0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
On HD, a number of neuronal model cells will similarly be used to
explore the overall changes of calcium signaling, again emphasizing the
response of the mitochondrial calcium transport system to the stress caused
by the changes in cytosolic calcium. As for AD, also in this case emphasis will
be placed on the function of the PTP, on its sensitivity to calcium in the
matrix, and on possible ways to control its opening.
2. Work plan
The project will concentrate on defects that are considered basic in neurodegenerative
diseases like AD and HD, i.e., the dysregulation of Ca 2+ homeostasis
and the dysfunction of mitochondria (for the general background of
the topic see the Presentation of the Collaboration). The idea behind the project
is that these defects, which are somehow triggered by deposition of conformationally
altered fragments of disease-related proteins, generate the general
scenarios of the diseases, which will begin with the dysfunction of the
synapse and will then inevitably progress to the global suffering of the neuron.
On the whole, the work foreseen will considered the dysregulation of Ca 2+ and
of mitochondrial function without differential focus on the synapse or the
neuronal soma. However, some of the work will be spatially specific, e.g.,
attempts will be made to develop Ca 2+ probes (i.e., recombinant aequorin)
specifically targeted to the synapse.
The major focus of the work will be Ca 2+ and the situation of mitochondrial
stress that Ca 2+ dysregulation generates. The stress is likely to be exacerbated
by an independent concomitant mitochondrial defect [Bezprozvanny
and Hayden 2004; Tang et al. 2005; Panov et al. 2002; 2005; Lim et al. 2008],
but the consequence of the defect will still have the dysregulation of Ca 2+ as
the basic result. Both transcriptional and non transcriptional events will be
studied in several distinct but related workpackages.
2.1. On AD we will study the ability of PS to form leak channels for Ca 2+ in
the endoplasmic reticulum (ER) and its control. For this part of the project,
the focus will be on the transcriptional role of the Ca 2+ -dependent gene
silencer DREAM [Carrion et al. 1999] in regulation of the putative PS channels.
The work will primarily use stable neuronal clones (SH-SY5Y neuroblastoma
cells) generated in the Unit, which overexpress the wild-type form of
DREAM or a mutant thereof that is unable to bind calcium and thus represses
target genes permanently. In addition, other neuronal cell types available in
the other Units of the Consortium will be investigated. RT-PCR experiments
will reveal whether the overexpression of DREAM (or, even more, of its Ca 2+ -
independent mutant) affects the transcription of the PS genes. In parallel
experiments, the cells will be transfected with recombinant aequorin targeted
to the ER and to other cell compartments [Brini et al. 1999], to obtain a complete
view of the role of DREAM in neuronal calcium homeostasis. SH-SY5Y
cells non over-expressing DREAM will be used as controls. Particular attention
will be devoted to the Ca 2+ dynamics in the ER, which will be studied in
detail by analyzing the kinetics of ER filling, as well as of the cytosolic transients
of Ca 2+ induced by stimulation of cells with InsP3-linked agonists. The
614 2009