0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

AFM.1 – A study on the effects of p66Shc on oxidative stress and mitochondrial damage…
Aim 2: Characterize the molecular mechanisms underlying
p66Shc-dependent pathways which mediates oxidative stress
and mitochondrial damage induced in cells by mutSOD1
Many candidate pathways might be involved in the generation of oxidative
stress and mitochondrial damage in ALS. However, we will focus on a
recently described, new pathological mechanism. It has been demonstrated
that mutant SOD1s expressed in human cell lines directly stimulate microglial
NADPH oxidase (Nox) by binding to Rac1, a member of the Rho family of
small GTPases, which controls intracellular levels of reactive oxygen species
(ROS), resulting in overproduction of damaging ROS. The binding of wildtype
SOD1 to Rac1 is redox sensitive, and can be cycled between bound and
unbound states depending on the redox state of Rac1. Binding to mutant
SOD1 inhibits this feedback regulation on Rac1, giving rise to a constitutive
active Rac1 (GTP-bound), which persistently stimulates ROS production. This
mechanism is clearly pathologically relevant, given that treatment with the
Nox inhibitor apocynin or elimination of Nox extends survival in ALS mice,
reviving the proposal that ROS mediate ALS pathogenesis [Harraz et al.
2008]. Although this mechanism is operative in microglia, a similar network
of functional and physical interaction between SOD1 and Rac1 controlling
the production of ROS can be envisaged even in neuronal cells [Boillee and
Cleveland 2008]. Given that Rac1 leads to phosphorylation-dependent
increase in stability and activity of the p66shc adaptor protein [Khanday et al.
2006], it is tempting to hypothesize that p66shc is a mediator of Rac1-induced
oxidative stress in neuronal cells.
To explore this possibility, we will take advantage of adenoviral vectors
that code for constitutive active (V12) or dominant-negative, inactive (N17)
mutants of Rac1, which are both in our availability.
In the cellular background that has been described in the previous paragraph,
we will:
Measure the state of activation of Rac1, by the use of specific pull-down
assays which utilize a GST–PAK protein to selectively bind the GTP-bound,
active form of Rac1. This assay has been extensively used by the proponent in
previous work [Cozzolino et al. 2003], and was shown to give confident results
in other ALS models [Harraz et al. 2008].
Evaluate the effects of Rac1 activation/inhibition on p66Shc-mediated
induction of oxidative stress and cell death. These experiments are expected to
clarify whether a cross-talk between Rac1 and p66Shc is actively operating in
conditions of oxidative stress induced by mutSOD1s.
Aim 3: Assess the effects of p66Shc genetic manipulation
on mitochondrial metabolism of cells
In the context of p66Shc manipulation, the effect of mutSOD1 overexpression
on the morphology and physiology of mitochondria will be thoroughly
evaluated. Alterations in mitochondria functionality in SH-SY5Y and NSC34-
derived cell lines will be studied by determination of different parameters,
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