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

Aß42/Aß40 ratios (Figure 2). A massive effect was
observed for A4CT-I45F (34-fold increase) making
this construct important for the generation of animal
models for Alzheimer‘s disease. Unlike the other mutations,
A4CT-V44F was processed mainly to Aß38, as
determined by mass spectrometry. Our data provide a
detailed model for the active site of γ-secretase (Figure
2). According to this model Aß40 is produced when
γ-secretase interacts with APP by binding to one side
of the alpha-helical transmembrane domain of APP.
Alternatively, Aß42 arises by binding of γ-secretase
to the opposite side (Fig. 2). Mutations in the transmembrane
domain of APP interfere with the interaction
between γ-secretase and APP and, thus, alter the
cleavage specificity of γ-secretase (Fig. 2).
Figure 2: Schematic representation of the amino acid positions
(P) of the ß-secretase product A4CT of APP relative to
the cleavage site of γ-secretase. Top: linear arrangement of the
residues relative to the cleavage site after residue 40 (Aß40)
and residue 42 (Aß42). The scissile peptide bond is constituted
by residues P1 and P1‘. Bottom: helical wheel arrangement
of amino acids 40 to 49 of A4CT with respect to the cleavage
sites after residues 40 and 42. Binding of γ-secretase to the
transmembrane domain giving rise to Aß40 or Aß42 has to
occur at opposite sides of the helix.
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IV. Regulation of exon 15 splicing of APP
premRNA
C. Bergsdorff, S. Kreger, K. Paliga
Alternative splicing of exon 15 of the amyloid precursor
protein (APP) pre-mRNA generates two APP
isoform groups APP(ex15) (containing exon 15) and
L-APP (without exon 15), which show a cell-specific
distribution in non-neuronal cells and neurons of rat.
Both APP isoforms differ in regard to functional properties
like post-translational modification, APP secretion,
and proteolytic production of Aß peptide from
APP molecules. Since Aß generation is an important
factor in the development of Alzheimer‘s disease, one
could anticipate that these major APP isoforms might
contribute differentially to the mechanisms underlying
neurodegeneration in Alzheimer‘s disease. We
established an APP minigene system in a murine cell
system to identify cis-acting elements controlling exon
15 recognition. A 12.5-kilobase pair genomic fragment
of the murine APP gene contained all cis-regulatory
elements to reproduce the splicing pattern of the
endogenous APP transcripts. By using this approach,
two intronic cis-elements flanking exon 15 were identified
that block the inclusion of exon 15 in APP transcripts
of non-neuronal cells. Point mutation analysis
of these intronic regions indicated that pyrimidinerich
sequences are involved in the splice repressor
function. Finally, grafting experiments demonstrated
that these regulatory regions cell-specifically enhance
the blockage of a chimeric exon in the non-neuronal
splicing system.
V. Physiological function of APP
A. Fossgreen, F. Reinhard, S. Scheurermann, P.
Soba, J. Stumm
In collaboration with B. Brückner and R. Paro, ZMBH,
we used Drosophila melanogaster as a model system
to analyze the function of APP by expressing wildtype
and various mutant forms of human APP in fly
tissue culture cells as well as in transgenic fly lines.
After expression of full-length APP forms, secretion
of APP but not of Aß was observed in both systems. By
using SPA4CT, a short APP form in which the signal
peptide was fused directly to the Aß region, transmembrane
domain, and cytoplasmic tail, we observed
Aß release in flies and fly-tissue culture cells. Consequently,
we showed a γ-secretase activity in flies.
Interestingly, transgenic flies expressing full-length
forms of APP have a blistered-wing phenotype. As
the wing is composed of interacting dorsal and ventral
epithelial cell layers, this phenotype suggests that
human APP expression interferes with cell adhesion/
signaling pathways in Drosophila, independently of
Aß generation. Wing morphogenesis in Drosophila is
characterized by the apposition of two epithelial cell
layers, the dorsal and ventral layer and may thus be
used as a model to analyze the role of APP in the interaction
of presynaptic and postsynaptic membranes at
the synapse. We presume that APP negatively interacts
with factors involved in the adhesion of the two wing
epithelia and the two synaptic membranes, respectively.
In Drosophila, some mutations deficient for the expression
of specific integrin subunits develop distinct wing
blisters whereas another one was shown to be involved
in short term memory. This provides an interesting
link between physiological APP functions, integrinmediated
adhesion processes and memory mechanisms.
Since integrins are heterodimeric receptors that
may be ligand-bound to extracellular matrix molecules
and may have a signaling function, integrins are
especially required for the function of the cell-matrixcell
junction, where one surface of a cell such as a
synaptic density adheres to the other. This function
of integrins supports our suggestion of APP’s involvement
in cell adhesion. We envisage that APP might
act as an antagonist for integrins, both in the developing
wing and at plasticity of the synapse. APP could
bind, either directly or via other molecules to the same
extracellular matrix sites as integrins. Indeed, in rat
primary neurons APP and integrins were colocalized
in structures that are reminiscent of synapses. However,
APP might be involved in other pathways resulting
in a deterioration of the adhesion or signaling
pathways supported by the integrins. For example,
expression of a mutant form of G protein involved in
cAMP signaling or the blistered gene encoding a protein
related to human serum response factors result
in Drosophila a comparable wing. Taken together,
Drosophila seems to be a valuable animal-model
system for studying the physiological and pathogenic
functions of human APP since it may be suited for
unraveling APP transmembrane signaling mechanisms
potentially related to Alzheimer‘s disease. By using
this fly model, testing genetic interactions between
APP-expressing lines producing the blistered phenotype
and other mutants engaged in wing morphology
offers the perspective to unravel those physiological
and pathogenic pathways linked to human APP.
VI. Regulation of APP-Aß metabolism by cholesterol
C. Bergmann, T. Hartmann, H. Runz, P. Semenfeld,
I. Tomic
Cholesterol metabolism and Alzheimer‘s disease are
genetically linked. The apoEε4 allele of the apolipoprotein
E gene is associated with higher cholesterol
levels and was shown to increase the risk of develop-
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