Visit our Expo - Redox and Inflammation signaling 2012

Session XII : Cell death and neurodegenerative diseases Poster XII, 21
ERK1/2 phosphorylation, I-NOS induction and chemokine secretion are three events
involved in the complex signaling of prion protein fragment 90-231 in microglial cells.
1Stefano Thellung, 1Alessandro Corsaro, 1Valentina Villa, 1Valentina Venezia, 1Mario
Nizzari, 1Michela Bisaglia, 2Claudio Russo, 1Gennaro Schettini, 3Antonio Aceto and
1Tullio Florio
1Pharmacology Dept. of Oncology, Biology & Genetics, University of Genova, Italy.
2Dept. Health Sciences, University of Molise, Campobasso Italy. 3Section of
Biochemistry, Dept. Biomedical Sciences, University G. D’Annunzio of Chieti, Italy.
E-mail: thellung@yahoo.com.
We show that PrP90-231, a neurotoxic prion protein fragment, activates the murine microglial
cell line N9 and describe the transduction mechanisms involved. PrP90-231 induced
phosphorylation of MAP kinase ERK1/2 and the expression of the inducible isoform of nitric
oxide synthase (I-NOS). Time course experiments, show that ERK1/2 activation is detectable
after 15 minutes of treatment, reaches a maximum at 25 minutes and is sustained up to 60
minutes of cell exposure to the peptide. Importantly, late onset and sustained ERK1/2
activation are typical events involved in cell differentiation rather than proliferation. Indeed,
we have observed by both MTT and [3H]-tymidine uptake assays, that PrP90-231 blocked N9
cell proliferation without inducing cell death. I-NOS induction was detectable after 6 hours of
cell exposure to PrP90-231 and reached its maximal value after 24 hours of treatment. We
excluded that I-NOS expression was dependent on ERK1/2 activation, since cell pretreatment
with the MEK inhibitor PD98059 did not prevent I-NOS activation by PrP90-231. Beside
nitric oxide, microglial cells produce and release several cytokines in response to
proinflammatory inputs. We observed that after 24 hours of stimulation with PrP90-231,
RANTES release is enhanced. By culturing N9 in chambers separated by a microporous
barrier we have observed that ERK1/2 activation and I-NOS expression were elicited by
PrP90-231 only if cells were directly exposed to the peptide. We conclude that these events
are not dependent on autocrine/paracrine loop triggered by the peptide and sustained by
diffusible mediators Rather, we suggest that a sustained physical interaction of PrP90-231
with the cell is required to keep microglia in an activated state.
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