0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

RF07.66 – The italian ALS genetic collaborative project: follow-up genetic association study…
Methods
We have already built up a collection of cell lines where expression of
wild type and mutant SOD1 is either low-level and constitutive (human neuroblastoma
SH-SY5Y) or inducible (mouse motoneuronal line NSC-34). The
use of an inducible expression system allows ruling out possible “ preconditioning
” effects due to the constitutive expression of the mutant SOD1, while
a low-level chronic expression in a human background has the advantage of
better mimicking the situation in patients.
Therefore, the two models are complementary and will be used in parallel
in this study.
Studies on mitochondria localization of SOD1 and on mitochondria functionality
(ATP production, membrane polarization, cytochome c release) will
be performed as described in detail for the same cell lines in our previous
papers [Carrì et al. (1997) Febs lett; Ferri et al. (2004) Faseb j; Cozzolino et al.
(2006) Neurobiol Disease; Ferri et al. (2006) PNAS]. Briefly, determination of
the state of mitochondria polarization in transfected cells will be carried out
using fluorescent dye JC-1 by cytofluorimetry; intracellular ATP production
will be assessed using ATP Bioluminescence Assay Kit (Sigma, St.Louis, MO,
USA); functionality of respiratory complexes will be performed by standard
spectrophotometric measurements.
Markers of oxidative stress (e.g. total reactive oxygen species, total protein
carbonyl content, reduce/oxidized glutathion) will also be determined by standard
techniques as previously reported [Gabbianelli et al. (1999); Beretta et al.
(2003); Ferri et al. (2004) Faseb j; Ferri et al. (2005) J Neurochem; Ferri et al.
(2006) PNAS]. Briefly, whole-cell and mitochondria reactive oxygen species
levels will be measured using the dyes 2’,7’-dichlorofluorescein diacetate
(DCF-DA) and dihydrorhodamine-123 (DRH). The level of fluorescence will
be quantified by FACScan. Cytosolic and mitochondrial carbonylated proteins
will be detected using an OxyBlot Kit (Intergen, Portland, OR) that employs
the reaction with 2,4-dinitrophenylhydrazine (DNP).
Proteins will be electrophoresed in SDS-PAGE, electrotransfered to a
polyvinylidene fluoride membrane, and immunodetected with anti-2,4-
dinitrophenyl antibodies. Total and mitochondrial glutathione content
(GSH/GSSG ratio) will be assayed by conversion of free amino groups to 2,4-
dinitrophenyl derivatives by the reaction with 1-fluoro-2,4-dinitrobenzene and
separation by HPLC.
Analysis of cytosolic and mitochondrial SOD1 aggregates will be carried
out by standard fluorescence microscopy on cells grown on polilysine coated
glass slides and fixed and by Western blot analysis of sedimentable particles
from whole cells or purified mitichondria as already reported by us [Beretta
et al. 2003; Cozzolino et al. (2007) J Biol Chem].
Viability of cell lines will be assessed, using various methods such as:
Hoechst staining for nuclear condensation and fragmentation (counting in
fluorescence microscopy), staining with propidium iodide (cytofluorimetry),
caspases activation (Western blots with commercially available antibodies
and determination of activity with fluorescent substrates, also commercially
2009 743