Amyloid pprotein precursors with kunitztvpe inhibitor domains and acetvlcXolinesterase in cerebrospinal fluid from patients with dementia of the Alzheimer tvPe Urakami K, Takahashi K, Saito H, Okada A, Nakamura S, Tanaka S, Kitaguchi N, Tokushima Y, Yamamoto S. Amyloid Dprotein precursors with kunitz-type inhibitor domains and acetylcholinesterase in cerebrospinal fluid from patients with dementia of the Alzheimer type. Acta Neurol Scand 1992: 85: 343-346.
We used the ELISA to measure the concentration of amyloid protein precursor with Kunitz type trypsin inhibitor domains (APPI) in C S F of dementia of the Alzheimer type (DAT) and examined the correlation of APPI with acetylcholinesterase (AChE) and somatostatin (SRIF). We found the APPI concentration in CSF of DAT to be significantly elevated compared with that of multi-infarct dementia and controls. We could significantly correlate APPI with AChE, but not correlate APPI with SRIF. The present results suggest that measurement of C S F APPI levels may be useful for diagnosis of DAT and the change of APPI may closely be associated with abnormality of acetylcholine system in DAT that has been reported.
Dementia of the Alzheimer type (DAT) is a progressive degenerative disorder and characterized by cerebral deposits of amyloid P-protein (P-AP). It has been suggested that the formation of senile plaque is one of the pathogeneses of DAT. Two types of mRNA coding for P-AP precursor protein (APP) with Kunitz type basic trypsin inhibitor domains (APPI), APP770 and APP751, as well as APP695 without APPI have been found (1-3). Soluble forms of APP were found in cerebrospinal fluid (CSF) (4) and it was of interest to quantify the amount of APP in CSF as possible diagnostic marker for DAT. There have been several reports on detection of APP in CSF by immunoblot analysis (4-9). However, these reports have given conflicting results. The apparent conflict data may, in part, be due to differences in the analytic methods. Kitaguchi et al. developed a new method of measuring APPI in CSF by enzyme linked immunosorbent assay (ELISA) (10). First purpose of our studies, we used the ELISA
’,
’,
K. Urakami ’, K. Takahashi H. Saito A. Okada’, S. Nakamura’, S.Tanaka3, N. Kitaguchi4, Y. Tokushima4, S.~ a m a r n o t o ~
’
Division of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, * Third Department of Internal Medicine, Faculty of Medicine, Hiroshima University, Department of Neurology, Faculty of Medicine, Kyoto University, Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry Co., Ltd., Shizuoka, Japan
Key words: amyloid fi protein precursor; acetylcholinesterase; dementia of The Alzheimer type K. Urakami, Division of Neurology, 1ns;irure cf Neurological Sciences, Faculty of M e d i n e . Tottori University, Nishimachi 86, Yonago-ski. Tottori-ken 683, Japan.
to measure the concentration of APPI or its biologically active forms in CSF of DAT. However, abnormalities of various neurotransmitters have also been reported in DAT. Especially, the acetylcholine (ACh) system and somatostatinergic system are important. There have been many reports on detection of acetylcholinesterase (AChE) and somatostatin (SRIF) in DAT CSF. CSF AChE levels in DAT is reported to be reduced (1 1-17), although not consistently (18-22). Reduced CSF AChE levels is presumably due to pathological changes in the cholinergic basal forebrain-cortical projections. Various abnormalities have also been reported in the neuropeptides, but reduction of CSF SRIF levels in DAT has been a consistent h d i n g (17,23-26). It is important to make clear the relation between APPI and neurotransmitters (AChE and SRIF). Second purpose of our studies, we examined the correlation ofAPPI concentration with AChE activity and SRIF concentration in CSF of DAT.
343
Urakami et al. Material and methods
The subjects consisted of 20 patients with DAT, 11 patients with multi-infarct dementia (MID) and 10 age-matched control subjects. Their profiles are shown in Table 1. Dementia was diagnosed according to the DSM-111-R diagnostic criteria (27) and NINCDS-ADRDA criteria (28). Dementia caused by other possible diseases was excluded. Assessments of these patients included carefully-examined medical history, physical examination, a drug inventory, a neurological examination, a comprehensive cognitive evaluation with the use of Functional Assessment Staging of Alzheimer’s disease (FAST staging), the Mini-Mental state examination (MMSE), Blessed dementia score, activity evaluation in daily living with Bartel index, assessment of patient’s environment, CT scanning and single photon emission tomography (SPECT) of the head, and routine laboratory tests, such as blood analysis, biochemical examination and electroencephalography. DAT was differentiated from MID according to Hachinslu’s ischemic score. We had the patients fast in the early morning and lie in bed at rest for 1-2 hours and collected CSF from them. The first 4 ml of CSF was subjected to routine examination (protein content, cell numbers and others), and the subsequent 4 ml was used as a test sample. The patients who showed abnormality in this routine examination were excluded from this study. Cell components were removed by refrigerated centrifugation and the supernatant was stored at - 80°C until measurement. We measured the AChE activity spectrophotometrically using the thiocholine method of Nakano et al. (16), and the SRIF concentration by the double antibody radioimmunoassay method developed by Arimura et al. (29), and the APPI concentration by the sandwich ELISA for detection of active (free) form of proteinase inhbitors by our using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microplate (10). The details of this method were described below. Fifty microliter solution (500 yglml) of porcine pancreatic trypsin (Sigma) in 20 mM Tris-HC1 pH 7.8 (buffer A) and 50 p1 aqueous solution of 1yo glutalaldehyde (GA) were added to each well of microtiterplate (Dynateck). The plate was shaken at room temperature (r.t.) for 45 min and washed with PBS. A hundred p1 of sample, ifnecessary diluted with buffer A, was incubated in the well at 37°C for 1 h and the plate was washed. In the case of the “denatured trypsin-plate ELISA” after fixation with 100 p1 of 0.5 % G A solution, trypsin-inhibitor complex was denatured with 100 pl of 62.5 mM Tris-HC1 buffer containing 0.15-2% SDS and 1-5% P-mercaptoethanol (pH 6.8) by incubation of the covered plate
344
Table 1. Profiles of subjects DAT
MID
Controls
No. (Male/Female)
2 0 (8/12)
Age (MeankSD)
71.9k7.6
11 (8/3) 68.7k7.6
10 (6/4 65.7k7.5
2.2k1.1
2.4k2.0
-
Duration of dementia (years)
ir
’I SF
II -,
P < 0.01
P< 0.05
0
8
t 0
tt
:
0 0
I
I
I
Controls
DAT
MID
Fig. I . Concentration of APPI in CSF of DAT, MID and Controls.
at 80°C for 30 min. Then the plate was washed and blocked with 2% bovine serum albumin (BSA), 0.1 % gelatin, 5 x 10 M nafamostat mesilate in PBS at 37°C for 1 hr, followed by washing with 0.05% Tween-20 in PBS. After incubation with 100 pl of 1:lOOO rabbit antiserum against denatured M ZtPI in PBS containing 1% BSA and 5 x nafamostat mesilate (buffer B) at r.t. for I hr and the subsequent wash, the plate was shaken at r.t. for 1 hr with 100 p1 of 1:lOOO anti rabbit IgG antiserum (ICN) conjugated with horse radish peroxidase in buffer B and then washed. o-Phenylenediamine and H,O, was added and absorption at 490nm was measured as usual. In the case of “the native trypsinplate ELISA, the 2nd GA treatment and the denaturation procedure were omitted”. ~
APPI and AChE in DAT AChE (n mol/min/ml)
200-
@
150 -
@
inn
lww
lor
0'
N=20 Y=10.1+2.4X r =0.66(Pc0.001) I
5
1
10 APPI (nM)
Fig. 2. Correlation between APPI and AChE in DAT.
Statistical analysis was done by Wilcoxon rank sum test. The correlations of APPI concentration with AChE activity and SRIF concentration were statistically evaluated by regression analysis. Results
The APPI concentration in CSF of DAT was found to be significantly elevated compared with that of MID and controls (p < 0.0 1, p < 0.05, respectively, Fig. 1). There was a significant correlation between APPI and AChE levels (p
We used the ELISA to measure the concentration of amyloid protein precursor with Kunitz type trypsin inhibitor domains (APPI) in C S F of dementia of the Alzheimer type (DAT) and examined the correlation of APPI with acetylcholinesterase (AChE) and somatostatin (SRIF). We found the APPI concentration in CSF of DAT to be significantly elevated compared with that of multi-infarct dementia and controls. We could significantly correlate APPI with AChE, but not correlate APPI with SRIF. The present results suggest that measurement of C S F APPI levels may be useful for diagnosis of DAT and the change of APPI may closely be associated with abnormality of acetylcholine system in DAT that has been reported.
Dementia of the Alzheimer type (DAT) is a progressive degenerative disorder and characterized by cerebral deposits of amyloid P-protein (P-AP). It has been suggested that the formation of senile plaque is one of the pathogeneses of DAT. Two types of mRNA coding for P-AP precursor protein (APP) with Kunitz type basic trypsin inhibitor domains (APPI), APP770 and APP751, as well as APP695 without APPI have been found (1-3). Soluble forms of APP were found in cerebrospinal fluid (CSF) (4) and it was of interest to quantify the amount of APP in CSF as possible diagnostic marker for DAT. There have been several reports on detection of APP in CSF by immunoblot analysis (4-9). However, these reports have given conflicting results. The apparent conflict data may, in part, be due to differences in the analytic methods. Kitaguchi et al. developed a new method of measuring APPI in CSF by enzyme linked immunosorbent assay (ELISA) (10). First purpose of our studies, we used the ELISA
’,
’,
K. Urakami ’, K. Takahashi H. Saito A. Okada’, S. Nakamura’, S.Tanaka3, N. Kitaguchi4, Y. Tokushima4, S.~ a m a r n o t o ~
’
Division of Neurology, Institute of Neurological Sciences, Faculty of Medicine, Tottori University, * Third Department of Internal Medicine, Faculty of Medicine, Hiroshima University, Department of Neurology, Faculty of Medicine, Kyoto University, Bio-Science Laboratory, Life Science Research Laboratories, Asahi Chemical Industry Co., Ltd., Shizuoka, Japan
Key words: amyloid fi protein precursor; acetylcholinesterase; dementia of The Alzheimer type K. Urakami, Division of Neurology, 1ns;irure cf Neurological Sciences, Faculty of M e d i n e . Tottori University, Nishimachi 86, Yonago-ski. Tottori-ken 683, Japan.
to measure the concentration of APPI or its biologically active forms in CSF of DAT. However, abnormalities of various neurotransmitters have also been reported in DAT. Especially, the acetylcholine (ACh) system and somatostatinergic system are important. There have been many reports on detection of acetylcholinesterase (AChE) and somatostatin (SRIF) in DAT CSF. CSF AChE levels in DAT is reported to be reduced (1 1-17), although not consistently (18-22). Reduced CSF AChE levels is presumably due to pathological changes in the cholinergic basal forebrain-cortical projections. Various abnormalities have also been reported in the neuropeptides, but reduction of CSF SRIF levels in DAT has been a consistent h d i n g (17,23-26). It is important to make clear the relation between APPI and neurotransmitters (AChE and SRIF). Second purpose of our studies, we examined the correlation ofAPPI concentration with AChE activity and SRIF concentration in CSF of DAT.
343
Urakami et al. Material and methods
The subjects consisted of 20 patients with DAT, 11 patients with multi-infarct dementia (MID) and 10 age-matched control subjects. Their profiles are shown in Table 1. Dementia was diagnosed according to the DSM-111-R diagnostic criteria (27) and NINCDS-ADRDA criteria (28). Dementia caused by other possible diseases was excluded. Assessments of these patients included carefully-examined medical history, physical examination, a drug inventory, a neurological examination, a comprehensive cognitive evaluation with the use of Functional Assessment Staging of Alzheimer’s disease (FAST staging), the Mini-Mental state examination (MMSE), Blessed dementia score, activity evaluation in daily living with Bartel index, assessment of patient’s environment, CT scanning and single photon emission tomography (SPECT) of the head, and routine laboratory tests, such as blood analysis, biochemical examination and electroencephalography. DAT was differentiated from MID according to Hachinslu’s ischemic score. We had the patients fast in the early morning and lie in bed at rest for 1-2 hours and collected CSF from them. The first 4 ml of CSF was subjected to routine examination (protein content, cell numbers and others), and the subsequent 4 ml was used as a test sample. The patients who showed abnormality in this routine examination were excluded from this study. Cell components were removed by refrigerated centrifugation and the supernatant was stored at - 80°C until measurement. We measured the AChE activity spectrophotometrically using the thiocholine method of Nakano et al. (16), and the SRIF concentration by the double antibody radioimmunoassay method developed by Arimura et al. (29), and the APPI concentration by the sandwich ELISA for detection of active (free) form of proteinase inhbitors by our using trypsin in place of the first antibody and by denaturation of APPI-trypsin complex in the microplate (10). The details of this method were described below. Fifty microliter solution (500 yglml) of porcine pancreatic trypsin (Sigma) in 20 mM Tris-HC1 pH 7.8 (buffer A) and 50 p1 aqueous solution of 1yo glutalaldehyde (GA) were added to each well of microtiterplate (Dynateck). The plate was shaken at room temperature (r.t.) for 45 min and washed with PBS. A hundred p1 of sample, ifnecessary diluted with buffer A, was incubated in the well at 37°C for 1 h and the plate was washed. In the case of the “denatured trypsin-plate ELISA” after fixation with 100 p1 of 0.5 % G A solution, trypsin-inhibitor complex was denatured with 100 pl of 62.5 mM Tris-HC1 buffer containing 0.15-2% SDS and 1-5% P-mercaptoethanol (pH 6.8) by incubation of the covered plate
344
Table 1. Profiles of subjects DAT
MID
Controls
No. (Male/Female)
2 0 (8/12)
Age (MeankSD)
71.9k7.6
11 (8/3) 68.7k7.6
10 (6/4 65.7k7.5
2.2k1.1
2.4k2.0
-
Duration of dementia (years)
ir
’I SF
II -,
P < 0.01
P< 0.05
0
8
t 0
tt
:
0 0
I
I
I
Controls
DAT
MID
Fig. I . Concentration of APPI in CSF of DAT, MID and Controls.
at 80°C for 30 min. Then the plate was washed and blocked with 2% bovine serum albumin (BSA), 0.1 % gelatin, 5 x 10 M nafamostat mesilate in PBS at 37°C for 1 hr, followed by washing with 0.05% Tween-20 in PBS. After incubation with 100 pl of 1:lOOO rabbit antiserum against denatured M ZtPI in PBS containing 1% BSA and 5 x nafamostat mesilate (buffer B) at r.t. for I hr and the subsequent wash, the plate was shaken at r.t. for 1 hr with 100 p1 of 1:lOOO anti rabbit IgG antiserum (ICN) conjugated with horse radish peroxidase in buffer B and then washed. o-Phenylenediamine and H,O, was added and absorption at 490nm was measured as usual. In the case of “the native trypsinplate ELISA, the 2nd GA treatment and the denaturation procedure were omitted”. ~
APPI and AChE in DAT AChE (n mol/min/ml)
200-
@
150 -
@
inn
lww
lor
0'
N=20 Y=10.1+2.4X r =0.66(Pc0.001) I
5
1
10 APPI (nM)
Fig. 2. Correlation between APPI and AChE in DAT.
Statistical analysis was done by Wilcoxon rank sum test. The correlations of APPI concentration with AChE activity and SRIF concentration were statistically evaluated by regression analysis. Results
The APPI concentration in CSF of DAT was found to be significantly elevated compared with that of MID and controls (p < 0.0 1, p < 0.05, respectively, Fig. 1). There was a significant correlation between APPI and AChE levels (p
