Proc. NatI. Acad. Sci. USA Vol. 89, pp. 2551-2555, April 1992 Medical Sciences

Decreased levels of soluble amyloid a8-protein precursor in cerebrospinal fluid of live Alzheimer disease patients (enzyme-linked immunosorbent assay/clinical dlagnoss/protease nexin 2)

WILLIAM E. VAN NOSTRAND*t, STEVEN L. WAGNER*t, W. RODMAN SHANKLE§, JEFFREY S. FARROW*4, MALCOLM DICK§, JOHANNA M. ROZEMULLER¶, MICHAEL A. KuIPERII, ERIK C. WOLTERS II, JAMES ZIMMERMAN§, CARL W. COTMAN**, AND DENNIS D. CUNNINGHAM* Departments of *Microbiology and Molecular Genetics and **Psychobiology and §Alzheimer's Disease Research Center, University of California, Irvine, CA 92717; and Departments of lNeuropathology and 'Neurology, Free University Hospital, Amsterdam, The Netherlands

Communicated by Richard F. Thompson, December 2, 1991

subjects (16, 19, 20). On the other hand, more recent reports indicated that the amounts of secreted APP in CSF were slightly decreased in AD patients (24-26). However, the measurements in those studies employed various antibodies prepared against synthetic peptides or expression products corresponding to various domains of APP. In light of these uncertainties and the importance of understanding the fate of APP in the central nervous system of live AD patients, the present study focused on quantitatively determining whether CSF levels of APP are selectively altered in these patients. We used a highly specific and sensitive monoclonal antibody (mAb) that was prepared against purified, native human PN-2[APP]. Enzyme-linked immunosorbent assays (ELISAs) revealed that the CSF levels of APP are markedly and selectively decreased in live patients with probable AD.

The amyloid 13-protein is deposited in senile ABSTRACT plaques and the cerebrovasculature in Alzheimer disease (AD). Since it is derived from proteolytic processing of its parent protein, the amyloid 13-protein precursor (APP), we investigated whether levels of the secreted forms of APP are altered in cerebrospinal fluid (CSF) of AD patients. Quantitative immunoblotting studies with the anti-APP monoclonal antibody P2-1 revealed that probable AD patients had markedly lower CSF APP levels than did demented non-Alzheimer-type patients and healthy control subjects. Using antibody P2-1 in an enzyme-linked immunosorbent assay, we measured CSF levels of APP in a larger population consisting of 13 patients diagnosed with probable AD, 18 patients diagnosed with dementia (non-Alzheimer type), and 16 nondemented, healthy controls. Mean CSF levels of APP were =3.5-fold lower in the live patients diagnosed with probable AD compared to the demented non-Alzheimer-type controls or the nondemented, healthy individuals. These findings suggest that abnormal metabolism of APP is reflected in the extracellular fluids of the central nervous system and that CSF levels of soluble APP provide a useful biochemical marker to assist in the clinical diagnosis of AD.

MATERIALS AND METHODS Materials. The anti-PN-2[APP] mouse mAb P2-1, which recognizes an amino-terminal epitope on all three major isoforms of APP, was prepared as described (14). PN-2[APP] was purified from serum-free culture medium from human foreskin fibroblasts (27). Tissue culture-treated 96-well microtiter plates were obtained from Corning. Affinity-purified goat anti-mouse IgG1 that was adsorbed with human serum was obtained from Sigma and was radiolabeled with Na125I by the chloroglycouril method (28). The anti-APP mouse mAb 22C11 (16) was purchased from Boehringer Mannheim. Patient Population. Thirteen probable AD patients, 18 demented non-Alzheimer-type patients, and 16 healthy, nondemented individuals comprised the study population. The probable AD patients, demented non-Alzheimer-type patients, and some nondemented individuals were from the AD Research Center at the University of California, Irvine. Additional samples from nondemented individuals were obtained from the Department of Neurology, Free University Hospital, Amsterdam. The probable AD group consisted of 5 men and 8 women, 53-85 years of age (mean, 69). The demented non-Alzheimer-type group consisted of 12 men and 6 women, 59-78 years of age (mean, 69). This group consisted of 14 probable vascular dementia patients, 2 patients diagnosed with frontotemporal-lobe dementia, 1 patient with alcoholic dementia, and 1 patient diagnosed as having transient global amnesia. The nondemented controls consisted of 5 men and 11 women, 29-82 years of age (mean, 62); none had

Alzheimer disease (AD) leads to a progressive and irreversible loss of memory and cognitive function in afflicted elderly individuals. One study has suggested that the disease afflicts as many as 4 million people in the United States alone (1). The hallmark event of AD is deposition of the amyloid }3-protein in senile plaques within brain parenchyma and in cerebral vessel walls of afflicted individuals (2-6). The amyloid (3-protein is a 4.2-kDa peptide proteolytically derived from a large precursor protein designated the amyloid 13-protein precursor (APP) (7-10). APPs can be translated from at least three alternatively spliced mRNAs, two of which contain an additional insert that encodes a domain homologous to Kunitztype serine protease inhibitors (11-13). The secreted form of APP that contains the Kunitz inhibitor domain is identical to the previously described protease inhibitor protease nexin 2 (PN-2) (14, 15). Soluble derivatives of APP are present in brain and cerebrospinal fluid (CSF), suggesting a normal function in the central nervous system (14, 16-20). Recent studies have suggested that abnormal proteolytic processing of APP leads to formation of the amyloid (3-protein (21-23), which in turn may contribute to the pathology of AD. Abnormal proteolysis associated with AD may, therefore, lead to altered levels of APP in the central nervous system. Previous studies suggested that CSF levels of secreted forms of APP were higher in AD patients than in control

Abbreviations: AD, Alzheimer disease; APP, amyloid a-protein precursor; PN-2, protease nexin 2; mAb, monoclonal antibody; CSF, cerebrospinal fluid. tTo whom reprint requests should be addressed. tPresent address: Salk Institute Biotechnology/Industrial Associates, Inc., 505 Coast Boulevard South, La Jolla, CA 92037.

The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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any acute or chronic illness and were diagnosed as being free from any neurological or psychological disease. Clinical Diagnoses. The diagnosis of probable AD conformed to the National Institute for Neurological and Communicative Disorders and Stroke (NINCDS)/AD and Related Disorders Association (ADRDA) criteria (29). The battery included the full Consortium to Establish a Registry for AD (CERAD) evaluation (30) with additional tests for a more comprehensive neuropsychological examination. Each patient's medical history was obtained and reviewed, a complete physical and neurological examination was performed, and '=30 psychometrics assessing global cognitive function, intelligence, language, memory, visual-spatial skills, and frontal-lobe skills were administered. In addition, biochemical analyses on blood and urine, a chest roentgenogram, and electrocardiogram were performed. These tests allowed the diagnosis of organic versus nonorganic causes of dementia. Each patient had a series of magnetic resonance imaging scans consisting of T1-weighted sagittal, T2-weighted axial, and inversion-recovery coronal planes of section. The coronal images through the temporal lobe were used to visualize the hippocampus, a structure particularly vulnerable to AD (31). Probable vascular dementia was diagnosed based on the presence of historical vascular risk factors including hypertension, hyperlipidemia, diabetes mellitus, heart disease, connective tissue disease, smoking, and stroke in addition to a subcortical dementia profile (preserved naming, impaired delayed free recall with relatively preserved delayed recognition, and slowing of thought process), with or without focal signs and extrapyramidal signs. The presence of large numbers of confluent T2-weighted magnetic resonance imaging signals in the periventricular and subcortical white matter, and the presence of multiple asymmetric defects of hexamethylpropyleneamine oxime (HMPAO) perfusion or xenon regional cerebral blood flow on single-photon emission computerized tomography scans, contributed to the diagnosis. If the patient history, neuropyschometrics, magnetic resonance imaging, and single-photon emission computerized tomography scans all suggested a vascular basis for the dementia, then the diagnosis of probable vascular dementia was made. The combined use of the magnetic resonance imaging and the single-photon emission computerized tomography scans suggested more patients with a vascular basis for the dementia than would either technique alone. Thus, our diagnosis of probable AD is more stringent than is typically found. Frontotemporal-lobe dementia versus AD was diagnosed based on a neuropsychometric profile with predominant frontal and temporal signs, a magnetic resonance image showing frontotemporal atrophy with relative sparing of the superior posterior temporal gyrms, a single-photon emission computerized tomography scan showing predominant frontotemporal hypoperfusion with a relative sparing of the parietal lobe, and a history of dementia at a relatively early age. Transient global amnesia was diagnosed by evidence of multiple episodes of transient disturbance of cognition followed by relatively complete recovery. CSF Collection. Without knowledge of the specific dementia, the neurologist collected CSF from each patient who did not appear too debilitated or fragile to safely undergo a lumbar puncture. Lumbar puncture was sterilely performed with a 20-gauge needle and 1% Xylocaine as a local anesthetic. Volumes of 3-5 ml were generally collected; however, 20-ml volumes were collected for one set of control samples. After samples were collected, cell counts and assays for glucose, total protein, and IgG levels were done to exclude any routine CSF abnormalities. Samples were aliquoted and stored at -70'C until assayed. Quantitative APP Immunoblot Analysis. Aliquots (2.5 ,ul) of CSF were subjected to quantitative immunoblotting with

Proc. Natl. Acad. Sci. USA 89 (1992)

mAb P2-1 as described (32). The bound mouse mAb on the immunoblots was detected with a solution of '251-labeled affinity-purified goat anti-mouse IgG1 (100 ng/ml; 1.5 X 105 cpm/pmol); immunoblots were exposed to x-ray film at -700C for 1-2 hr. To quantitate autoradiograms, five readings were made for each band with a slit width of 5 mm in an LKB scanning laser densitometer. Quantitative APP ELISAs. Analyses of the CSF samples were conducted as blind experiments. Triplicate samples containing 5 p1 of CSF or known quantities of purified PN-2[APP] were diluted to 100 ul in phosphate-buffered saline and coated on tissue culture-treated 96-well microtiter plates (Corning) overnight at 40C. It was necessary to use these particular cationically charged microtiter plates to achieve enhanced adsorption of APP from the CSF. The CSF was removed by aspiration and the remaining unoccupied sites in the wells were blocked with 1% ovalbumin in phosphate-buffered saline for 30 min at room temperature. This blocking solution was removed and the wells were washed three times with phosphate-buffered saline containing 0.05% Tween 20. After washing, 100 1.l of a solution of mAb P2-1 (10 ,ug/ml) was added to each well and incubated at 370C for 1 hr with shaking. In some parallel ELISAs the mouse mAb 22C11 (10 ,ug/ml) was used as the primary antibody. The mAb solution was removed and, after washing, the bound mouse mAb was detected with a solution (1:400) of biotinylated goat anti-mouse IgG that was adsorbed with human serum (Sigma) and a solution (1:800) of streptavidinhorseradish peroxidase conjugate (Amersham) in phosphatebuffered saline containing 1% ovalbumin. The above washing protocol was included after each step. The assay was developed by the addition of 100 ,ul of 10 mM o-phenylenediamine/ 0.1 M sodium citrate, pH 4.5/0.012% H202 per well. The reactions were quenched with 50 pul of 2 M H2SO4 per well. The absorbance at 492 nm was recorded with a Titertek Multiskan (Flow Laboratories). Values obtained from the CSF samples were compared with standard curves generated from known quantities of purified PN-2[APP].

RESULTS Classification of Demented Patients. This study focused on a thoroughly characterized population of live normal and demented patients. CSF samples from less characterized sources were avoided because a valid clinical diagnosis of AD versus other dementias (non-Alzheimer type) was essential to the analysis. All of our analyses were performed on CSF samples that were collected by lumbar puncture. Each patient evaluated in the AD Research Center had to meet the NINCDS/ADRDA (29) and CERAD (30) criteria, in addition to undergoing magnetic resonance imaging analysis and single-photon emission computerized tomography scanning for the diagnosis of probable AD. The probable AD patients presented in this study exhibited strong indications for typical AD and did not present evidence for another basis of their dementia. Patients that had a clear history of risk factors, a neuropsychometric profile inconsistent with AD, and evidence of vascular and/or other involvement based on the imaging and scanning analyses were classified as probable nonAlzheimer-type dementia. By definition, these patients are also designated as possible AD and a subset of the population is most likely afflicted with both forms of dementia. These patients were classified as probable non-Alzheimer-type dementia based on the compelling evidence provided by the imaging and scanning analyses. Quantitative Immunoblotting of APP in CSF Samples. APP levels were markedly reduced in representative CSF samples from probable AD patients compared with demented nonAlzheimer-type patients and healthy controls (Fig. 1A).

Proc. Natl. Acad. Sci. USA 89 (1992)

Medical Sciences: Van Nostrand et al.

A

Non- Demented (non- Probable demented Alzheimer) Alzheimer r

kDa

I

I

I

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2

3

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FIG. 1. Quantitative immunoblot analysis of APP in CSF. (A) Aliquots (2.5 ,A) of CSF samples were subjected to nonreducing SDS/PAGE and quantitative immunoblotting with mAb P2-1. (B) Results of the laser densitometric scan of the autoradiogram in A.

Quantitative densitometric scanning of the immunoblot (Fig. 1B) showed that the non-demented control group (lanes 1-3) and the demented (non-Alzheimer type) group (lanes 4-6) exhibited similar immunoreactivity on the immunoblot. In contrast, the probable AD group (lanes 7-9) showed =3.5fold lower immunoreactivity on the immunoblot. Our immunoblotting pattern, which appears as a single APP species, differs from previous studies that have reported two distinct APPs (Kunitz protease inhibitor domaincontaining and -lacking forms) in CSF (17-20, 24-26). mAb P2-1 recognizes forms of APP that contain or lack the Kunitz protease inhibitor domain (32). mAb P2-1 was prepared against native PN-2[APP] and will not recognize APP that has been treated with reducing agents such as 2-mercaptoethanol. Parallel immunoblots were conducted with mAb 22C11, which was prepared against an APP bacterial fusion protein (16). This particular mAb recognized two distinct CSF APP bands under reducing electrophoretic conditions. However, only one APP band was detected under nonreducing conditions (W.E.V.N., unpublished data), identical to our immunoblots shown in Fig. 1A. Thus, this different immunoblotting pattern is the result of electrophoretic conditions.

A r

:0.999

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Format of Quantitative CSF APP ELISA. To readily measure CSF APP levels in a larger number of samples, we developed a quantitative ELISA using mAb P2-1. This antibody did not crossreact with any other proteins in the CSF samples (Fig. 1A), thus validating its use in an ELISA format. The ELISAs using mAb P2-1 and purified PN-2[APP] coated on tissue culture-treated 96-well microtiter plates showed a strong linear correlation between reaction absorbance at 492 nm and quantity of antigen (Fig. 2A). To determine whether other CSF proteins would interfere with our ability to measure APP, we conducted ELISAs with known quantities of PN-2[APP] in the presence or absence of control and AD CSF. Even in the presence of control and AD CSF, we recovered total amounts of added PN-2[APP] indicating that under these conditions total CSF APP is adsorbed to the microtiter plates (Fig. 2B). Quantitation of CSF APP by ELISA. The ELISAs with mAb P2-1 showed that the mean CSF levels of APP were markedly lower in the probable AD patients (0.8 + 0.4 Ag/ml) than in the demented non-Alzheimer-type controls (2.9 + 1.0,ug/ml) and the nondemented controls (2.7 + 0.7 pug/ml) (Table 1 and Fig. 3). The demented non-Alzheimer-type group had APP levels similar to those observed in the nondemented control group. In contrast, the mean CSF levels of APP in the probable AD patients were approximately 3.4-fold and 3.6fold lower than the levels in the demented non-Alzheimertype group and the nondemented control group, respectively. We did not observe a general decrease in the CSF levels of APP with respect to age in any of the three populations. In fact, some of the older nondemented and demented nonAlzheimer-type individuals had the highest CSF APP levels. The CSF total protein concentrations were similar in all three populations, demonstrating that the decreased APP levels in the probable AD patients were not due to a decrease in total protein content. Together, these studies indicate that determining the CSF levels of APP can aid in distinguishing between probable AD patients and demented non-Alzheimertype controls. Our results show a more pronounced decrease in CSF APP levels in AD patients than have other recent reports (25, 26). To understand the basis for these differences, we conducted parallel ELISA studies on purified PN-2[APP] and representative CSF samples using our mAb P2-1 and mAb 22C11, which was used in one of the other studies (25). mAb 22C11 recognized purified PN-2[APP] in a dose-dependent fashion but with 3-fold less intensity than mAbP2-1 (Fig. 4A). Similar results were obtained in ELISAs with APP proteins purified from control and AD CSF (W.E.V.N., unpublished data). However, when ELISAs were conducted with CSF samples as described in Materials and Methods, mAb 22C11 yielded even lower signals than parallel ELISAs with mAb P2-1 (Fig. 4B). Moreover, the differences in CSF APP levels observed

B CnrCSF

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.0

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FIG. 2. APP ELISA. Known quantities of purified PN-2[APP] were coated on tissue culture-treated microtiter plates in the absence (@) or of 5 1l of AD (A) or control (m) CSF and ELISA was conducted with mAb P2-1.

presence

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Proc. Natl. Acad Sci. USA 89 (1992)

Table 1. Summary of ELISA quantitation of APP in CSF from nondemented controls, demented non-Alzheimer-type patients, and probable AD patients Age, APP,* Total protein, APP/total protein,

Population

years

,ug/ml

mg/dl

Nondemented (n = 16) 62 ± 16 2.7 ± 0.7 Demented non-Alzheimer (n = 18) 69 ± 6 2.9 ± 1.0 Probable AD(n = 13) 69 ± 9 0.8 ± 0.4 *CSF APP concentrations determined by ELISAs and based on purified PN-2[APP].

between AD patients and the demented non-Alzheimer-type patients and nondemented controls were much less in the ELISAs with mAb 22C11 (Fig. 4B). These findings suggest that the previously reported smaller differences in CSF APP levels in AD patients compared with nondemented controls and demented non-Alzheimer-type patients may be the result of the sensitivity and specificity of the antibodies employed to conduct these measurements.

DISCUSSION Altered proteolysis of APP leads to formation of the amyloid P-protein, the major constituent of senile plaques and cerebrovascular deposits in AD. Since these abnormal proteolytic events occur in the brain parenchyma and in the walls of cerebral blood vessels, they may result in altered levels of APP in CSF. To further evaluate the CSF levels of APP in live AD patients, we employed a highly specific and sensitive ELISA and supportive quantitative immunoblots. Our studies employed mAb P2-1, which was prepared against purified, native human PN-2[APP] (14). The immunoblot in Fig. 1A demonstrates the specificity and sensitivity of mAb P2-1. As

,ug/mg

40 ± 12 6.8 39 ± 12 7.4 38 ± 11 2.1 comparison to standard curves of

little as 2.5 ,ul of CSF contains significant immunoreactivity detectable on immunoblots. Importantly, only APP was detected in the CSF samples, thus validating its use in the ELISAs. The sensitivity of mAb P2-1 facilitated the use of very small volumes (5 ,l) of CSF, without prior manipulations, to determine the concentrations of APP in ELISAs. The nondemented control and demented non-Alzheimertype groups had mean CSF concentrations of APP that were approximately 3.5-fold higher than those of the probable AD patients (Table 1). When the concentrations of APP were standardized to total CSF protein the differences were very similar. This demonstrates that the decreases in CSF APP levels in the probable AD patients were not due to a decrease in total protein. The CSF levels of APP in this study are higher than those previously reported. The results presented in Fig. 4 suggest that the CSF APP values may be higher in our assays due to a combination of the sensitivity of our monoclonal antibody for the native protein and its specificity for APP in complex mixtures of proteins such as CSF.

E C

Non Demented Probable demented (non-Alzheimer) Alzheimer

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_I FIG. 3. CSF levels of APP in 16 nondemented controls, 18 demented non-Alzheimer-type patients, and 13 probable AD patients. CSF APP concentrations were determined based on comparison to standard-curve ELISAs with purified PN-2[APP].

2

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FIG. 4. Comparative APP ELISAs using mAb P2-1 and mAb 22C11. (A) ELISAs were conducted with known quantities of purified PN-2[APP] and mAb P2-1 (o) or mAb 22C11 (0). (B) ELISAs were conducted on representative CSF samples from nondemented controls (bars 1-3), demented non-Alzheimer-type patients (bars 4-6), and probable AD patients (bars 7-9) with mAb P2-1 (hatched bars) or mAb 22C11 (filled bars). CSF APP concentrations were determined based on comparison to respective standard-curve ELISAs with purified PN-2[APP] for each mAb.

Medical Sciences: Van Nostrand et al. Together, our findings suggest the potential usefulness of mAb P2-1 in the quantitative CSF APP ELISA to provide biochemical information as an adjunct to other clinical criteria employed in the diagnosis of AD. Our results with AD patients show a striking parallel to individuals afflicted with hereditary cerebral hemorrhage with amyloidosis (Icelandic-type). Individuals with this disease have a point mutation in the gene encoding cystatin C, a cystiene protease inhibitor, which leads to altered proteolysis of the protein, deposition of a resulting amyloid fragment in the cerebral vessel walls, and decreased CSF levels of cystatin C (33-35). Lower CSF APP levels in AD may result from excessive proteolysis of variant or abnormally processed APP, and this mechanism may represent a common feature in certain amyloidoses. Note Added in Proof. We have extended our quantitative analyses to APP proteins in CSF samples from patients with hereditary cerebral hemorrhage with amyloidosis, Dutch type (HCHWA-D), another disorder that involves deposition of the amyloid f-protein in the brain parenchyma and cerebrovasculature. Similar to our present findings with probable AD patients, HCHWA-D patients exhibited a 3- to 4-fold decrease in CSF APP levels.

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