0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

PFIZER.2 – Effects of the phosphodiesterase type X (PDE10) inhibitor on R6/2 mouse model of HD
inhibitor therapy? We propose to shed light on these issues with the following
studies.
We will measure by immunohistochemical (striatum and cortex) and
Western blot analyses (striatum) the levels of PDE10A protein in brains from
R6/2 mice as a function of disease progression. Time points for analysis will
be 4, 8, and 12 weeks, when the animals are pre-, immanently-, fully-symptomatic,
respectively. In the immunohistochemical analysis, we will pay particular
attention to loss of PDE10A immunoreactivity in different compartments,
namely, the striatal parenchyma, the terminal fields in globus pallidus, and
the perinulear region of the striatal medium spiny and cortical pyramidal
neurons. This latter compartment is of particular interest, since PDE10A
located in the nuclear compartment may be specifically involved in regulation
of CREB-mediated transcription in striatal as well as cortical neurons. Thus,
it may be that loss of PDE10A highly expressed in the dendritic and axon/
terminal compartment of the medium spiny neurons may serve as a biomarker
of disease progression, whereas it is PDE10A in a restricted compartment associated
with the nucleus that is the target of PDE10A inhibitor therapy.
We also propose to measure changes in PDE10A levels by Western blot
analyses in striata obtained from parallel groups of treated with rolipram and
TP-10. This analysis will inform whether loss of PDE10A protein is slowed or
halted by pharmacologically distinct therapies, which is critical in considering
PDE10A as a viable biomarker. These studies will utilize the PDE10A antibody
24F3.F11 [Seeger et al. 2003] in a modified protocol specifically
designed for use of mouse monoclonal antibodies on mouse tissue.
Specific Aim 4: Cortical neuron toxicity. In the rat QA lesion model, TP-10
treatment had a significant effect on cortical neuron survival. This may have
resulted secondarily due to the effect on striatal neuron survival. However, it
is also possible that this effect was directly the result of inhibition of the small
pool of PDE10A protein observed in the perinulear region of cortical neurons
[Seeger et al. 2003; Coskran et al. 2006; see discussion above]. Determining
whether the effect of PDE10A inhibition on cortical neuron survival is direct
or indirect may be important in considering this approach relative to others
that may have a more obvious rationale for an impact on cortical degeneration.
Furthermore, demonstration of a direct effect of PDE10A inhibitors of
cortical neuron survival opens the possibility for the use of these compounds
in other neurodegenerative diseases such as AD. The study proposed uses as a
model system stereotaxic injection of NMDA into cortex. Analogous to
Giampà et al., we will compare the effect of the PDE10A inhibitor TP-10 with
that of rolipram, which also causes an upregulation of CREB phosphorylation
and BDNF expression in cortex [DeMarch et al. 2007; DeMarch et al. 2008].
Thus, this study may elucidate a more general role for activation of the
cAMP/PKA/CREB/BDNF pathway in cortical neuron survival and offer a
broader perspective on potential new targets to treat neurodegenerative diseases.
Rats will be administered NMDA into the cortex using a stereotaxic apparatus
and then animals will be treated for 4 weeks with TP-10, rolipram or
2009 669