69
Neuroscience Letters, 124 (1991) 69-73 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100008U NSL 07602
Quantitative changes in the amyloid flA4 precursor protein in Alzheimer cerebrospinal fluid Reinhard Prior I , Ursula M6nning 1, Ursula Schreiter-Gasser z, Andreas Weidemann 1, Kai Blennow 3, Carl G. Gottfries 3, Colin L. Masters 4 and K o n r a d Beyreuther 1 1Centerfor Molecular Biology, University of Heidelberg, Heidelberg ( F.R.G. ), 2Central Institute for Mental Health, Mannheim (F. R.G. ), 3Department of Psychiatry and Neurochemistry, University of G6teborg, St. J6rgen's Hospital, Hisings Backa (Sweden) and 4Department of Pathology, University of Melbourne, Parkville, Vic. (Australia) (Received 2 November 1990; Revised version received 10 December 1990; Accepted 11 December 1990)
Key words: Alzheimer's disease; Cerebrospinal fluid; flA4 amyloid precursor; Quantitation; ELISA; Densitometry The major three secretory isoforms of Alzheimer flA4 amyloid precursor protein (APP) were quantified in cerebrospinal fluid (CSF) using (1) a newly developed enzyme-linked immunosorbent assay (ELISA) and (2) densitometric analysis of CSF Western blots. The protease inhibitor-containing APP751/770 isoforms represented an average of 10.5% of total APP in CSF of patients with Alzheimer's disease (AD, n = 22), multi-infarct dementia (MID, n = 5 ) and normal controls (n= 10). APP levels in CSF did not depend on total CSF protein. Both findings are inconsistent with a hematogeneous origin of APP in CSF and suggest an intracerebral source. Total APP, APP695 and APP751/770 were significantly decreased in the AD and in the MID groups, but were not correlated to the ages of patients or controls.
Intracerebral and cerebrovascular amyloid deposits are a prominent neuropathological feature of Alzheimer's disease (AD), Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). The amyloid protein (flA4) has a molecular weight of 4.5 kDa [4, 13]. It is derived from a larger glycosylated precursor protein (APP) that resembles a cell surface receptor and is controlled by the PAD (precursor of amyloid in Alzheimer's disease and Down's syndrome) gene located on chromosome 21 [7, 25]. Three major alternatively spliced mRNAs code for molecules with 695,751 and 770 amino acid residues which all contain the complete and partially membrane-inserted flA4sequence [7, 9, 20, 30]. APP695 is found predominantly in brain, whereas APP751 and APP770, which contain a Kunitz type II protease inhibitor domain, are the most common isoforms in peripheral organs [5, 8]. Secretory forms of APP695, 751 and 770 are produced from transmembrane APP by proteolysis within the flA4 sequence [3, 27] and are present in cerebrospinal fluid (CSF) [17, 32] and blood [19].
Correspondence: R. Prior, Zentrum ffir Molekulare Biologie, Laboratory for Molecular Neuropathology, Im Neuenheimer Feld 282, D6900 Heidelberg, F.R.G.
Various pathogenetic mechanisms may operate during the synthesis and the processing of APP and participate in the intracerebral deposition of flA4. In Down's syndrome, APP overproduction due to the presence of an additional copy of the chromosome 21 might be a relevant pathogenetic factor [24]. However, little is known about possible pathogenetic mechanisms in familial and sporadic AD, which account for the overwhelming majority of cerebral flA4 amyloidoses. There have been several investigations of the APP mRNA splicing pattern describing the APP-isoform ratio in AD and normal brains. The results are controversial and range from the absence of changes [l l, 12] to selective increases in APP695 [16], APP751 [6] and APP770 [29]. Since ADassociated changes of intracerebral production, processing or secretion of APP might be reflected by alterations of the APP levels and isoform ratios in the CSF, we have recently developed an enzyme-linked immunosorbent assay (ELISA) that measures the concentration of total APP in the CSF [21]. We used this ELISA to analyze the CSF content of APP in 22 AD and 15 control patients. In addition, we measured the quantities of APP695 and APP751/770 in the CSF by densitometric analysis of CSF Western blots. CSF samples were obtained from three groups of patients: (I) Patients with early onset 'probable Alz-
70
heimer's disease' (n = 22) according to the diagnostic criteria of the NINCDS-ADRDA work group [14]. The mean age in the AD group was 62 years (range 55-70 years). (II) A second group of patients (n = 5) was classified as having multi-infarct dementia according to the DSM-III-R and on the basis of a clear temporal relationship between the vascular events (TIA/stroke) and the evolution of dementia symptoms; the mean age in this group was 71 years (range 63-76 years). (III) As reference group (n= 10) we selected neurologically normal patients admitted for minor urologic or orthopedic surgery in spinal anesthesia with a mean age of 68 years (range 62-77 years). All CSF samples were frozen at 70°C within 1 h of lumbar puncture. For the ELISA procedure we used a double sandwich technique with the monoclonal antibody 22C11 as first antibody. Mab 22C11 was produced against purified fusion protein (Fd-APP) containing the APP695 protein and the Fd fragment of the murine B 1-8 IgM immunoglobulin heavy chain. As second antibody we used the IgG-fraction of polyclonal rabbit anti-Fd-APP antiserum. The preparation of Fd-APP and antibodies and details of the ELISA procedure have been described previously [21, 32]. For Western blot analysis (Fig. 1), the CSF samples (60/tl) were vacuum dried, the pellet was resuspended in 30/tl 2 x Laemmli buffer and boiled for 10 min. The whole sample (30/A) was then applied to SDS-PAGE on 8% polyacrylamide gels. The separated proteins were electroblotted onto nitrocellulose filter paper (0.45/tm), which was blocked with phosphate-buffered saline + 3% bovine serum albumin for 3 h at 37°C. The filters were incubated for 3 h at 37°C with Mab22Cll ascitic fluid diluted with 10 mM Tris-HC1, -
C S F A P P W e s t e r n blot AD-
-
+ -
+ -
+-
+ + + + + . . . .
+ +
112 kD
~PP751/ 770
91 led
APP695
Fig. 1. Secretory APP detected in CSF by Western blot analysis. The major reaction product with a molecular weight of 91 kDa corresponds to APP695, whereas APP751 and APP770 are included in a minor band of 112 kDa molecular weight. An identical pattern is detected in CSF samples from both AD patients ( + ) and neurologically normal control cases ( - ) . After exposure of the ~25I-labelled nitroceUose illters, the APP content was analyzed by densitometric evaluation of the autoradiographs.
150 mM NaC1, 0.05% Tween 20 (TBST) to a final antibody concentration of 0.5 /Lg/ml. After washing with TBST, 0.1 /tC1/ml of x2SI-labelled sheep anti-mouse Ig (Amersham, spec. act. 5-20/tCi//tg) was applied for 2 h at 37°C and the filters were washed 5 times in TBST. The filters were then exposed to Kodachrom X-Omat AR film for 48h at -70°C and the band intensity was determined by transmission densitometry using an Elscript 400 Scanner (Hirschmann). All CSF samples were examined for their total protein content by the method of Bradford [1]. The results of the ELISA were well correlated to the results of the densitometric analysis of Western blots of the same CSF samples confirming the reliability of the quantitative assays (linear regression analysis of all CSF samples for total APP: correlation coefficient 0.76, P
Neuroscience Letters, 124 (1991) 69-73 © 1991 Elsevier Scientific Publishers Ireland Ltd. 0304-3940/91/$ 03.50 ADONIS 030439409100008U NSL 07602
Quantitative changes in the amyloid flA4 precursor protein in Alzheimer cerebrospinal fluid Reinhard Prior I , Ursula M6nning 1, Ursula Schreiter-Gasser z, Andreas Weidemann 1, Kai Blennow 3, Carl G. Gottfries 3, Colin L. Masters 4 and K o n r a d Beyreuther 1 1Centerfor Molecular Biology, University of Heidelberg, Heidelberg ( F.R.G. ), 2Central Institute for Mental Health, Mannheim (F. R.G. ), 3Department of Psychiatry and Neurochemistry, University of G6teborg, St. J6rgen's Hospital, Hisings Backa (Sweden) and 4Department of Pathology, University of Melbourne, Parkville, Vic. (Australia) (Received 2 November 1990; Revised version received 10 December 1990; Accepted 11 December 1990)
Key words: Alzheimer's disease; Cerebrospinal fluid; flA4 amyloid precursor; Quantitation; ELISA; Densitometry The major three secretory isoforms of Alzheimer flA4 amyloid precursor protein (APP) were quantified in cerebrospinal fluid (CSF) using (1) a newly developed enzyme-linked immunosorbent assay (ELISA) and (2) densitometric analysis of CSF Western blots. The protease inhibitor-containing APP751/770 isoforms represented an average of 10.5% of total APP in CSF of patients with Alzheimer's disease (AD, n = 22), multi-infarct dementia (MID, n = 5 ) and normal controls (n= 10). APP levels in CSF did not depend on total CSF protein. Both findings are inconsistent with a hematogeneous origin of APP in CSF and suggest an intracerebral source. Total APP, APP695 and APP751/770 were significantly decreased in the AD and in the MID groups, but were not correlated to the ages of patients or controls.
Intracerebral and cerebrovascular amyloid deposits are a prominent neuropathological feature of Alzheimer's disease (AD), Down's syndrome and hereditary cerebral hemorrhage with amyloidosis of the Dutch type (HCHWA-D). The amyloid protein (flA4) has a molecular weight of 4.5 kDa [4, 13]. It is derived from a larger glycosylated precursor protein (APP) that resembles a cell surface receptor and is controlled by the PAD (precursor of amyloid in Alzheimer's disease and Down's syndrome) gene located on chromosome 21 [7, 25]. Three major alternatively spliced mRNAs code for molecules with 695,751 and 770 amino acid residues which all contain the complete and partially membrane-inserted flA4sequence [7, 9, 20, 30]. APP695 is found predominantly in brain, whereas APP751 and APP770, which contain a Kunitz type II protease inhibitor domain, are the most common isoforms in peripheral organs [5, 8]. Secretory forms of APP695, 751 and 770 are produced from transmembrane APP by proteolysis within the flA4 sequence [3, 27] and are present in cerebrospinal fluid (CSF) [17, 32] and blood [19].
Correspondence: R. Prior, Zentrum ffir Molekulare Biologie, Laboratory for Molecular Neuropathology, Im Neuenheimer Feld 282, D6900 Heidelberg, F.R.G.
Various pathogenetic mechanisms may operate during the synthesis and the processing of APP and participate in the intracerebral deposition of flA4. In Down's syndrome, APP overproduction due to the presence of an additional copy of the chromosome 21 might be a relevant pathogenetic factor [24]. However, little is known about possible pathogenetic mechanisms in familial and sporadic AD, which account for the overwhelming majority of cerebral flA4 amyloidoses. There have been several investigations of the APP mRNA splicing pattern describing the APP-isoform ratio in AD and normal brains. The results are controversial and range from the absence of changes [l l, 12] to selective increases in APP695 [16], APP751 [6] and APP770 [29]. Since ADassociated changes of intracerebral production, processing or secretion of APP might be reflected by alterations of the APP levels and isoform ratios in the CSF, we have recently developed an enzyme-linked immunosorbent assay (ELISA) that measures the concentration of total APP in the CSF [21]. We used this ELISA to analyze the CSF content of APP in 22 AD and 15 control patients. In addition, we measured the quantities of APP695 and APP751/770 in the CSF by densitometric analysis of CSF Western blots. CSF samples were obtained from three groups of patients: (I) Patients with early onset 'probable Alz-
70
heimer's disease' (n = 22) according to the diagnostic criteria of the NINCDS-ADRDA work group [14]. The mean age in the AD group was 62 years (range 55-70 years). (II) A second group of patients (n = 5) was classified as having multi-infarct dementia according to the DSM-III-R and on the basis of a clear temporal relationship between the vascular events (TIA/stroke) and the evolution of dementia symptoms; the mean age in this group was 71 years (range 63-76 years). (III) As reference group (n= 10) we selected neurologically normal patients admitted for minor urologic or orthopedic surgery in spinal anesthesia with a mean age of 68 years (range 62-77 years). All CSF samples were frozen at 70°C within 1 h of lumbar puncture. For the ELISA procedure we used a double sandwich technique with the monoclonal antibody 22C11 as first antibody. Mab 22C11 was produced against purified fusion protein (Fd-APP) containing the APP695 protein and the Fd fragment of the murine B 1-8 IgM immunoglobulin heavy chain. As second antibody we used the IgG-fraction of polyclonal rabbit anti-Fd-APP antiserum. The preparation of Fd-APP and antibodies and details of the ELISA procedure have been described previously [21, 32]. For Western blot analysis (Fig. 1), the CSF samples (60/tl) were vacuum dried, the pellet was resuspended in 30/tl 2 x Laemmli buffer and boiled for 10 min. The whole sample (30/A) was then applied to SDS-PAGE on 8% polyacrylamide gels. The separated proteins were electroblotted onto nitrocellulose filter paper (0.45/tm), which was blocked with phosphate-buffered saline + 3% bovine serum albumin for 3 h at 37°C. The filters were incubated for 3 h at 37°C with Mab22Cll ascitic fluid diluted with 10 mM Tris-HC1, -
C S F A P P W e s t e r n blot AD-
-
+ -
+ -
+-
+ + + + + . . . .
+ +
112 kD
~PP751/ 770
91 led
APP695
Fig. 1. Secretory APP detected in CSF by Western blot analysis. The major reaction product with a molecular weight of 91 kDa corresponds to APP695, whereas APP751 and APP770 are included in a minor band of 112 kDa molecular weight. An identical pattern is detected in CSF samples from both AD patients ( + ) and neurologically normal control cases ( - ) . After exposure of the ~25I-labelled nitroceUose illters, the APP content was analyzed by densitometric evaluation of the autoradiographs.
150 mM NaC1, 0.05% Tween 20 (TBST) to a final antibody concentration of 0.5 /Lg/ml. After washing with TBST, 0.1 /tC1/ml of x2SI-labelled sheep anti-mouse Ig (Amersham, spec. act. 5-20/tCi//tg) was applied for 2 h at 37°C and the filters were washed 5 times in TBST. The filters were then exposed to Kodachrom X-Omat AR film for 48h at -70°C and the band intensity was determined by transmission densitometry using an Elscript 400 Scanner (Hirschmann). All CSF samples were examined for their total protein content by the method of Bradford [1]. The results of the ELISA were well correlated to the results of the densitometric analysis of Western blots of the same CSF samples confirming the reliability of the quantitative assays (linear regression analysis of all CSF samples for total APP: correlation coefficient 0.76, P
