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M. d’Erme - Epigenetic modifications in neurodegenerative diseases
ment. All the experiments were carried out with an
Abeta 25-35 fragment dissolved in PBS and left at 37
°C overnight (aggregated form) or dissolved immediately
before use (not aggregated form).
The results indicate that aggregated Abeta 25-35
fragment activates PARP around 30-40% after 1h
incubation. It is of note that the not aggregated form
activates the enzyme activity up to 60% in the same
assay conditions. The activation of the enzyme is
reduced in the presence of the new PARP inhibitor
MC2050.
A similar enzyme activation is seen when SH-SY5Y
cells are treated with aggregated or not aggregated
Abeta 25-35 which enhance endogenous PARP activity
up to 30 or 40%, respectively, within 4 hours of
treatment. The increase in PARP activity is prevented
by pre-treatment with MC2050.
In order to verify whether PARP activation is mediated
by Reactive Oxygen Species (ROS) generated by
Abeta fibrils formation, SH-SY5Y cells were treated
with aggregated or not aggregated Abeta 25-35 and
the intracellular ROS generation assessed by flow
cytometry. The Abeta peptide was able to induce a
marked rise in intracellular ROS production within
the first two hours of treatment, particularly in the
not aggregated form. These data allow to hypothesize
that the ROS production induced by the fibrillization
process is the main responsible for PARP
activation.
This study also demonstrate that MC2050 is highly
active in the micromolar range (IC 50 = 25 mM)
compared to the well known inhibitor 3-ABA. Cell
viability assay revealed that this compound is not
cytotoxic at the tested concentrations.
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Second objective
Recent studies show that MPP + and rotenone
induce α-synuclein overexpression and aggregation
(Lee et al., J Biol Chem. 2002, 277:5411) and this latter
event is considered to be one of the pathological
hallmarks of PD. Hence preliminary experiments
were devoted to investigate whether the treatment
with CysDA affects α-synuclein expression. Western
blots of cell lysates show an increase of the level of
this protein 8-24 hrs after CysDA treatment.
Confirming previous data, the accumulation of
α-synuclein has also been demonstrated by fuorescence
microscopy.
Since oxidative DNA damage is able to alter the
enzymatic methylation at the CpG sites, thereby
playing an important role in the regulation of gene
expression, we investigated the methylation status of
the α-synuclein promoter. Methylation level was
assessed by the bisulfite genomic sequencing, which
revealed no change of DNA methylation pattern
under the experimental conditions.
Further experiments will be dedicated to assess the
effect of Poly(ADP-ribosylation) on the Abeta signal
transduction and on the expression and aggregation
of α-synuclein. This goal will be achieved with the
use of the new PARP inhibitor MC2050.
Selected publications
Mattiussi S, Tempera I, Matusali G, Mearini G,
Lenti L, Fratarcangeli S, Mosca L, D'Erme M,
Mattia E. Inhibition of Poly(ADP-ribose)polymerase
impairs Epstein-Barr Virus lytic cycle progression.
Infect Agent Cancer 2007, 2:18.