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Evaluation of perspective agents for Alzheimer disease therapy by means of in vitro test with beta amyloid protein
Kri?tofiková Z., Klaschka J., Majer E.* and Fales E.*
Prague Psychiatric Centre and *Psychiatric Hospital, Ustavni 91, 181 03 Prague 8 - Bohnice, Czech Republic
9th International Conference of Biological Psychiatry, Luhacovice, June 16.-19.,1999.
527 KA (18-03-1) 
Divider
Introduction
Brains of patients with Alzheimer disease (AD) are characterized among others by the presence of amyloid deposits in selected brain regions. Amyloid beta peptides (Abeta) are proteolytically derived from a larger transmembrane amyloid precursor protein. The Abeta 1-40 is the predominant form of cerebrovascular soluble amyloid produced by different cell types and normally present in the circulation. It is capable of forming stable amyloid fibres but the rate of aggregation is a very slow process in the absence of nucleating agents. On the contrary, the rate of aggregation is increased for the longer Abeta 1-42 that is the major constituent of insoluble amyloid fibres of senile plaques (1).
Recent research suggests important physiological functions for Abeta under normal conditions (2). However, depending on the degree of aggregation, higher concentrations can induce apoptotic or necrotic neuronal degeneration (1, 2). It seems that one of possible mechanism of neurotoxicity could be mediated by reactive free radicals. Concentrations of Abeta 25-35 as low as 10 nM intercalate into the membrane and inhibit the lipid peroxidation (LPO) in a dose- and time-dependent manner as a result of physicochemical interactions of amphiphilic peptide with the membrane bilayer (3). On the contrary, higher concentrations of different fragments significantly increase membrane fluidity and induce the LPO (1, 2, 4).
At the present time, great attention concentrates on the therapy of AD and a possible administration of some plant or animal proteases is suggested. There are several reasons for their therapeutic testing: i) Abnormal metabolisms of endogenous proteases and their inhibitors play a role in the ethiology of senile plaques, ii) Abnormally phosphorylated tau proteins are major components of neurofibrillary tangles. An important role for proteolytic enzyme calpain in tau metabolism is suggested, iii) An increased vulnerability of basal forebrain cholinergic neurons to Abeta during AD has been observed. A potential role of acetylcholinesterase in the stimulation of the Abeta aggregation is assumed. In all the three above-mentioned cases, some proteolytic mechanisms could be influenced by exogenous proteases. iv) Inflammatory mechanisms play a very important role in AD pathogenesis. Exogenously administered proteolytic enzymes act as anti-inflammatory, anti-edematous and immunostimulating agents through the regulation of cytokines (5). And finally, v) proteases can increase the permeability of the blood-brain barrier to some exogenous drugs. Therefore, the application of proteases as supportive agents together with other medicaments is tested now in neurology (e.g., cerebrospinal sclerosis).
The aim of this study is to evaluate in vitro experiments with Abeta and plant proteolytic enzymes (bromelain - BRO, papain - PAP) using the thiobarbituric acid-test for the determination of the efficiency of the proteases to influence the effects of Abeta on LPO in the hippocampal rat and human tissue.
Materials and methods
i) Brain tissue: hippocampal animal tissue (male and female 1-, 3- and 23-month old Wistar rats of the Konarovice breed) and hippocampal human tissue (cornu Ammoni et gyrus parahippocampalis of men and women, demented patients with AD or multi-infarct dementia (MID) as controls)
ii) Chemicals: Abeta (amyloid beta-protein fragment 1-40, Sigma), BRO (Ananas Comosus, Mucos Pharma), PAP (Carica papaya , Mucos Pharma) and 10% DMSO (dimethyl sulfoxide, Sigma) as a solvent
iii) Methods: thiobarbituric acid test by Ohkawa et al. (6)
iv) Data analysis: ANOVA and Student's t-test (separate variance)
* p < 0.05, ** p < 0.01, *** p < 0.001
data presented as the means ± S.D.
Results
i) The effect of Abeta on LPO in the hippocampal tissue of young 3-month animals was significantly time- and dose-dependent. No changes were observed for 10 nM concentration in the case of the short (15-30 min) as well as long incubation (60-120 min) (Fig. 1). However, the decrease of LPO after the application of a small Abeta concentration could be hidden behind the DMSO effect that acts as a OH radical scavenger (7). 100-500 nM concentrations increased the LPO in the case of the longer 60 min incubation (Fig. 2). The effect of 1mM Abeta was comparable for 30 and 60 min incubations (Tables I-IV).
ii) The effect of Abeta on the LPO was signficantly age-dependent (Tables II and III). The higher effect of 1 mM Abeta was found in the brain tissue of old compared to young animals.
iii) The effects of both plant proteases on the LPO in hippocampal tissue of 3-month old animals were more rapid than those of Abeta. No significant differences between 30 and 60 min incubation were found (Tables I-III). Concentrations lower than 100 mg/ml did not influence significantly the LPO (Fig. 3). 100 mg /ml concentration increased the LPO (Tables I-IV).
iv) The effects of both proteases and especially of BRO were significantly age-dependent. The higher LPO after application of proteases was found in the brain tissue of young animals (Table III).
v) Both proteases eliminated the effect of Abeta previously applied to hippocampal homogenates of young animals (Table IV, experiment A). Abeta added to samples previously incubated with both proteases did not significantly increase the LPO (Table IV, experiment B).
vi) The lower basal levels of lipid peroxides and the higher effects of Abeta and of both proteases were found in the AD compared to the MID group (Table V).
Table I: The effects ofAbeta and of plant proteases on LPO in rat brain during 30 min incubation
age of animals:
1-month
(n)
3-month
(n)
23-month
(n)
buffer
22.1 ± 1.0
(18)
22.4 ± 1.4
(7)
23.1±1.6
(8)
1 mM Abeta
23.1±1.6
(8)
23.8 ± 1.0
(7)
22.8 ± 1.4
(8)
100 mg/ml BRO
26.4 ± 1.9***
(10) 
25.6 ± 0.5***
(7)
24.3 ± 0.7
(8)
100 mg/ml PAP
24.6 ± 1.9**
(10) 
23.3 ± 1.2
(7)
24.0 ± 2.1
(8)
ANOVA:
p< 0.001
p< 0.001
p= 0.1771
The experiments were performed on mixed hippocampal homogenates of eleven male 1-month, six female 3-month and six male 23-month old animals. The values are expressed as nmoles of thiobarbituric acid-reactive products per g of tissue. All samples contained 0.9% DMSO.
Table II: The effects of Abeta and of plant proteases on LPO in rat brain during 60 min incubation
age of animals:
1-month
(n)
3-month
(n)
23-month
(n)
buffer
22.4 ± 1.3
(8)
23.3 ± 1.4
(8)
22.5 ± 0.6
(8)
1 mM Abeta
21.1±1.5
(8)
23.3 ± 1.5
(8)
23.8 ± 0.8**
(8)
100 mg/ml BRO
27.4 ± 1.7***
(8)
27.8 ± 1.6***
(8)
25.4 ± 1.2***
(8)
100 mg/ml PAP
23.1±1.1
(8)
25.3 ± 0.9**
(8)
23.6 ± 1.2*
(8)
ANOVA:
p< 0.001
p< 0.001
p< 0.001
The experiments were performed on mixed hippocampal homogenates of thirteen female 1-month, six female 3-month and seven male 23-month old animals. The values are expressed as nmoles of thiobarbituric acid-reactive products per g of tissue. All samples contained 0.9% DMSO.
Table III: The effects of age on LPO in rat brain influenced by Abeta and plant proteases
age of animals
Abeta
BRO
PAP
i) 30 min incubation
1-month
104.5 ± 7.3
119.7 ± 8.4
111.4±8.6
3-month
105.9 ± 4.2
114.1 ± 2.4
103.7 ± 5.5*
23-month
98.6 ± 5.9
105.1 ± 3.1***
103.8 ± 8.8
ANOVA:
p= 0.0589
p< 0.001
p-0.0838
ii) 60 min incubation:
1-month
94.2 ± 6.8
122.6 ± 7.4
103.4 ± 4.7
3-month
100.0 ± 6.3
119.3 ± 6.9
108.4 ± 4.0*
23-month
105.7 ± 3.6**
112.9 ± 5.4*
105.0 ± 5.0
ANOVA:
p= 0.0026
p = 0.0246
p= 0.1100
The levels of thiobarbituric acid-reactive products from Tables I and II were related to the levels with buffer and expressed in %.
Table IV: Shared effects of Abeta and both plant proteases on LPO in rat brain
Experiment A
Experiment B
composition
n
nmoles/g of tissue
composition
n
nmoles/g of tissue
Abeta
5
31.0 ± 2.2
Abeta
6
34.0 ± 1.7
BRO
5
27.5 ± 1.0
BRO
6
21.9 ± 2.2
PAP
5
29.0 ± 1.1
PAP
6
31.3±3.2
Abeta + BRO
5
28.3 ± 1.2*
BRO + Abeta
6
23.5 ± 2.5***
Abeta + PAP
5
27.5 ± 1.3*
PAP + Abeta
6
30.2± 1.1**
ANOVA:
p = 0.0053
ANOVA:
p< 0.001
Experiment A: samples were firstly preincubated for 30 min with 1 mM Abeta or 1.7% DMSO, subsequently proteases (100 mg/ml) were added and incubated for further 30 min, the experiment was performed on mixed homogenates of four male 3-month old rats.
Experiment B: samples were firstly preincubated for 30 min with 100 mg/ml proteases or 1.7% DMSO, subsequently 1 mM Abeta was added and incubated for further 30 min, the experiment was performed on mixed homogenates of five male 3-month old rats t-test was calculated with respect to the samples with Abeta.
Table V: The effects of Abeta and plant proteases on LPO in human brain of patients with Alzheimer disease (AD) and multi-infarct dementia (MID) during 60 min incubation
composition
AD (n=4)
nmoles/g of tissue
MID (n=4)
nmoles/g of tissue
buffer
32.6 ± 4.5
38.7 ± 0.5
1 mM Abeta
38.2 ± 1.1
37.9 ± 1.8
100 mg/ml BRO
41.0 ± 1.1*
41.0 ± 1.1*
100 mg/ml PAP
37.5 ± 2.0
38.6 ± 3.5
ANOVA:
p = 0.0049
p=0.2311
The experiments were performed on mixed hippocampal homogenates. All samples obtained 0.9 % DMSO. Statistical significance (t-test) between AD and MID for samples with buffer: p = 0.0724.
group
n
sex
(M/F)
age
(years)
postmortem interval
(hours)
brain weigh
(g)
AD
3
0/3
86.3 ± 2.1
5.3 ± 0.6
983.3 ± 104.1
MID
3
2/1
83.7 ± 6.4
6.0 ± 1.0
1100.0 ± 0.0
Fig. 1: The effect of time on LPO for Abeta and DMSO

% DMSO: related to DMSO at 15 min                 ANOVA for DMSO: p=0.0062
% Abeta: related to corresponding DMSO             ANOVA for Abeta: p=0.2056
Fig. 2: The effect of Abeta on LPO

ANOVA for 30 min: p=0.0085
ANOVA for 60 min: p=0.0002
Fig. 3: The effect of proteases on LPO

ANOVA for BRO: p=0.1045
ANOVA for PAP: p=0.1653
Discussion
Increased levels of lipid peroxides in the individual brain regions of patients with AD have been observed for many times (8). A marked role for Abeta in the induction of oxidative stress is suggested. The effect of normal aging on the susceptibility of brain tissue to undergo the LPO has been also found (e.g., 9). It is well known that some nootropic drugs act as free radical scavengers (10, 11). Therefore, a question of whether drugs increasing the LPO can be applied to patients with AD should be raised here.
Our results with BRO and PAP are in accordance with the other studies where the enhanced release of reactive oxygen species after application of some plant proteases in vivo and in vitro have been found (e.g., 12). It is suggested that this release is one of the mechanism of anticancer activity of proteolytic enzymes. Regarding the AD therapy, some of our experiments in vitro suggest certain positive circumstances for the BRO and PAP human application in vivo in future. Firstly, the effects of Abeta and of both proteases on LPO are significantly age-dependent with antagonistic trends. While the modified crosslinked brain proteins of aging animals are more resistant to proteolysis (13, Tables I-III), the effect of Abeta is more pronounced in old animals (Tables II and III). Secondly, both proteases are able to eliminate the negative effects of Abeta on LPO (Table IV). However, more detailed experiments and especially the application in vivo to young and old rats must be further performed.
Our experiments on demented patients suggest that oxidative impairment of hippocampal tissue in AD group can be lower (decreased basal levels of lipid peroxides, higher effects of proteases) when compared with the MID group. On the contrary, the higher sensitivity to Abeta have been found in AD patients (Table V).
References
1. Cotman CW and Pike CJ (1994): Alzheimer Disease (Terry RD, Katzman R and Bick KL, eds.). New York, Raven Press, pp. 305-315.
2. Auld DS, Kar S and Quirion R (1998): Trends Neurosci. 21, 43-49.
3. Walter MF, Mason PE and Mason RP (1997): Biochem. Biophys. Res. Commun. 233, 760-764.
4. Avdulov NA, Chochina SV, Igbavboa U, O'Hare EO, Schroeder F, Clearly JP and Wood WG (1997): J. Neurochem. 68, 2086-2091.
5. Klaschka J (1997): Systémová Enzymoterapie. Praha, Dum Medicíny, pp. 34-85.
6. Ohkawa H, Ohishi N and Yagi K (1979): Anal. Biochem. 95, 351-358.
7. Ueda T, Toyoshima Y, Kushihashi T, Hishida T and Yassuhara H (1993): J. Toxicol. Sci. 18, 239-244.
8. Markesbery WR and Ehmann WD (1994): Alzheimer Disease (Terry RD, Katzman R and Bick KL, eds.). New York, Raven Press, pp. 353-367.
9. Kri?tofiková Z, Majer E, Fales E, Pekn? I and Klaschka J. (1998): Dement. Geriatr.Disord.9, 6-12.
10. Bene?ová 0, Krejci I and Pavlík A (1991): Nootropic Drugs. Prague, Avicenum, pp. 22-25.
11. Stone TW (1994): Anti-dementia Agents (Nicholson D., ed.). London, Academic Press, pp.229-249
12. Zavadova E, Desser L and Mohr T (1995): Cancer Biotherapy 10, 147-152.
13. Burcham PC and Kuhan YT (1997): Arch. Biochem. Biophys. 340, 2331-2337

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