J. Clin. Biochem. Nutr., 14, 133-139, 1993

Enzymatic Changes in Cerebrospinal Fluid of Patients with Alzheimer-Type Dementia

Takaaki AOYAGI,I* Takao WADA,2 Fukiko KOJIMA,2 Machiko NAGAI,2 Shigeko HARADA,2 Tomio TAKEUCHI,2 Kunihiro ISSE,3 Mitsuo OGURA,3 Makoto HAMAMOTO,3 Kuniaki TANAKA,3 and Takehiko NAGAO3

1Showa College of Pharmaceutical Sciences , Machida 194, Japan 2Institute of Microbial Chemistry , Shinagawa-ku, Tokyo 141, Japan 3 Tokyo Metropolitan Tama Geriatric Hospital , Higashi-Murayama 187, Japan

(Received October 29, 1992)

Summary Previously, we reported protease changes in cerebral tis- sues, as well as in serum, of patients with Alzheimer-type dementia. In the present study, we investigated enzymatic activities in their cerebrospinal fluid. In the Alzheimer patients the activities of dipeptidyl peptidase III (DPP-III), DPP-IV, and B were significantly increased when compared with those of the control subjects. In vascular dementia pa- tients the activities of Leu-AP and cathepsin B were increased but the activity of DPP-III was decreased. These results, as well as those of a multivariate study on these enzymatic changes, suggest the independency of the cerebral changes in the Alzheimer patients from those in vascular dementia. The results may also be of use for clinical diagnoses of these pathologic states.

Key Words: Alzheimer's dementia, vascular dementia, proteases, plas- min,

There are quite a few papers indicating that the accumulation of abnormal proteins in the brain is pathogenetically related to Alzheimer's disease [1-4]. Injection of a protease inhibitor into animal brains was shown to induce the formation of lysosome-associated granular aggregates (dense bodies) closely resem- bling the ceroid-lipofuscin that accumulates in certain disease states and in the process of aging [5]. Thus the abnormality of protease activities may play some important roles in the development of Alzheimer's disease. In agreement with such

*To whom correspondence should be addressed .

133

134 T. AOYAGI et alt a hypothesis, we previously found deficiency in the cerebral tissues of patients with Alzheimer-type dementia [6]. In addition to the changes in the cerebral tissues, we found that the activities of and urokinase were significantly lower in serum of the Alzheimer patients [7]. These previous findings prompted us to study the protease changes in cerebrospinal fluid (CS F) of the patients.

MATERIALS AND METHODS

Cerebrospinal fluid of control subjects and patients with dementia. We selected 10 cases of Alzheimer-type dementia (primary degenerative dementia of the Alzheimer type, 73.7±2.9 SE years old), 10 cases of vascular dementia (78.0+ 4.7 [mean+SE] years old), and 21 control subjects (78.5± 1.6 years old) so that their ages were matched between the three groups. Alzheimer-type dementia was diagnosed by DSM-III-R criteria [8]; and vascular dementia, by NINDS criteria [9]. Several milliliters of cerebrospinal fluid were taken for examination. Substrates for assay. The sources of substrates were as follows (see Table 1) [11-19]: Arg • NA, Leu •NA, Pro • NA, Lys-Ala • NA, Arg-Arg • NA, and Gly-Pro • NA from Bachem Feichemi-kalien AG, Budendorf, Switzerland; Pro- Phe-Arg •MCA and Boc-Val-Pro-Arg •MCA from Institute Inc., Osaka; Z-Lys • S-Bzl from Sigma Chemical Company, St. Louis, MO. Z-Arg-Arg • NA was synthesized in our laboratory according to the method of Zervas et al. [10].

Table 1. List of the proteases measured and their substrates.

Abbreviations used: Arg • NA, L-arginine fl-naphthylamide hydrochloride; Leu • NA, L-leu- cine /3-naphthylamide hydrochloride; Pro. NA, L- /3-naphthylamide hydrochloride; Lys-Ala • NA, L-lysyl-L-alanine f3-naphthylamide; Arg-Arg • NA, L-arginyl-L-arginine /3-naph- thylamide; Gly-Pro • NA, glycyl-L-proline /3-naphthylamide; Z-Arg-Arg • NA, benzyloxycar- bononyl-L-arginyl-L-arginine /3-naphthylamide; Pro-Phe-Arg • MCA, L-prolyl-L-phenylala- nyl-L-arginine 4-methylcoumaryl-7-amide; Boc-Val-Pro-Arg • MCA, t-butyloxycarbonyl-L- valyl-L-prolyl-L-arginine 4-methylcoumaryl-7-amide; Z-Lys • S-Bzl, N-a-benzyloxycarbonyl- L-lysine thiobenzyl ester.

J. Clin. Biochem. Nutr. CSF PROTEASES IN DEMENTIA 135 Determination of enzyme activities. The cerebrospinal fluids were dispensed into microwell plates (nunclon, F96, Copenhagen, Denmark) for aminopeptidases and , to which the respective substrates were added, followed by incubation for 1 h at 3TC. For the aminopeptidase (AP) assay, 50 p 1 of 2.5 mM /3-naphthylamide derivative was used as substrate, and the absorbance at 525 nm was determined by a microplate reader model 3550 (BIO-RAD, Richmond, VA). For the assay, 20 ,u1 of 2.5 mM 4-methylcoumaryl-7-amide derivative was used, and the fluorescence (Ex 380 nm, Em 460 nm) was determined with a HITACHI MDF-4 fluorimeter (Hitachi). The references for the assay methods and substrates used are listed in Table 1. All the enzyme assays in the CSF were done in the range with linearity of the time-concentration relationship. The assays were done in triplicate, and their standard deviations were within 10% of the average values [6, 11]. For the assays, the units of enzyme activities were expressed as nmols of reaction products generated during one minute of incubation per ml (CSF). The time-course of the enzyme reaction in the CSF increased linearly for at least 60 min, and the enzymatic activities in the CSF were linear with respect to the CSF volume in the assay medium [18]. The synthetic substrates used for the assay of endopeptidase activities can be cleaved by miscellaneous different from the target enzymes. In order to exclude such nonspecific effects, we utilized bestatin to suppress extra reactions [6]. This maneuver enabled us to secure the linearity of the endopeptidase assays. Statistical analysis. Comparisons among three groups were made by the analysis of variance (ANOVA). The enzymes found to be significant with ANOVA were further analyzed by Bonferroni's method for differentiation between the two groups. The computations were done with a statistical software distributed by Kyoritsu Shuppan Co., Tokyo [20].

RESULTS

In Table 2 are compared the enzymatic activities in CSF among the three groups of subjects examined. In the Alzheimer patients, the activities of DPP-III, DPP-IV, and cathepsin B were significantly higher than in the controls. In the patients with vascular dementia, the activity of Leu-AP was significantly higher, but that of DPP-III was significantly lower, than in the controls. There was no significant difference as to age. Although the activity of PEP was also examined in some samples, no significant difference was seen. In order to examine these enzymatic changes from the multivariate point of view, we used factor analysis. Three main factors were extracted from the matrix of correlations among various enzymatic activities. Table 3 shows the factor loadings indicating the degree of correlation of such factors to each of the enzymes examined. One should note that this table contains only those with values of 0.5 or more. Factor 1 (Fl) was most closely related to the activities of kallikrein,

Vol. 14, No. 2, 1993 136 T. AOYAGI et al.

Table 2. Enzymatic changes in cerebrospinal fluid of patients with dementia .

*p<005; **p<0.01; according to Bonferroni's test; SE, standard error of mean.

Table 3. Relation of three main factors to various enzymes in cerebrospinal fluid .

Fig. 1. Comparison of enzyme network in cerebrospinal fluid on the plane of three main factors.

J. Clin. Biochem. Nutr. CSF PROTEASES IN DEMENTIA 137 Leu-AP, and LSP. Factor 2 (F2) was closely related to DPP-III, AP-B, and age; whereas factor 3 (F3) was moderately related to cathepsin B. Figure 1 shows the relationship among the three groups of the subjects examined on the scales of the above three factors: Fl, F2, and F3. Here, the ellipsoids represent the mean and SE of the factor scores for each group of subjects examined. In the direction of F2 on the scales, Alzheimer patients and control subjects were close to each other, but the group of vascular dementia was well separated from them. This is compatible with the above-mentioned fact that the activity of DPP-III was decreased in vascular dementia. On the scales of F 1 and F3, and on the scales of F2 and F3, one can see that, exclusively in the direction of F3, the differences between the controls and Alzheimer patients are larger than those between the controls and the patients with vascular dementia. This is compatible with the larger increase in the activity of cathepsin B in the group of Alzheimer patients, as shown in Table 2.

DISCUSSION

Alzheimer's disease is characterized by extracellular deposits of amyloid beta/ A4 protein fibrils in senile plaque cores and vessel walls in the brain. Amyloid beta-protein precursor (APP) represents a family of transmembrane glycoproteins containing amyloid beta-protein, and part of this protein is included in the transmembrane sequence [21]. Cole et al. [22] reported that stimulated platelets release membrane fragments containing both immunoreactive C-terminal and N-terminal portions of APP that should contain the intact beta/A4 sequence. They confirmed that human platelets can be stimulated by (or ionomy- cin) to secrete soluble truncated amyloid beta-protein precursor and particulate membrane fragments that contain immunoreactive C-terminal and N-terminal portions of amyloid beta-protein precursor. In agreement with previous reports suggesting the role of PEP in the produc- tion of amyloid-beta protein in the brain [23], we found that the PEP activity in the brain of Alzheimer patients was significantly increased [6]. In the present study, we failed to detect any significant changes in the activity of this enzyme in cerebrospinal fluid. It is to be noted, however, that in the present study the increase in the activity of cathepsin B was more related to Alzheimer's dementia than to vascular dementia. Moreover, the decrease in the kallikrein activity found in the cerebral tissues [7] was not reflected in the fluid. This discrepancy may be due to the property of the barrier between the cerebral tissues and cerebrospinal fluid. Vascular dementia was characterized more by an increase in the activity of Leu-AP than in that of cathepsin B. Thus the examination of cerebrospinal fluid may be utilized as a supplementary means for the differentiation between the above two types of dementia.

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