0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

ERANET – Calcium homeostasis dysfunction and mitochondrial damage in Alzheimer’s and…
analysis of ER (and cytosolic) Ca 2+ homeostasis will then be extended to cell
overexpressing PS.
The second line of work on PS, which will be performed in the second
year of the project, will explore the possible non-transcriptional role of
DREAM on the release of Ca 2+ from the ER, i.e., its direct interaction with PS.
The rationale for the work stems from controversial claims that one variant of
DREAM residing in the cytoplasm (originally termed calsenilin) would interact
with PS, influencing its ability to release Ca 2+ from neuronal ER
[Buxbaum et al. 1998]. Both DREAM and PS will thus be transiently
expressed in SH-SY5Y cells also expressing aequorin targeted to the ER, and
the calcium dynamics in the ER will be evaluated. From the debate in the literature,
it could be expected that the co-expression would alter the dynamics
of Ca 2+ homeostasis, i.e., of Ca 2+ release. This would be an indirect indication
for the interaction of the two proteins, and the project foresees tests of the
interaction by performing GST pull-down experiments with specific antibodies,
using as probes portions of the PS that are known to face the cytosol,
where DREAM could reasonably come in contact with them. Once the role of
PS in the homeostasis of Ca 2+ in the ER and its interplay with DREAM has
been established, in the 2 nd and 3 rd year of the project the work will be
extended to PS variants carrying mutations linked to AD.
It will be important to assess whether the altered Ca 2+ homeostasis could
have an impact on mitochondrial metabolism. The effects on metabolism will
be evaluated by measuring the ATP concentration with a specific recombinant
probe, i.e., mitochondria-targeted luciferase [Jouaville et al. 1999]. The targeted
Ca 2+ and ATP probes are routinely employed in the Unit, and probes to
monitor Ca 2+ concentration are also available in lentiviral vectors that will be
used to transduce primary neuronal cells.
2.2. On AD we will also investigate another aspect of the PS function, which
is related to its g-secretase activity. The PS-containing g-secretase complex is
an unusual membrane-embedded protease that processes a large variety of
integral membrane proteins. The PS is the catalytic core of the complex that
produces the Ab peptide. The latter has been recently localized also in mitochondria,
where it promotes the opening of the PTP [Moreira et al. 2001].
Furthermore, we have lately identified a novel defect in the APP gene
(A673V) that, unlike all other mutations linked to AD, shows a recessive
mendelian trait of inheritance: only the homozygous state results in disease
while heterozygous carriers are not affected, even in advanced age. In vitro
data have shown that this mutation remarkably increases the fibrillogenic
properties of Ab. However, co-incubation of mutated and wild-type Ab peptides
– a situation that mimics the in vivo heterozygous state – hinders
amyloidogenesis [Di Fede et al. submitted]. We have already generated four
lines of transgenic mice expressing the A673V-mutated human APP under
the control of the Thy1 promoter. The animals are now been crossed with
APP knock-out mice to obtain Tg mice expressing only the human V673-
APP variant. These mice will be analyzed to determine the in vivo consequences
of the mutation, including those on the synaptic transmission,
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