Oligomerization and neurotoxicity of the amyloid ADan peptide

implicated in familial Danish dementia

Gillian Gibson,* Nicola Gunasekera,* Maria Lee,* Victor Lelyveld,* Omar M. A. El-Agnaf,*

Andrew Wright� and Brian Austen*

*Neurodegeneration Unit, Department of Basic Medical Sciences, St George’s Hospital Medical School, London, UK

�PP19, MRIC, Northeast Wales Institute, Wrexham, UK

Abstract

Familial Danish dementia (FDD) is a rare neurodegenerative

disorder, which is pathologically characterized by widespread

cerebral amyloid angiopathy, parenchymal protein deposits

and neurofibrillary degeneration. FDD is associated with

mutation in the BRI gene. In FDD a decamer duplication

between codons 265 and 266 in the 3¢ region of the BRI gene

originates an amyloid peptide named ADan, 11 residues longer

than the wild-type peptide produced from the normal BRI gene.

ADan deposits have been found widely distributed in the CNS

of FDD cases. The deposits of ADan are predominantly non-

fibrillar aggregates. We show here that synthetic ADan forms

oligomers in vitro, seen by Tricine–PAGE and gel filtration, and

higher aggregates, which are seen by atomic force spectros-

copy and electron microscopy as carrot-shaped objects that

bunch together. Here we report that oligomeric ADan is toxic to

neuronal cell lines. We find that the soluble non-fibrillar oligo-

meric species of both the reduced and oxidized forms of ADan

are toxic. These results support the idea that the non-fibrillar

soluble aggregates are the pathogenic species, which may

play a central role in the pathogenesis of FDD, and imply that

similar mechanism may also be involved in other neurode-

generative diseases associated with amyloid deposits.

Keywords: ADan, amyloid, BRI, Danish dementia, neuro-

toxicity, oligomers.

J. Neurochem. (2004) 88, 281–290.

Familial British dementia (FBD) and familial Danish

dementia (FDD) are rare autosomal dominant neurodegen-

erative disorders that share features of Alzheimer’s disease

(AD), including a pathology of amyloid plaques surrounded

by astrocytes and microglia, neurofibrillary tangles, neuronal

loss and progressive dementia (Vidal et al. 1999, 2000). FBD

is clinically characterized by the onset of dementia in the

fourth decade, progressive spastic tetraparesis and cerebellar

ataxia. FDD also involves cataract formation and auditory

loss. FBD and FDD are distinguished from AD and other

dementing disorders by plaque deposition in the cerebellum

and the accompanying cerebellar ataxia. These conditions

were previously reported as atypical forms of familial AD

(Worster-Drought et al. 1933; Aikawa et al. 1985; Plant

et al. 1990). Histological studies show that some of the

amyloid deposits in FBD patients exhibit yellow–green

birefringence under polarized light after staining with Congo

red, indicating the presence of amyloid-like fibrils with

crossed b-sheet structure (Plant et al. 1990; Holton et al.

2002). Immunohistochemical and biochemical analysis of

plaques and vascular amyloid of FBD and FDD brains

revealed that �4 kDa peptides named ABri and ADan,

respectively, are the main components of the insoluble

amyloid deposits likely to be involved in pathogenesis (Vidal

et al. 1999, 2000).

The ABri and ADan peptides were determined to be

fragments derived from a larger, membrane-anchored

Received June 10, 2003; revised manuscript received July 29, 2003;

accepted September 9, 2003.

Address correspondence and reprint requests to Brian Austen.

E-mail: sghk200@sghms.ac.uk.1Present address for Omar M. A. El-Agnaf is Department of Biological

Sciences, Lancaster University, Lancaster LA1 4YQ, UK.

Abbreviations used: AD, Alzheimer’s disease; ADDL, Ab-deriveddiffusible ligands; CAA, cerebral amyloid angiopathy; DTT, dithio-

threitol; ES-MS, electrospray mass spectroscopy; FBD, familial British

dementia; FDD, familial Danish dementia; HPLC, high-performance

liquid chromatography; LDH, lactate dehydrogenase; MALDI–TOF,

matrix-assisted laser desorption ionization time of flight; MEM, modified

essential medium; MTT, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetra-

zolium bromide; oxi-ADan, oxidized form of ADan peptides; PBS,

phosphate-buffered saline; red-ADan, reduced form of ADan peptides;

SDS, sodium dodecyl sulphate; SEC, size exclusion chromatography;

TFA, trifluoroacetic acid; WT, wild-type; zVAD-FMK, benzyloxycar-

bonyl valylalanyl aspartic acid fluoromethyl ketone .

Journal of Neurochemistry, 2004, 88, 281–290 doi:10.1046/j.1471-4159.2003.02134.x

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290 281

precursor protein termed BRI precursor protein, encoded by

the BRI gene on chromosome 13 (Vidal et al. 1999, 2000).

Wild-type (WT) BRI protein has 266 amino acid residues. A

decamer duplication between codons 265 and 266 in the 3¢region of the BRI gene originates the ADan peptide that is

associated with the pathology of FDD (Vidal et al. 2000;

Fig. 1).

In the mutant form, the resulting reading frame is extended

to 277 amino acid residues, and cleavage by furin releases a

peptide of 34 residues, which is identical to ABri and WT in

its N-terminal 22-residues, but contains a distinct C-terminal

10-residues composed of mainly hydrophobic residues (Vidal

et al. 2000). Staining of post-mortem sections of FDD

patients with an antibody to ADan revealed widespread

cerebral amyloid angiopathy (CAA), and widespread depos-

its of ADan in the leptomeninges, blood vessels and paren-

chyma. Congo red staining of adjacent sections revealed that

the majority of parenchymal lesions were non-fibrillar

(Holton et al. 2002), although deposits elsewhere were bi-

refringent. Previously, we have shown that solutions of the

oxidized form of ABri (oxi-ABri), which contain non-

fibrillar oligomeric species, are toxic to neuronal cells in

culture, whereas solutions of the WT peptide are not toxic to

cells (El-Agnaf et al. 2001a). In this study we investigated

the biophysical and neurotoxic properties of both reduced

(red-ADan) and oxidized forms (oxi-ADan) of ADan pep-

tides. In contrast to ABri, soluble oligomers of red-ADan are

more neurotoxic than those of oxi-ADan.

Materials and methods

Materials

The reduced form of ADan (red-ADan) peptide was synthesized

using the Fmoc/tBu chemistry recently optimized for the synthesis

of amyloid peptides (El-Agnaf et al. 2000). The crude peptide was

purified on a preparative Vydac C4 (250 · 22 mm) 214TP1022column with a gradient of acetonitrile in 0.1% trifluoroacetic acid

(TFA). Purity of red-ADan peptide was judged to be > 95% by

analytical high-performance liquid chromatography (HPLC) on a

column (250 · 4.6 mm) of Phenomenix Jupiter C8, and by

electrospray (ES-MS) and matrix-assisted laser desorption ioniza-

tion time of flight (MALDI-TOF) mass spectrometry. The mass/

charge of red-ADan was found to be 4065, identical to its calculated

mass (Fig. 2). Syntheses of the WT and ABri peptides have been

described previously (El-Agnaf et al. 2001b).

As the sequence of the BRI protein predicts the possibility of an

intramolecular disulphide bond in its C-terminal region, we

produced the cyclic form of ADan (oxi-ADan) by air oxidation.

HPLC-purified red-ADan was dissolved in 0.1 M ammonium

bicarbonate at pH 8.0 and 0.1 mg/mL and stirred for 2 days at

room temperature in contact with air, then lyophilized. The

oxidation was monitored by analytical HPLC from the disappear-

ance of the reduced form and appearance of the faster eluting

oxidized form, and by the loss of 2 mass units seen by MALDI-TOF

mass spectrometry (m/z ¼ 4062.6) (Fig. 2). Tryptic digests were

performed on 0.1 mg of the peptide in 0.1 M ammonium bicarbonate

(pH 8), in 0.2% sodium 3-[2-methyl-2-undecyl-1,3-dioxolan-4-

yl)methoxy]-1-propanesulphonate (Waters, Milford, MA, USA) as

solubilizing agent with 0.005 mg modified trypsin (Promega,

Madison, WI, USA) at 37�C for 12 h, then incubated with 20 mMHCl at 37�C for 1 h to hydrolyse the solubilizing agent, prior to

spotting on a MALDI plate. MALDI analyses were performed after

co-spotting and drying samples with an equal volume of 10 mg/mL

solution of a-cyano-4-hydroxycinnamic acid in 50% (v/v) acetonit-

rile in 0.1% TFA on a Kratos Axima MALDI mass spectrometer.

Calibration was with angiotensin I, angiotensin II and neuropeptide

Y on an adjacent sample spot.

Dimers of oxi-ADan and red-ADan were analysed in solutions in

0.01 M Tris–HCl (pH 7.4) by ES-MS. A sample (20 lL) of oxi-ADan (1 mg/mL) was diluted with 40 lL of methanol and 1 lL ofconcentrated formic acid (to improve solubility of the peptide).

Samples of red-ADan in 0.01M Tris–HCl (pH 7.4) either with

fourfold molar excess of dithiothreitol (DTT) or without added

reducing agent, were aged for 3 days at 37�C, then precipitatedmaterial was dissolved in formic acid (5 lL), methanol (200 lL)and water (100 lL) as above. Samples were centrifuged and used instatic nanospray mode on a Thermo LCQ Deka XP mass

spectrometer.

Preparation of aged solutions of ADan peptides

Solutions of red-ADan were made in sterile 0.1 M Tris–HCl

(pH 7.4) at the required concentrations. oxi-ADan was first

dissolved in sterile water, and then sterile 1 M Tris–HCl (pH 7.4)

was added to give a final 0.1 M concentration of Tris–HCl. Aging

was performed by incubation at 37�C for the number of days

recorded below.

E A SNC F A IR H F E N K F AVE TLIC S R T V KK N II EE N

E A SNC F A IR H F E N K F AVE TLIC F N L F LN S O E K HY

ABri

AD an

W T

Transmembranedomain

N

Extracellular domain100 200 244 AG A 277

C

In AD anDuplicate TTAATTGT

In ABri substituteStop TGA

Fur in

Fig. 1 Schematic presentation of the BRI precursor protein and pep-

tides ABri and ADan released in the brains of patients with FBD and

FDD. In FDD, there is a 10-base duplication occurring between

codons 265 and 266 of the BRI gene, 1 codon before the normal stop

codon, resulting in a frame-shift in the gene and a 277-residue

extended precursor protein. In patients with FBD, there is a single-

base substitution at the stop codon of the BRI gene, which again

generates a 277-residue precursor protein. The arrow indicates where

furin cleavage releases the 34-residue peptides ADan and ABri from

the mutated proteins, or the 23-residue WT-peptide.

282 G. Gibson et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290

Size exclusion chromatography (SEC) system

At various time points, aliquots (200 lL) of solutions of ADanpeptides incubated in 0.1 M Tris–HCl at 2.5 or 1.0 mg/mL were

loaded onto a Superdex 75 column (10 · 270 mm) equilibrated andeluted with 0.1 M Tris–HCl (pH 7.4) at a flow rate of 0.45 mL/min.

Elution was monitored at 215 nm.

Congo red bi-refringence

Aged 50 lL aliquots of aged ADan solutions were added to 950 lLof Congo red solution [1% w/v solution in phosphate-buffered saline

(PBS) pH 7.4]. This mixture was incubated for 1 h at room

temperature and then centrifuged at 336 000 g in a TL-100

Beckman centrifuge (Beckman, Palo Alto, CA, USA). The resultant

pellet was placed on a slide and examined under polarizing light

using an Nikon Optiphot light microscope under a ·10 lens fittedwith polarizers.

Gel electrophoresis

Samples were electrophoresed on precast 16.5% Tris–Tricine gels

(Bio-Rad Laboratories, Hercules, CA, USA). Fresh and aged

solutions of oxi-ADan peptide samples were diluted 1 part with 2

parts of sample buffer [glycerol, 40% (w/v); sodium dodecyl

sulphate (SDS), 2% (w/v); 0.2 M Tris–HCl, pH 6.8 and Coomassie

G-250, 0.005% (w/v)], prior to electrophoresis; the concentration of

Coomassie G-250 in the sample was reduced 10-fold from that

recommended by the gel manufacturers, because co-migration with

low molecular weight peptides obstructed visualization after

staining of the gel. Red-ADan was treated with DTT (50-fold

molar excess) in sample buffer, whereas oxi-ADan was applied in

the absence of DTT. Samples were not boiled. The gels were loaded

with 150 nmole of peptides, and run in tricine buffer [Tris base,

1.31% (w/v), tricine 1.8% (w/v) and SDS 0.1% (w/v)], at 20 mA per

gel on the Bio-Rad Mini-Gel. The gels were fixed and stained in

methanol, 40% (v/v), acetic acid 10% (v/v) solution containing

Coomassie blue R-250 0.1% (w/v) for 3 h. De-staining was

performed in the methanol/acetic acid solution without Coomassie

blue.

Atomic force microscopy

Red-ADan (1 mg/mL) was dissolved in sterile semiconductor grade

water (18 MOhm), made 0.1 M in Tris–HCl (pH 7.4) by addition of

sterile 1 M Tris–HCl (pH 7.4) and agitated for several minutes. A

few drops were placed onto a freshly cleaved square of mica and a

few minutes were allowed for any materials to adsorb before it was

gently washed off with a light stream of ultra-pure water. The

sample, after removing the excess around the edges, was allowed to

dry under a cover. AFM using AC (or tapping) mode at 300 kHz

was done using a type 1620 Si tip with tip radius of 20 nm or less.

The mica was held down onto magnetic holders using conductive

double-sided adhesive discs (Agar). The Setpoint for AC was 50%,

proportional gain 1, integral 0.5 and differential ¼ 0.75.

Electron microscopy

A solution (1 mg/mL) of either red-ADan or oxi-ADan in sterile

0.1 M Tris–HCl (pH 7.4), were either analysed immediately or

incubated at 37�C for either 3 or 7 days. Solutions were subse-

quently diluted to 0.1 mg/mL in sterile distilled water. Then, 5 lL ofthe diluted solutions were spotted onto Parloidin-covered EM grids

and allowed to air dry for 5 min. Staining of the samples was

achieved using 2% uranyl acetate, with removal of excess using

filter paper (Whatman, Maidstone, UK). Grids were examined using

a Zeiss 900 transmission EM.

Cell culture

SH-SY5Y cells (European Collection of Cell Cultures, Porton

Down, UK) were cultured in Dulbecco’s modified essential medium

(MEM)/Nutrient Mix F-12 (1 : 1; Gibco–BRL, Gaithersburg, MD,

USA) containing (10 IU penicillin/mL, 100 lg streptomycin/mL,15% foetal calf serum, 1% non-essential MEM amino acid

supplement, and 2 mM freshly prepared glutamine), at 37�C in a

humidified incubator with 5% CO2/95% air.

Measurement of cell viability

The effect of ADan peptides on cell viability was assessed by

measuring lactate dehydrogenase (LDH) release, and residual

cellular redox activity with 3-(4,5-dimethylthiazol-2-yl)-2,5-diphe-

nyltetrazolium bromide (MTT; Sigma, St Louis, MO, USA) as

previously described (El-Agnaf et al. 1998). Briefly, cells were

plated at 7500 cells/well in 96-well plates in 100 lL of fresh

medium. After 24 h, the medium was replaced with 200 lL of freshOPTI-MEM (Gibco–BRL) serum-free medium, containing freshly

prepared or aged solutions (20 days) of the peptides, alone or in the

Mass / Charge

100

90

80

70

60

50

40

30

20

10

03070 3080 3090 4000 4010 4020 4030 4040 4050 4060 4070 4080 4090 4100 4110 4120 4130

4063

.0

4066

.0

spot 2 Adan Reduced0001, spot 3Adan Redoxised0001Kratos PCAxima CFR V2.1.0

%Int.

2.6 mV 0.4 mV

Fig. 2 The mass spectrometry (MALDI-

TOF) of red-ADan (broken line) oxi-Adan

(continuous line), shows loss of two protons

upon oxidation.

Oligomerization and neurotoxicity in FDD 283

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290

presence of fourfold molar excess of DTT. Cells were then incubated

at 37�C in 5% CO2 for 2 days for LDH assay or 24 h for MTT

assay. An index of cell death was also obtained by measuring LDH

in cell supernatants using a cytotoxicity detection kit (Boehringer

Mannheim, Mannheim, Germany). For MTT assay, 20 lL of stockMTT (6 mg/mL) was added to each well, and the plates were

incubated at 37�C for 4.5 h. The medium-MTT solution was

carefully removed, and cell lysis buffer (100 lL per well; 15% (w/v)SDS/50% (v/v) N,N-dimethylformamide, pH 4.7) was added, and

the plate incubated overnight at 37�C, and the absorbance values at570 nm were measured.

Caspase activation

The caspase activities were measured by means of the binding of

the carboxyfluorescein derivative of benzyloxycarbonyl valylalanyl

aspartic acid fluoromethyl ketone (zVAD-FMK; Caspatag, Inter-

gen, Oxford, UK) which is a potent general inhibitor of caspase

activity. Briefly, SH-SY5Y cells were seeded in fresh media at

50 000 cells/mL, and 0.45 mL/well in eight-well chambered cover

glass (Nalgene Nunc Int., Naperville, IL, USA), and incubated for

24 h. The media were then replaced with fresh serum-free OPTI-

MEM containing ADan peptides, the incubation was continued for

8 h, and Caspatag was then added at 10 lM, and incubated for 1 h.After one wash with PBS, cells were visualized under a Carl Zeiss

LSM using a narrow pass 515–565 nm filter and 488 nm laser

irradiation.

Results

In view of our previous findings that the oxidized form of

ABri was more neurotoxic than the reduced form (El-Agnaf

et al. 2001a), some of the synthetic reduced form of ADan

peptide was converted into its oxidized form containing an

internal disulphide linkage by air oxidation. The oxidation

was monitored by HPLC and confirmed by mass spectrom-

etry. The oxidized form product gave one peak on MALDI–

TOF mass spectrometry of 4062.6 Da (Fig. 2), showing a

loss of two protons. To demonstrate that this change was not

due to intermolecular disulphide scrambling into oligomers,

the preparation was re-analysed after tryptic digestion.

The reduced form produced tryptic fragments with

reduced Cys residues at m/z of 1009.3 Da (data not shown),

corresponding to residues 1–9 (calculated 1010.5 Da), a

fragment (F/6) of m/z of 2771.9 Da corresponding to

residues 10–32 (calculated 2772.39 Da; Fig. 3), F/7 at

3072.3 Da residues 10–34 (calculated m/z of 3072.51 Da)

and a fragment F/8 of m/z of 3763.94 Da containing residues

1–32 with (calculated 3763.84 Da), the oxidized material

gave a large peak at 3125.0 Da, coincident with a peptide

containing residues 1–9 and 15–33 containing the intact

disulphide between residues 5 and 22 (calculated mass

3124.6 Da) (Fig. 3), showing that the Cys residues were

engaged in intramolecular cyclisation and not intermolecular

bonding. The same peaks were seen after trypsin digestion of

aged samples, showing that no scrambling of the disulphide

occurred during incubation at neutral pH.

As with the oxidized form of ABri (El-Agnaf et al.

2001a), solutions of oxi-ADan were shown to contain non-

covalent dimers by ES-MS (Fig. 4). A peak of m/z 1625.09

corresponded to a dimer with 5+ charges (Fig. 4a), as

confirmed by a high-resolution scan that showed a separ-

ation between the isotopes of 0.17 Da (Fig. 4b). Moreover,

ms2 of this species showed the appearance of a monomeric

% Int.

100

F/62772.39

80

60

40

20

02700 2900 3100 3300 3500 3700 3900 4100 4300

4062.7

4050.8

3763.5

3586.04064.6

3763.84F/3

F/44064.97

M/z 3125 Residues 1-9 S-S to 15-33

F/73072.51

2771.9

Mass/Charge

8Fig. 3 MALDI-TOF anaysis of tryptic frag-

ments of oxi-ADan (upper trace) and red-

ADan (lower trace). Fragments predicted

from the sequence are shown by the

dashed lines.

Fig. 4 Mass spectrometry of static nanosprayed solutions of oxi-ADan

(a–c) and red-ADan (d and e). Whole scan (300–1800 m/z units) of

oxi-ADan is shown in (a). (b) A zoom-scan of the peak at 1626,

showing the splitting (0.17) expected from a 6+-charged species. ms2

at low (20%) collision energy of the peak at 1626.14 is shown in (c).

Whole scan (400–2000 m/z units) of red-ADan is shown in (d), and

ms2 of the peak at 1162. 1 m/z units in red-ADan at low (20%) collision

energy is shown in (e).

284 G. Gibson et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290

tm/z = 1355.

1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000

1000800600400 1200 1400 1600 1800 2000

m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

1626

1355

Adancorrect1pptinformicdilutedfull #3RT:0.06AV:1NL:3.90E6T:+ c Full ms [ 390.00-2000.00]

m/z

05

101520253035404550556065707580859095

100

Rel

ativ

e A

bund

ance

1017.1

1355.4

1162.1

1626.31374.2

1196.21021.3904.2 1375.31220.5503.1 813.6 1636.71109.4 1229.1 1527.9793.8 1653.7678.5 1015.5 1741.9415.1 520.2 1843.9

Monomer 4+

Monomer 3+

Dimer 5+

Covalent dimer 7+

Full MS scan of Red AdanAdancorrect1pptinformicdiluted1162msms#10RT:0.20AV:1NL:5.47E4T:+ p sid=25.00 Full ms2 1162.00@25.00 [ 390.00-2000.00]

400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 1600 1700 1800 1900 2000m/z

0

5

10

15

20

25

30

35

40

45

50

55

60

65

70

75

80

85

90

95

100 1159.5

1142.9

1186.71280.8

1318.01081.21007.9935.7 1428.2822.4447. 1366.4 1918.5878.9 1441.7 1505.6 1584.1791.5 1884.51668.9 1764.5 1934.0592.1470.1 709.1

800 1000 1200 1400 1600m/z

0

10

20

30

40

50

60

70

80

90

100

Rel

ativ

e A

bund

ance

1016.62813.66

1355.27

897.49

1625.69

Dimer5+ 5+

Monomer 3+

Monomer 4+Monomer 5+

10

20

Rel

ativ

e A

bund

ance

1623.5 1624.0 1624.5 1625.0 1625.5 1626.0 1626.5 1627.0 1627.5 1628.0 1628.5

30

40

5565

85

1626.14

1626.541625.74

1626.341625.551625.36

1626.591625.19

(a) (b)

(d)(c)

(e)

Rel

ativ

e A

bund

ance

Oligomerization and neurotoxicity in FDD 285

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290

triple-charged species at m/z ¼ 1355 (Fig. 4c), confirming

a non-covalent dimer. The low yield of dimers found by

this technique may have been due to the use of organic

solvents in the nanospray injector. Analyses of pre-incuba-

ted solutions of red-ADan also showed the presence of the

non-covalent dimeric peaks, and in addition showed the

presence of a covalent 7+ charged dimer of 1162.1 m/z

(Fig. 3d). Ms2 at 1162.1 did not yield a monomeric species,

but generated fragments from b-type and y-type fragmen-

tation (Fig. 3e). The same dimers were present in samples

incubated in the absence or presence of DTT; showing that

the intermolecular disulphide was not readily accessible to

DTT.

Non-covelent oligomers in solutions of both oxi-ADan

and red-ADan were also shown by electrophoresis on highly

cross-linked SDS–PAGE (Fig. 5). Fresh solutions of red-

ADan after DTT treatment gave rise to an oligomer of about

32 kDa, and a dimer of 8 kDa, plus aggregated material not

entering the separating gel, whereas oxi-ADan fresh solu-

tions showed a strong dimeric species, as well as trimer and

quadramer (depicted by arrows; Fig. 5), but no higher

aggregates.

Aged solutions showed only dimer and aggregated

material not entering the separating gel.

Gel filtration chromatography of fresh preparations of red-

ADan solutions showed oligomers of molecular weight

higher than the monomeric form (Fig. 6).

A peak eluting at 41 mins from the column was indicative

of an oligomer of about 30 kDa (Fig. 6), as well as peak with

MW > 70 kDa, eluting rapidly at 19 min from the column

were also present, in keeping with the SDS–PAGE. However,

when the red-ADan solutions were incubated at 37�C for

21 days, only peaks containing high MW oligomers

(> 70 kDa) were detected. Incubation for 9 days converted

oxi-ADan from a dimer into high MW soluble oligomers

> 70 kDa eluting at 24 min (data not shown).

Aged preparations of oxi-ADan were very weakly

bi-refringent after treatment with Congo red, whereas aged

red-ADan was not birefringent.

ADan on Tris-Tricine PAGE

Freshoxidised

Agedoxidised

Freshreduced

Agedreduced

>70K>70K

32K

8K-Dimer8K Dimer8K

Octamer

Quadrimer

Trimer

Dimer

Monomer

34.3k

26k

17.9k

8k

4k

Fig. 5 SDS–PAGE (16% acrylamide) ana-

lysis of self-oligomerization of fresh and

aged red- and oxi-ADan peptides using

Tricine buffers. Sample preparation is des-

cribed in Methods. Only the reduced sam-

ples contained DTT.

10.00 20.00 30.00 40.00 50.00 60.00Elution time (min)

OD230nm

1.00

0.50

0.00

Oligomer>75kDa Oligomer 30kDa

Monomer

–0.50

Fig. 6 Size exclusion chromatography of red-ADan, A freshly pre-

pared solution of red-ADan (1 mg/mL; continuous line) or aged or 24 h

(dotted line) or 21 days (dashed-dotted line) was eluted from a column

of Superdex 75 in 0.1 M Tris–HCl t pH 7.4 at 0.5 mL/min. Insoluble

material was removed by centrifugation prior to loading.

2000 nm

1000 nm

0 nm0 nm 1000 nm 2000 nm

13.15 nm

0.00nm

Fig. 7 Atomic force microscopy of a fresh solution of red-ADan

(1 mg/mL).

286 G. Gibson et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290

Microscopy

When viewed by atomic force microscopy, fresh red-ADan

suspensions appeared as carrot-shaped objects each with a

head and elongated body of about 130 nm in length and

70 nm in maximum diameter (Fig. 7). The objects can be

seen stacked together in uniform orientation with heads

uppermost to form larger aggregates.

Electron microscopy of oxi-ADan preparations revealed

that after aging for 3 days, regular-shaped electron-dense

oligomers adhering to each other in arrays were formed,

whereas after 7 days the aggregated material appeared as

a matrix. Clusters of oligomeric spheroids were seen in

red-ADan preparations immediately after dissolution, which

formed three-dimensional matrices after 3–7 days (Fig. 8).

Neurotoxicity

The neurotoxicity of the reduced and oxidized forms of BRI

peptides on human neuroblastoma SH-SY5Y cells was

investigated. Cells were treated with solutions of BRI peptides

(200 lM), diluted from fresh or aged (20 day) solutions of

1 mg/mL. After incubation for 24 h, cell viability was

measured by LDH released to the media by the cells. Fresh

ABri andADan solutions were more toxic than aged solutions.

In contrast to ABri, red-ADan was more toxic than oxi-ADan.

The WT BRI peptides showed very little toxicity, either in

aged, fresh, oxidized or reduced states, as reported previously.

(Fig. 9). Similar relative results were obtained when toxicities

were measured by MTT uptake (Fig. 9, open bars) or LDH

release (Fig. 9, black shaded bars).

DTT at fourfold molar excess included with red-ADan to

retain it in the sulphydryl form did not change the levels of

toxicity recorded (data not shown).

We further investigated the mechanisms involved in the

neurotoxicity induced by ADan and ABri peptides. Using

FAM-VAD-FMK green fluorescent caspase inhibitor, we

investigated caspase induction following treatment of cells

oxi-Adan (0days aging) oxi-Adan (3 days aging)

red-Adan (3 days aging)red-Adan (0 days aging)

oxi-Adan (7 days aging)

red-Adan (7 days aging)

Fig. 8 Electron micrographs of prepara-

tions of (1 mg/mL) red-ADan and oxi-ADan;

the white tab is equivalent to 100 lm.

LDH

MTT

LDH

Peptides at 200µM

% Toxicity

100

80

60

40

20

0

Red

AB

ri

Red

Ada

n

Red

AB

riFr

esh

OxA

Bri

Fres

h

Fres

h

Fres

h

Oxi

d A

Bri

OxA

Dan

Oxi

d W

T

OxW

T

Age

d

Age

d

Age

dAge

d

MTT

Fig. 9 Neurotoxicity of aged (20 days; Ag)

and fresh (Fr) solutions of BRI peptides at

200 lM, determined by LDH release (h) or

MTT uptake (j) in SHSY-5Y cells in cul-

ture.% toxicity with the LDH assay is the

enzyme relased compared to that released

by 0.1% Triton X-100, and toxicity of the

MTT assay is the colour reduction com-

pared to that achieved with 100 lM campt-

othecin. Results are those averaged from

six readings on separate batches of cells.

Standard deviations were less than 10% of

averages recorded.

Oligomerization and neurotoxicity in FDD 287

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290

with fresh solutions of ADan and ABri peptides. Our results

show that at 200 lM concentration red-ADan led to panac-

tivation of caspases in cells, as shown by increased binding

of green fluorescent FAM-VAD-FMK as compared to control

cells (Fig. 10). At this concentration, red-ADan produced

more caspase activation than oxi-ABri. Altogether, these data

suggest that red-ADan oligomer-induced caspase activation

might represent a seminal event of ADan-induced neuronal

cell death.

To elucidate atomic interactions between ADan molecules

that might give rise to oligomer formation, we modelled

oxi-ADan by residue replacement in the previously mod-

elled ABri (El-Agnaf et al. 2001b) and docked two of these

molecules together using Swiss PBD Viewer. The resulting

model (Fig. 11) was subjected to energy-minimization steps.

The model shows that the monomers are held together by

electrostatic bonding between Arg (blue) and Glu (red)

residues in a largely hydrophobic environment provided by

other side-chains in ADan. Although the Cys–Cys (marked

1.42) intramolecular disulphide is involved in holding two

strands together in oxi-ADan, the triple-stranded sheet may

be stable in its absence, suggesting that red-ADan would

also form non-covalent oligomers readily. Examination of

the molecular model shows that the presence of an

intermolecular disulphide in a dimer of red-ADan, as

detected by ES-MS, would add to the hydrophobic and

(a) Control (Cells alone)

(b) 200 µM Oxi-ABri (c) 10 µM Oxi-ABri

(d) 200 µM Oxi-ADan (e) 10 µM Oxi-ADan

(f) 200 µM Red ADan (g) 10 µM Red ADan

Fig. 10 (a–g) Apoptotic effects of reduced

and oxi-ADan and oxi-ABri, detected with

FAM-VAD-FMK (Caspatag).

Fig. 11 A model of an ADan dimer mod-

elled in Swiss-PDB-viewer.

288 G. Gibson et al.

� 2003 International Society for Neurochemistry, J. Neurochem. (2004) 88, 281–290

electrostatic forces annealing the oligomeric structure and

could explain the enhanced neurotoxicity of red-ADan over

oxi-ADan.

Discussion

In this study we have investigated the biophysical and the

neurotoxic properties of ADan, the peptide which is found

deposited in the brain of patients suffering from FDD. Using

SDS-PAGE, SEC and ES-MS we found that the reduced

form of ADan red-ADan forms stable soluble oligomers

containing both covalent and non-covalent oligomers more

readily than the oxidized form, oxi-ADan. Higher molecular-

weight forms were visualized as adherent elongated sphe-

roids of about 50 lm in diameter, by atomic force microcopyand electron microscopy. In our previous report we have

shown that the oxidized form of the ABri, which is released

in the brains of a British family with dementing disease,

forms very toxic soluble oligomers, whereas the reduced

form did not form soluble oligomers and was not toxic to

neuronal cells (El-Agnaf et al. 2001a). To investigate

whether ADan is toxic to neuronal cells, we tested the

toxicity of both peptide forms the red-ADan and oxi-ADan.

Peptide solutions prepared freshly or aged for 20 days were

added to the cells and LDH released by cells to the media

were measured. Remaining cells were also measured by dye

binding (MTT). Interestingly, our results showed that the

fresh and aged preparations of red-ADan induced cell death

more than oxi-ADan as measured by LDH release. Fresh

preparation were more toxic than aged preparations. The

cell death induced by ADan involves the activation of

caspases. Interestingly, these results are in contrast with our

recent studies on ABri peptide, where we found that the

oxidized form was toxic whereas the reduced form was not

toxic to neuronal cells (El-Agnaf et al. 2001a). However, in

that study we have found that fresh solutions of the

oxidized form of ABri contained a series of soluble

oligomers, whereas the reduced form solution did not

contain a series of oligomers. The results for the ADan and

ABri peptides are in agreement with the notion that the

non-fibrillar aggregates ‘soluble oligomers’ formed by

amyloid peptides are the pathogenic species, which cause

neurodegeneration.

We hypothesize that the non-fibrillar aggregates of ADan

deposited in the brain are the cause of FDD. In support of

this hypothesis, the pathological studies of FDD brains have

shown that the hippocampal deposits can be recognized with

anti-ADan antibody but are negative to Congo red staining,

suggesting that ADan is deposited predominantly in a non-

fibrillar form (Vidal et al. 2000; Holton et al. 2002).

Interestingly, recent studies have shown that small oligomers

of many amyloid proteins, associated with neurodegenerative

disease, are the pathogenic species that drive neurodegener-

ation and neuronal cell death (Chiesa et al. 1998; Saudou

et al. 1998; Goldberg and Lansbury 2000; Conway et al.

2000; Masliah et al. 2000; Sian et al. 2000; Van der Putten

et al. 2000; El-Agnaf et al. 2000, 2001a; Walsh et al. 2002).

Ab-derived diffusible ligands (ADDLs) were shown to betoxic by Lambert et al 1998, whereas the experiments of

Bucciantini et al. (2002) suggested that some proteins,

previously considered non-pathogenic, were toxic in soluble

oligomeric form. Support for the idea that soluble aggregates

of amyloidogenic proteins are pathogenic and play a central

role in the development of several neurodegenerative

diseases also came from the detection of soluble Abaggregates in cerebrospinal fluid (CSF) from AD patients

(Pitschke et al. 1998) and prion protein aggregates in CSF

from patients with Creutzfeldt–Jakob disease (Bieschke et al.

2000; Giese et al. 2000) but not in control patients.

Identifying the molecular structure of the amyloid aggregate

species leading to neurodegeneration is an important step for

the design of novel drugs for neurodegenerative disorders

caused by amyloid deposits.

In summary, in this study we have shown for the first time

that both reduced and oxidized forms of ADan peptide

associated with FDD are toxic to neuronal cells, and that

fresh solutions containing soluble oligomers are more toxic

than aged solutions containing insoluble aggregates. Soluble

aggregates appear to activate caspases and apoptosis in

neuronal cells. Hence, an understanding of the role of ADan

in the pathogenesis of the FDD may shed light on causes of

neurodegeneration in more common neurological disorders

associated with amyloid deposition, including Alzheimer’s

disease.

Acknowledgements

We are grateful to the Wellcome Trust for their support and to

Dr Ted Tarelli for help with mass spectrometry.

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