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

ing the disease. To study the influence of cholesterol
on APP metabolism we collaborated with M. Simons,
K. Simons and C.G. Dotti (EMBL) and used primary
cultures of rat hippocampal neurons infected with
recombinant Semliki Forest virus carrying APP and
APP mutants. This system has been used previously
by us to study the intracellular transport and processing
of human APP. Inhibition of cholesterol biosynthesis
and extraction of cholesterol from neuronal membranes
was achieved by a combination of statin treatment
and methyl-ß-cyclodextrin extraction. Statins
such as lovastatin or simvastatin allow in the presence
of low amounts of mevalonate the control of the synthesis
of cholesterol by inhibiting 3-hydroxy-3-methylglutaryl-CoA
(HMG-CoA) reductase. Mevalonate is
required for the synthesis of non-steroidal products
and essential to prevent inhibition of the proteasome
by lovastatin. ß-Cyclodextrin has been shown to very
efficiently and selectively extract cholesterol from the
plasma membrane.
The combined treatment of rat hippocampal neurons
with statins and methyl-ß-cyclodextrin resulted in
reduced intracellular Aß production and Aß secretion
(Fig. 3), however APP levels and cell viability
remained unaffected in both treated and control cells
during the time course of the experiments.
Since Aß generation occurs in two steps, we employed
the two APP constructs APP695 and A4CT (SPC99)
to analyze which Aß cleavage is inhibited by cholesterol
lowering drugs. The first cleavage of APP by
ß-secretase generates the 10-kDa fragment C99 that is
further cleaved within the transmembrane domain by
γ-secretase to produce Aß. When antibodies recognizing
the C-terminal domain of APP were used to immunoprecipitate
APP fragments from the cell lysates of
cholesterol-depleted and control rat hippocampal neurons
expressing human APP695 a dramatic inhibition
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of ß-cleavage was detected while the production of the
fragment generated by α-secretase was unperturbed.
To analyze the effect of lovastatin treatment on the
cleavage of γ-secretase, neurons expressing SPC99
were used since removal of the transient signal
sequence of SPC99 generates C99 which is identical
with the C-terminal ß-secretase product of APP. Conversion
of C99 to Aß requires γ-secretase. Both lovastatin
treatment and methyl-ß-cyclodextrin treatment
- alone and together - of neurons expressing SPC99
inhibited Aß production as well suggesting that cholesterol-lowering
regiments inhibit also γ-secretase
cleavage. Thus our combined results suggest that cholesterol
is required for APP cleavage by ß-secretase
and γ-secretase but not for α-cleavage that produces
αAPPsec. We conclude that lowering of cholesterol
affects amyloidogenic processing of APP while allowing
nonamyloidogenic cleavage to proceed.
These findings raise the question how cholesterol
affects Aß formation. One possibility is that reduc-
Figure 3: Cholesterol-lowering of hippocampal neurons
reduces intracellular (lysate) production and secretion
(media) of Aß40 and Aß2. Neurons were grown for 4 days
in the presence of lovastatin/mevalonate and after infection
with Semliki Forest virus/APP-expression vectors were
treated with 5mM methyl-ß-cyclodextrin for 20 min.
tion in membrane cholesterol changes the intracellular
transport of APP so that the protein does not reach the
cellular sites where ß- and γ-secretase cleavage takes
place. If this is the case, cholesterol is required as a
sorting platform for inclusion of APP protein cargo
destined for delivery to the apical membrane in nonneuronal
cells and to the axonal membrane in neurons.
Alternatively, the ß- and γ-secretase require cholesterol
for their activity.
VII. Aß as biological marker of Alzheimer’s
disease
A. Diehlmann, T. Hartmann, N. Ida
All mutations known to cause familial Alzheimer‘s
disease act by increasing the levels of soluble ß-amyloid
peptide, especially the longer form, Aß42. However,
in vivo elevation of soluble Aß in sporadic
Alzheimer‘s disease has so far not been shown. In collaboration
with M. Jensen and L. Lannfelt (Karolinska),
we used our monoclonal antibodies specific for
Aß40 and Aß42 in an enzyme-linked immunosorbent
assay to investigate cerebrospinal fluid from sporadic
Alzheimer‘s disease at different stages of disease
severity, to clarify the roles of Aß42 and Aß40 during
disease progression. We also evaluated three other
groups, one group of patients with mild cognitive
impairment who were at risk of developing dementia,
a cognitively intact, nondemented reference group
diagnosed with depression, and a perfectly healthy
control group. We found that Aß42 is strongly elevated
in early and mid stages of AD, and thereafter
it declines with disease progression. On the contrary,
Aß40 levels were decreased in early and mid stages
of AD. The group of cognitively impaired patients
and the depression reference group had significantly
higher levels of Aß42 than the healthy control group,
implying that Aß42 is increased not only in AD, but
in other central nervous system conditions as well.
Our data also point out the importance of having thoroughly
examined control material. The initial increase
and subsequent decrease of Aß42 adds a new biochemical
tool to follow the progression of AD and
might be important in the monitoring of therapeutics.
Acknowledgements: We acknowledge the stimulating
and productive collaboration with among others
the group of Gerd Multhaup (ZMBH), Colin L. Masters
(The University of Melbourne, Australia), Renato
Paro (ZMBH), Kai Simons and Carlos G. Dotti
(EMBL), Thomas A. Bayer (Psychiatry, University of
Bonn, Germany), Michael Hennerici and Klaus Fassbender
(Neurology, University Heidelberg at Mann–
heim), Lars Lannfelt (Karolinska Institute, Stockholm,
Sweden); Joachim Schröder (Psychiatry, University
Heidelberg), Bart De Strooper (University Leuven,
Belgium), Rudolph E. Tanzi and Ashley I. Bush
(Harvard University, Cambridge, USA), Hans Peter
Schmidt (Neuropathology, University Heidelberg),
Hans Förstl (Psychiatry, TU Munich, Germany) and
Christian Haass (Biochemistry, University Munich).
External Funding
Our research summarized in this report would not
have been possible without the following grants: an
institutional grant of the Minister for Science and Arts
of the State of Baden-Württemberg, by grants and project
grants of the Deutsche Forschungsgemeinschaft,
of the Landes Forschungsschwerpunktprogramm of
Baden-Württemberg, the European Community, of the
German-Israelic-Foundation, of the Humboldt-Foundation,
of the Fonds of the Chemical Industry of Germany
and by generous donations.
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