Zelluläre Modulatoren der Ektodomänenspaltung von APP

The processing of APP occurs in two alternative ways: upon release
of the ectodomain by α-secretase, the neuroprotective
APPsα-fragment is produced. But if APP is cleaved by the
β-secretase the Aβ-peptide can be produced. To be able to influence
the production of Aβ-peptides, it is essential to understand how it
is decided if cleavage occurs by α- or β-secretase. At present
little is known about the control of the alternate processing.
Until now, the molecular mechanisms and especially the responsible
cellular modulators are not understood in detail or not yet
identified. To get a better understanding of cellular regulatory
processes and to identify novel cellular modulators of APP
ectodomain shedding, the present work chose two approaches: on the
one hand cellular mechanisms of TMEM59-mediated inhibition
ectodomain shedding of APP were investigated. On the other hand a
genome-wide RNAi screening in Drosophila cells was performed in
order to identify novel cellular modulators of APP ectodomain
shedding in human cells. TMEM59 was identified as a novel modulator
of APP ectodomain shedding in a cDNA expression screening in the
lab (Neumann et al., 2006; Schobel et al., 2008; Schobel et al.,
2006). TMEM59 is a Golgi protein that inhibits on the one hand
processing and maturation of APP and on the other hand Golgi
glycosylation reactions (Fischer, 2008). My own work could verify
these effects of TMEM59 and its homolog TMEM59L on processing and
maturation of APP. In particular, it was shown that these effects
are not only true for transiently expressed APP but also for
endogenous levels of APP. In detailed immunofluorescence studies it
was shown that TMEM59 colocalizes with different markers of the
Golgi subcompartments and that therefore TMEM59 is present
throughout the whole Golgi apparatus. This finding points to a more
general modulation of Golgi glycosylation reactions by TMEM59. To
test if TMEM59-dependet modulation of Golgi glycosylation reactions
also affects APP secretases ADAM10 and BACE1, which are also
glycosylated proteins, the activities of these proteases were
investigated. It was shown that proteolytic activities were not
changed, ruling out that impairment of secretase activities by
TMEM59 could cause the observed inhibition of APP processing. But
interestingly, studies of intracellular APP transport could show
that TMEM59 caused retention of APP in the Golgi apparatus and
blockage of transport towards the cell surface and into endosomal
compartments. Since APP is cleaved by α-secretase at the plasma
membrane and by β-secretase in endosomes it is likely that a
TMEM59-dependent APP transport block causes the observed inhibition
of APP ectodomain shedding. For further validation of TMEM59 and
its homolog TMEM59L as modulators of APP ectodomain shedding, a
double knockdown study was performed. In this approach effects on
APP ectodomain shedding could also be established, affirming TMEM59
and its homolog TMEM59L as modulators of APP ectodomain shedding
with novel cellular mechanisms. In order to identify novel cellular
modulators of APP ectodomain shedding a genome wide RNAi screening
in Drosophila cells was performed and candidate genes were
investigated in human cells in present work. Initially a suitable
Drosophila reporter cell line expressing a reporter construct of
APP ectodomain shedding (HRP-APP) was established. Other constructs
were used to monitor general secretion (GLuc) and transfection
efficiency (FLuc). Using Kuzbanian, the α-secretase in Drosophila
(Sapir et al., 2005), as a positive control guaranteed that
transfection of cDNAs into Drosophila cells did not interfere with
uptake of dsRNAs or efficiency of RNAi and that the reporter
construct HRP-APP is normally produced and processed in reporter
cells. After successful establishment of the reporter cell line the
genome wide RNAi was performed in two steps: a primary screening
revealed approx. 300 candidate genes out of which 43 could be
confirmed in a secondary screening to be modulators of APP
ectodomain shedding. The RNAi screening was verified by the
several-fold appearance of Kuzbanian among the top modulators. For
further investigation of the top candidates human ortholog genes
were identified. The 30 human candidate genes were investigated in
RNAi studies in human SH-SY5Y cells. In these cells, APP is
processed by α-secretase ADAM10 as well as by β-secretase BACE1.
Therefore effects on both shedding products (APPsα and APPsβ) were
investigated upon depletion of candidate genes using siRNAs. It is
known that siRNAs produce a high rate of off target effects, to
this end a robust validation strategy was developed. Candidate
genes were first depleted with two different siRNA pools and their
effects on APP shedding were compared. Afterwards the remaining 12
candidate genes were depleted using single siRNA sequences and the
effects were compared to those of the siRNA pool. Only when a
reproduction of effects was obtained in a next step correlation of
knockdown and phenotype were assessed. Using these steps of
validation 5 candidate genes could be verified as modulators of APP
shedding in human cells: next to genes coding for a histone protein
(HIST1H4C), a ribosomal protein (RPL36AL), a protein of the minor
spliceosom (ZMAT5), an unknown gene (METTL16) and the gene VPS24
(„vacuolar protein sorting-associated protein 24“), coding for a
protein of intracellular protein transport, were identified. VPS24
was chosen for further validation by a pathway analysis. VPS24
belongs to the ESCRT machinery („endosomal sorting complex required
for transport“) and therefore participates in endosomal-lysosomal
protein transport. In further RNAi studies other members of the
ESCRT machinery were depleted in human cells and effects on APP
shedding were compared to VPS24 depletion. For most of the ESCRT
members a consistent reduction in APPsβ production could be
observed. To engross these results VPS24 was depleted by using an
alternative RNAi system. With this stable knockdown approach, the
knockdown phenotype could be confirmed. This stepwise validation
strategy for candidate genes of the initial Drosophila RNAi
screening verified VPS24 as a modulator of APP ectodomain shedding
in human cells.