Poster
Recent research suggests that some forms of beta amyloid
peptides (Ab) inhibit the high-affinity choline uptake in
rat hippocampus in vitro [1,2]. It seems that the effect
could lead to autocannibalism of cholinergic neurons observed
in Alzheimer disease. The specific binding of (3H)hemicholinium-3
is closely associated with substrate recognition site on
the high-affinity choline carrier [3]. However, the in vitro
effect of Ab on this binding site has not been evaluated
yet.
Our experiments on hippocampal synaptosomes of male
and female 3-month old Wistar rats suggest that lipophilic
Ab 1-40 eliminates depolarization effects and reminds actions
of ethanol [4]. However, 100 nM concentration incubated 10-20
min has evoked no significant effects on the high-affinity
transport and specific binding under basal conditions. Plant
cysteine proteolytic enzymes bromelain and papain (100 mg/ml,
short-term 10-20 min preincubation), two anti-inflammatory
drugs [5] and perspective supportive agents for Alzheimer
disease therapy, have decreased the uptake and specific binding
under both basal and stimulated conditions. Membrane-localized
carrier protein seems to be cleft by both proteases independently
of its actual functional state, i.e. active as well as inactive
carriers. It appears that papain influences rather external
part of carrier and surrounding membrane in comparison with
a more specific action of bromelain. The high-affinity transport
measured on hippocampal synaptosomes of young rats and previously
inhibited by Ab 1-40 is more sensitive to the effects of
bromelain and papain in vitro.
[1] D.S.Auld et a., TINS, 21: 43-49, 1998.
[2] S.Kar et al., J Neurochem, 70: 2179-2187, 1998.
[3] S.S.G.Ferguson et al., J Neurochem, 63: 1328-1337,
1994.
[4] Z.Krištofiková et al., Neurochem
Res, 23: 923-929, 1998.
[5] K.L.Lee et al., Biochem J, 327: 199-202, 1997.
unpublished paper
Introduction
Brains of patients with Alzheimer disease (AD) are characterized
among others by the presence of amyloid deposits in selected
brain regions (1). Recent research suggests important physiological
functions for amyloid beta peptides (Abeta) proteolytically
derived from a larger amyloid precursor protein (2). However,
depending also on the fragment length and on the degree of
aggregation, higher concentrations of Abeta can induce apoptotic
or necrotic neuronal degeneration (1,2).
Enhanced vulnerability of cholinergic forebrain system
under normal and pathological aging (cholinergic hypothesis
of AD) has been described for many times (e.g., 3). Recent
research suggests direct actions of Abeta on cholinergic
neurons leading probably to their autocannibalism under pathological
conditions. While higher concentrations of Abeta (mM) incubated
for a sufficiently long time (hours) increase generally the
lipid peroxidation (1,2) and the leakage of choline across
cell membranes (2,4,5), low concentrations (pM-nM) incubated
for a short time (minutes) influence significantly cholinergic
neurotransmission (2,6). Some fragments of Abeta inhibit
among others the hippocampal and cortical high-affinity choline
uptake (HACU) that is a rate-limiting step in acetylcholine
synthesis (6). However, effects of Abeta on the specific
binding of (3H)hemicholinium-3 ((3H)HC-3, specific and competitive
inhibitor of HACU) have not been evaluated yet.
At the present time, great attention concentrates
on the therapy of AD and a possible administration of plant
cysteine proteases as supportive agents is suggested. It
seems that different endogenous proteases or their inhibitors
play a role in the ethiology of senile plaques and neurofibrillary
tangles. Some of these mechanisms could be influenced by
exogenous proteases. Moreover, the proteolytic enzymes act
as anti-inflammatory agents and increase the permeability
of the blood-brain barrier. Our preliminary results of experiments
in vitro indicate certain advantages for a perspective application
in vivo to demented patients especially for bromelain (BRO)
and papain (PAP).
Aim of the study
• To evaluate in vitro effects of Abeta on the
specific binding of (3H)HC-3 and to contribute to the more
detailed elucidation of action mechanism on hippocampal presynaptic
cholinergic nerve terminals.
• To evaluate in vitro effects of BRO and PAP
on HACU and (3H)HC-3 binding.
• To compare these results with our preliminary
data and to assess possible risks and advantages for a protease
application in AD therapy.
Material and methods
• Brain tissue: Hippocampal synaptosomes were
isolated from 68 male and female 3-month old Wistar rats
of the Konárovice breed.
• Chemicals: Abeta (amyloid
beta-protein fragment 1-40, Sigma), BRO (Ananas Comosus,
Mucos Pharma), PAP (Carica papaya, Mucos Pharma), (3H)choline
(NEN), (3H)HC-3 (NEN) and HC-3 (Sigma) were used.
• Methods: HACU and (3H)HC-3
binding were measured in accordance with our previous work
(7). Abeta and proteases were incubated for 10 min (HACU)
or for 20 min ((3H)HC-3 binding) at 37°C before measurement.
Concentration of proteins was estimated by a method of Bradford
(8).
• Data analysis: ANOVA and Student's t-test
(separate variance) were applied. Data are presented as the
means ± S.D.
Results
• 100 nM Abeta influenced significantly the
HACU and (3H)HC-3 specific binding only under stimulated
conditions. More detailed analysis revealed that the decrease
of transport is linked to the change in KM, the decrease
of binding to the change in both Bmax and Kd. |
