Potential implications of endogenous aldehydes in β-amyloid misfolding, oligomerization and fibrillogenesis

Potential implications of endogenous aldehydes in b-amyloidmisfolding, oligomerization and fibrillogenesis

Kun Chen,* Jason Maley� and Peter H. Yu*

*Neuropsychiatry Research Unit, Department of Psychiatry; and �Saskatchewan Structural Sciences Centre, University of

Saskatchewan, Saskatoon, Saskatchewan, Canada

Abstract

Aldehydes are capable of inducing protein cross-linkage. An

increase in aldehydes has been found in Alzheimer’s disease.

Formaldehyde and methylglyoxal are produced via deamina-

tion of, respectively, methylamine and aminoacetone cata-

lyzed by semicarbazide-sensitive amine oxidase (SSAO, EC

1.4.3.6. The enzyme is located on the outer surface of the

vasculature, where amyloidosis is often initiated. A high SSAO

level has been identified as a risk factor for vascular disorders.

Serum SSAO activity has been found to be increased in Alz-

heimer’s patients. Malondialdehyde and 4-hydroxynonenal

are derived from lipid peroxidation under oxidative stress,

which is also associated with Alzheimer’s disease. Aldehydes

may potentially play roles in b-amyloid aggregation related to

the pathology of Alzheimer’s disease. In the present study,

thioflavin-T fluorometry, dynamic light scattering, circular

dichroism spectroscopy and atomic force microscopy were

employed to reveal the effect of endogenous aldehydes on

b-amyloid at different stages, i.e. b-sheet formation, oligo-

merization and fibrillogenesis. Formaldehyde, methylglyoxal

and malondialdehyde and, to a lesser extent, 4-hydroxynon-

enal are not only capable of enhancing the rate of formation of

b-amyloid b-sheets, oligomers and protofibrils but also of

increasing the size of the aggregates. The possible relevance

to Alzheimer’s disease of the effects of these aldehydes on

b-amyloid deposition is discussed.

Keywords: Alzheimer’s disease, b-amyloid oligomerization,

atomic force microscopy, formaldehyde, methylglyoxal,

semicarbazide-sensitive amine oxidase.

J. Neurochem. (2006) 99, 1413–1424.

Extracellular b-amyloid (Ab) deposition in the brain is amajor hallmark of Alzheimer’s disease (reviewed in Selkoe2002; Tanzi & Bertram 2005). Ab peptides, comprisingheterogeneously truncated fragments from amyloid precursorprotein, readily form b-sheets, which assemble to oligomers,protofibrils, and fibrils, and subsequently coaggregate withother proteins, such as lipoproteins, to form senile plaques(Ma et al. 1994; Wisniewski et al. 1994; Cotman et al. 2000;Taylor et al. 2003). Ab is toxic and capable of triggering acascade of events including induction of oxidative stress andinflammatory responses leading to neurodegeneration (Looet al. 1993; Morishima et al. 2001). Recently, Ab oligomers(5–10 Ab molecules) have been shown to be the mostcytotoxic (Kayed et al. 2003; Klein et al. 2004). The roles ofthe advanced Ab aggregates, i.e. fibrils and plaques, in theneurodegeneration of Alzheimer’s disease have not beenclearly defined.

A large body of literature suggests that vasculardisorders are involved in the pathogenesis of vasculardementia and Alzheimer’s disease (see reviews by Jellinger2002; Kalaria and Ballard. 1999). Indeed, a variety of

types of microvascular changes have been observed inaging and in Alzheimer’s disease brains (Farkas & Luiten.2001). Ab depositions were found closely associated withthe degenerated cerebral vasculature (Coria et al. 1988;Kawai et al. 1993). Ab isolated from the vascular tissuescontains significantly less isomerized and racemizedaspartyl residues than those Ab in the neuritic plaques,suggesting that the vascular amyloid is ‘younger’ (Roheret al. 1993). The cerebral vascular basement membrane haseven been considered to play an important role in

Received April 12, 2006; revised manuscript received July 16, 2006;accepted July 17, 2006.Address correspondence and reprint requests to Peter H. Yu, Neuro-

psychiatry Research Unit, University of Saskatchewan, Saskatoon,Saskatchewan, S7N 5E4 Canada. E-mail: [email protected] used: Ab, b-amyloid; AFM, atomic force microscope;

CD, circular dichroism spectroscopy; DLS, dynamic light scattering;FA, formaldehyde; HFIP, 1,1,1,3,3,3-hexafluoro-2-propanol; HNE,4-hydroxy-2-nonenal; MG, methylglyoxal; MDA, malondialdehyde;PBS, phosphate-buffered saline; SSAO, semicarbazide-sensitive amineoxidase; ThT, thioflavin-T fluorescence assay.

Journal of Neurochemistry, 2006, 99, 1413–1424 doi:10.1111/j.1471-4159.2006.04181.x

� 2006 The AuthorsJournal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 1413–1424 1413

amyloidosis and to act as a nidus for plaque formation(Perlmutter and Chui. 1990; Inoue 2001). Alzheimer’sdisease may be a result of a series of cerebral microin-farctions (Kalaria and Ballard 1999). Interestingly, Alzhei-mer’s disease shares common pathological features andrisk factors with other vascular disorders, such as athero-sclerosis and diabetes mellitus (Messier 2003). Cholesterol,an important risk factor for vascular disorder, is alsorelated to the pathophysiology of Alzheimer’s disease(Wolozin 2004).

Semicarbazide-sensitive amine oxidase (SSAO), anenzyme bound on the outer membrane of vascular smoothmuscle and endothelial cells (Trevethick et al. 1981), isprobably involved in the pathogenesis of vascular disordersassociated with atherosclerosis, diabetic complications, etc.(Yu and Zuo 1993; Yu and Deng 1998). It has beenrecognized as a potential risk factor for vascular disorders(Boomsma et al. 2000; Yu et al. 2003). SSAO catalyzes theproduction of toxic formaldehyde and methylglyoxal viadeamination of methylamine and aminoacetone, respectively.

SSAO

CH3NH2 þ O2 þ H2O! HCHOþ H2O2 þ NH3

CH3COCH2NH2þO2þH2O!CH3COCHOþH2O2þNH3

SSAO is also known as an adhesion molecule (VAP-1)regulating lymphocyte trafficking and involved in the processof inflammation (Salmi & Jalkanan 2001). Formaldehyde,derived from an SSAO-mediated reaction, has been shown tocross-link with proteins in vivo (Gubisne-Haberle et al.2004). The enzyme was found to be over-expressed on thesurface of meninges in the Alzheimer’s brain (Ferrer et al.2002) and its activity is apparently elevated in the serum ofpatients of Alzheimer’s disease (del Mar Hernandez et al.2005). The discrete localization of this enzyme on the outersurface of the vasculature as well as its generation of reactivealdehydes and involvement in inflammatory process suggestthat it may play a role in vascular-related disorders such asAlzheimer’s disease (Yu 2001).

Reactive species of carbonyl metabolites are generatedfrom various pathways. Polyunsaturated fatty acids followingfree radical attacks under oxidative stress conditions canproduce lipid peroxides, which would be fragmented tonumerous small carbonyl compounds such as malondialde-hyde, 4-hydroxy-2-nonenal, acrolein, etc. (Esterbauer et al.1991; Leonarduzzi et al. 2000). These carbonyls are alsoextremely reactive and capable of interacting with proteinsand inducing aggregation. Oxidative stress and lipid perox-idation are associated with aging and Alzheimer’s disease(Subbarao et al. 1990). Increased levels of 4-hydroxy-2-nonenal and malondialdehyde have been detected in plasmafrom Alzheimer’s disease patients (McGrath et al. 2001; Dibet al. 2002). Cross-linkage of these carbonyl compounds

with proteins has even been shown histochemically in theamyloid plaques (Kamalvand and Ali-Khan 2004).

Methylglyoxal, despite its production via deamination ofaminoacetone, could be derived from several pathways andsignificantly contributes to advanced protein glycation indiabetes (Thornalley 2002). Diabetes mellitus is a well-known risk factor for Alzheimer’s disease (Luchsinger et al.2001). Interestingly, CSF obtained from Alzheimer’s diseasepatients exhibits significantly higher levels of methylglyoxalthan CSF in control subjects and has been implicated inamyloidosis (Kuhla et al. 2005).

In the present study we investigated the interaction ofreactive aldehydes with Ab. Four aldehydes generated fromSSAO-mediated deamination and oxidative stress associatedto Alzheimer’s disease were chosen for the study. Differentmethodologies, i.e. thioflavin-T fluorescence assay (ThT),dynamic light scattering (DLS), circular dichroism spectro-scopy (CD) and atomic force microscope (AFM), wereemployed. The effects of these aldehydes on Ab b-sheetformation, oligomerization and fibrillogenesis in vitro wereassessed.

Materials and methods

Materials

Ab1)40 was purchased from BioSource (Camarillo, CA, USA).

1,1,1,3,3,3-Hexafluoro-2-propanol (HFIP) and methylglyoxal were

from Sigma-Aldrich (St. Louis, MO, USA). Formaldehyde, mal-

ondialdehyde and 4-hydroxynonenal (4-hydroxy-2-nonenal) were

obtained from BDH Inc. (Toronto, ON, Canada), Fluka (Buchs,

Switzerland) and Oxis (Portland, OR, USA), respectively. 96-well

microfluor black plates were purchased from Dynex Technologies

Inc. (Chantilly, VA, USA). Specific anti-Ab antibodies were

obtained from Biosource.

Preparation of seed-free Ab1)40

To ensure a seed-free monomer solution of Ab1)40 the peptide was

pretreated immediately before each experiment. Ab1)40 was

dissolved in 100% HFIP (1 mg/mL) and incubated in a water bath

sonicator at 4�C for 2 h. The HFIP was removed under a gentle

stream of nitrogen. The treated Ab1)40 crystals were dissolved in

nanopure water and used immediately. The purity of Ab monomers

free of oligomers was ensured by testing using an oligomer specific

antibody. The final Ab concentration was determined using

Bradford protein assay (purchased from Bio-Rad Laboratories).

Interactions of Ab1)40 with aldehydes

Freshly prepared seed-free Ab1)40 (1 mg/mL) was incubated in the

presence or absence of various concentrations (ranging from 1 lM to

10 mM) of formaldehyde, methylglyoxal, malondialdehyde or

4-hydroxy-2-nonenal in phosphate-buffered saline (PBS) (pH 7.4,

20 mM) in 0.2 mL Eppendorf tubes at 37�C without shaking or

pipetting. For the AFM imaging experiment, to overcome the

problem of salt crystallization, ammonia/formic acid (20 mM,

pH 7.4) volatile buffer was used instead of PBS buffer.

1414 K. Chen et al.

Journal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 1413–1424� 2006 The Authors

ThT fluorometry

ThT fluorescence assays reveal the early stage of Ab1)40 aggrega-

tion, i.e. b-sheet formation. Ab (final concentration at 10 lg/mL)

was incubated in the presence or absence of aldehydes in a total

reaction mixture of 200 lL (in 50 mM glycine-NaOH buffer,

pH 9.0). Aliquots of the reaction solution were transferred to the

black microfluor plates at different time intervals for fluorescence

readings. Fluorescence was monitored at an excitation wavelength

of 450 nm and an emission wavelength of 482 nm using a Spectra

Max Gemini XS fluorescence reader (Molecular Devices, Sunny-

vale, CA, USA).

DLS

DLS measurements were carried out on a Dyna-Pro 99 MS800

instrument (Protein Solutions, Lakewood, NJ, USA) at 25�C using a

824.8-nm (55 mW) Anodisk with a fixed catering angle of 90�. Theprotein solution was filtered with a 0.2-lm Anodisk filter, and

placed in a 12-lL cuvette (b ¼ 1.5 nm). Data acquisition time was

5 s and S/N Threshold was 2. The size distributions were analyzed

with Dynamic V5.26.60 (Protein Solutions). The count rates (signal

intensity), which are proportional to the amount of photons reflected

from the sample solution, were assessed at different time points of

the incubations.

CD spectroscopy

CD measurements were carried out on a PiStar-180 spectrometer

(Applied Photophysics, Surrey, UK) at room temperature using a

0.1-cm cell optical pathlength cuvette (Hellma, 106-OS). The

sample solutions were scanned from 260 to 190 nm in 0.5-nm steps

at a scan rate of 10 nm per min and with 4 nm bandwidth. CD

spectra of the PBS buffer containing the appropriate aldehydes were

obtained and subtracted from the protein solutions. Protein

secondary structure analysis was conducted using CDNN software

(CD Spectra Deconvolution v 2.1) (Bohm et al. 1992).

AFM imaging

For AFM imaging under ambient conditions, 1-lL aliquots of the

samples previously described above were placed on freshly cleaved

mica (Structure Probe Inc., West Chester, PA, USA) for 1 min until

dry. Solutions containing malondialdehyde and 4-hydroxy-2-none-

nal were also imaged under wet conditions, where 10-lL aliquots

were placed onto freshly cleaved mica in a sample well for 2 min,

and subsequently imaged in 250 lL PBS (20 mM, pH 7.4). AFM

measurements were carried out on a Pico-SPM (Molecular Imaging

Inc., Tempe, AZ, USA) with an AFMM scanner operating in MAC

mode. Its specifications include a force constant of approximately

1.2–5.5 N/m, and a resonant frequency of approximately 60–

90 kHz under ambient conditions. All measurements were taken

with the ratio of the set-point oscillation amplitude to free air

oscillation amplitude of 0.80. In addition, all measurements were

performed with the instrument mounted in a vibration isolation

system. The scan rate was 1–2 lines/s (256 pixel per line) for all

images. At least five positions on the mica for each sample were

randomly chosen for scan and imaging. Each image was conducted

in two opposite directions simultaneously and the final image was

averaged. Size distribution within scanning area and the final

3-dimensional image were calculated and generated by software

from Visual SPM Molecular Imaging Inc.

Statistics

The results were assessed using one-way analysis of variance

(ANOVA) followed by multiple comparisons (Newman-Keuls). The

null hypothesis used for all analyses was that the factor has no

influence on the measured variable and significance was accepted at

> 95% confidence level.

Results

Effect of aldehydes on b-sheet formation of AbAs can be seen in Fig. 1, ThT fluorometry reveals that bothformaldehyde and malondialdehyde significantly enhance theformation of b-sheets. The increase is in a time- andconcentration-dependent manner. After prolonged incuba-tion, i.e. around day 5, the fluorescence intensity reached aplateau and even began to reduce slightly thereafter.

The effects of other aldehydes of interest on b-sheetformation of Ab1)40 were also assessed. Methylglyoxal hasbeen shown equally potent in enhancing the Ab1)40 b-sheetformation (results not shown). However, HNE only exhibitsa small but significant increase under the same experimentalconditions.

Molecular assembly of Ab assessed by DLS

DLS data can provide useful information regarding thedistribution of molecular sizes of polymerized Ab1)40 insolutions. As can be seen in Fig. 2, aldehydes clearly induceshifts of the molecular sizes in the early phases ofpolymerization. After incubation for 24 h, untreated Abforms oligomers with sizes around 10 nm and protofibrilsabout 100 nm. In the presence of formaldehyde the moleculesizes increase to about 50 nm and 200 nm.

Count rate derived from the DLS experiment reflects thepredominant molecular sizes in a solution (Chayen et al.2004). As shown in Fig. 3, all aldehydes tested significantlyincreased the count rate in comparison with the controlgroup. However, the count rates also reached plateaus after48 h, when protofibrils began to assembly into fibrils. In thisstudy a significantly lower concentration of formaldehydewas used. It probably became a limiting factor for themolecular assemble. These results are consistent with earlierobservations, namely, these aldehydes significantly enhancedAb assembly.

The above results demonstrated that aldehydes are capableof inducing the initiation of Ab aggregation. We furtherexamined whether aldehydes affect Ab aggregation in a moreadvanced stage. Ab was preincubated for 48 h for theformation of aggregation. Aldehydes were then added tothese 2-day aged Ab solutions. As can be seen in Fig. 4,formaldehyde can further increase the count rate, whichreached the plateau, but the difference remained in allmeasurements in a concentration-dependent manner.

Endogenous aldehydes and b-amyloid aggregation 1415

� 2006 The AuthorsJournal Compilation � 2006 International Society for Neurochemistry, J. Neurochem. (2006) 99, 1413–1424

CD spectroscopy

CD spectra can provide information not only on b-sheets butalso a-helix and random coils in a protein solution. As can beseen in Fig. 5, formaldehyde affects the CD spectra of Ab ina concentration-dependent manner. The negative peak at200 nm of the control group (representing a mixture ofa-helices and random coils) was gradually shifted to around218 nm (the peak of b-sheet structure) following formalde-hyde treatment. Formaldehyde reduced the amplitude of thenegative peak. This suggests that the b-sheet content isincreased. Formaldehyde itself exhibited no effect on thebackground. CDNN software was used to calculate thepercentage of each secondary structure in each sample.Overall, the formaldehyde treatment resulted in an increaseof antiparallel and parallel b-sheets as well as a decrease ina-helices and random coils compared with the control group.This result further confirmed the observations of ThT

spectroscopy and DLS. Methylglyoxal and malondialdehydehad a similar effect to formaldehyde. 4-Hydroxy-2-nonenalhad a limited effect in comparison with other aldehydes(results not shown).

AFM imaging

The above spectrophotometric techniques merely revealthe effects of aldehydes on the conformation and size of theAb1)40 aggregates. AFM was employed to examine themorphological changes of the Ab1)40 oligomers andprotofibrils following incubation with aldehydes. As canbe seen in Fig. 6, formaldehyde alters the size of Ab1)40oligomers, as revealed by the dry AFM method. Theaverage Ab1)40 oligomer sizes of the untreated Ab1)40,based on the heights of the AFM scans, is about 5 nm andincreases to 6–7 nm in the presence of formaldehyde 6 hafter incubation.

Fig. 1 Kinetic effect of formaldehyde (FA)

and malondialdehyde (MDA) on Ab1)40

b-sheet formation by ThT fluorescence.

Ab1)40 was incubated in the presence or

absence of various concentrations of form-

aldehyde (upper panel) and malondialde-

hyde (lower panel) for a period of up to

6 days. The fluorescence was measured at

different time points. The measurement was

carried out in a total of 200 lL solution

(2 mM ThT in 50 mM glycine-NaOH buffer,

pH 9.0) containing Ab (10 lg/mL). kex ¼450 nm, kem ¼ 482 nm. The background

fluorescence was subtracted. Data repre-

sent means ± SD of a representative

experiment out of three. *p < 0.05, com-

pared to corresponding control values.

1416 K. Chen et al.


Similar to formaldehyde, methylglyoxal is capable ofincreasing the rate of formation and size of Ab1)40 oligomersand protofibrils. Figure 7 shows the effect of methylglyoxalon Ab1)40 oligomerization using a 3-dimensional presenta-tion. The average Ab1)40 oligomer size, based on the heights,in the scan areas for the controls and the methylglyoxal-treated Ab1)40 is about 5 nm and 7–8 nm, respectively, 6 hafter incubation (Fig. 7a). After prolonged incubation, i.e.48 h, the average size (or height) increases to 10 nm and20 nm, respectively, for the controls and the treated Ab1)40(Fig. 7b).

4-Hydroxy-2-nonenal by itself would crystallize at drynessand therefore was not suitable for the dry AFM scan. A wetAFM method was adopted. The appearances of the imagesobtained are somewhat different from the images derivedfrom the above dry method. Although 4-hydroxy-2-nonenal

increases the size of the Ab oligomers, the effect on the rateof aggregation appears to be less pronounced in comparisonwith those induced by formaldehyde and methylglyoxal(Fig. 8).

Discussion

Proteins fold to form functional tertiary configurations. Theamino acid sequence determines how proteins fold (Anfinsen1973). Ab peptides not only fold but also aggregate amongthemselves and subsequently with other proteins and becomeinvolved in Alzheimer’s disease pathology (reviewed inSelkoe 2001; Tanzi & Bertram 2005). Although it is unclearwhether this process is a metabolic accident, the aggregateproducts, such as Ab oligomers (often comprising 5–16 Abmonomers), have been found toxic towards neurons (Walsh

Fig. 2 Effect of aldehydes on the size dis-

tribution of Ab1)40 assessed by DLS. (a) Ab

(1 mg/mL) alone; (b) Ab plus formaldehyde

(5 lM); (c) Ab plus methylglyoxal (MG)

(5 lM); (d) Ab plus malondialdehyde (5 lM);

(e) Ab plus 4-hydroxy-2-nonenal (HNE)

(5 lM). The incubation was carried out at

37�C in 20 mM PBS (pH 7.4) without stirring

for 24 h. Each RH distribution was normal-

ized to 100%. Aggregated Ab accounts for

the majority of the scattering intensities.

The scattering intensity from the buffer

(RH < 0.2 nm) is not shown. The magni-

tudes of intensities of each experiment were

independent and therefore not comparable.

Fig. 3 Effect of aldehydes on DLS count

rate with respect to polymerization of Ab.

Ab1)40 (1 mg/mL) was incubated in the

presence or absence of different aldehydes

(5 lM) at 37�C in PBS buffer (20 mM,

pH 7.4) without stirring. Count rates of PBS

and aldehydes were subtracted from cor-

responding Ab count rate for comparison.

Data represent means ± SD of a represen-

tative experiment out of three. *p < 0.05,

compared to corresponding control values.



et al. 2002; Demuro et al. 2005). At this time it is unclearhow harmful these toxic dumps are to the neuronal network.Although Ab deposition plays a critical role in the pathologyof Alzheimer’s disease and the peptide readily aggregates byitself, it is unclear why or how such aggregation is initiatedand advanced in the Alzheimer’s brain. There is littleevidence to indicate that over-production of Ab is respon-sible for the majority of cases of late-onset Alzheimer’sdisease (Selkoe 2001). The increase in Ab deposition may bedue to impaired Ab clearance (Zlokovic 2004), failure ofdegradation (Mukherjee and Hersh 2002), or perhaps factors

enhancing aggregation (Zhang et al. 2004). Interestingly,CSF from Alzheimer’s disease patients has been shown to becapable of enhancing Ab fibrillogenesis (Ono et al. 2005).

Factors such as pH, metal ions, cholesterol, reactiveoxygen species, and ApoE4 have been shown to influencethe Ab aggregation process. Indeed, ApoE4 is well known tobe deposited in the Ab plaques and is a genetic risk factor forAlzheimer’s disease (Roses et al. 1994). Cholesterol isanother risk factor and plays an important role in thepathogenesis of Alzheimer’s disease (Wolozin 2004). React-ive carbonyl metabolites generated via cholesterol ozonolysisduring inflammation (Wentworth et al. 2003) have beenshown to be capable of covalently modifying and acceler-ating Ab aggregation (Zhang et al. 2004).

Formaldehyde, methylglyoxal, malondialdehyde and4-hydroxy-2-nonenal were chosen in the present investiga-tion. These aldehydes are derived from different pathwaysand represent products from oxidative deamination, lipidperoxidation and hyperglycemia, which are all known to berisk factors for Alzheimer’s disease. It is also interesting tonote that formaldehyde is a potent inflammatory agent (Tao& Johns 2002; Damas & Liegeois 1999), and inflammation iswell known to be involved in Alzheimer’s disease (McGeer& McGeer 2001).

An increase in 4-hydroxy-2-nonenal was found inAlzheimer’s disease (Sayre et al. 1997). However, 4-hyd-roxy-2-nonenal protein adducts are localized to neuronalcytoplasm and neurofibrillary tangles but not amyloidplaques (Montine et al. 1997). This does not explain whyAb deposition takes place primarily extracellularly orassociated with cerebral vasculature.

ThT fluorometry only reveals the formation of b-sheetstructures, which takes place in the initial stage in Ab

Fig. 4 Effect of formaldehyde on 48-h Ab

DLS count rate. Ab of control group as well

as other groups were preincubated alone

for 48 h at 37�C in PBS (20 mM, pH 7.4).

Formaldehyde was added to different

groups at a final concentration of 1, 10 and

50 lM, respectively, and further incubated

for up to 48 h. Data represent the

means ± SD of a representative experi-

ment out of three. *p < 0.05, compared to

corresponding control values.

Fig. 5 Effect of formaldehyde on Ab CD spectrum. Ab (1 mg/mL) was

incubated in the absence and presence of different concentrations of

formaldehyde for 12 h in PCR tubes at room temperature (20�C)

without stirring. Samples were degassed before measurement. Each

sample was measured five times and an average was obtained.

Corresponding backgrounds with respect to formaldehyde in PBS

were subtracted for final plotting and comparison. [h]MRW ¼ mean

residue weight ellipticity.

1418 K. Chen et al.


aggregation. Prolonged incubation may cause advancedaggregation, i.e. to Ab protofibrils and fibrils, by which timeb-sheets would probably no longer be detectable by the ThTmethod. This observation is consistent with a previous report(Stanyer et al. 2004). The effect of aldehydes on b-sheetformation has been further confirmed by CD spectra. ThT orCD methods are suitable for the measurement of b-sheets butnot for advanced Ab assembly.

The DLS method, which can measure molecular sizedistributions, was therefore employed. The molecularsizes are estimated via an indirect measurement, i.e. thehydrodynamic radius (RH), which was calculated based onthe Stokes-Einstein equation. The shift of relative molecularsizes caused by formaldehyde is consistent with its effect onAb deposition assessed by other methods. Methylglyoxal and4-hydroxy-2-nonenal are also capable of increasing the size

Control (6 h) Formaldehyde (10 mM) (6 h)

Formaldehyde (10 mM) (24 h)

Formaldehyde (10 mM) (72 h)Control (72 h)

Control (24 h)

Fig. 6 AFM imaging of the effect of for-

maldehyde on Ab1)40 aggregation. Ab

(1 mg/mL) was incubated in the absence or

presence of formaldehyde. At each time

point, aliquots of samples were diluted 100

times before imaging. A 1-lL diluted sample

was imaged on freshly cleaved mica. Five

positions on a mica were randomly picked

for each sample. Upper row: incubation for

48 h; middle row: incubation for 72 h; lower

row: incubation for 5 days.



Methylglyoxal (10 µM) (6 h)Control (6 h)

(a)

Methylglyoxal (10 µM) (48 h)Control (48 h)

(b)

Fig. 7 Three-dimensional AFM images

showing the effect of methylglyoxal on

Ab1)40 aggregation. The condition of treat-

ment is described in legend in Fig. 6. Upper

row: incubation time 6 h; lower row: incu-

bation time 48 h.

1420 K. Chen et al.


of Ab aggregates. DLS assessment is designed for globularproteins and thus is suitable for analysis of Ab oligomeri-zation. Formaldehyde and malondialdehyde exhibited asimilar effect on b-sheet formation in the ThT assay, yetmalondialdehyde appears to be more effective, as shown inthe DLS experiment. Such an effect suggests that in additionto an increase in b-sheet formation, malondialdehyde may bedifferent in enhancing inter-molecule interactions than otheraldehydes. This observation is consistent with an earlierreport (Esterbauer et al. 1991).

The AFM imaging method not only reveals the effect ofaldehydes on the morphological changes of the oligomersand protofibrils to fibrils, but also estimates their rates ofreaction and the aggregate sizes based on the heights of thescans. As Ab oligomers are water soluble, rinsing the micawith buffer during the preparation process would removethe oligomers attached on the mica, affecting the imaging.By omitting the rinsing, the salts in the buffer wouldcrystallize, which interferes with the AFM scan. Thisproblem was overcome by using a volatile buffer andbringing samples to dryness without rinsing. Subsequently,a wet AFM method was applied. We used this wet methodfor examination of the effects of malondialdehyde and4-hydroxy-2-nonenal because these aldehydes are in salt

form and thus form crystals at dryness. As indicated in theresults, aldehydes not only increase the number but also thesize of the oligomers.

Control (12 h)

Control (12 h) HNE (10 µM) (12 h)

HNE (10 µM) (12 h)

Fig. 8 Effect of 4-hydroxy-2-nonenal and

malondialdehyde on Ab1)40 aggregation

using the wet AFM method of imaging.

Ab1)40 (1 mg/mL) was incubated in the

absence or presence of 4-hydroxy-2-none-

nal (10 lM) for 12 h. Size distribution was

statistically calculated based on heights.



Formaldehyde reacts with the free amino group of lysineor arginine and forms Schiff bases. As can be seen in thescheme below, two adjacent Schiff bases would crosslink toeach other and form a methylene bridge.

Formaldehyde, methylglyoxal and malondialdehyde exhi-bit a similar effect on Ab oligomerization. The effect of4-hydroxy-2-nonenal, however, is limited under the presentconditions. The longer aliphatic chain of 4-hydroxy-2-nonenal may somewhat hinder the kinetic property ofinteracting with proteins. This limited effect of 4-hydroxy-2-nonenal on Ab oligomerization is consistent with an earlierreport (Stanyer et al. 2002).

Formaldehyde generated via SSAO-mediated deaminationwas shown to crosslink with proteins in vivo (Yu and Zuo1996). Lysine and arginine are the primary sites forformaldehyde interaction (Gubisne-Haberle et al. 2004).We confirmed that lysine residues of Ab are also subject tointeraction with formaldehyde (result not shown). Abpossesses two lysines (Lys16 and Lys28) and an arginine(Arg5) residue and therefore readily interacts with aldehydes.Between Lys16 and Lys28 is a lipophilic domain, whichinteracts and folds with another lipophilic domain at theN-terminal of Ab (Petkova et al. 2002). Alteration of thelysine residues by formaldehyde would change the lipophi-licity and flexibility of the lipophilic domain of Ab infavor of stabilizing the folding. Subsequently, it may formintramolecular and intermolecular methylene bridges whichstabilize the aggregation. Although a lysine residue is themost favorable target for formaldehyde, it can also interactwith other amino acid residues of proteins in a much morecomplicated manner, which depends on the ambient condi-tions (Metz et al. 2004). The concentrations of aldehydesin vivo, which are generated via deamination of short chainaliphatic amines, lipid peroxidation or glucose metabolism,are likely insufficient to cause massive protein cross-linkageor acute cytotoxicity. The concentrations of Ab in the brainare not well established. Its distribution is probably uneven.The cell membrane surface, where Ab will be subject totranslocation and clearance, might accumulate more Ab. Thelocal Ab concentrations could be much higher. Alsoinformation on the concentrations of formaldehyde andmethylglyoxal in the brain is limited. Malondialdehyde and4-hydroxy-2-nonenal concentrations can be as high as 0.8and 100 lM, respectively (Benedetti et al. 1984; Tomitaet al. 1990). However, its interaction with adjacent proteinsand effect on protein misfolding can proceed in a chronicprogressive, irreversible and accumulative manner followinga pseudo first order kinetic mode. Indeed, amyloidosis is avery slow process occurring in chronic diseases such asatherosclerosis, diabetes mellitus, Alzheimer’s disease, etc.

The present study provides evidence that reactive alde-hydes are capable of interacting with Ab, which canpotentially affect its nucleation as well as the subsequentfibrillogenesis. The induction of Ab aggregation by alde-

hydes, may explain why amyloid plaques occur extracellu-larly and particularly on the outer surface of the vascularcompartment. SSAO is discretely located on the outer surfaceof vascular smooth muscle and endothelial cells. As a matterof fact, in the brain the cerebral vasculature is the onlylocation where SSAO is present (Zuo and Yu 1993).Therefore, formaldehyde and methylglyoxal, produced fol-lowing deamination of methylamine and aminoacetone,readily form adducts in situ with adjacent proteins, includingAb, associated to the cerebral vasculature. Intracellularaldehydes formed via different pathways may be quicklymetabolized by aldehyde dehydrogenase (with sufficientNAD cofactor) before reacting with other proteins. SSAO-mediated production of aldehydes on the cell surface wouldnot be readily metabolized. The compartments where alde-hydes are formed are therefore crucially important to decidewhether or not they form long-lasting protein adducts.

In conclusion, aldehydes, which are derived from oxida-tive deamination of short aliphatic amines, lipid peroxidationand glucose metabolism, are capable of enhancing Abb-sheet formation, oligomerization and fibrillogenesis andadvanced stable aggregates with other proteins. An increasein these carbonyl compounds may potentially play animportant role in protein misfolding and amyloidosis inchronic diseases including Alzheimer’s disease. Currentinformation supports the hypothesis that an increase incarbonyl compounds may be a risk factor for Alzheimer’sdisease.

Acknowledgements

The authors wish to thank the Canadian Institutes of

Health Research, the Alzheimer’s Society of Saskatchewan,

and the Saskatchewan Health Research Foundation for their

continuing financial support. The authors have no conflicting

financial interests.

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Potential implications of endogenous aldehydes in β-amyloid misfolding, oligomerization and fibrillogenesis