Analysis and Quantitation of the β‐Amyloid Precursor Protein in the Cerebrospinal Fluid of Alzheimer's Disease Patients with a Monoclonal Antibody‐Based Immunoassay

Henriksson, Thomas; Barbour, Robin; Braa, Sonia; Ward, Pamela J.; Fritz, Lawrence C.; Johnson-Wood, Kelly; Chung, Hyung D.; Burke, William J.; Reinikainen, Kari; Riekkinen, Paavo; Schenk, Dale

doi:10.1111/j.1471-4159.1991.tb02026.x

Cited by 46 publications

(18 citation statements)

References 36 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…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)(18)(19)(20)(24)(25)(26). mAb P2-1 recognizes forms of APP that contain or lack the Kunitz protease inhibitor domain (32).…”

Section: Resultscontrasting

confidence: 82%

“…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).…”

Section: Resultssupporting

confidence: 51%

“…On the other hand, more recent reports indicated that the amounts of secreted APP in CSF were slightly decreased in AD patients (24)(25)(26). However, the measurements in those studies employed various antibodies prepared against synthetic peptides or expression products corresponding to various domains of APP.…”

mentioning

confidence: 99%

See 2 more Smart Citations

Decreased levels of soluble amyloid beta-protein precursor in cerebrospinal fluid of live Alzheimer disease patients.

Nostrand

Wagner

Shankle

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

195

View full text Add to dashboard Cite

The amyloid 13-protein is deposited in senile 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.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)(17)(18)(19)(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 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 variou...

show abstract

Section: Resultscontrasting

confidence: 82%

Section: Resultssupporting

confidence: 51%

See 1 more Smart Citation

Decreased levels of soluble amyloid beta-protein precursor in cerebrospinal fluid of live Alzheimer disease patients.

Nostrand

Wagner

Shankle

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

195

View full text Add to dashboard Cite

show abstract

“…For the detection of secreted APP, the monoclonal antibody 1G5 [29] was used, and the blots were incubated overnight at room temperature. Detection was carried out by incubation with horseradish peroxidase-conjugated goat antimouse IgG (Kirkegaard and Perry Laboratories, Gaithersburg, MD, U.S.A.) for 1 h. The blots were then washed extensively and sAPP visualized using an enhanced chemiluminescence method (Amersham).…”

Section: Immunodetection Of Sappmentioning

confidence: 99%

Secretory processing of amyloid precursor protein is inhibited by increase in cellular cholesterol content

Racchi

Baetta

Salvietti

et al. 1997

Biochemical Journal

150

View full text Add to dashboard Cite

Plasma-membrane composition plays a crucial role in most of the cellular functions that depend on membrane processes. In virtually all cell types the proteolytic processing of Alzheimer amyloid precursor protein (APP) to generate soluble APP (sAPP) is believed to occur at the plasma membrane or in its immediate proximity. Alteration of this metabolic pathway has been linked to the pathogenesis of Alzheimer's disease. We analysed the effect of membrane cholesterol enrichment on APP metabolism. Incubation of COS cells with increasing concentrations of nonesterified cholesterol carried by rabbit β-very low-density lipoprotein caused a dose-dependent inhibition of sAPP release : 70 % inhibition with 10 µg\ml non-esterified cholesterol. A less pronounced inhibitory effect was observed on treatment with

show abstract

“…Immunoblots were probed with (i) affinitypurified rabbit antibodies to bacterially expressed recombinant proteins of either jAPP4"_592 [aB5 (14); numbering according to BAPP695 (5) Ihara, Tokyo University). A monoclonal antibody to recombinant BAPP"44592 (1GS) has been described (18). Two antibodies to APP were used: a high-titer rabbit antiserum (1280) to synthetic AfBP1_40, produced as described (19) for our antiserum Y to Af3P1-38, and a monoclonal antibody (6C6) to synthetic AiP1_28, from D. Schenk (Athena Neurosciences).…”

mentioning

confidence: 99%

Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels.

Tamaoka

Kalaria

Lieberburg

et al. 1992

Proc. Natl. Acad. Sci. U.S.A.

119

View full text Add to dashboard Cite

Altered proteolysis of the (3-amyloid precursor protein (PAPP) (36 and 38 yr), and 18 non-AD subjects (fetal-78 yr). Frozen nonneural tissues (renal cortex, heart, spleen, and adrenal) and fresh or frozen animal brains (rat, guinea pig, rabbit, and cow) were also used to prepare microvessels. Microvessels were purified at 40C by a modification of the method of Tsuji et al. (9). Cerebral gray matter (usually 5-10 g of wet weight) was minced into -2 mm3 blocks, suspended in 5-10 vol of Tris-buffered saline (TBS) (50 mM Tris/150 mM sodium chloride/2 mM EDTA/0.1 mM diisopropyl fluorophosphate/ leupeptin at 1 Ag/ml/pepstatin at 0.1 ug/ml/7-amino-1-chloro-3-tosylamide-2-heptanone at 1 tig/ml, pH 7.6), and homogenized with a motor-driven, loosely fitting Teflon pestle (5-10 up-and-down strokes). The homogenate was sieved through 73-,.m nylon mesh. The flow-through fraction was saved as the nonvascular brain fraction, and the mesh was washed in TBS buffer to resuspend the retained material.This suspension was placed on a column of 425-to 600-Iumdiameter glass beads (Sigma) with a sheet of 73-,um nylon mesh at the bottom. The column was washed with 5-10 vol of TBS, and its contents were placed in a beaker of 50-100 ml of TBS. Microvessels were separated from the glass beads by agitation with a metal rod. The beads settled, and the microvessel suspension was decanted and centrifuged at 10,000 x g for 30 min. The resultant pellet was resuspended in 0.25 M sucrose, homogenized (five strokes), and layered on a discontinuous sucrose-density gradient (8 ml each of 1.0 M, 1.3 M, and 1.5 M sucrose in H20). After centrifugation at 58,000 x g in an SW 28 rotor for 120 min, the microvessel fraction was recovered as a pellet from the bottom of the tube and resuspended in 1-2 ml of TBS. Some microvessel fractions were prepared from fresh brain tissue by an alternate but comparable purification method described by Kalaria and Harik (10).To separate large from small microvessels, the suspension retained on the 73-pum mesh was sieved through a 153-,um nylon mesh, and the latter was washed in TBS to resuspend the retained material. This suspension was centrifuged at 10,000 x g for 30 min at 40C, and the resultant pellet was layered on a sucrose gradient as described above. For purification of small microvessels, the flow-through fraction off the 153-,um mesh was successively processed over the glass bead column and the sucrose gradient as described above.Purified microvessel fractions were homogenized (10 strokes) in a Teflon homogenizer, mixed 3:1 with 4x SDS Abbreviations: 8APP, 13-amyloid precursor protein; FL-f3APP, fulllength fAPP; A,8P, amyloid ,1-protein; AD, Alzheimer disease. 1345The 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.

show abstract

Analysis and Quantitation of the β‐Amyloid Precursor Protein in the Cerebrospinal Fluid of Alzheimer's Disease Patients with a Monoclonal Antibody‐Based Immunoassay

Cited by 46 publications

References 36 publications

Decreased levels of soluble amyloid beta-protein precursor in cerebrospinal fluid of live Alzheimer disease patients.

Decreased levels of soluble amyloid beta-protein precursor in cerebrospinal fluid of live Alzheimer disease patients.

Secretory processing of amyloid precursor protein is inhibited by increase in cellular cholesterol content

Identification of a stable fragment of the Alzheimer amyloid precursor containing the beta-protein in brain microvessels.

Contact Info

Product

Resources

About