sheet propensity (CD signal at 218 nm versus time) is shown in Figure 2  Effect of L17A and F19A on the Structural Stability of Ab 40

and its Mutations
Previously we replaced residues L17 and F19 with alanine, resulting in an increase in structural stability and reduction of toxicity [37]. We used the same strategy [37] to test the effect of L17A and F19A on structural stability and toxicity of the most toxic FAD mutant, Arctic-type Ab peptide [Ab 40

Aggregation Kinetics of the Ab Peptide
We further investigated the aggregation kinetics for Ab 40   Solid lines show the best fit curves using equation (1). The aggregation process of Ab40(L17A/F19A) as shown in Figure 3 (B) contained a lag phase through the whole incubation period, suggesting that the aggregation ability of Ab40(L17A/F19A) was reduced compared to that of wild-type Ab40. Furthermore, the Th-T intensity of Ab40(E22G) at the origin point was higher than that of others [ Figure 3 (C)], and the aggregation profile of Arctictype Ab40(E22G) was most likely a hyperbolic curve instead of the typical sigmoidal curve.
Under the same conditions, the aggregation profiles of Ab40 and Ab 40 (L17A/F19A/E22G) [ Figure 3 (B) and (D)] were the typical sigmoidal shape, although the aggregation profile of Ab40 lacked the nucleation stage. The results show that both Ab40 and Ab40(L17A/F19A/E22G) underwent typical nucleation-dependent aggregation processes. This demonstrates that, similar to Ab40(L17A/F19A), alanine replacement of L17 and F19 can reduce the aggregation rate of Arctic-type Ab40(E22G). In Table 1, the aggregation rate of Ab40(E22G) is 200-fold higher than that of Ab40 and Ab 40 (L17A/F19A/E22G), indicating that the aggregation of Arctic-type Ab40(E22G) was faster than other Ab 40 peptides and may have undergone a nucleation-dependent polymerization.

Molecular State of Ab Peptides
We further examined the molecular state of Abs using western blot analysis. Figure 4 (A) shows the blot of Arctic-type Ab40(E22G) and other Ab 40 peptides at day 0, day 1, day 2, and day 3. Consistent with the aggregation profiles, Ab40(E22G) aggregated into more severe polymorphologies than other Ab 40 peptides. Compared with Ab 40 , Ab 40 (L17A/F19A), and Ab 40 (L17A/F19A/E22G), Ab40(E22G) showed a smeared band at day 1 in the western blot. This phenomenon even becomes obvious at day 3 for Ab40(E22G). For Ab 40 (L17A/F19A), the aggregation profile was less obvious, even at day 2 when western blotting showed slight smearing. The aggregation profiles for Ab 40 and Ab 40 (L17A/F19A/E22G) were approximately similar.
The structural state of these Ab 40 peptides at day 3 was further confirmed using FT-IR spectra. The FT-IR spectrum area from 1400-1800 cm 21 were curve fitted to determine the status of possible b-sheet, random coil, and a-helix structures [43]. The spectrum area of b-sheet/aggregated is at 1610-1640 cm 21 and a-helix/unordered is at 1660-1685 cm 21 [14]. As shown in Figure 4 (B), the 1665 cm 21 peak of Ab40(E22G) showed a significant shift to a 1626 cm 21 peak, while for Ab 40 (L17A/ F19A), the 1665 cm 21 peak of Ab40(E22G) did not shift to this wavelength. The shift of the 1665 cm 21 peak to 1626 cm 21 for Ab 40 and Ab 40 (L17A/F19A/E22G) was less obvious than that of Ab40(E22G). Taken together, the results confirmed that Ab40(E22G) aggregates more than other Ab 40 peptides. These results also show that L17 and F19 mutants can reduce the rate of aggregation. Figure 5 shows the results of TEM for Ab40, Ab40(L17A/ F19A), Ab40(E22G), and Ab40(L17A/F19A/E22G) from day 1 to day 6. TEM morphologies of Ab40, Ab40(E22G), and Ab40(L17A/F19A/E22G) were all aggregated into fibrils. In contrast, no fibrils were observed for Ab40(L17A/F19A), even at day 6. The Arctic-type Ab40(E22G) was the fastest to form fibrils at day 2, Ab40(L17A/F19A/E22G) formed fibrils at day 4, while wild-type Ab40 did not form obvious fibrils until day 5. This is consistent with aggregation characterized by the other assays. The morphologic study clearly demonstrated that replacement of L17 and F19 with alanine reduced the aggregation ability of wild-type Ab40 and Arctic-type Ab40(E22G).

Cell Viability
As shown previously in this report, replacement of L17 and F19 with alanine reduced the aggregation ability and stabilized the conformation for not only wild-type Ab40, but also for Arctic-type Ab40(E22G). Because the conformational state and aggregation ability are linked with toxicity, the effects of L17 and F19 on toxicity induced by Arctic-type Ab40(E22G) was further investigated. Comparative cell viability of Ab40 peptides, including wild-type Ab40, Ab40(L17A/F19A), Arctic-type Ab40(E22G), and Ab40(L17A/F19A/E22G), was studied as shown in Figure 6. The results show the comparative cell viabilities after treatment with 30 mM of Ab40, Ab40(L17A/F19A), Arctic-type Ab40(E22G), and Ab40(L17A/F19A/E22G) peptides at 48 hours and 72 hours. Both at 48 hours and 72 hours, the toxicity induced by Ab40(E22G) was more severe than with other peptides. Similar to a previous study [37], Ab40(L17A/F19A) showed less toxicity than other peptides. However, toxicity induced by Ab40(L17A/ F19A/E22G) was approximately the same as Ab40, suggesting that alanine replacement of L17 and F19 reduced the cytotoxicity induced by Ab40(E22G), which is consistent with the aggregation rate and structural stability. Cell viability at 48 hours and 72 hours was on the order of Ab40(L17A/F19A) .Ab40(L17A/F19A/ E22G) = Ab40.Ab40(E22G).

Effect of Ab on ROS
Excessive ROS generation from dysfunctional or damaged mitochondria may trigger autophagy which removes the damaged mitochondria [44]. Flow cytometry shows that the rate of cell death was greatest for Arctic-type Ab40(E22G) [ Figure 7(A)], which was consistent with the cell viability assay. Flow cytometry also showed that treatment with Ab40(L17A/F19A/E22G) resulted in decreased ROS production (Figure 7(B), and that treatment with Arctic-type Ab40(E22G) resulted in higher ROS that caused cell death faster than Ab40(L17A/F19A/E22G). The same results were obtained for wild-type and Ab40(L17A/F19A) [ Figure 7(B)]. Together, the results indicated that L17A/F19A mutants could reduce ROS-induced oxidative DNA damage which could cause cell death.

Discussion
Aggregation and toxicity of Ab is highly correlated with its conformational states. Conformational change is a key step in the Ab-aggregation cascade, during which the conformation of Ab undergoes a structural conversion from either a-helix or random coil to b-sheet [15,16]. Numerous studies have shown that prevention of conformational change can reduce Ab aggregation. Furthermore, there have been many studies to elucidate the contribution of individual amino acid residues to helix stabilization [18,30]. The effects of amino acid sequence variations on the conformational change of Ab may provide useful information for further understanding the molecular mechanism of Ab aggregation.
Previous study has shown that L18A/F19A/F20A mutant can stabilize a-helix conformation [30,31,33,34]. Interestingly, our previous study identified that the amino acids L17 and F19 play a key role in the stabilization of the Ab40 conformation, leading to the reduction of its aggregation and cytotoxicity [37]. Therefore, we would further test the effects of L17 and F19 on the conformation, aggregation, and toxicity of other Ab40 mutants. We used the same methodologies as the previous study, constructing a rational mutation Ab40(L17A/F19A/E22G) of Arctic-type Ab40(E22G), and studied its conformation, aggregation ability, and cytotoxicity.
Although, unlike Ab40(L17A/F19A), where alanine replacement of L17 and F19 of wild-type Ab40 could completely inhibit the aggregation and toxicity, the rate of conformation conversion, aggregation ability, and toxicity of Ab40(L17A/F19A/E22G) were greatly reduced in comparison with those of Arctic-type Ab40(E22G), which is known to be the most toxic FAD species. Interestingly, regarding the rate of conformation conversion, aggregation ability, and induced neurotoxicity, the related properties of Ab40(L17A/F19A/E22G) were approximately the same as wild-type Ab40. Previously, several studies have shown that Ab aggregation can be blocked by helix inducing reagents, such as trifluoroethanol and sodium dodecyl sulfate [45]. Moreover, it has been shown that residues 16-23 have been predicted as the region of discordant helix [30][31][32][33][34], and any factors which stabilize the conformation of this discordant helix region could prevent the aggregation of Ab40. Our previous study identified 17-25 as the discordant helix region [36]. In another study we reported that instead of V18, F19, and F20, only L17 and F19 may be enough to stabilize the conformation of Ab40 and inhibit its aggregation, showing that residues L17/F19 are important residues that can significantly increase the helical potential [37]. In the present study, we further demonstrated that residues L17 and F19 play crucial roles in the stabilization of Ab40 structure. This is consistent with the hypothesis of discordant helix that residues 17-20 constitute the most sensible region for environmental changes, playing an important role in the conformational stabilization of not only Ab40, but also other mutants such as Ab40(E22G).
Beta-amyloid can cause cell death through the well-known process of oxidative stress. This process is an imbalance between the systemic manifestations of RNS and ROS [12,13]. Many neurodegenerative diseases have high levels of oxidative stress, especially those affected by ROS. Our present results showed that treatment with Arctic-type Ab40(E22G) results in more ROS than Ab40(L17A/F19A/E22G). We also showed that treatment with Ab40(L17A/F19A/E22G) can reduce ROS production to a level approximately equal to that produced by Ab40. These results again demonstrate that L17 and F19 are important residues in the reduction of cytotoxicity.
The amyloidogenic differences between Ab40(L17A/F19A) and Ab40(L17A/F19A/E22G) further indicate that the propensity of the discordant helix is highly dependent on the sequence, because the inhibition ability of L17A/F19A on aggregation and toxicity of Ab40(L17A/F19A/E22G) was weaker than that of Ab40(L17A/ F19A). This is obviously affected by the E22G mutation. Mutation of E22 with G22 may increase the propensity of the discordant helix of Arctic-type Ab40(E22G) in a stronger manner than that of wild-type Ab40. This provides a possible explanation why Arctictype Ab40(E22G) is more toxic than wild-type Arctic-type Ab40, because replacement of glutamate with glycine may increase the hydrophobicity. Therefore the helix propensity is less stable for the discordant helix region of Ab40(E22G), and as a consequence, Arctic-type Ab40(E22G) more easily aggregates and is more toxic than wild-type Ab40. In conclusion, our results confirm that the discordant helix region of Ab is located at residues 17-25, which is sensitive to environmental changes. Furthermore, our study further demonstrates that residues L17 and F19 of this discordant helix may play a crucial role in the stabilization of Ab conformation. Changes such as alanine replacement of L17 and F19 may stabilize the conformation, to diminish aggregation and reduce the neurotoxicity of Ab40 and even Ab40(E22G). Thus, our results provide important information about structural parameters involved in the Ab-aggregation cascade.