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Frameshifted APP (APP+1) is a secretory protein and the level of

APP+1 in cerebrospinal fluid is linked to Alzheimer pathology

Elly M. Hol1, Renske van Dijk1, Lisya Gerez2, Jacqueline A. Sluijs1, Barbara Hobo1, Martijn

T. Tonk1, Annett de Haan2, Wouter Kamphorst3, David F. Fischer1, Rob Benne2 and Fred W.

van Leeuwen1.

1Graduate School for Neurosciences Amsterdam, Netherlands Institute for Brain Research,

2Department of Biochemistry, Academic Medical Centre, University of Amsterdam and

3Department of Pathology, Free University Hospital, Amsterdam, The Netherlands.

Correspondence should be addressed to:

Elly M. Hol, PhD

Netherlands Institute for Brain Research

Meibergdreef 33, 1105 AZ Amsterdam,

The Netherlands

e-mail: e.hol@nih.knaw.nl tel: +31-20-5665503 fax: +31-20-6961006

Running title: APP+1 and Neurofibrillary Pathology

Copyright 2003 by The American Society for Biochemistry and Molecular Biology, Inc.

JBC Papers in Press. Published on August 4, 2003 as Manuscript M302295200 by guest on January 31, 2018

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Summary

Molecular misreading of the β-amyloid precursor protein (APP) gene generates mRNA with

dinucleotide deletions in GAGAG-motifs. The resulting truncated and partly frameshifted

APP protein (APP+1) accumulates in the dystrophic neurites and the neurofibrillary tangles in

the cortex and hippocampus of Alzheimer patients. In contrast, we here show that neuronal

cells transfected with APP+1 proficiently secreted APP+1. Since various secretory APP

isoforms are present in cerebrospinal fluid (CSF), this study aimed to determine whether

APP+1 is also a secretory protein that can be detected in CSF. Post-mortem CSF was obtained

at autopsy from 52 non-demented controls and 122 Alzheimer patients, all subjects were

staged for neuropathology (Braak score). Unexpectedly, we found that the APP+1 level in the

CSF of non-demented controls was much higher (1.75 ng/ml) than in CSF of AD patients

(0.51 ng/ml) (p<0.001) and that the level of APP+1 in CSF was inversely correlated with the

severity of the neuropathology. Moreover the earliest neuropathological changes are already

reflected in a significant decrease of the APP+1 level in CSF. These data show that APP+1 is

normally secreted by neurons, preventing intra-neuronal accumulation of APP+1 in brains of

non-demented controls without neurofibrillary pathology.

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Introduction

Alzheimer’s disease (AD) is a progressive neurodegenerative disease and is the most

common form of dementia in aged populations (1). The major constituent of the extra-cellular

plaques in the brains of AD patients is amyloid β (Aβ), which is cleaved from the β-amyloid

precursor protein (APP) by β and γ secretases. Patients suffering from the hereditary forms of

AD either carry a mutation in the APP gene or in one of the presenilin genes. These

mutations cause an alteration in the proteolytic processing of APP, resulting in the formation

of more Aβ40 and Aβ42, which are both prone to aggregate and precipitate in the plaques

(2,3).

Genomic APP mutations are not the only source of aberrant APP proteins in AD. We

(4) and others (5) have reported that small frameshift mutations occur in APP transcripts near

short simple repeats. The observed dinucleotide deletions, such as ∆GA, in the GAGAG

motif of exon 9 or 10 of APP result in the translation of an aberrant APP protein, i.e. APP+1,

which accumulates in the neurofibrillary tangles, neuropil threads and dystrophic neurites of

the neuritic plaques of AD and Down syndrome patients (4,6). APP+1 is a truncated APP

protein of 348 amino acids (AA) with a wild-type N-terminus and an aberrant C-terminus

translated in the +1 reading frame.

The different isoforms of full-length APP are type I transmembrane proteins, which

are proteolytically cleaved by secretases (7,8) to form secretory APPs and Aβ40 or Aβ42 and

p3 (9,10). These secretory APP proteins, sAPPα and sAPPβ (11-13), and Aβ (14) are

detectable in cerebrospinal fluid (CSF) as well as in human brain homogenates (15). The

present study focuses on APP+1 that consists of the N-terminal 329 AA of the APP695,

including the signal peptide, and a unique 19 AA C-terminus (Fig. 1). A major difference

between APP+1 and APP, except for their distinct C-termini, is the lack of the membrane

anchor (AA 625-648 of APP695) and the lack of the Αβ sequence in APP+1. The presence of

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the signal peptide and the absence of the membrane anchor make it very likely that APP+1 is

also a secretory protein. Indeed, it has been shown that an APP+1–enhanced green fluorescent

protein fusion protein is readily secreted from rat neuronal cell lines (16). The aim of the

present study was to determine whether the endogenous human APP+1 is a secretory protein

and can be detected in human CSF.

FIGURE 1

Experimental procedures

Antibodies

The AMY6 peptide (YNVPGHERMGRGRTSSKELA) represents the final 19 aminoacids of

APP+1 ∆GA exon 9 (see Fig. 1). The peptide was coupled to thyroglobulin by glutaraldehyde

and a rabbit was immunized with a mixture of the coupled peptide and complete Freunds

adjuvans (1:1). Several bleedings were collected and the immunoreactivity of the serum was

tested with a spot blot. To the N-terminus of the AMY6 peptide an additional tyrosine (Y)

was added to allow iodination. 22C11 is a monoclonal antibody directed against amino acids

66 to 81 of the N-terminus of APP (17). Antibody specificity was tested by staining

recombinant APP+1 protein on a Western blot with pre-immune serum, the AMY6 antibody

and pre-adsorbed AMY6 antibody.

Cell lines and transfections

The human neuronal SH-SY5Y cell line (ATCC: CRL-2260) (18) was cultured in high-

glucose Dulbecco's Modified Eagle Medium supplemented with 10% v/v fetal calf serum,

100 IU/ml penicillin and 100 µg/ml streptomycin (all media and chemicals from Life

Technologies, Grand Island, NY). For Western blot analysis the cells were seeded in 6 cm

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dishes (Nunc, Roskilde, Denmark). The next day cells were transfected with 10 µg of plasmid

DNA per 6 cm dish, according to the calcium-phosphate transfection method. The human

APP 695 isoform (kindly provided by Dr. T. Hartmann, Heidelberg, Germany) and APP+1

(APP 695 ∆GA exon 9) were cloned into pcDNA3 (Invitrogen, Carlsbad, CA, USA). The full

sequence of both constructs was confirmed by sequencing.

Human cerebrospinal fluid

Post-mortem non-haemolytic ventricular cerebrospinal fluid (CSF) was obtained from 50

non-demented controls and 122 neuropathologically confirmed AD patients. In addition,

brain homogenates from 4 non-demented controls and 8 AD patients were obtained. The CSF

samples and brain tissues were collected by the Netherlands Brain Bank, Amsterdam

(coordinator Dr. R. Ravid). Sex, age, brain weight, post-mortem delay, pH of CSF and

clinico-pathological data of the patients described in this study can be found on our web site:

http://www.nih.knaw.nl/ (See enclosed additional table). The concentration of protein content

of the CSF was determined with a Bradford assay (19).

Recombinant His-tagged APP+1

Full length human APP+1 cDNA was cloned into pQE31 (Qiagen, Hilden Germany). Xl1blue

E. coli were transformed with pQE31-APP+1 and recombinant protein production was induced

with 1 mM IPTG. The recombinant 6x His-tagged APP+1 was purified over a Ni-NTA column

and used as a positive control on the Western blot and for determining the specificity of the

APP+1 radio-immunoassay (see below).

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Western blotting

Cell lines: Approximately 24 h after transfection the medium was collected and the cells were

resuspended in 0.1 M NaCl, 0.01 M Tris-HCl, 1 mM EDTA pH 7.6, containing the protease

inhibitors Phenylmethylsulfonyl fluoride (PMSF; 100 µM) and leupeptin (10 µg/ml). Human

post-mortem CSF: 15 µl CSF was directly mixed with 15 µl loading buffer (50 mM Tris-HCl

pH 6.8, 100 mM DTT, 2% SDS, 0.1% bromo-phenol-blue, 10% glycerol) and put on gel. Cell

culture medium, cell lysates and CSF were fractionated on SDS-PAGE (7.5% gel) and

transferred semi-dry to nitrocellulose (Protran BA85 Schleicher & Schuell). The blots were

incubated overnight at 4°C with a polyclonal rabbit anti-human APP+1 (AMY6 bleeding

050897) 1:1000 in supermix (50 mM Tris, 2.5 g/l gelatin, 0.15 M NaCl 0.5% Triton-X-100,

pH 7.4) or 22C11 (17) 1:100 in supermix. Subsequently, the blots were washed 3x in Tris-

buffered saline-Tween 20 (TBS-T, pH 7.5; 65 mM Tris, 150 mM NaCl, 0.05% Tween 20)

and incubated with anti-rabbit HRP (DAKO; 1:1000 in supermix) or anti-mouse HRP

(DAKO; 1:1000 in supermix) at room temperature for 1 hr. After three final washings in

TBS-T the labelled proteins were visualized with Lumi-Light ECL (Boehringer, Mannheim).

Liquid phase pre-adsorption of the AMY6 antibody (1:1000 in supermix) was performed by

adding an excess of AMY6 peptide. Prestained molecular weight markers used are

MultiMark® Multi-Colored Standard of Invitrogen and Rainbow Marker of Amersham

Biosciences.

Radioimmunoassay

Fifty µl of cerebrospinal fluid was directly measured in the radio-immunoassay (RIA) in

duplicate. Standard peptides and samples were diluted in RIA buffer (50 mM Tris, 140 mM

NaCl, 10 mM EDTA and 10 g/l BSA, pH 8.0). Fifty µl standard or sample was incubated at

4°C for 48 h with 50 µl antiserum (AMY6 1:10,000) and 10 µl (=10,000 cpm) 125I- AMY6

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peptide. AMY6 peptide was iodinated by the chloramine T method. To precipitate the

antibody peptide/protein complex, 50 µl cellulose coated with a secondary antibody against

rabbit IgG’s (Saccel; IDS LTD, Boldon, England) was added and incubated at 4°C for 1 h.

The samples were centrifuged at 5,000 rpm for 15 min and the pellets were counted in a

COBRA-γ-counter for 5 min. The sensitivity of the RIA is 15 pg APP+1/50 µl.

Characteristics of APP+1 RIA

The AMY6 peptide was iodinated and subsequently purified on a sepharose G25 column to

separate free 125I-Na, non-iodinated AMY6 and 125I-AMY6. The binding capacity of five

different bleedings of the AMY6 antibody and the pre-immune serum to 125I-AMY6 was

tested. A maximum binding of 80% could be reached with an antibody dilution of 1:1000. A

clear increase in binding capacity was observed between the first bleeding (170697) and the

later bleedings of the AMY6 serum. Pre-immune serum did not bind to 125I-AMY6 peptide at

all. All subsequent RIAs were done using AMY6 bleeding 050897.

The optimal antibody-peptide binding for the APP+1 RIA was reached with AMY6

bleeding 050897 at a dilution of 1:20,000. We performed displacement curves by adding an

increasing amount of non-labelled AMY6 peptide or recombinant 6xHis-APP+1 to AMY6

(dilution 1:20,000, bleeding 050897; results not shown). The slope of the graphs obtained

with peptide and the recombinant protein are identical, indicating that this assays reliably can

measure full length APP+1. In our subsequent experiments we used the AMY6 peptide in the

standard curves of the RIA.

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Statistical Analysis

The Kruskal-Wallis nonparametric analysis of variance with multiple comparison of groups

(20) was used to test differences between groups (program developed by J.M. Ruijter (Dept.

of Anatomy and Embryology, Academic Medical Centre, Amsterdam).

Results

APP+1 is secreted by human neuronal cells

To determine whether APP+1 is a secretory protein, like sAPPα and sAPPβ, we transfected

human neuronal SH-SY5Y cells with the APP and APP+1 pcDNA3 constructs. The

expression of APP and APP+1 was driven by the CMV promotor to ensure high expression.

The cells and their supernatants were harvested one day after transfection. Western blots of

the cell pellets and the supernatants were probed with the 22C11 antibody (Fig. 2A), directed

against residues 66 to 81 of APP, as well as APP+1. The AMY6 antibody (Fig. 2B), directed

against the unique C-terminus of APP+1, was used to detect specifically APP+1. The Western

blot analysis revealed the presence of both APP (#) and APP+1 (*) in the cell lysate (Fig. 2A:

lys) and in the cell culture medium (Fig. 2A: med) with the 22C11 antibody. In addition,

endogenous APP is visible in all transfection conditions (arrow heads). A second blot of the

same experiment was probed with APP+1 specific antibody AMY6 and showed only staining

of the APP+1 proteins (*) in the cell lysate (Fig. 2B: lys) and in the cell culture medium (Fig.

2B: med). Recombinant-APP (from the transfected cells, Fig. 2A #), endogenous APP and

sAPP (Fig. 2A, arrow heads) stained with 22C11 and not at all with the APP+1 specific

antibody AMY6. The intracellular APP+1 is present in two forms, probably reflecting the

non-glycosylated and an O-glycosylated form of APP+1. Such a doublet has also been

observed by others for APP695, and they also argue that the doublet consists of an O-

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glycosylated and non-O-glycosylated form (21). In addition, Hersberger et al. (16) have also

discussed the O-glycosylation of APP+1. Only the higher band of APP+1 is present in the cell

culture medium, which is likely to be the fully processed, glycosylated form of APP+1. This

band does not run exactly at the same height as the higher APP+1 band in the cell pellet, due to

the presence of albumin in the cell culture medium. In the lanes of the mock transfected cells,

as well as the APP transfected cells, two bands, that represent proteins with an apparent

weight between 50 and 60 kD, reacted with the 22C11 antibody. These proteins might be

degradation products of APP or N-terminal fragments. Since AMY6 does not react with these

bands, it is excluded that these proteins do represent endogenous APP+1.

FIGURE 2

Quantification of APP+1

The radioimmunoassay (RIA) developed to measure APP+1 in CSF was validated by

analyzing temporal and frontal cortex homogenates of 4 non-demented controls and 8 AD

patients (Table 1). The latter have a confirmed GA-deletion in either exon 9 or exon 10 in part

of the APP transcripts (4). In the cortex homogenates of AD patients a 3.4-fold increase in

intracellular APP+1 levels could be measured compared with the non-demented control group

(Fig. 3A). To establish the presence of APP+1 in CSF and to determine the differences

between non-demented controls and AD patients, we assayed post-mortem ventricular CSF

collected by the Netherlands Brain Bank. In contrast with the data on the cortex

homogenates, we measured significantly lower levels of APP+1 in CSF samples of AD

patients (Fig. 3B). CSF of non-demented controls contained 3.4 times more APP+1. With

respect to the age of the subject and pH of the CSF there was no significant difference

between the control subjects and the Alzheimer patients. The value of the pH of CSF is an

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indication for the agonal state of the patients (22,23). A significant difference between the

groups was found in the post-mortem delay, however no correlation was found between post-

mortem delay and APP+1 levels or protein levels. Furthermore, a significant decrease in brain

weight of AD patients, which is an inevitable characteristic of the disorder, as well as a

significant decrease in CSF total protein content were observed (Table 2).

FIGURE 3

Western analysis of APP+1 in CSF

The nature of the APP+1 immunoreactivity in CSF was determined by a Western blot. First,

we determined whether the AMY6 antibody, which is directed against the AMY6 peptide

will stain purified 6xHis-APP+1 on a Western blot. This protein is produced in a prokaryotic

expression system and therefore no glycosylation of APP+1 takes place. Furthermore, 6

histidines are added to the N-terminus and the signal peptide sequence is not cleaved off from

6xHis-APP+1. Consequently, 6xHis-APP+1 (lane 3, fig 4A) will run at a slightly higher

molecular weight than the secreted endogenous APP+1 (~ 50-60 kD band indicated with an

asterisk; lane 2 Fig 4B). The same blot probed with a buffer without the first antibody or with

pre-adsorbed AMY6 showed no signal at all (Fig. 4A, lane 1 and 2). In human CSF of a non-

demented control (NBB92-030, female, 78 year-old, ApoE 33) a banding pattern was

observed after staining with the AMY6 antibody (Fig. 4B, lane 2). The band of ~ 50-60 kD

(asterisk) is most likely the APP+1 band, since this band has been significantly reduced in the

blot probed with pre-adsorbed antibody (Fig. 4B, lane 1). In agreement with the findings in

the culture medium of the APP+1 transfected SH-SY5Y cells, only the fully processed form of

APP+1 protein is secreted. Figure 4C provides a direct comparison between 6xHis-APP+1,

APP+1 in lysate and APP+1 in cell culture medium of APP+1 transfected cells. On the same gel

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the two different pre-stained molecular weight markers used in the study were loaded, i.e. in

the first lane the Multimark® Multi-Colored Standard of Invitrogen and in the last lane the

Rainbow marker of Amersham. It is clear from this blot that the several different forms of

APP+1 all have an apparent molecular weight between 50 and 60 kD.

FIGURE 4 APP+1 levels in CSF and association with neuropathology

The analysis of APP+1 in CSF showed a particularly striking inverse correlation between the

concentration of APP+1 in the CSF and the neuropathological stage of the patients, i.e. the

Braak stages (24). Braak staging of the autopsy brain material was performed previously by

W. Kamphorst. Fig. 5A shows that a higher Braak stage, i.e. more severe cortical Alzheimer

changes, is strongly related to a decline in the amount of APP+1 in the CSF. No such

correlation was found in total protein content, although a significant decrease was measured

in the CSF samples of AD patients (Braak 4-6) compared to non-demented controls (Braak 0-

2) (Fig. 5B). After correction for protein content of the CSF, the amount of APP+1 per mg

total protein shows a rapid and significant decline in APP+1 levels between Braak stage 0 and

1, to remain constant at higher Braak stages (Fig. 5C). These findings imply that measuring

APP+1 levels in CSF indeed can be used as an ante-mortem test to diagnose early AD changes

before the onset of clinical manifestation of the disease.

FIGURE 5

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Discussion

In this study we demonstrate that the APP+1 protein is normally secreted by neuronal cell

lines transfected with APP+1. Furthermore, we show that APP+1 is present in CSF of non-

demented controls with no neuropathology and that the concentration of APP+1 is decreased in

patients with AD pathology. In earlier studies we have shown that APP+1 accumulates in the

neurons in the cortex and hippocampus of AD patients (4), indicating that the neuronal

secretion of APP+1 in these patients has stopped. This process of intraneuronal retention and

accumulation of APP+1 already starts in clinically non-demented controls with initial

neuropathology (Van Leeuwen, unpublished observations). Moreover, we show that the

concentration of APP+1 in CSF is closely related to the grade of neurofibrillary pathology in

AD patients. Even minor pathological changes are reflected in a decrease in the APP+1 level

in CSF. Hence, the APP+1 RIA can potentially be used for diagnosing AD in patients with

initial neuropathology.

APP is a type I trans-membrane protein, which is cleaved by secretases, resulting in

secretory forms of APP that are either secreted by the constitutive (25) or, as a recent study

suggests, by the regulated pathway (26). Given that APP+1 consists of the first 329 N-terminal

amino acids of APP and therefore contains the signal peptide moiety, we anticipated that

APP+1 would also be secreted by neurons and could even be detected in CSF. In the present

study, we show by transfecting human neuronal cells with APP+1 that APP+1 is indeed a

secretory protein with an apparent molecular weight between 50 and 60 kD. The calculated

molecular weight of APP+1, however, is 38 kD. Still this difference in calculated and

observed molecular weight is expected, since a similar deviant SDS-PAGE migration pattern

has been observed for the several different splice-forms of APP (27). The aspartate /

glutamate rich acidic region at amino acid position 230-260 causes the aberrant migration

pattern on the SDS-PAGE gel. In our earlier study (4) on APP+1 in human brain homogenate

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we have reported on a 38 kD APP+1 band. In this study we used a different APP+1 antibody

(AMY1) to detect the protein on a Western blot. The antibody used in the present study

(AMY6) is much more sensitive on a Western blot. By the current transfection studies we

have proven that the observation of the 38 kD band in the earlier study does not represent the

full length APP+1. It might be a C-terminal fragment of APP+1, similar to the 38 kD band in

Fig 4A and B. Despite the observation that neuronal cells transfected with APP+1, secrete

APP+1 readily we were surprised to find APP+1 in CSF of non-demented controls. Thus far

we had only observed the presence of APP+1 mRNA in hippocampus and cortex of AD and

Down syndrome patients and the APP+1 protein in the neuritic plaques and neurofibrillary

tangles of these brain areas in AD and Down syndrome patients (4,6).

The failure to detect a GA deletion in the mRNA of APP in non-demented controls

can be explained by the relatively low sensitivity of the immunoscreening assay we used in

our earlier study (4). In the cDNAs from APP mRNA isolated from cortex and hippocampus

of AD brains, we found between 2 and 12 APP+1 immunopositive clones out of 20,000. It is

therefore conceivable that we missed the mutation in the 2 non-demented control patients we

screened in that study. Recent extensive studies on the frequency of molecular misreading of

APP in cell lines and temporal cortex of non-demented control, AD and Down syndrome

patients, showed that a low frequency of GA deletions in APP mRNA, i.e. 1:100,000, occur

in all studied tissues (Gerez, de Haan and Benne, unpublished observations). Furthermore,

our group did report on the presence of dinucleotide deletions in mRNAs of ubiquitin B RNA

isolated from the cortex of elderly non-demented controls, indicating that molecular

misreading is not restricted to AD patients (4). Recent stainings of vibratome sections of

cortex and hippocampus of non-demented controls also showed that APP+1 immunoreactivity

is present in beaded fibers in these brain areas. (Van Leeuwen et al. unpublished

observations). This immunocytochemical technique is more sensitive compared to the

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staining on paraffin sections that was applied in our initial study on APP+1 (4). Moreover, with

the vibratome technique we also observed APP+1 in the neurofibrillary tangles and neuritic

fibers in plaques of clinically non-demented controls with initial neuropathology. Therefore,

the accumulation of APP+1 in the neuropathological hallmarks of AD is likely to be caused by

a deficiency in the secretion of this protein, given our present findings and the earlier

publication of Hersberger et al. (16) that APP+1 and eGFP-tagged APP+1 are secreted.

The analysis of the CSF samples of non-demented controls and AD patients showed

clearly that the APP+1 level was significantly decreased in neuropathologically confirmed AD

patients (Braak score 4-6). The RIA technique to measure APP+1 is highly specific, since there

is direct competition between endogenous APP+1 and iodinated-peptide for the same antibody.

Figure 4B shows that the APP+1 antibody used in this study, AMY6, recognized several

different proteins on Western blot, but preadsorption with the same peptide as used in the

RIA showed a diminished intensity of mainly a ~50-60 kD band, indicating that this band

likely represents the APP+1 which is measured in the RIA. The decrease in APP+1 in CSF of

AD patients supports the idea that neuropathology is preceded by a deficiency in protein

secretion by neurons. Also other hormones and secretory proteins are decreased in CSF of

AD patients, such as melatonin (28) and sAPP (12). Another explanation of the drop in the

concentration of APP+1 is, that the enlargement of the ventricles may cause a subsequent

dilution of CSF proteins, since the volume of ventricular CSF in AD patients has been shown

to be twice as much as in non-demented controls (29). Furthermore, ventricular dilatation and

reduced CSF production will presumably result in a progressive reduction in CSF turnover

during aging (30). An alternative reason for the reduced levels of APP+1 in CSF of AD

patients could, therefore, be the proteolytic breakdown of APP+1 in CSF. From the patients

we analysed, there is no information available on the degree of ventricular dilatation.

Therefore, we decided to measure the total protein content of the CSF, which will provide

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information on the dilution of CSF and therefore, these data might reflect the degree of

ventricular dilatation. As can be observed in Figure 5B, the protein content of CSF obtained

from AD patients with Braak stage 4–6 is significantly less compared to the patients with

Braak 0-2. It is also clear from figure 5 that dilution of the CSF is not the cause of the

decrease in APP+1 levels. A strong argument in favor of a specific reduction of APP+1

secretion in patients with early AD changes comes from the Braak stage 1 group. In these

controls less than half of the APP+1 levels of the Braak stage 0 group has been found (Fig.

5A), but no decline in total protein was observed (Fig. 5B). It is highly unlikely that a

ventricular dilatation by more than a factor two would already have occurred in these non-

demented controls with only very mild AD changes.

The clinical diagnosis of probable and possible AD is largely based on

neuropsychological examinations. Although the accuracy of the clinical diagnosis has

improved, a definite diagnosis still can only be made after autopsy (1). A biomarker that can

aid the clinical diagnosis, and even can detect early neuropathological changes would be

extremely valuable (31-34). In this respect, CSF markers are expected to be useful, since the

biochemical processes in the brain are likely to be reflected by the proteins that are present in

the CSF. Aβ42 and sAPP levels have often been reported to be decreased in CSF of AD

patients, while Aβ40 levels were unchanged. The former finding also indicates that there is an

initial problem with protein secretion in neurons. Another protein that plays an important role

in the pathogenesis of AD is the microtubule associated protein tau (35). The level of tau

protein is increased in CSF of AD patients (36) and patients with mild cognitive impairments

(37), probably reflecting neuronal death. Recently, it has been shown that altered tau and

Αβ42 concentration can help to diagnose AD patients in subjects with mild cognitive

impairments (38). In post-mortem ventricular CSF it has been shown before that the

melatonin concentration is closely correlated to the Braak stage. In patients with more severe

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neuropathology a lower concentration of melatonin was measured (28), which is in

agreement with our findings. In conclusion, the measurement of AD related proteins in CSF,

including APP+1, can be of great use to improve the clinical diagnostic accuracy of AD (24),

which is presently only definite after autopsy.

In this manuscript we show that APP+1 is a ~50-60 kD secretory protein. Furthermore,

we provide evidence that APP+1 is already retained in neurons in the brains of non-demented

controls with initial AD pathology. This retention probably reflects an impaired capacity of

protein secretion and other early pathological changes in affected neurons or dying neurons.

Measuring levels of secretory proteins, like APP+1, in CSF can help to reveal these early

deficits in the function of neurons. Above all, the strong correlation between APP+1 levels in

CSF and observed pathological changes could help in diagnosing AD at an early stage.

Acknowledgements

The authors like to thank Dick Swaab (Netherlands Institute for Brain Research, Amsterdam)

for helpful discussions, Marina Kahlman, José Wouda, Anne Holtrop, Michiel Kooreman and

Rivka Ravid (coordinator) of the Netherlands Brain Bank for providing the CSF and patient

information, Jan Ruijter (Dept. of Anatomy and Embryology, University of Amsterdam) for

help with the statistical analysis, Tobias Hartmann (ZMBH Heidelberg, Germany) for

providing the 22C11 antibody and APP 695 construct, The research was supported by

“Nederlandse Hersenstichting” H00.06, “Platform Alternatieven voor Dierproeven

”PAD98.19, EU grant QLRT-1999-02238, “Jan Dekkerstichting en Dr. Ludgardine

Bouwmanstiching” 99-17, “Van Leersumfonds KNAW” and Human Frontier Science

Program Organization RG0148 / 1999B.

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Figure legends

Fig. 1 - Schematic representation of the APP and APP+1 proteins. The epitopes recognized

by the several different APP antibodies used in this study are indicated. The APP+1 molecule

depicted consists of 348 AA and is the +1 protein of the 695 splicing variant, thus without the

Kunitz fragment. The distinct +1 C-terminal aminoacid sequence is indicated in italics. TM:

transmembrane domain; SP: signal peptide; 22C11 is a monoclonal APP antibody directed

against amino acid 66 to 81 (17). The antibody directed against the AMY6 peptide is used for

the RIA and Western blot. As this peptide contains an additional N-terminal tyrosine it can

easily be iodinated.

Fig. 2 – APP and APP+1 in neuronal cells. Western blot of lysates (lys) and cell culture

medium (med) of human neuronal SH-SY5Y cells transfected with pcDNA3-CMV-APP695

(APP) or pcDNA3-CMV-APP+1 (APP+1). The blots were probed with the 22C11 antibody (A)

or the AMY6 antibody (B). APP (A, #) and APP+1 (A, *) are stained with 22C11 in cell lysate

and culture medium. A specific staining for APP+1 is observed with the AMY6 antibody (B,

*). Endogenous APP and sAPP are only detected by the 22C11 staining (arrow heads).

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Fig. 3 – Quantification of APP+1. Presence of APP+1 in brain homogenate (A) and CSF (B)

of non-demented controls (open boxes) and AD patients (grey boxes) as measured with a

radioimmunoassay (RIA). The antibody used in the RIA was directed against the AMY6

epitope (Fig. 1). The box depicts the the 25th and 75th percentile values, the median value is

given by the horizontal line in the box. The whiskers range from the 10th to the 90th

percentiles. Total protein was measured with a Bradford assay (19). The amount of APP+1 is

corrected for protein content. In A: CON n=4; AD n=8; Mann-Whitney (20), # p=0.042. In B:

50 CSF samples from 22 female and 28 male non-demented controls (CON; median age: 78 )

and 122 CSF samples from 90 female and 32 male AD patients (AD, median age: 79.5) were

analysed; Significance is tested with a Mann-Whitney (20) nonparametric test, ** p<0.001.

Fig. 4 – Western blot CSF APP+1 and pre-adsorbtion. (A) 6xHis-APP+1 was detected on a

Western blot. Lane 1: no 1st antibody; lane 2: pre-adsorbed AMY6 and lane 3: AMY6

antibody. One specific band was clearly visible with an apparent molecular weight of ~ 50-60

kD. (B) Endogenous APP+1 detection in CSF with the AMY6 antibody showed a pattern of

several bands, the band assigned with the asterisk (*) is APP+1, since this band specifically

disappears in a blot probed with pre-adsorbed AMY6. (C) 6xHis-APP+1, cell lysate of APP+1

transfected SH-SY5Y cells and the culture medium of these cells were put on one gel to

compare directly the differences in molecular weight between these different APP+1 proteins.

The blot is stained with the AMY6 antibody. The left molecular weight marker is the

MultiMark® Multi-Colored Standard of Invitrogen and the right molecular weight marker is

the Rainbow marker of Amersham Biosciences.

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Fig. 5 – APP+1 and Braak staging. Absolute APP+1 concentration (A), protein content (B)

and APP+1 concentration corrected for protein content (C) in CSF of non-demented controls

(open boxes) and AD patients (grey boxes). The controls and patients were subdivided in

Braak stages (24), independently and prior to CSF detection of the APP+1 levels. The non-

demented control group consisted of 19 Braak stage 0 (no neurofibrillary changes), 19 Braak

stage 1 and 12 Braak stage 2 patients (neurofibrillary changes confined to transentorhinal

region), the AD group consisted of 19 Braak stage 4 (severe neurofibrillary changes in

entorhinal and transentorhinal regions), 56 Braak stage 5 and 47 Braak stage 6 patients

(isocortical destruction). Braak 3 patients were left out of these analyses because they already

have senile cortical changes and some of them suffer from dementia. The box depicts the 25th

and 75th percentile values, the median value is given by the horizontal line in the box. The

whiskers range from the 10th to the 90th percentiles. The statistical analysis of APP+1

concentration and protein content in CSF samples are shown in the cross-tables, Kruskal-

Wallis (20); *: p<0.05; NS: not-significant. For example: In Fig. 5A the Braak 0 group shows

a significantly higher concentration of APP+1 in CSF compared to Braak stages 4, 5 and 6. No

significant difference was found between the Braak stage 0 group and Braak stages 1 and 2.

This multiple comparison of the different Braak stages showed a significant decrease of the

absolute APP+1 concentration Braak 4, 5 and 6 (see cross-table in A) and a significant

decrease in the APP+1 levels corrected for protein content in Braak 1 through 6 (see cross-

table in C).

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Table 1 – Determination of APP+1 in brain homogenates of non-demented controls and AD

patients. More detailed information on the clinicopathological data of the patients in this

study can be found on our web-site www.nih.knaw.nl. Data are depicted as median values.

PMD = post-mortem delay

CON

AD

Mann-Whitney; p-value

Total number patients

n = 4

n = 8

male

n = 3

n = 4

female

n = 1

n = 4

age (years)

61.5

75.5

0.306

PMD (min)

392.5

217.5

0.027 *

pH

6.59

6.56

0.349

brain weight (g)

1332.5

1100

0.089

APP+1 (ng/mg protein)

0.75

2.55

0.042 *

* statistical significant difference between non-demented controls and AD patients.

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Table 2 - Determination of APP+1 in CSF of non-demented controls and AD patients. More

detailed information on the clinicopathological data of the patients in this study can be found

on our web-site www.nih.knaw.nl. Data are depicted as median values. PMD = post-mortem

delay.

CON

AD

Mann-Whitney; p-value

Total number patients

n = 50

n = 122

male

n = 28

n = 32

female

n = 22

n = 90

age (years)

78.0

79.5

0.057

PMD (min)

385

255

<0.001 *

pH

6.70

6.62

0.325

brain weight (g)

1302

1105

<0.001 *

protein (µg/ml)

439

272

<0.001 *

APP+1 (ng/ml)

1.75

0.51

<0.001 *

APP+1 (ng/mg protein)

3.34

1.95

0.003 *

* statistical significant difference between non-demented controls and AD patients.

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Clinicopathological information of the non-demented controls and Alzheimer patients

Non-demented controls (to be published on our web site: www.knaw.nl/nih)

NBB

Age

(years)

sex

apoE

Age at disease onset (years)

GDS

Braak stage

PMD

(hours)

pH

of CSF

Brain

weight (g)

Cause of death

94-119(2) 51 f 33 - na 0 07:40 7.1 1156 Sepsis 95-092 63 f 43 - na 0 06:25 6.67 1216 Mamacarcinoma, euthanasia 97-042 65 f 33 - na 1 12:50 6.94 1030 Hypoxia in combination with metabolic acidosis and

dehydration 96-013 68 f 33 - na 2 10:30 6.83 1122 Hematemesis and melena secondary to esophageal

varices amongst others 96-021 68 f 33 - na 1 05:45 6.89 1194 Unknown 95-101 73 f 33 - na 1 05:30 6.38 1304 Heart failure 94-063 74 f 33 - 1 0 05:35 7.04 982 Mesenterial thrombosis 97-156 77 f 33 - na 1 02:40 6.37 1235 Septic shock; Metastasized pancreas carcinoma 97-088 78 f 33 - na 1 04:15 6.2 1351 Pneumonia, CVA, insults 96-084 78 f 43 - na 2 07:30 6.6 1330 Terminal pulmonary emphysema 95-110 81 f 32 - na 1 22:15 6.94 1052 Coronary shock/ Pulmonal arterial Pressure 100/40

mmHg 94-057 81 f 33 - na 0 07:15 6.2 1350 Bleeding in the upper abdominal stoma area 98-016 82 f na - na 1 10:45 6.33 1078 Post PTCA complication: Haemorrhage in the groin 98-056 83 f na - na 1 23:31 7.3 1000 Euthanasia, coloncarcinoma with possible liver and brain

metastasis 94-074 85 f 33 - na 0 05:11 6.95 925 Pneumonia 96-019 86 f 43 - na 1 05:15 6.7 1196 Euthanasia 96-078 87 f 33 - na 2 08:00 6.91 1315 Cardiac failure; Cheyne Stokes breathing 97-008 88 f 33 - na 1 03:45 6.74 1159 Cachexia 93-035 89 f 33 - na 2 04:20 6.68 1152 Respiratory and heart failure 95-097 89 f 43 - na 1 06:25 7.11 1220 Aspiration pneumonia 96-044 90 f 33 - na 2 05:50 7 1101 Unknown 90-083 90 f 43 - na 1 04:30 6.7 1040 Metabolic acidosis due to insufficient cardiac output 96-132 94 f 33 - na 1 06:10 6.4 1118 Unknown 97-162 38 m 33 - na 0 10:45 6.71 1618 M. Wegener; aluminium intoxication 97-159 48 m 32 - na 0 05:30 6.88 1500 On the patient's request euthanasia was performed 98-006 50 m 43 - na 0 08:30 6.65 1436 Cardiac arrest 89-079 51 m 33 - na 0 05:15 6.67 1540 Myocardial infarction 94-125(1) 51 m 43 - na 0 06:00 6.5 1518 Progressive liposarcoma and ileus 95-007 54 m 32 - na 0 09:10 6.96 1440 Bleeding from the right artery carotis communicans 96-010 63 m 32 - na 0 10:20 6.37 1250 Acute enlargement of an old myocardial infarction in the

posterior and lateral left ventricular wall, severe lung emphysema and bronchopneumonia on both sides

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NBB

Age

(years)

sex

apoE

Age at disease onset (years)

GDS

Braak stage

PMD

(hours)

pH

of CSF

Brain

weight (g)

Cause of death

93-133 64 m 22 - na 0 08:09 6.9 1448 Leukemia, thrombocytopenia, subarachnoidal bleeding and bleeding in cerebellum

93-112 67 m 33 - na 0 09:20 7.2 1275 Heart failure as well as respiratory insufficiency 97-043 68 m 33 - na 2 10:10 7.08 1547 Heart attack 97-157 69 m 33 - na 0 05:55 6.41 1475 Serious prostate cancer with metastasis 96-129 70 m 32 - na 0 07:30 6.4 1280 Pancreas carcinoma 93-005 72 m 33 - na 0 04:30 6.83 1196 Anuria and cardiogenic shock 90-079(1) 72 m 43 - na 2 04:25 6.51 1330 Anuria with hypotensia 96-024 73 m 32 - na 1 06:00 6.44 1392 Coloncarcinoma with liver metastases 95-106 74 m 32 - na 0 08:00 6.75 1317 Heart failure due to myocardial infarction 93-071 76 m 43 - na 2 04:35 6.47 1401 Combined cardiac and respiratory insufficiency due to a

bronchus carcinoma. 94-076 78 m 33 - na 2 08:25 6.56 1442 Cardiac arrhythmia 95-093 78 m 33 - na 1 07:00 6.96 1440 Decompensatio cordis, fever as a result of pulmonary

embolism 95-098 78 m 33 - na 0 04:40 7.17 1259 Lung emphysema 97-144 78 m 43 - na 2 04:00 6.43 1160 Pulmonary carcinoma 97-025(2) 79 m 32 - na na 07:05 6.7 1158 From the bladder ascending pyelonephritis, probably

with sepsis, and cordial decompensation with a status post double CABG with old and recent myocardial infarctions.

97-143 79 m 33 - na 1 06:00 6.51 1392 Extensive metastases of known adenocarcinoma of the prostate and specifically diffuse tumour embolisms in both lungs. No indication for sepsis.

95-062 80 m 33 - na 2 04:30 6.22 1400 Renal insufficiency with metabolic acidosis and hyperkalemia

97-116 80 m 33 - na 0 06:56 6.6 1380 Respiratory insufficiency, lung emphysema 95-006 81 m 33 - na 1 08:25 6.7 1301 Cardiogenic shock 94-053 83 m 33 - na 1 08:50 6.7 1120 Decompensatio cordis 96-085 84 m 33 - na 1 09:00 6.2 1367 Heart failure by uremia 98-049 87 m na - na 2 07:25 6.8 1379 Cardiac arrest -

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Alzheimer patients NBB

Age

(years)

sex

apoE

Age at disease onset (years)

GDS

Braak stage

PMD

(hours)

pH

of CSF

Brain

weight (g)

Cause of death

91-092 54 f 33 49 6 5 03:15 6.32 1055 Cachexia 97-110 54 f 33 na 4 6 03:55 6.14 1089 Pneumonia 97-033 59 f 43 53 5 6 03:55 6.48 1074 Morphine (4 dd 30 mg i.m.) 97-048 61 f 43 55 2 5 03:45 6.45 1070 Respiratory insufficiency 95-045 62 f 44 52 7 6 05:15 6.6 913 Aspiration pneumonia 97-136 62 f 44 50 7 6 04:25 6.53 914 Dehydration 92-103 63 f 33 51 5 6 04:55 6.5 934 Myocardial infarction 93-013 63 f 33 54 7 6 03:20 6.64 980 Asystole, cachexia 95-014 66 f 44 54 7 6 03:24 7.04 825 Dehydration 95-023 66 f 44 55 7 6 04:15 6.53 1134 Pneumonia 96-109 66 f 33 54 6 6 03:35 6.42 1105 Marusmus seniles due to dementia 98-026 67 f na 61 7 5 03:30 6.73 1119 Severe deterioration secondary to meningeoma 95-087 69 f 33 62 6 6 04:00 7.28 1150 Pneumonia 94-117 71 f 33 68 7 5 04:20 6.23 1150 Cachexia and dehydration with decubitus 97-056 71 f 43 63 7 5 03:30 7.1 1024 Dehydration 93-004 72 f 44 60 7 6 04:00 6.54 1254 cachexia en dehydration 91-094 73 f 44 62 7 5 04:05 6.45 1106 Dehydration. Insufficient circulation 97-076 73 f 44 65 7 5 04:25 6.48 1073 Pneumonia (aspiration) and cardiac decompensation 93-138 74 f 43 65 0 6 04:30 6.37 1070 Pneumonia; cachexia 95-035 74 f na 67 7 5 06:30 6.6 1403 Cachexia, decubitus. 96-089 74 f 33 60 7 5 06:05 7.36 934 Heart failure 94-083 75 f 33 75 6 5 04:15 6.58 1040 Rectal bleeding, unknown 96-058 75 f 43 68 0 6 03:35 6.49 1012 Respiratory insufficiency 96-122 75 f 32 71 0 5 04:19 7.14 1270 Dehydration and cachexia, pneumonia and COPD. 97-063 75 f 44 65 7 5 05:40 6.34 1110 Aspiration pneumonia 97-125 75 f 33 74 7 4 03:25 6.59 1100 Cachexia- Pneumonia with dementia 98-007 75 f 44 64 7 6 03:50 6.55 1066 Cachexia and cardiac decompensation 95-071 76 f 43 72 6 5 04:25 6.32 1046 Septicaemia 92-100 77 f 43 72 7 5 04:12 6.43 1151 Cachexia, severe decubitus, decompensatio cordis left 92-104 77 f 44 68 6 6 04:45 6.74 1116 Unknown 93-033 77 f 44 69 7 6 03:00 6.95 1368 Decubitus/cachexia 94-014 77 f 33 na 6 5 03:35 6.67 1235 Pneumonia 95-088 77 f na 74 0 5 03:35 6.3 1220 Uraemia, decompensatio cordis 97-026 77 f 33 65 7 6 02:20 6.9 927 Dehydration 94-007 78 f 43 65 7 6 05:00 6.92 887 Pneumonia

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NBB

Age

(years)

sex

apoE

Age at disease onset (years)

GDS

Braak stage

PMD

(hours)

pH

of CSF

Brain

weight (g)

Cause of death

95-015 78 f 43 65 7 6 05:15 6.75 1005 Aspiration pneumonia 97-087 78 f 43 69 7 5 03:10 6.72 1019 Cachexia 95-053 79 f 43 73 7 6 04:10 6.13 1201 Cachexia and dehydration 96-133 79 f 44 69 7 6 04:25 6.54 934 Pneumonia, cachexia 94-002 80 f 33 71 6 6 06:40 6.16 1128 Cachexia 96-110 80 f 43 74 0 6 05:45 6.51 1195 Pneumonia 98-040 80 f na 68 0 6 05:20 6.75 1175 Infection of lungs 90-015(2) 81 f 44 75 6 na 02:53 6.6 1020 Decompensatio cordis 93-106 81 f na 69 7 5 03:00 6.75 915 Bronchopneumonia, cachexia 94-045 82 f 44 64 7 6 04:00 6.66 1110 Mors subita with unknown cause. Cachexia. 96-063 82 f 43 na 0 5 09:00 7.45 1385 Unknown 97-167 82 f 44 76 7 5 04:00 6.29 1123 Sepsis 96-070 83 f 43 75 7 5 04:00 6.73 1007 Dehydration 93-045(2) 83 f 33 69 7 na 04:55 6.47 1005 Cachexia: urinary tract infection, CVA, insufficient cordis 96-102 83 f 42 79 7 5 04:15 6.67 1122 Respiratory insufficiency 95-066 84 f 43 80 na 5 04:40 6.28 1080 Cachexia and dehydration 96-061 84 f 33 79 0 4 03:50 6.64 1196 Heart failure. 96-072 84 f 33 81 6 5 03:20 6.68 1021 Cachexia and dehydration 97-012 84 f 33 74 5 5 03:40 6.43 1218 Marasmus due to urinary tract infection among others. 97-015 85 f 43 83 7 5 03:10 6.9 1109 Bronchopneumonia 97-038 85 f 32 75 6 4 03:40 6.59 1065 Cachexia, uremic coma 97-091 85 f 43 75 7 5 02:00 7.25 1100 Cachexia and dehydration 97-112 85 f 33 76 7 5 04:35 6.82 1051 Complete deterioration 98-046 85 f na 82 6 4 02:45 6.73 1247 Infection of lungs 94-016 86 f 33 79 7 5 05:15 7 1195 Pneumonia 97-134 86 f 43 na 7 4 05:20 6.12 1136 Cachexia 92-093 87 f 33 na 0 5 02:45 6.83 1033 Exhaustion 93-030 87 f 43 82 6 6 03:00 6.5 1132 Bronchopneumonia due to influenza 93-140 87 f 33 na 6 4 04:55 7.35 1210 Urosepsis; cachexia 94-023 87 f 43 83 6 6 04:00 6.8 1048 Decompensatio cordis 95-115 87 f 43 60 0 5 06:20 6.25 1115 Acute heart failure 96-054 87 f 43 70 na 5 06:55 6.96 1010 Cardiac insufficiency 97-020 87 f 43 80 7 5 02:55 6.85 1092 Dehydration. 98-015 87 f na 71 7 6 06:15 6.84 956 Cardiac decompensation. 95-024 88 f 43 81 na 6 03:40 6.55 1144 Cardiac asthma, cardiac decompensation. 96-045 88 f 33 na 0 4 05:35 6.65 1112 Dehydration, cachexia. 97-132 88 f 33 82 7 5 03:05 6.38 1090 Dehydration with SDAT, dysphagia. 98-045 88 f na 84 7 5 03:00 6.29 1101 Cachexia, dehydration 97-009 89 f 33 83 0 5 05:55 7.95 1035 Pneumonia and cachexia

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NBB

Age

(years)

sex

apoE

Age at disease onset (years)

GDS

Braak stage

PMD

(hours)

pH

of CSF

Brain

weight (g)

Cause of death

94-006 90 f 44 78 6 5 02:35 6.93 1047 Unknown 96-006 90 f 43 85 7 5 03:10 6.99 1154 Cachexia and dehydration by influenza 96-035 90 f 33 78 7 4 04:11 6.62 1015 Cardiac decompensation 96-049 90 f 43 80 7 5 03:50 7.02 942 Dehydration 98-052 90 f na 78 4 4 22:31 6.63 1040 Dehydration and atrial fibrillation with tachycardia 96-066 91 f 43 74 7 6 04:35 6.55 949 pneumonia en cachexia 93-011 92 f 33 80 7 5 04:45 6.18 939 Uremia 93-048(1) 92 f 33 89 7 4 04:00 6.74 896 Cachexia and uremia 96-115(1) 92 f 33 85 7 5 02:50 6.64 964 Dehydration and cachexia 98-032 92 f na 90 6 4 03:50 6.96 1043 Cachexia 93-001 93 f 43 84 6 4 05:35 6.65 1194 Cachexia; dysphagia 97-071 93 f 33 88 6 5 03:00 6.62 1139 Cachexia and dehydration 95-082 94 f 43 87 7 4 05:50 6.7 1295 Breathing Depression. Cacherix dehydration. Serious

decubilus 95-096 95 f 43 83 7 6 02:50 6.87 1016 Pneumonia and abstention of therapy 94-071 96 f 33 86 7 6 05:45 6.36 949 Cachexia 97-119 96 f 33 92 6 5 03:30 6.35 997 Urinary tract infection (for two days) 93-038 97 f 42 86 7 6 03:15 6.84 952 Broncho pneumonia 97-086 101 f 33 80 7 4 04:25 6.93 1016 Mors subita as a result of cachexia and dehydration 89-057(1) 40 m 32 na na 5 02:50 6.46 1410 Unknown 90-102 49 m na 43 7 6 04:25 6.17 1426 Unknown 95-111 57 m 43 50 na 6 05:00 7 1690 Pneumonia, urosepsis 95-105 58 m 43 46 7 6 06:25 6.42 1273 Pneumonia 96-020 58 m 33 50 7 6 05:10 6.99 1290 Cachexia 92-054(1) 61 m 33 58 6 6 06:70 6.7 1180 Fever, unknown 98-011 62 m 43 55 7 5 03:30 6.5 1287 Pneumonia. 94-037 63 m 44 57 7 6 06:00 6.39 1251 Acute agranulocytosis 94-082 64 m 44 58 3 6 05:55 7.01 1218 Aspiration pneumonia 88-073(2) 66 m 33 53 7 na 03:15 6.5 1270 Cachexia and possible sepsis (decubitus) 97-124 67 m 33 59 7 5 04:20 6.62 1094 Pneumonia, cachexia, M. Alzheimer 95-069 68 m 44 60 6 6 04:15 6.65 1446 Aspiration pneumonia 95-103 69 m 44 62 7 5 02:55 7.2 1362 Bronchopneumonia. 93-047(1) 70 m 44 58 na 6 04:23 6.39 1325 Ileus picture with urinary tract infection 94-028 70 m 33 55 7 6 04:40 6.54 1267 Dehydration 93-019 72 m 43 60 na 5 05:15 6.6 1520 Pneumonia 95-077 72 m 33 64 7 5 04:45 6.75 1092 Cardiac failure 94-086 75 m 43 61 5 5 05:30 6.59 1233 Cachexia and dehydration 94-126 75 m 33 69 4 6 04:20 6.91 1390 Pneumonia. 93-026 76 m 44 73 6 6 04:55 6.55 1055 Cardiac pulmonary insufficiency

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NBB

Age

(years)

sex

apoE

Age at disease onset (years)

GDS

Braak stage

PMD

(hours)

pH

of CSF

Brain

weight (g)

Cause of death

93-034 78 m 43 na 0 4 04:30 6.64 1315 cachexia 98-022 78 m na 73 2 5 06:00 6.72 1425 Urinary tract infections, diabetes mellitus, shock 97-148 79 m 43 70 5 4 05:45 6.41 959 Pneumonia 93-087 81 m 43 75 6 5 04:10 7.08 1088 The consequences of an infection from an unknown

source 94-081 82 m 44 72 7 6 04:55 6.45 1184 Pneumonia 92-088 83 m 33 79 7 4 03:40 6.55 1378 Pneumonia with lungcarcinoma 96-097 84 m 43 76 7 6 04:35 6.67 1085 Dehydration due to swallowing problems. 94-121 85 m 33 82 4 4 06:15 6.3 1155 Kidney insufficiency and pyelonefritis. 90-117 86 m 33 76 7 5 04:10 7.41 1303 Uremia 97-093 86 m 43 81 3 5 05:35 6.39 1315 CVA / Myocardial infarction 91-086 88 m 33 84 na 4 04:41 6.38 1058 Decompensatio cordis 97-003 88 m 43 79 7 5 05:10 6.45 1044 Pneumonia 97-133 95 m 32 92 6 5 03:05 6.4 1203 Cachexia following urinary tract infection.

NBB = Netherlands Brain Bank, na = not available GDS = Global Deterioration Scale (B. Reisberg, F.H. Ferris, M.J. DeLeon and T. Crook, Am. J. Psych. 139, 1136, 1982). PTCA = Percutaneous Transluminal Coronary Angiopathy COPD = Chronic Obstructive Pulmonary Disease CVA = CerebroVascular Accident SDAT = Senile Dementia of the Alzheimer Type CABG = Coronary Artery Bypass Grafting M = Morbus (1) CSF and brain homogenate (2) Only brain homogenate

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van LeeuwenW.T. Tonk, Annett de Haan, Wouter Kamphorst, David F. Fischer, Rob Benne and Fred

Elly M. Hol, Renske van Dijk, Lisya Gerez, Jacqueline A. Sluijs, Barbara Hobo, Martijncerebrospinal fluid is linked to alzheimer pathology

in1) is a secretory protein and the level of APP+1Frameshifted APP (APP+

published online August 4, 2003J. Biol. Chem. 

  10.1074/jbc.M302295200Access the most updated version of this article at doi:

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