0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
Two different cellular models of SOD1-dependent ALS will be used:
1) Mouse motoneuronal NSC34 cells that express a wide panel of mutant
SOD1s (mutSOD1s) under control of the inducible Tet-On promoter. These
cells have been extensively characterized in the lab of the proponent [Cozzolino
et al. 2008a; Ferri et al. 2006].
2) SH-SY5Y human neuroblastoma cells transiently infected with adenoviruses
coding for wt and G93A mutant SOD1. These cells have been successfully
used in the lab of the proponent [Carrì et al. 1997; Cozzolino et al.
2006; Gabbianelli et al. 1999]. Adenoviral vectors coding for the expression of
mutant SOD1 have been previously produced and they are able to induce the
appearance of a clear apoptotic phenotype in SH-SY5Y cells (see preliminary
results).
NSC34-derived and SH-SY5Y-derived cells will be stably transfected with
plasmids coding for the different p66Shc proteins. Five to eight independent
clones will be selected for each conditions (e.g. 5-8 clones of NSC34 and SH-
SY5Y for mouse and human p66Shc wild-type, respectively, etc.). This part is
supposed not to give rise to any difficulties, since the selection of stable transfectants
has been proved feasible for both cell lines [Cozzolino et al. 2006;
Ferri et al. 2006], and stable clones of SH-SY5Y expressing the S36A mutant
Shc have been already obtained (see preliminary results).
In these systems, three experimental readouts will be evaluated:
p66Shc phosphorylation/mitochondrial accumulation. Phosphorylation of
p66Shc on serine 36 will be analysed by Western blot using a phosphorylationspecific
antibody, both on total protein extracts or on immunoprecipitates with
an antibody recognising Shc proteins. The accumulation of p66Shc in mitochondria
will be assessed by subcellular fractionation and Western blot in conditions
which we have already set up on the same cell lines [Ferri et al. 2006].
Accumulation of oxidative stress will be analysed in cells expressing
wild-type and mutSOD1s, by using probes specifically sensitive to oxidative
stress (dihydro-dichlorofluorescein diacetate, dihydroethium) to be analysed
by spectrofluorimetric and cytofluorimetric techniques. Moreover, HPLC
quantification of the amounts of oxidized (GSSG) and reduced (GSH) glutathione,
which is an accurate readout for cellular oxidative balance, will be
used as previously described [Ferri et al. 2006].
The effects of the genetic activation or inhibition of p66Shc on the
apoptotic phenotypes induced by mutSOD1s will be studied. A panel of apoptotic
markers, such as release of mitochondrial pro-apoptotic proteins
(cytochrome c, EndoG), caspase3 activation, nuclear morphology (Hoechst
staining) will be examined, with methods that have been successfully utilised
in the lab of the proponent on the same cellular models [Cozzolino et al. 2004;
Cozzolino et al. 2006].
These parameters will be evaluated in cells either expressing or nonexpressing
wild-type and mutSOD1s. All the results will be compared to control
conditions, i.e. cells treated with known activators of p66Shc-dependent
oxidative stress and apoptosis, such as hydrogen peroxide an ATP.
566 2009