Complement activation in chromosome 13 dementias: Similarities with

Alzheimer’s disease

Agueda Rostagno1, Tamas Revesz3, Tammaryn Lashley3, Yasushi Tomidokoro1, Laura

Magnotti1, Hans Braendgaard4, Gordon Plant5, Marie Bojsen-Moller6, Janice Holton3,

Blas Frangione1, 2, and Jorge Ghiso1, 2.

Departments of 1Pathology and 2Psychiatry, New York University School of Medicine,

New York, U.S.A; 3Queen Square Brain Bank and Department of Molecular

Pathogenesis, Division of Neuropathology, Institute of Neurology, UCL, London, U.K.;

Departments of 4Neurology and 6Neuropathology, Århus University Hospital, Århus,

Denmark; and 5National Hospital for Neurology and Neurosurgery, London, U.K.

Running title: Chromosome 13 dementia and complement activation

Keywords: amyloidosis, ABri, ADan, familial British dementia, familial Danish

dementia, classical complement pathway, alternative complement pathway

Corresponding author: Agueda Rostagno, Ph.D. Department of Pathology New York University School of Medicine 550 First Avenue, TH-435 New York, NY 10016 rostaa02@popmail.med.nyu.edu

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ABSTRACT

Chromosome 13 dementias - Familial British Dementia (FBD), and Familial Danish

Dementia (FDD) - are associated with neurodegeneration and cerebro-vascular

amyloidosis, with striking neuropathological similarities to Alzheimer’s disease (AD). In

spite of the structural differences among the amyloid subunits (ABri in FBD, ADan in

FDD, and A in AD), these disorders are all characterized by the presence of

neurofibrillary tangles and parenchymal and vascular amyloid deposits co-localizing with

markers of glial activation, suggestive of local inflammation. Proteins of the complement

system and their pro-inflammatory activation products are among the inflammation

markers associated with AD lesions. Immunohistochemistry of FBD and FDD brain

sections demonstrated the presence of complement activation components of the classical

and alternative pathways as well as the neo-epitope of the membrane attack complex.

Hemolytic experiments and ELISAs specific for the activation products iC3b, C4d, Bb

and C5b-9 indicated that ABri and ADan are able to fully activate the complement

cascade at levels comparable to those generated by A 1-42. ABri and ADan specifically

bound C1q with high affinity and formed stable complexes in physiological conditions.

Activation proceeds ~70-75% through the classical pathway while only ~25-30% seems

to occur through the alternative pathway. The data suggest that the chronic inflammatory

response generated by the amyloid peptides in vivo might be a contributing factor for the

pathogenesis of FBD and FDD and, in more general terms, to other neurodegenerative

conditions.

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INTRODUCTION

The classical hallmark lesions of Alzheimer’s disease (AD)1, cerebral senile plaques and

neurofibrillary tangles (NFTs), have been known for nearly a century. During the past

two decades, a wide range of inflammatory markers - typically absent or significantly

reduced in the normal elderly population - were reported in AD brains (1), and

accumulating evidence suggests that sustained brain inflammation might be an essential

co-factor in AD pathogenesis (2,3). In this sense, immunologic factors and inflammation

mediators, including complement proteins and pro-inflammatory peptides generated at

different stages of complement activation (4) as well as various cytokines (5) have been

implicated in accelerating the progression of AD.

The complement system is a highly regulated, powerful effector mechanism of the

immune system that destroys and clears deleterious substances. It is composed of more

than twenty proteins that become sequentially activated in a proteolytic cascade.

Originally, activation of the complement system was thought to occur only by binding of

immune complexes to C1q, the recognition component of the classical pathway.

However, it became then evident that the complement system can directly be activated, in

the absence of antibody, by interaction of certain foreign molecules with C3 (alternative

activation pathway), C1q (antibody-independent classical activation pathway) or by

specific lectins on the surface of certain microorganisms (lectin activation pathway)

(3,6,7).

The first step in the classical complement pathway involves the binding of an

activator to C1q resulting in the subsequent conversion of the serine proesterases C1r and

C1s to their active forms and, in turn, in the activation of C4, C2 and then C3. The

alternative pathway differs from the classical pathway in that activation begins at the

level of C3 and involves factors B, D, and Properdin. Proteolytic modification of C3 by

either pathway leads to the cleavage of C5 and the incorporation of C6, C7, C8, and

multiple molecules of C9 resulting in the formation of the membrane attack complex

(MAC), C5b-9, a transmembrane channel capable to produce cell lysis (8).

1 AD, Alzheimer’s disease; CVF, cobra venom factor; ELISA, enzyme-linked immunosorbent assay; FBD, familial British dementia; FDD, familial Danish dementia; HPLC, high performance liquid chromatography; MAC, membrane attack complex; NFTs, neurofibrillary tangles; NHS, normal human serum

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Activation-derived proteins of both the classical and alternative pathways have

been demonstrated in association with AD lesions by numerous groups (3). We

investigated the complement activation cascade in chromosome 13 dementias, two

hereditary conditions - Familial British Dementia (FBD) and Familial Danish Dementia

(FDD) - also associated with neurodegeneration and amyloid deposition in the central

nervous system. FBD has been described in members of three British pedigrees and is

characterized clinically by dementia, cerebellar ataxia and spastic paraparesis with the

disease onset typically in the fourth to fifth decade of life and death occurring some ten

years later (9). FDD is a disease associated with a single Danish family with the onset of

cataracts in patients before the age of thirty. Affected family members subsequently

develop sensory hearing loss, cerebellar ataxia, psychosis and dementia leading to death

between the ages of fifty and sixty years (10). The neuropathology in both diseases is

remarkably similar to that seen in AD, including cerebral amyloid angiopathy, pre-

amyloid lesions, amyloid plaques of various types and NFTs, ultrastructurally composed

of paired helical filaments with an electrophoretic pattern of abnormal

hyperphosphorilated tau indistinguishable from that observed in AD. Activated

microglia expressing the major histocompatibility class II antigens that are characteristic

of inflammatory processes, can be demonstrated around amyloid plaques and dystrophic

neurites but not in preamyloid lesions in both FBD and FDD (10,11) in a topographical

distribution similar to that found in AD. In these disorders the deposited amyloid

proteins, ABri in FBD and ADan in FDD, are proteolytic fragments of a larger precursor

molecule BriPP codified by a multiexonic gene BRI2 (also known as ITM2B) located on

the long arm of chromosome 13 (12-14). The amyloid peptides originate as a result of

two different genetic defects namely a Stop-to-Arg mutation in FBD and a ten-nucleotide

duplication-insertion immediately before the stop codon in FDD. Regardless of the

nucleotide changes, the final outcome is common to both diseases: the ordinarily

occurring stop codon is not in frame causing the genesis of an extended precursor

featuring a C-terminal piece that does not exist in normal conditions. These de novo

created amyloid peptides, with no sequence identity to any known amyloid protein, are

both 34-residues long, share 100% homology of the first 22 residues and have a

completely different 12 amino acid C-terminus. When deposited, both ABri and ADan

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feature pyroglutamate at their N-terminus, a post-translational modification also

identified in some truncated amyloid species isolated from Alzheimer’s disease brains,

i.e. A pE3 and A pE11. (15-17).

The results presented here show i) the co-localization of complement activation

products with amyloid plaques and cerebrovascular amyloid deposits in FBD and FDD

and ii) the activation of both the classical and the alternative pathways of the complement

system by synthetic peptides representing the main species deposited in both disorders,

activation that proceeds to the terminal stages with the generation of C5b-9. The data

suggest that chronic inflammation driven by in vivo complement activation may be a

contributing factor to disease progression and pathogenesis in both FBD and FDD.

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MATERIALS AND METHODS

Materials

Synthetic peptides: The following peptides were synthesized at the W. M. Keck facility at

Yale University and further purified by high-performance liquid chromatography

(HPLC): ABri1-34 (EASNCFAIRHFENKFAVETLICSRTVKKNIIEEN), ADan1-34

(EASNCFAIRHFENKFAVETLICFNLFLNSQEKHY), ABri1-23 (EASNCFAIRHFEN

KFAVETLICS), ABri24-34 (RTVKKNIIEEN), ADan23-34 (FNLFLNSQEKHY), as

well as full length variants containing N-terminal pyroglutamate, oxidized cysteines at

positions 5 and 22, and serine residues replacing the wild type cysteines at both positions.

Peptide A 1-42, DEFRHDSGYEVHHQKLVFFAEDVGSNKGAIIGLMVVGGVVIA,

used as a control in all the experiments, was also synthesized at the same facility, purified

and characterized as described (18).

Complement Reagents: Pooled normal human serum (NHS), C1q-depleted serum,

purified C1q protein, polyclonal goat anti-C1q antiserum, and ELISA kits for the

quantitation of the activation products C4d, Bb, iC3b, and SC5b-9 were purchased from

Quidel, Inc., Mountain View, CA. EZ diagnostic kit for the assay of total complement

activity (CH50) was obtained from Diamedix Corporation, Miami, FA.

Immunohistochemistry reagents: Antibodies immunoreactive with ABri (Ab 338) and

ADan (Ab 5282) molecules were raised in New Zealand white rabbits by immunization

with synthetic peptides comprising positions 22-34 of the respective amyloid molecules,

as previously described (12,14). Polyclonal rabbit anti-C1q was purchased from Dako

(Carpinteria, CA), polyclonal anti-C5b-9 (neo-epitope) and monoclonals anti-C4d, and

anti-Bb were obtained from Quidel, Inc.

Immunohistochemical Studies

Sequential paraffin sections from the hippocampus including the dentate fascia were used

for C1q immunostaining in 5 cases with FBD (mean age at death 62.4 years, range 59-68

years) and 3 cases with FDD (mean age at death 53.7 years, range 43-60 years). For the

immunohistochemical detection of C4d, C5b-9, and Bb sequential frozen sections, fixed

in acetone, were used. Sections from 4 controls (mean age at death 64.3 years, range 33-

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88 years) without a neurological disease and also 7 cases with Alzheimer’s disease (mean

age at death 78.2 years, range 63-92 years) were also stained in a similar manner.

Depending on the antibody, a number of different pretreatments were employed for the

paraffin embedded tissues, which are detailed in Table I. After pretreatment, the tissue

sections were incubated with the pertinent primary antibodies, followed by sequential

incubations with either biotinylated anti-mouse or anti-rabbit secondary antibodies, as

appropriate, and the corresponding ABC complex (Dako, Denmark). Color was

developed using diaminobenzidine/ H2O2 followed by hematoxylin counterstaining.

Characterization of amyloid peptides

Structural analysis. Secondary structure was assessed by circular dichroism (CD)

spectrometry in the far UV (190-250 nm) at 24 oC using a Jasco J-720 spectropolarimeter

(Jasco, Tokyo, Japan) as previously described (19).

Peptide solubilization and aggregation. The different ABri and ADan peptides were

solubilized in 10 mM NaHCO3, pH 9.6, aliquoted and lyophilized. Before use, each

aliquot was dissolved in distilled water at a concentration of 1 mg/ml and immediately

used in the complement activation assays. A 1-42 was dissolved in distilled water, and

added of concentrated PBS to reach a final concentration of 150 mM NaCl. Solutions of

1mg/ml were allowed to aggregate for 7 days at 37 oC, as described (19).

HPLC purification. Fractions enriched in ABri and ADan monomers were obtained via

reverse-phase HPLC (Applied Biosystems 130A Separation system) on a 0.21 x 25 cm

Vydac C4 TP52 microbore column (Vydac, Hesperia, CA), using isocratic conditions at

25% acetonitrile in 0.1% trifluoroacetic acid for 10 minutes followed by a 30 minutes

linear gradient of 25 to 40 % acetonitrile in 0.1% trifluoroacetic acid at a flow-rate of 200

µl/min. Fractions were collected in accordance to the absorbance at 220 nm.

Western Blot analysis. The degree of oligomerization of the various peptides was

assessed by western blot. Aliquots of 100 – 150 ng of each peptide freshly dissolved or

after 1 h incubation in either PBS pH 7.4 or Tris-HCl pH 7.4 containing 2.5 mM Ca+2, 1

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mM Mg+2, 75 mM NaCl were separated in 16% tris-tricine SDS-PAGE gels and

transferred for 45 min at 400 mA to polyvinylidene difluoride (PVDF) membranes

(Immobilon-P, Millipore, Bedford, MA) using CAPS (3-cycloexylamino-1-

propanesulfonic acid, Sigma, St Louis, MO) buffer pH 11, containing 10 % methanol.

After transference, membranes were blocked with 5% non-fat milk in PBS pH 7.4,

containing 0.1 % Tween 20 and incubated with the corresponding primary antibody [anti-

ABri, antibody 338 (1 µg/ml IgG); anti-ADan, antibody 5282 (2 µg/ml IgG)] for 3 h at

room temperature followed by horse-radish peroxidase conjugated anti-rabbit

immunoglobulins [Amersham Pharmacia Biotech, Piscataway, NJ (1:3000)].

Immunoreactivity was assessed by enhanced chemiluminescence (ECL) after developing

with ECL western blotting detection reagent (Amersham Pharmacia Biotech) according

to the manufacturer’s specifications.

Assays for Complement activation/consumption

1) Hemolytic Assays. Total functional activity of the classical pathway (CH50). The

assay relies on the lysis of sensitized sheep erythrocytes by human serum following

activation of the Ca+2- and Mg+2

-dependent classical pathway. Briefly, in order to induce

complement activation, constant volumes (10 µl) of NHS were separately incubated for

45 min at 37oC with ten µl of ABri, ADan and A 1-42 solutions containing increasing

concentrations [0-1000 µg/ml in Tris-buffered saline pH 7.4 containing 5 mM CaCl2 and

2 mM MgCl2 (TBS)] of the amyloid peptides. The reaction was stopped on ice and

immediately analyzed for total complement activity (CH50) employing EZ diagnostic kit

in accordance to the manufacturer’s specifications. Results were expressed as percentage

of lysis compared to controls of 100% complement activity (no complement

consumption) in which NHS was incubated with buffer in the absence of amyloid

peptides.

2) Quantitation of activation products by ELISA. NHS was incubated with identical

volumes of the different amyloid peptides in increasing concentrations (0-1000 µg/ml in

TBS) as described above, and the reaction stopped by the addition of EDTA to a 5 mM

final concentration. The generation of complement activation products was analyzed by

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capture-ELISA employing C4d, iC3b, Bb, and SC5b-9 kits from Quidel in accordance to

the manufacturer’s instructions. Samples were diluted of 1:100 for the quantitation of

C4d and Bb, 1:300 for iC3b, and 1:200 for SC5b-9 prior to the analysis by ELISA.

In order to estimate the contribution of the alternative complement pathway to the

total activation of the system, in a separated set of experiments and using identical

conditions as above, the amyloid peptides were incubated with C1q depleted serum

(reconstituted with Ca+2 and Mg+2 ions to a 10 mM final concentration) instead of NHS,

and the generation of SC5b-9 assessed by ELISA. Confirmation of the contribution of the

alternative pathway to the total activation was achieved by quantitating the SC5b-9

generated by incubation of the amyloid peptides with NHS in the presence of

Mg+2/EGTA.

3) Controls of complement activation.

a) Aggregated IgG: As positive control for classical pathway activation in both, CH50

and ELISA tests, NHS was separately added of identical volumes of aggregated IgG

solutions of increasing concentrations (0-1000 µg/ml) and incubated at 37oC for 45

minutes. The NHS was subsequently analyzed for total remaining complement hemolytic

activity (CH50) and generation of complement activation products by ELISA as

described above. Aggregated IgG was prepared by incubating a 5 mg/ml solution of

human polyclonal IgG (Cohn fraction II; Miles Laboratories, Inc., Kaukakee, IL) at 63 C

for 15 minutes as described (20,21). Following centrifugation at 2,000 x g for 10 minutes,

the pelleted larger aggregates were discarded and the soluble IgG aggregates aliquoted

and stored at –70 oC.

b) Cobra Venom Factor (CVF). Positive control for the generation of activation products

by the alternative pathway consisted of NHS incubated for 1 hr at 37 oC with cobra

venom factor (CVF, naja naja kaouthia; Sigma Chem. Co., St. Louis, MO) at a ratio of

2µg/100 µl serum (22). The reaction was stopped by cooling on ice to 4 oC, and the serum

immediately analyzed for the presence of complement activation products by ELISA, as

described above.

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Solid-phase binding assays. Binding of C1q to immobilized amyloid peptides

ELISA microtiter wells were coated for 2 h at 37 oC with ABri1-34, ADan1-34, ABri1-23

and A 1-42 peptides at a concentration of 400 ng/0.1 ml of 0.1M NaHCO3, pH 8.6. After

blocking with 1% bovine serum albumin for one hour at room temperature, variable

concentrations (0-20 nM in TBS) of C1q were incubated with the peptide-coated wells

for one hour at room temperature. Bound C1q was detected with goat polyclonal anti-C1q

antiserum (1:1000 in TBS containing 0.1% Tween 20 and 0.1% BSA [TBST]) followed

by alkaline phosphatase-labeled swine anti-goat IgG (1:5000 in TBST; BioSource

International, Camarillo, CA). The reaction was developed with p-nitrophenyl phosphate

in diethanolamine buffer (BioRad, Richmond, CA) stopped with 0.4 M NaOH and the

absorbance at 405 nm quantitated in a Spectracount ELISA microplate photometer

(Packard, Meriden, CT). Binding data were analyzed by non-linear regression using

GraphPad Prism (GraphPad Software, Inc., San Diego, CA).

ABri-C1q and ADan-C1q Complex formation

The formation of the ABri/ADan-C1q complexes was assessed via amino acid sequence

analysis. Ten µg of C1q in TBS were incubated with 20 µg of either ABri or ADan

peptides for 1 h at 37 oC. The resulting complexes were separated by electrophoresis on

1% agarose gels in 75 mM veronal buffer, pH 8.6 containing 2 mM sodium lactate and

contact-transferred to PVDF membranes. Transferred proteins were stained for 1 minute

with 0.125% Coomassie Blue R-250 in 47% methanol, membranes distained, extensively

washed with water and the bands of interest excised and subjected to N-terminal

sequencing on a Procise 494 protein sequencer (Applied Biosystems, Foster City, CA).

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RESULTS

Immunohistochemistry

Figure 1 (Panel A) shows that antibody 338 specific for the carboxyl-terminal region of

ABri strongly labels amyloid plaques and amyloid laden blood vessels, including affected

small arteries, arterioles and capillaries in FBD. Both the vascular and parenchymal

amyloid lesions were strongly labeled with anti-C1q (Panel B), anti-C4d (Panel C), anti-

C5b-9 (neoepitope) (Panel D), and anti-Bb (Panel E) in a staining pattern similar to that

seen for ABri immunohistochemistry. Diffuse deposits, defined as Congo red and

Thioflavin S negative or weakly positive ABri parenchymal lesions (11), were only

faintly stained (not shown). The FDD lesions, mainly vascular amyloid and parenchymal

preamyloid plaques were highlighted by antibody 5282 recognizing the carboxyl-terminal

end of ADan (Panel F). The anti-complement antibodies (anti-C1q (panel G), anti-C4d

(Panel H), anti-C5b-9 (neoepitope) (Panel I), and anti-Bb (Panel J) labeled amyloid

primarily deposited in small arteries, arterioles and capillaries. The immunoreactivity

with these antibodies was weak or absent in the parenchymal lesions which have been

shown to be composed primarily of protein in preamyloid conformation (Congo red and

Thioflavin S negative or weakly positive deposits) (10). Immunohistochemical analysis

of AD cases, which were used as positive controls, showed labeling of both vascular A

amyloid deposits and parenchymal A -positive plaques by anti-C1q, anti-C4d and anti-

C5b-9 antibodies. Smaller numbers of the A -positive lesions were also stained with anti-

Bb antibody (not shown). Two of the normal controls were entirely negative for

complement proteins, while in the two normal control cases with age at 81 and 88 years,

occasional A -positive plaques immunoreacted only with the anti-C1q antibody (not

shown).

Classical pathway of Complement activation

In view of the presence of complement activation products in the amyloid lesions of FBD

and FDD, we investigated whether their co-localization reflected a secondary

phenomenon or a specific interaction between complement proteins and the deposited

peptides. As an initial step, we tested in hemolytic assays the ability of ABri and ADan

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peptides to activate the classical pathway. As shown in Figure 2A, incubation of NHS

with increasing concentrations of ABri1-34 and ADan1-34 resulted in a concomitant

decrease in the remaining complement activity (CH50) compared with NHS incubated

under the same conditions in the absence of the amyloid peptides. Under our

experimental conditions, both peptides consumed complement to approximately the same

extent, with remaining complement values that reached a minimum of 18% for ABri1-34

and 23% for ADan1-34 at the maximal concentration tested (final concentration: 500

µg/ml; ~ 120 nmol/ml). The consumption of complement induced by the ABri and ADan

peptides was also similar to that of 7-days aggregated A 1-42 that, under the conditions

tested, reduced the complement activity to 23% of the values obtained in the absence of

peptide, in agreement with previously published data (23). As a positive control for the

classical pathway activation, Figure 2A also depicts the decrease in complement activity

induced by incubating NHS with aggregated IgG, a known activator of the classical

pathway. As it can be deduced from the data, aggregated IgG is a more potent activator of

the complement system than ABri and ADan, achieving similar levels (~ 30% of the

original complement activity) at a much lower molar ratio (500 µg/ml; 3.3 nmol/ml), as

described (24). No differences in activation were observed among ABri or ADan

peptides bearing different post-translational modifications, i.e. N-terminal glutamate or

pyroglutamate, oxidized cysteines 5 and 22, or peptides containing serine residues

replacing cysteines 5 and 22 (not shown).

In view of the values obtained in the CH50 hemolytic assay, we quantitated the in vitro

formation of the activation products C4d, iC3b, and SC5b-9 via specific capture ELISAs

employing specific monoclonal antibodies directed against neo-epitopes originated in the

activation-derived fragments (8). The C4d levels generated by incubation of NHS with

ABri and ADan peptides are shown in Figure 2B. C4d, together with C4c, are the

physiological degradation products of C4b as a result of proteolytic cleavage by the

complement regulatory protein Factor I in the presence of either C4-binding protein or

complement receptor 1 (CR1) (25,26). The ability of both amyloid peptides to generate

C4d in a dose response manner indicates that activation of the complement system

occurred through C1 activation since the conversion of the proenzyme C1s is solely

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needed to produce the C4 cleavage. Proteolytic fragments of C3 (Figure 2C) and the

soluble terminal complex SC5b-9 (Figure 2D), on the other hand, may originate by

activation of both the classical and the alternative pathways. Quantitation of iC3b was

used to estimate C3b generation since, once produced, C3b is rapidly inactivated by

Factor I in conjunction with either Factor H or CR1 as cofactors (26,27). As indicated in

Figure 2C, both ABri and ADan were able to generate iC3b in a dose-dependent manner.

The assembling of SC5b-9 shown in Figure 2D, determined by a widely used standard

method to assess complement activation (24,28), confirmed the ability of ABri and ADan

to in vitro trigger the complement cascade to full completion, including the terminal

stages. The terminal cytolytic C5b-9 complex generated by the assembly of C5b, C6, C7,

C8 and multiple C9 molecules in the absence of a target membrane (as in the case of

these experiments) binds to the naturally occurring serum S protein (vitronectin) resulting

in the formation of the soluble, non-lytic form of the MAC, SC5b-9. The data in Figure 2

also indicates that incubation of NHS with ABri and ADan peptides results in the

production of activation-generated fragments of the complement proteins C4 and C3 as

well as the complex SC5b-9 to levels comparable to those induced by incubation with

aggregated A 1-42, in agreement with the CH50 findings (Figure 2A). In addition, the

levels of SC5b-9 produced by incubation of A 1-42 with NHS confirm previously

reported data acquired under similar experimental conditions (29). Similarly to the results

obtained using the hemolytic assay, aggregated IgG produced a comparable level of

activation-generated fragments at a lower molar ratio. In the presence of EDTA, a

chelator of both Ca+2 and Mg+2 ions essential for the activation of the complement

cascade, none of the activation products C4d, iC3b and SC5b-9 were generated, as

expected (not shown).

Alternative pathway of Complement activation

The ability of the ABri and ADan peptides to trigger the alternative pathway was

assessed by measuring the generation of Bb by ELISA, a method that provides a direct,

specific indication of alternative pathway activation (30). As shown in Figure 3A both

ABri and ADan are able to induce the production of Bb to similar levels as A 1-42

following incubation with NHS. These values are significantly different from those

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originated spontaneously by incubation of NHS with buffer in the absence of amyloid

peptides that are also shown in the figure for comparison purposes. Although the Bb

levels that result from incubation with the amyloid peptides are similar to those reported

in certain infectious pathological conditions that take place with activation of the

alternative pathway (31), they are significantly lower that those induced by incubation of

NHS with CVF, a potent activator of the pathway. CVF, a functional analog of human

C3b, forms stable C3/C5 convertases that are not inactivated by the plasma regulatory

proteins factor H and factor I. It binds factor B rendering it available for cleavage by

factor D and initiating in this way a potent alternative pathway activation.

To estimate the contribution of the alternative pathway to the total activation of

the complement system, we quantitated the levels of SC5b-generated by incubation of the

peptides with NHS under conditions in which both pathways are activated (presence of

Ca+2 and Mg+2 ions) and compared them with those originated by activation of the AP

only (presence of Mg+2/ EGTA or substitution of NHS by C1q depleted serum). As

shown in Figure 3B, whereas addition of EDTA resulted in complete absence of SC5b-9

corroborating that activation of the system is required to produce SC5b-9, the incubation

with Mg+2/EGTA reduced the levels to an average of 25%, 32%, and 31% of the total

values of SC5b-9 for ABri, ADan, and A 1-42, respectively. Corroborating these results,

incubation with C1q depleted serum reduced SC5b-9 levels to similar values (an average

of 35% for ABri, 24% for ADan, and 28% for A 1-42). Therefore, the classical pathway

appears to be the major route of activation of the complement system for all the amyloid

peptides tested here, representing 70-75% of the total activation while the alternative

pathway accounts for the remaining ~25-30%.

Complex formation with C1q

All the above data strongly suggested that the trigger of the complement cascade by ABri

and ADan mainly proceeds through the classical activation pathway, most likely through

a direct binding interaction of the peptides to C1q. In a set of solid phase binding

experiments, incubation of either ABri-, ADan-, or A 1-42-coated microtiter wells with

increasing concentrations of C1q (0-20 nM) resulted in a dose-response relationship that

reached saturation (Figure 4A). Non-linear regression analysis of the binding data

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estimated the dissociation constants for ABri and ADan as 1.9 ± 0.4 nM and 1.2 ± 0.3

nM, respectively, within the same range to that of A 1-42 (Kd: 2.4 ± 0.5 nM). These

high affinity interactions correlated well with the pronounced capability of the amyloid

peptides to activate the classical complement cascade in hemolytic assays (Figure 2A). In

addition, these high affinity values suggested that the ABri or ADan peptides likely form

complexes with C1q. In vitro complex formation was performed in a 50-fold molar

excess of the peptides, to assure that most (if not all) the C1q was part of the complex.

To visualize the final product, we took advantage of the characteristic cathodic

electrophoretic mobility of C1q in agarose gels and the mostly anodic migration of the

peptides in the same system. As indicated in Figure 4B, Coomassie blue staining of the

PVDF-transferred material shows C1q (lane 2) with its characteristic cathodic migration,

matching the very end of the gamma region in the human serum profile shown for

comparison purposes (lane 1). When complexed with ABri or ADan, the electrophoretic

mobility shifted noticeable towards more anodic positions (lanes 3 and 4, respectively).

To corroborate the formation of the complexes, the bands were excised and subjected to

limited N-terminal sequence analysis. Three identifiable sequences were recovered in

each case (see Figure 4B for details); two sequences corresponded to the A and C chains

of C1q while the other matched the N-terminus of the ABri or the ADan peptides. No

sequence was retrieved for the B chain of C1q known to contain a blocked N-terminal

glutamine residue (pyrrolidone carboxylic acid) [Protein ID: P02746; Swiss-Prot

database of the Swiss Institute of Bioinformatics; http://www.expasy.org].

Mapping the complement-activation activity of ABri and ADan

In order to map the complement activating activity to a specific region of the ABri

and ADan molecules we employed synthetic peptides representing different regions of

the amyloid subunits in the CH50 hemolytic assay. The peptides tested consisted of the

common N-terminal region of the amyloid subunits (ABri /ADan1-23), as well as the C-

terminal fragments from both molecules ABri24-34 and ADan23-34. Since the molecular

mass of the peptides corresponding to different regions of the molecules differ

significantly, identical molar concentrations of the peptides were used in the experiments.

For comparison purposes, the molar concentrations of the full-length ABri and ADan

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peptides used were equivalent to the peptide concentrations displayed in Figure 2 (e.g. 12

nmol/ml, 48 nmol/ml, and 120 nmol/ml, corresponding to 50, 200 and 500 µg/ml,

respectively). As indicated in Figure 5, both C-terminal fragments lack ability to activate

and consume complement proteins whereas the common N-terminal region of the

amyloid molecules retains the functional activity (27% at a concentration of 120 nmol/ml

for ABri/ADan1-23 compared to 18% for ABri and 23% for ADan). The localization of

the complement-activating site to the common N-terminal fragment correlates with the

similar behavior of both ABri and ADan peptides in inducing almost identical levels of

complement activation. In addition, the mapping of the complement-activating activity to

the ABri/ADan1-23 region correlates with the capacity of the N-terminal peptide to bind

C1q with almost identical affinity as the full-length peptides (ABri1-23 Kd: 1.45 ± 0.3

nM).

Peptide oligomerization and complement activation

An important element in the activation of the complement system by amyloid

peptides seems to be directly related to their degree of oligomerization. In our

experimental conditions, the activation of the classical pathway takes place with freshly

solubilized peptides, reaching complement activation values similar to those obtained

with 7-day aggregated A 1-42. In order to clarify this issue, we examined the secondary

structure and the oligomerization state of ABri and ADan peptides immediately after

solubilization and after 45-minutes incubation at 37oC, the incubation time required for

the hemolytic assays and the ELISA experiments. As shown in Figure 6A, full-length

ABri and ADan rendered CD spectra compatible with high -sheet content whereas the

respective C-terminal fragments (ABri24-34 and ADan23-34) showed a random coil

configuration suggesting a role for -sheet structure in the complement activating ability

of the peptides. Both peptides were already heavily aggregated at the starting conditions

(Figure 6 panels B and C, lanes 1) although the oligomerization was even more evident

after the incubation required for all the complement assays (panels B and C, lanes 2).

Similar results were observed with the N-terminal peptide ABri/ADan1-23 (not shown).

These findings indicate that the ABri/ADan peptides have a higher tendency to form

oligomers than A 1-42 and suggest a faster aggregation kinetics. Of note, although both

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peptides share a similarly high -sheet content, ABri (panel B) seems to aggregate even

faster than ADan (panel C) as indicated by the presence of higher molecular mass

oligomers under the same experimental conditions. In spite of this apparently different

aggregation kinetics, both full-length peptides trigger complement activation to

practically the same extent suggesting that other factors besides the degree of

oligomerization (i.e. the secondary structure) play a role in the activation mechanism.

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DISCUSSION

Although viewed for many years as an immune-privileged organ, the CNS

contains many immune system components among them proteins of the complement

system that are synthesized by astrocytes, microglia and neurons. Whereas the pathogenic

role of complement is well recognized in systemic disorders its contribution to

neurodegenerative diseases has only recently emerged. One of these pathologic entities in

which the activation of the complement system was studied in more detail is Alzheimer’s

disease, the most common form of human dementia. Proteins of the classical pathway

and their activation fragments - namely C1q, C3b, and C4b - as well as the terminal MAC

have all been identified in senile plaques, cerebrovascular amyloid deposits, and in

association with dystrophic neurites and neurofibrillary tangles, indicating that the

complete cascade can be fully triggered in vivo (4,5,23,32-35). Of note, the presence of

C5b-9 was demonstrated in AD but not in non-demented elderly control cortices (36),

suggesting that complement-induced injury and the chronic inflammation resulting from

the system activation may be at least partially responsible for the progression of the

disease. Although originally not described, both mRNA and proteins of the alternative

pathway have more recently been demonstrated in AD brains together with their specific

activation fragments (37). Our data demonstrates that in other unrelated

neurodegenerative disorders resulting in dementia – namely FBD and FDD - complement

proteins of both the classical and alternative pathways co-localize with parenchymal

plaques and cerebrovascular amyloid deposits, closely resembling the findings in AD.

Complement immunoreactivity in FBD and FDD was mainly associated with Congo

Red/Thioflavin positive amyloid deposits rather than Congo Red/Thioflavin negative

parenchymal pre-amyloid lesions.

Activation of the classical complement pathway in an antibody independent

manner was demonstrated for various non-immune substances such as C-reactive protein,

serum amyloid P component, DNA (38-40), amyloid A (23), and neurofibrillary tangles

(24). Aggregated A peptides in vitro are able to directly activate the classical

complement system both in fluid phase and immobilized onto solid matrices by binding

to the recognition component C1q (23,28,29,41). Our results indicate that both ABri and

ADan peptides are also able to induce activation of the classical complement system

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through a similar mechanism. The data from the hemolytic assays, the quantitation of

activation fragments by ELISA, and the immunohistochemical analysis demonstrate that

both amyloid molecules can trigger the activation of the classical pathway and proceed to

the terminal stages with the in vitro and in vivo generation of the terminal MAC. Their

direct high affinity binding to C1q and the corresponding complexes formation achieved

under physiologic conditions strongly suggest that both peptides trigger the classical

pathway of complement activation mainly through direct interaction with the recognition

protein C1q.

Activation of the alternative pathway may be initiated by a variety of elements or

cellular surfaces including pathogenic bacteria, parasites, viruses and virus infected cells.

Different studies demonstrated some degree of in vitro activation of this pathway by A

aggregates leading to the production of the activation fragments C3b, which remained

covalently bound to the fibrillar A , as well as of the alternative pathway-specific Bb

fragment (29,42,43). The results presented here demonstrate that ABri and ADan are also

able to trigger the alternative pathway resulting in the production of Bb in comparable

levels to A 1-42. However, the classical pathway appears to be the major route of

activation of the complement system accounting for ~70-75% of the total activation as

indicated by the concentration of SC5b-9 generated under specific conditions for

alternative pathway activation. These findings coincide with previous reports for A

peptides in which 70% of the C3 convertase activity formed by incubation of aggregated

A 1-42 with NHS originated from the classical pathway (42).

The degree of oligomerization of the amyloid peptides represents an important

element in the activation of the complement system. In the case of AD, fibrillar or

aggregated A species activate complement in vitro while non-aggregated peptides do

not (28,43). Although both A 1-40 and A 1-42 are able to trigger the activation, on a

molar basis A 1-42 was found to be a more potent activator (41,43), a difference that

most likely reflects the ability of A 1-42 to aggregate more readily and at lower

concentrations than A 1-40 (44). In vivo, complement activation components co-localize

with parenchymal and vascular A amyloid deposits and are almost absent in the non-

fibrillar (pre-amyloid) lesions (3,45) and in “cotton wool” plaques seen in a variant form

of Alzheimer’s disease due to a deletion of exon 9 of presenilin 1 (46). In the case of

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FBD and FDD, components of the complement activation cascade also co-localize with

fibrillar but not with non-fibrillar deposits in vivo. In vitro, ABri and ADan peptides

form spontaneous -sheet-rich structures that exhibit very fast aggregation kinetics,

modifying their degree of oligomerization even after the short incubation time required

for the various assays. Under these conditions, both peptides achieve complement

activation values similar to those obtained with 7-day aggregated A 1-42. Although both

ABri and ADan are able to activate complement in vitro to practically the same extent,

the in vivo accumulation of activation components in FDD parenchymal lesions is

significantly lower than in FBD parenchymal deposits. This most likely reflects the fact

that FDD parenchymal lesions are mainly of pre-amyloid, non-fibrillar nature (Congo-

Red negative) and thus unable to achieve high levels of complement activation, as

demonstrated by the in vitro studies. In spite of their similarities FBD and FDD present

striking differences in their respective CNS pathology including the more severe

neocortical involvement in FDD and the nature of most of the hippocampal and

neocortical lesions showing features of amyloid in FBD (11) and of preamyloid in FDD

(10). The difference in the aggregation/fibrillization state between these two types of

lesions with the concomitant difference in their ability to activate the complement system

most likely accounts for the different topographical distribution of associated

complement proteins. Aggregated/fibrillar deposits translate in the presence of activation

derived components in association with vascular amyloid in both diseases and with FBD

parenchymal amyloid plaques, and in their absence in non-fibrillar lesions. However, the

paucity of complement-derived proteins observed in the parenchymal, preamyloid lesions

in FDD suggests that activation of the complement system may not be the only critical

factor in neurodegeneration. This is also supported by similar observations in the cotton

wool variant of familial AD, in which the characteristic morphological feature is the

presence of plaques largely composed of Congo red negative preamyloid A species

unassociated with complement activation (46-48).

The importance of inflammation in neurodegenerative processes, in particular in

Alzheimer’s disease, has become clear over the last years. The presence of dementia

invariably correlates with the detection of inflammatory markers, activated microglia and

reactive astrocytes, and increased levels of cytokines and complement products around

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amyloid plaques and dystrophic neurites (3). Epidemiological studies have found that

anti-inflammatory drugs reduce the risk of AD (49) while animal models of sustained

CNS inflammation loose cholinergic nerve cells in the hippocampus and exhibit memory

and learning impairments (50,51). These findings, together with the in vitro

demonstration that complement activation can lead to cell death in both rat hippocampal

and neuronal cell lines (52,53) point to the importance of chronic inflammation in the

pathogenesis of AD dementia. Supporting this notion, complement activation products

and inflammatory mediators have also been found in Down’s syndrome in association

with A amyloid deposits (54) and in animal models of AD (55). Immune and

inflammatory responses in the CNS are also observed in other chronic and acute

neurological conditions among them multiple sclerosis, myasthenia gravis, head trauma

and stroke, as well as in animal models of some of these disorders (3,7,56,57).

Complement activation proteins have been also demonstrated in association with Lewy

and Pick bodies in Parkinson and Pick’s disease, respectively (58,59), with dystrophic

neurites and early stage extracellular neurofibrillary tangles in the Parkinsonism-

dementia complex of Guam (60), with clusters of degenerating axons in amyotrophic

lateral sclerosis (61) as well as around AL and TTR amyloid deposits in peripheral nerves

in both acquired and hereditary neuropathy (51,62). In Creutzfeldt-Jacob and Gerstmann-

Straussler-Scheinker diseases (63), complement activation products have been found

associated with amyloid plaques, and it has been recently demonstrated that the

complement system plays a role in the early prion pathogenesis in transmissible

spongiform encephalopathies (64,65). In this case, follicular dendritic cells participate in

the prion replication before the infective agent moves through the nerves into the spinal

cord or brain stem, and finally into the brain. Mice bearing deficiencies of either one of

the early complement factors or complement receptors present significant delays in both

the onset of disease symptoms and the splenic accumulation of the pathological prion

protein following injection of infective scrapie strains indicating that activation of

complement is most likely involved in the initial trapping of prions in lymphoreticular

organs.

The activation of the complement system demonstrated in all these neurological

diseases and the concomitant generation of opsonins and anaphylotoxins that drive

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numerous inflammatory mechanisms, including scavenger cell activation, chemotaxis,

frustrated phagocytosis, and the secretion of cytokines, chemokines and reactive oxygen

and nitrogen species (1) may contribute to some of the neuropathological features of the

different disorders. Our studies suggest that the chronic inflammatory response generated

by the amyloid peptides in vivo might be a contributing (although not the solely

responsible) factor to the pathogenesis of FBD and FDD and, in more general terms, to

other neurodegenerative disorders. Therapeutics directed toward controlling complement

activation to prevent or ameliorate the course of the disease may provide a useful

approach in the management of these pathological entities.

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ACKNOWLEDGEMENTS

Supported by National Institute of Health grants AG05891, AG08721, NS38777, the

Alzheimer’s Association, the Brain Research Trust and CRDC of RF and NCMS-UCLH.

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FIGURE LEGENDS

Figure 1: Immunohistochemical identification of complement activation products in

FBD (panels A-E) and FDD (Panels F-J). In the FBD case used for illustration the age

at death was 68 years preceded by an 11-year-long period of cognitive decline. The FDD

patient, whose case is demonstrated, died at age of 43 years and had dementia in the final

3 years of life. Deposition of ABri (antibody 338) and ADan (antibody 5282) in blood

vessels and parenchymal lesions in the hippocampus of FBD and FDD cases is shown in

panels A and F, respectively. In both FBD (panels B-E) and FDD (panels G-J) the anti-

C1q, anti-C4d, anti-C5b-9 neoepitope, and anti-Bb antibodies prominently label amyloid

depositing in blood vessels, including small arteries and arterioles (arrow) as well as

capillaries (double arrows) with a staining pattern similar to that seen in ABri and ADan

immunohistochemistry. While in FBD the parenchymal amyloid lesions are also strongly

labeled with the anti-complement antibodies, in FDD the primarily preamyloid deposits

(arrowhead) are either negative or much less intense stained. Scale bar represents 100

µm on panels A-D and F-I and inserts on panels B, C, D, E (top left) and G. On panels E

and J and in the inserts of panels E (bottom right), H and I it denotes 50 µm.

Figure 2: Classical complement pathway activation by ABri and ADan peptides.

Constant volumes (10 µl) of reference NHS were separately incubated for 45 min at 37oC

with 10 µl of ABri, ADan and A 1-42 solutions containing increasing concentrations (0-

1000 µg/ml TBS) of the amyloid peptides. The serum was immediately analyzed for total

hemolytic activity (EZ diagnostic kit; Diamedix Corporation, Miami, FA) and

quantitation of complement activation products by capture ELISA (Quidel, Inc.,

Mountain View, CA). In all cases, for comparison purposes, the complement activation

induced by incubation of NHS with identical concentrations of aggregated IgG is also

shown. Panel A: Remaining complement hemolytic activity (CH50). Results are

expressed as percentage of lysis compared to controls of 100% complement activity (no

complement consumption) in which NHS was incubated with Tris-buffered saline in the

absence of amyloid peptides. The data displayed represent the mean ± SD of seven

independent experiments. Panel B: Generation of C4d. Panel C: Generation of iC3b. D.

Generation of SC5b-9. In all the ELISA studies results represent mean ± SD of three

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independent experiments. In all cases peptide final concentrations are: :0 µg/ml; : 50

µg/ml; : 200 µg/ml; :500 µg/ml

Figure 3. Activation of the alternative pathway by ABri and ADan peptides.

Panel A: Generation of Bb. To determine activation of the alternative pathway, NHS was

incubated for 45 min at 37 oC with ABri, ADan, and A 1-42 peptides at a concentration

that resulted in maximum classical pathway activation (final concentration: 500 µg/ml).

The concentration of Bb was subsequently assessed by ELISA employing Bb quantitation

kit (Quidel, Inc.) as described in Methods. For comparison purposes the levels of Bb

produced by incubation of NHS with CVF and in the absence of any activators, are also

indicated. Results show mean ± SD of three independent experiments. Panel B:

Generation of SC5b-9. To estimate the contribution of the classical and alternative

pathways to the total complement activation, ABri, ADan, and A 1-42 peptides (500

µg/ml) were incubated with NHS i) under conditions in which both pathways are active

(presence of Ca+2 and Mg+2 ions), ii) under conditions in which only the alternative

pathway may be activated (presence of Mg+2 ions/ EGTA), iii) in the presence of EDTA

in which no complement activation takes place. Additionally the peptides were incubated

in the presence Ca+2 and Mg+2 ions but substituting NHS with C1q depleted serum to

corroborate the activation induced by the alternative pathway. In all cases the

concentration of SC5b-9 produced was analyzed by ELISA. Results represent mean ± SD

of two independent experiments. : EDTA; : Ca+2 / Mg+2; : Mg+2/ EGTA; : C1q

depleted serum.

Figure 4: ABri- and ADan-C1q complexes formation. Panel A: Binding isotherm.

ELISA microtiter wells were coated with 400 ng of ABri1-34, ADan1-34 and A 1-42

peptides and incubated for 1 hour at room temperature with increasing concentrations (0-

20 nM in TBS) of C1q. Bound C1q was detected with goat polyclonal anti-C1q antiserum

(Quidel, Inc.; 1/1000 in TBST) followed by alkaline phosphatase labeled swine anti-goat

IgG (BioSource International; 1/5000 in TBST). The reaction was developed with p-

nitrophenyl phosphate and the Absorbance at 405 nm quantitated. Data represent the

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means ± SD of three independent experiments performed in duplicate. Binding data were

analyzed by non-linear regression using GraphPad Prism (GraphPad Software, Inc.).

ABri; ADan; A . Panel B: ABri-C1q and ADan-C1q complex formation. C1q was

incubated with either ABri or ADan peptides in the presence of Ca+2 and Mg+2 as

described in Material and Methods. The resulting complexes were separated by

electrophoresis on 1% agarose gels, transferred to PVDF membranes, stained with

0.125% Coomassie Blue R-250, and the bands of interest were subjected to N-terminal

sequencing. Lane 1: NHS used as a control for the electrophoretic separation on agarose;

lane 2: purified C1q; lane 3: ABri-C1q complex; lane 4: ADan-C1q complex. The amino

acid sequences yielded by the different bands are shown in one letter code. In both ABri-

C1q and ADan-C1q complexes, the N-terminal sequences corresponded to those of the A

and C chains of the C1q in addition to the N-terminal of the ABri or ADan peptides,

respectively. No sequence information was retrieved for the B chain of C1q that begins

with a blocked N-terminal glutamine.

Figure 5: Localization of the complement-activating site within ABri and ADan

molecules. To promote complement activation, NHS was incubated with increasing

concentrations (0-120 nmol/ml in TBS) of the full-length ABri and ADan amyloid

peptides, the common N-terminal 1-23 peptide, and the C-terminal fragments of both

molecules ABri 24-34 and ADan 24-34. The remaining total complement lytic activity

(CH50) was assessed as described in Figure 2A. Since the molecular mass of the

peptides corresponding to different regions of the molecules differ significantly,

identical molar concentrations of the peptides were tested. For comparison purposes, the

molar concentrations of the full-length ABri and ADan peptides employed are

equivalent to those in Figure 2A (e.g. 12, 48, and 120 nmol/ml corresponds to 50, 200,

and 500 µg/ml, respectively. : 0 nmol/ml; : 12 nmol/ml; : 48 nmol/ml; :120

nmol/ml. Results represent the mean ± SD of three independent experiments.

Figure 6: Peptide oligomerization and complement activation. Panel A: Circular

dichroism spectrometry in the far u.v. (190 to 250 nm) of ABri and ADan peptides. Both

full-length peptides exhibit similar -sheet rich structure whereas the C-terminal

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fragments ABri24-34 and ADan23-34 show random coil configuration. Dotted lines: C-

terminal fragments; solid lines: full-length peptides. Red color: ABri; Blue color: ADan.

Panels B and C: The degree of aggregation of ABri (Panel B) and ADan (Panel C)

peptides employed in the complement activation assays was assessed by Western Blot

analysis after SDS-PAGE. The synthetic peptides either immediately after solubilization

(lanes 1) or following incubation 45 min at 37oC under the same conditions employed to

determine their ability to activate the complement proteins (lanes 2) were loaded on 16%

Tris-tricine gels, transferred to Immobilon-P membranes and analyzed as described in

Methods. The fluorograms showed were developed by ECL.

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REFERENCES

1. Akiyama, H., Barger, S., Barnum, S., Bradt, B., Bauer, J., Cole, G. M., Cooper, N. R., Eikelenboom, P., Emmerling, M. R., Fiebich, B. L., Finch, C., Frautschy, S., Griffin, W. S. T., Hampel, H., Hull, M., Landreth, G., Lue, L.-F., Mrak, R. E., Mackenzie, I. R., McGeer, E. G., O'Banion, M. K., Pachter, J., Pasinetti, G. M., Plata-Salaman, C., Rogers, J., Rydel, R., Shen, Y., Streit, W., Strohmeyer, R., Tooyama, I., Van Muiswinkel, F. L., Veerhuis, R., Walker, D. G., Webster, S., Wegrzyniak, B., Wenk, G., Wyss-, and Coray, T. (2000) Neurobiol. Aging 21, 383-421

2. Eikelenboom, P., Biewenga, J., Rozemuller, M., Kraal, G., Stam, F. C., McBride, P. A., Bruce, M. E., and Frazer, H. (1991) Virchows Arch. B Cell Pathol. 60, 329

3. Emmerling, M. R., Watson, M. D., Raby, C. A., and Spiegel, K. (2000) Biochim.

Byophys. Acta 1502, 158-171

4. Eikelenboom, P., and Stam, F. C. (1982) Acta neuropathol. 57, 239

5. Berkenbosch, F., Biewenga, J., Bouns, M., Rozemuller, M., Strijbos, P., and van Dam, A. (1992) Res. Immunol. 143, 657

6. Thiel, s., Vorup-Jensen, T., Stover, C. M., Schwaeble, W., Laursen, S. B., Poulsen, K., Willis, A. C., Eggleton, P., Hansen, S., Holmskov, U., Reid, K. B., and Jensenius, J. C. (1997) Nature 386, 506-510

7. Tenner, A. J. (2001) Neurobiol. Aging 22, 849-861

8. Morgan, B. P. (2000) Complement Methods and Protocols, 2000 Ed. Methods in Molecular Biology, Humana Press, Totowa, New Jersey

9. Mead, S., James-Galton, M., Revesz, T., Doshi, R. B., Harwood, G., Pan, E. L., Ghiso, J., Frangione, B., and Plant, G. (2000) Brain 123, 975-986

10. Holton, J., Lashley, T., Ghiso, J., Braendgaard, H., Vidal, R., Guerin, C., Gibb, G., Hanger, D. P., Rostagno, A., Anderton, B., Strand, C., Ayling, H., Plant, G., Frangione, B., Bojsen-Moller, M., and Revesz, T. (2002) J. Neuropathol. Exp.

Neurol. 61, 254-267

11. Holton, J., Ghiso, J., Lashley, T., Rostagno, A., Guerin, C., Gibb, G., Houlden, H., Ayling, H., Martinian, L., Anderton, B., Wood, N., Vidal, R., Plant, G., Frangione, B., and Revesz, T. (2001) Am. J. Pathology 158, 515-526

12. Vidal, R., Frangione, B., Rostagno, A., Mead, S., Revesz, T., Plant, G., and Ghiso, J. (1999) Nature 399, 776-781

13. Ghiso, J., Revesz, T., Holton, J., Rostagno, A., Lashley, T., Houlden, H., Gibb, G., Anderton, B., Bek, T., Bojsen-Moller, M., Wood, N., Vidal, R., Braendgaard, H., Plant, G., and Frangione, B. (2001) Amyloid: J. Protein Folding Disord. 8, 277-284

- 28 -

by guest on Decem

ber 11, 2020http://w

ww

.jbc.org/D

ownloaded from

14. Vidal, R., Revesz, T., Rostagno, A., Kim, E., Holton, J., Bek, T., Bojsen-Moller, M., Braendgaard, H., Plant, G., and Frangione, B. (2000) Proc. Natl. Acad. Sci. USA 97, 4920-4925

15. Saido, T., Yamao-Harigaya, W., Iwatsubo, T., and Kawashima, S. (1996) Neuroscience Letters 13, 173-176

16. Mori, H., Takio, k., Ogawara, M., and Selkoe, D. (1992) J. Biol. Chem. 267, 17082-17086

17. Tekirian, T., Saido, T., Markesbery, W., Russell, M., Wekstein, D., Patel, E., and Geddes, J. (1998) J. Neuropathol. Exp. Neurol. 57, 76-94

18. Matsubara, E., Frangione, B., and Ghiso, J. (1995) J. Biol. Chem. 270, 7563-7567

19. Miravalle, L., Tokuda, T., Chiarle, R., Giaccone, G., Bugiani, O., Tagliavini, F., Frangione, B., and Ghiso, J. (2000) J. Biol. Chem. 275, 27110-27116

20. Salvarrey, M., and Rostagno, A. (1989) Clin. Exp. Immunol. 76, 92-96

21. Wener, M. H. (1997) in Manual of Clinical Laboratory Immunolology (Rose, N. R., Conway de Macario, E., Folds, J. D., Lane, H. C., and Nakamura, R. M., eds), 1997 Ed., pp. 208-216, American Society of Microbiology, Washington, D. C.

22. Parker, C. J. (1999) in Current Protocols in Immunology (Cologan, J. E., Kruisbeek, A. M., Margulies, D. H., Shevach, E. M., and Strober, W., eds) Vol. 3, pp. 13.15.17-13.15.10, 3 vols., John Wiley and Sons, Inc., New York, NY

23. Rogers, J., Cooper, N. R., Webster, S., Schultz, J., McGeer, P. L., Styren, S. D., Civin, W. H., Brachova, L., Bradt, B., Ward, P., and Lieberburg, I. (1992) Proc. Natl.

Acad. Sci. USA 89, 10016

24. Shen, Y., Lue, L.-F., Yang, L.-B., Roher, A., Kuo, Y.-M., Strohmeyer, R., Goux, W. J., Lee, V., johnson, G. V. W., Webster, S., Cooper, N. R., Bradt, B., and Rogers, J. (2001) Neuroscience Letters 305, 165-168

25. Nagasawa, S., Ichihara, D., and Stroud, R. M. (1980) J. Immunol. 125, 578

26. Ross, G. D., and Medof, M. E. (1985) Adv. Immunol. 37, 217

27. Weiler, J. M., Daha, M. R., Austen, K. F., and Fearon, D. T. (1976) Proc. Natl. Acad.

Sci. USA 73, 3268

28. Webster, S., Bradt, B., Rogers, J., and Cooper, N. (1997) J. Neurochem. 69, 388-398

29. Bradt, B., Kolb, W. P., and Cooper, N. R. (1998) J. Exp. Med. 188, 431-438

- 29 -

by guest on Decem

ber 11, 2020http://w

ww

.jbc.org/D

ownloaded from

30. Rabson, A. R. (1997) in Manual of Clinical Laboratory Immunology (Rose, N. R., Conway de Macario, E., Folds, J. D., Lane, H. C., and Nakamura, R. M., eds), 1997 Ed., pp. 187-191, American Society of Microbiology, Washington, D. C.

31. Narkio-Makela, M., Hellwage, J., TAhkokallio, O., and Meri, S. (2001) Clin.

Immunol. 100, 118-126

32. Ishii, T., and Haga, S. (1984) Acta neuropathol. 63, 296

33. McGeer, P. L., Akiyama, H., Itagaki, S., and McGeer, E. G. (1989) Can. J. Neurol.

Sci. 16, 516

34. Rozemuller, M., Bots, G. T., Roos, R. A., and Eikelenboom, P. (1992) Neurosc. Lett. 140, 137-140

35. Verbeek, M. M., Otte-Holler, I., Veerhuis, R., and Ruiter, D. J. (1998) Acta

Neuropathol. 96, 628-636

36. Webster, S., Lue, L.-F., Brachova, L., Tenner, A. J., McGeer, E. G., Terai, K., Walker, D. G., Bradt, B., Cooper, N. R., and Rogers, J. (1997) Neurobiol. Aging 18, 415-421

37. Strohmeyer, R., Shen, Y., and Rogers, J. (2000) Molec. Brain Res. 81, 7-18

38. Jiang, H., Cooper, B., Robey, F. S., and Gewurz, H. (1992) J. Biol. Chem. 267, 25597

39. Jiang, H., Robey, F. S., and Gewurz, H. (1992) J. Exp. Med. 175, 1373

40. Ying, S.-C., Gewurz, A. T., Jiang, H., and Gewurz, H. (1993) J. Immunol. 150, 169

41. Jiang, H., Burdick, D., Glabe, C. G., Cotman, C. W., and Tenner, A. J. (1994) J.

Immunol. 152

42. Emmerling, M. R., Spiegel, K., and Watson, M. D. (1997) Immunopharmacology 38, 101-109

43. Watson, M. D., Roher, A., Kim, K. S., Spiegel, K., and Emmerling, M. R. (1997) Amyloid: J. Protein Folding Disord. 4, 147-156

44. Jarrett, J. T., Berger, E. P., and Lansbury, P. T. (1993) Ann. New York Acad. Sci. 695, 144-148

45. Afgah, A., Cummings, B. J., Cribbs, D. H., Cotman, C. W., and Tenner, A. J. (1996) Exp. Neurol. 138, 22-32

46. Crook, R., Verkkoniemi, A., Perez-Tur, J., Mehta, N., Baker, M., Houlden, H., Farrer, M., Hutton, M., Lincoln, S., Hardy, J., Gwinn, K., Somer, M., Paetau, A., Kalimo, H., Ylikoski, R., Poyhonen, M., Kucera, S., and Haltia, M. (1998) Nature Med. 4, 452-455

- 30 -

by guest on Decem

ber 11, 2020http://w

ww

.jbc.org/D

ownloaded from

47. Steiner, H., Revesz, T., Neumann, M., Romig, H., Grim, M. G., Pesold, B., Kretzschmar, H. A., Hardy, J., Holton, J., Baumeister, R., Houlden, H., and Haass, C. (2001) J. Biol. Chem. 276, 7233-7239

48. Houlden, H., Baker, M., McGowan, E., Lewis, P., Hutton, M., Crook, R., Wood, N., Kumar-Singh, S., Geddes, J., Swash, M., Scaravilli, F., Holton, J., Lashley, T., Tomita, M., Hashimoto, T., Verkkoniemi, A., Kalimo, H., Somer, M., Paetau, A., Martin, J. J., Van Broeckhoven, C., Golde, T. E., Hardy, J., Haltia, M., and Revesz, T. (2000) Ann. Neurol. 48, 806-808

49. Rogers, J., Kirby, L. C., Hempelman, S. R., Berry, D. L., McGeer, P. L., Kaszniak, A. W., Zalinski, J., Cofield, M., Mansukhani, L., and Willson, P. (1993) Neurology 43, 1609-1611

50. Hauss-Wegrzyniak, B., Dobrzanski, P., Stoehr, J. D., and Wenk, G. (1998) Brain Res. 780, 294-303

51. Heyser, C. J., Masliah, E., Samimi, A., Campbell, I. L., and Gold, L. H. (1997) Proc.

Natl. Acad. Sci. USA 94, 1500-1505

52. Schultz, J., Schaller, J., McKinley, M., Bradt, B., Cooper, N., May, P., and Rogers, J. (1994) Neurosc. Lett. 175, 99-102

53. Shen, Y., Halperin, J. A., and Lee, C.-M. (1995) Brain Res. 671, 282-292

54. Stoltzner, S. E., Grenfell, T. J., Mori, C., Wisniewski, K. E., Wisniewski, T. M., Selkoe, D., and Lemere, C. A. (2000) Am. J. Pathology 156, 489-499

55. Matsuoka, Y., Picciano, M., Malester, B., LaFrancois, J., Zehr, C., Daeschner, J.-A. M., Olschowka, J. A., Fonseca, M. I., O'Banion, M. K., Tenner, A. J., Lemere, C. A., and Duff, K. (2001) Am. J. Pathology 158, 1345-1354

56. Spiegel, K., Barnum, S., and Emmerling, M. R. (1997) (Wood, P. L., ed), pp. 129-176, Humana Press, Totowa, NJ

57. Gasque, P., Dean, Y. D., McGreal, E. P., VanBeek, J., and Morgan, B. P. (2000) Immunopharmacology 49, 171-186

58. Iseki, E., Marui, W., Akiyama, H., Ueda, K., and Kosaka, K. (2000) Neurosc. Lett. 286, 69-73

59. Yasuhara, O., Aimi, Y., McGeer, E. G., and McGeer, P. L. (1994) Brain Res. 652, 346-349

60. Schwab, C., Steele, J. C., and McGeer, P. L. (1997) Acta Neuropathol. 94, 486-492

61. Kawamata, T., Akiyama, H., Yamada, T., and McGeer, P. L. (1992) Am. J. Pathology 140, 691-707

- 31 -

by guest on Decem

ber 11, 2020http://w

ww

.jbc.org/D

ownloaded from

62. Haffer-Macko, C., Dyck, P., and Koski, C. (2000) J. Peripheral Nervous System 5, 131-139

63. Ishii, T., Haga, S., Yagishita, S., and Tateishi, J. (1984) Appl. Pathol. 2, 370-379

64. Mabbott, N. A., Bruce, M. E., Botto, M., Walport, M. J., and Pepys, M. B. (2001) Nature Med. 7, 485-487

65. Klein, M. A., Kaeser, P. S., Schwarz, P., Weyd, H., Xenarios, I., Zinkernagel, R. M., Carroll, M. C., Verbeek, J. S., Botto, M., Walport, M. J., Molina, H., Kalinke, U., Acha-Orbea, H., and Aguzzi, A. (2001) Nature Medicine 7, 488-492

- 32 -

by guest on Decem

ber 11, 2020http://w

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.jbc.org/D

ownloaded from

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Table I: List of the antibodies used for immunohistochemical studies

Antibody Dilution Species Paraffin/frozen sections

& pretreatment

Source

5282 (anti-ADan) 1:1000 Rabbit anti-human

Paraffin Formic Acid 99% for 10 minutes

Reference 14

338 (anti-ABri) 1:2000 Rabbit anti-human

Paraffin Formic Acid 99% for 10 minutes

Reference 12

anti-C1q 1:100 Rabbit anti-human

Paraffin Trypsin 15 minutes

Dako

anti-C4d 1:1000 Mouse anti-human

Frozen section fixed in acetone Quidel

anti-C5b-9 1:50 Rabbit anti-human

Frozen section fixed in acetone Quidel

anti-Bb 1:50 Mouse anti-human

Frozen section fixed in acetone Quidel

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F

G

A

B

C

D

E

H

I

J

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0

20

40

60

80

100

ABri ADan A 42 aggIgG

Rem

ain

ing C

H50

(%

of

contr

ol)

A

0

10

20

30

ABri ADan A 42 aggIgGC

4d

ge

ne

rati

on

(g/m

l)

B

0

100

200

300

400

ABri ADan A 42 aggIgG

C3

bi

ge

ne

rati

on

(g/m

l)

C

0

5

10

15

20

ABri ADan A 42 aggIgG

SC

5b

-9 g

en

era

tio

n(

g/m

l)

D

Figure 2

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A

B

ABri ADan Aβ1−420

5

10

SC

5b

-9 g

e

(g

/ml)

15

20

ne

rate

d

50

100

150

200B

b g

en

era

ted

(g

/ml)

0ADan Aβ1-42Buffer ABri CVF

Figure 3

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A

0 5 10 15 200.0

0.2

0.4

0.6

0.8

1.0

Kd ABri = 1.9± 0.4 nΜKd ADan = 1.2± 0.3nΜKd Aβ42 = 2.4± 0.5 nΜ

C1q added (nM)

A 4

05 n

m

B

EDLXRAPDGK

NTGXYGIPGM

EASNXFAIXX ABri

1

1

C1q (A and C chains)

EDLXRAPDGK

NTGXYGIPGM

EASNXFAIRX ADan

1

1

1 2 3 4

C1q (A and C chains)

Figure 4

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������������������������������������������������������������������������

������������������������������������������������������������������������

������������������������������������������������������������������������

ABri ADan ABri1-23ABri-Ct ADan-Ct

0

20

40

60

80

100

120

Rem

ain

ing C

H50

(%

of contr

ol)

Figure 5

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A

-50

20

-40

-20

0

190 250200 220 240

CD

[m

deg

]

Wavelength [nm]

1 2

3

6.5

14

21.5

30

46

B1 2

3

6.5

14

21.5

30

46

C

Figure 6

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C1q

C4d

Alzheimer

C5b-9

Bb

Aβ

C1q

Normal

C4d

C5b-9

Bb

Antibodies to C1q, C4d, C5b-9 and Bb label Aβ-positive lesions in Alzheimer’s disease, but not in normal controls. Scale bar on panels A, B and E-H represents 100µm, while on panels C, D and I it represent 200 µm.

A

B

C

D

E

F

G

H

I

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Frangione and Jorge GhisoMagnotti, Hans Braendgaard, Gordon Plant, Marie Bojsen-Moller, Janice Holton, Blas

Agueda Rostagno, Tamas Revesz, Tammaryn Lashley, Yasushi Tomidokoro, Lauradisease

Complement activation in chromosome 13 dementias: Similarities with Alzheimer's

published online October 17, 2002J. Biol. Chem. 

  10.1074/jbc.M206448200Access the most updated version of this article at doi:

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