0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
chondria, where it binds to cytochrome c and acts as an oxidoreductase,
generating reactive oxygen species (ROS) in the form of H 2
O 2
and leading to
organelle dysfunction and cell death [Giorgio et al. 2005; Pinton et al. 2007].
The H 2
O 2
generated by p66Shc is ~30% that of the total pool of intracellular
H 2
O 2
and is biologically relevant, as shown by p66Shc ability in vitro and in
vivo to induce mitochondrial permeability transition and by the findings
that cells and tissues derived from p66Shc-/- mice accumulate significantly
less oxidative stress. Most importantly, p66Shc-/- mice exhibit a 30%
extended lifespan, demonstrating that p66Shc acts as a key molecular sentinel
that controls cellular stress responses and mammalian lifespan [Migliaccio
et al. 1999]. In addition, p66Shc-generated H 2
O 2
regulates insulin signaling
and fat development, indicating that the oxidative signaling pathway
depending upon p66Shc regulates energetic metabolism [Berniakovich et al.
2008].
On the basis of these considerations, and in view of the preliminary
results that are shown in the next paragraph, we propose to study the p66shcdependent
pathways as a possible valuable target for ALS therapy.
Previous work/ Preliminary results
1) In the recent past we have built a collection of inducible cell lines,
derived from mouse motoneuronal line NSC34 (motoneuron x neuroblastoma
hybrid), that express a wide panel of mutant SOD1s (mutSOD1s) under control
of the inducible Tet-On promoter [Ferri et al. 2006]. We have included in
the selection of mutant SOD1s mutant proteins with widely differing biophysical
properties, i.e. mutations in metal ligands in the active site (H46R, H48Q,
H80R), substitutions in the electrostatic loop (S134N, D124V and D125H),
mutations affecting metal binding (G85R); mutants in residues located at the
dimer interface (A4V); mutants in residues critical for maintenance of the
b-barrel structure (G37R, D90A, G93A); one mutant shortened by truncation
of the C-terminal end in a loop connecting two b strands (E133d). These cell
lines proved to be a valid model to study intracellular properties of mutant
SOD1s generally, and to analyze the involvement of mitochondria specifically.
In fact, the expression of mutSOD1s in these cells induce clear mitochondrial
phenotypes, ranging from a change in the redox state of these organelles in
terms of a shift in the ratio reduced glutathione/oxidized glutathione, to the
impairment in the activity of respiratory complexes and ATP production
[Ferri et al. 2006].
2) We have accumulated clear evidence that p66Shc is recruited in the
molecular pathways activated by mutSOD1 in cells. p66Shc is phosphorylated
on a serine residue in position 36 in response to cellular stress induced
by various stimuli, and this event is crucial to p66Shc-mediated oxidative
stress and apoptosis [Migliaccio et al. 1999]. As an initial step to investigate
the role of p66Shc in mutSOD1-induced cell stress, mouse motoneuronal
NSC34 cell stably transfected with plasmids coding for wild-type or the
G93A mutSOD1 under the control of a tetracycline-responsive element were
treated with doxycycline for various period of time. To determine whether
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