ZMBH J.Bericht 2000 - Zentrum für Molekulare Biologie der ...

APP catalyzed the reduction of H 2 O 2 and oxidation of
Cu(I) to Cu(II) in a „peroxidative“ reaction in vitro.
The resulting bound copper-hydroxyl radical intermediate
[APP-Cu(II) (•OH)] then likely participated in
a Fenton type of reaction with radical formation as a
prerequisite for APP-Cu(I) complex degradation. Evidence
from two observations suggests that the reaction
takes place in two phases. Bathocuproine, a trapping
agent for Cu(I), abolished the initial fragmentation
of APP, and chelation of Cu(II) by DTPA (diethylenetriaminepentaacetic
acid) interrupted the reaction
cascade induced by H 2 O 2 at later stages. WT cortical,
cerebellar and hippocampal neurons were significantly
more susceptible than their respective APP 0/0 neurons
to toxicity induced by physiological concentrations
of copper but not by zinc or iron. There was no difference
in copper toxicity between APLP2 0/0 and WT
neurons, demonstrating specificity for APP-associated
copper toxicity. Treatment of neuronal cultures with a
peptide corresponding to the human APP copper binding
domain (APP142-166) potentiated copper but not
iron or zinc toxicity. Incubation of APP142-166 with
low density lipoprotein (LDL) and copper resulted
in significantly increased lipid peroxidation compared
to copper and LDL alone. Substitution of the
copper co-ordinating histidine residues with asparagines
(APP142-166 H147N, H149N, H151N ) abrogated the
toxic effects. A peptide corresponding to the zinc binding
domain (APP181-208) failed to induce copper or
zinc toxicity in neuronal cultures. These data support a
role for the APP copper binding domain in APP-mediated
Cu(I) generation and toxicity in primary neurons.
To test the hypothesis that biometals such as copper
can contribute to the amyloid pathology in AD we are
currently investigating the dietary exposure of copper
and its chelators to APP transgenic mice and to APP
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knock-out mice. The effects of dietary supplementation
is studied by ICP-MS (inductively coupled plasma
mass-spectrometry) to determine the levels of Cu, Zn
and Fe. The AD pathology is analyzed by conventional
methods. We expect that Cu has an effect on the
AD pathology and metal-chelation can delay amyloid
Aß deposition in APP transgenic mice.
II. The role of copper and zinc in the normal
function of the amyloid precursor protein
(APP)
C. Schmidt, A. Simons, M. Strauss, A. Schlicksupp,
G. Multhaup (in collaboration with D.
Ha-Hao, R. Cappai, A. White, C.L. Masters and
K. Beyreuther)
The expression of APP and APLP2 in the brain
suggests they could have an important direct or indirect
role in neuronal metal homeostasis. In APP and
APLP2 knockout mice copper levels were significantly
elevated in both APP 0/0 and APLP2 0/0 cerebral
cortex (40% and 16%, respectively) and liver (80% and
36%, respectively) compared with matched wild-type
(WT) mice. These findings indicate APP and APLP2
expression specifically modulates copper homeostasis
in the liver and cerebral cortex, the latter being a
region of the brain particularly involved in AD. Perturbations
to APP metabolism and in particular, its secretion
or release from neurons may alter copper homeostasis
and explain a disturbed metal-ion homeostasis
observed in AD.
Zinc up to concentrations of 50µM or the presence of
1,10-phenanthroline specifically increased the level of
secreted APP in APP transfected CHO-K1 cells. By
contrast, the level of secreted APP in copper-resistant
CHO-CUR3 cells remained unaffected. APP holoprotein
increased dramatically in CHO-CUR3 cells com-
pared with CHO-K1 cells. The large decrease of Aß
release seen in both cell lines at elevated extracellular
zinc levels was due to specific inhibition of secretion.
These results indicate that a disturbed zinc-homeostasis
may be an important factor influencing APP production,
transport and processing.
Figure 3: The α- and β-secretase protease activities cleave
APP within its ectodomain. The remaining membranebound
C-terminal fragment p3CT (after α-secretase cleavage
of APP) is further cleaved by γ-secretase in the middle
of the putative transmembrane domain, yielding p3 (α- and
γ-secretase activities). Amyloid Aβ is produced by β- and
γ-secretase activities; CuBD-I, copper binding domain 1.
Adding copper to APP-transfected CHO cells greatly
reduced the levels of ß-amyloid (Aß) peptide in both
parental CHO-K1 and in copper resistant CHO-CUR3
cells which have lower intracellular copper levels.
Copper also caused an increase in the secretion of the
APP ectodomain indicating that the large decrease in
Aß release was not due to a general inhibition in protein
secretion. There was an increase in intracellular
full-length APP levels which paralleled the decrease
in Aß generation suggesting the existence of two distinct
regulating mechanisms, one acting on Aß production
and the other on APP synthesis. Thus, our
findings suggest that copper or copper agonists might
be useful tools to discover novel targets for anti-
Alzheimer drugs since copper promoted the non-amyloidogenic
pathway of APP.
Our current understanding is that copper and/or zinc
binding is central to the normal cellular function of
APP. We therefore want to identify agonists of APP
ligand binding sites (e.g. of the copper-binding site)
that are able to inhibit amyloidogenic proteolytic processing
of APP. Lead compounds will be discovered
followed by a further screen for small APP ligands.
Our research program combines biochemical, immunohistochemical
and cellular approaches to start a
risk-benefit analysis of divalent metal binding agonists
of APP in vitro.
III. Dimerization and stability of APP isoforms:
influence of APP ligands on relative
stability and metabolism
M. Strauss, C. Schmidt, A. Simons, A. Schlicksupp,
C. Elle, G. Multhaup (in collaboration with
C. Haass, C.L. Masters and K. Beyreuther)
The highly conserved nature and tissue specificity of
the eight APP isoforms provide circumstantial evidence
that functional differences among isoforms may
exist in vitro and in vivo. When APP and Aß are central
to AD pathogenesis, then molecules which influence
the conformation and the stability of APP isoforms
and that interact with specific APP forms could
differentially alter their metabolism/activity and thus
represent risk factors for AD.
For example, heparin and Zn(II) were found to augment
the ability of full-length and secreted KPI-APP to
inhibit FXIa. In contrast, both compounds heparin and
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