0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
U.O. 5 – Laboratorio di Neurochimica
Alberto Ferri
Specific contribution of the unit to the project
Vulnerability of motor neurons in ALS likely arises from a combination of
several mechanisms, including protein misfolding and aggregation, mitochondrial
dysfunction, oxidative damage, defective axonal transport, excitotoxicity,
insufficient growth factor signalling, and inflammation. In particular, oxidative
stress and protein aggregates are found in motor neurons in models for
fALS linked to mutation in the gene coding for SOD1, and in ALS patients as
well, and they may be linked in that aggregation of mutant SOD1 in the cytoplasm
and/or into mitochondria has been repeatedly proposed as a main culprit
for the degeneration of mitochondria in motor neurons [Ferri et al. (2006)
PNAS; Cozzolino et al. (2007) J Biol Chem].
Increasing evidence indicates that cellular functions impaired in familial
ALS as a consequence of the expression of mutant SOD1 converge on pathways
that could be activated in sporadic ALS by other toxic factors [Boilée et
al. (2005) Neuron]. For instance, recent work suggests that under certain circumstances
(e.g. oxidative stress) wild type SOD1 may behave and aggregate
similarly to mutant SOD1 [Banci et al. (2007) PNAS; Gruzman et al. (2007)
PNAS]. Therefore, what is demonstrated true for SOD1-linked familial ALS
may prove true also in non-SOD1 linked fALS and in the sporadic form of the
disease.
The contribution of this unit will be to set up and use models in vitro to
test whether candidate gene products identified in sporadic ALS patients
induce a degenerating phenotype in neuronal cell, similarly to the one
induced by mutant SOD1.
To this aim, we will use cell lines derived from mouse motoneuronal
line NSC-34 (motor neuron x neuroblastoma hybrid) and cell lines derived
from human neuroblastoma SH-SY5Y. Those cell lines will be manipulated
for the silencing of candidate genes through RNA interference or for the
overexpression of candidate genes through transfection with expression
plasmids, and used in comparison to the same cell lines expressing mutant
SOD1, to test several parameters of oxidative stress, mitochondria functionality
and protein aggregation that are associated with ALS. Tests will be
performed also under conditions mimicking environmental factors (e.g.
oxidative stress, herbicides) that may contribute to the onset of sporadic
ALS.
Thus, this study will allow a further screening and a validation of those
genetic factors that may be involved in the pathogenesis of ALS and allow the
individuation of novel pathways modified in the course of the disease. Results
from this study will hopefully provide suggestions of other candidate factors,
to be translated back to studies in patients.
742 2009