0-TESTO COMPLETO.pdf - Fondazione Santa Lucia

Sezione III: Attività per progetti
Specific Aim 1: Analysis of the effect of TP-10 treatment on CREB phosphorylation
and BDNF levels in the R6/2 mice from the current study.
Specific Aim 2: Analysis of whether TP-10 treatment had an effect on cortical
neuron loss in the R6/2 mice in samples from the current study.
Specific Aim 3: Quantify the loss of PDE10A in striatum as a function of
disease progress in the R6/2 mice and the effect of TP-10 on this potential biomarker.
Specific Aim 4: Investigate the direct effects of TP-10 treatment on cortical
neuron toxicity.
DETAILED PROPOSAL FOR NEW STUDIES
Specific Aim 1: CREB and BDNF. We hypothesize that beneficial effects
of PDE10A inhibition in HD may stem from activation by phosphorylation
of the transcription factor CREB and a resultant upregulation of CREBregulated
transcription of genes critical for neuronal survival. Indeed, we
observed that the beneficial effect of rolipram treatment in the rat QA model
and the R6/2 mice [DeMarch et al. 2007; DeMarch et al. 2008] and the effect
of TP-10 treatment in the rat QA model [Giampà et al. 2009] were accompanied
by increased CREB phosphorylation in both striatum and cortex. There
was also an increase in BDNF levels, which is a key neurotrophic factor regulated
by CREB signaling. We propose to quantify the level of phosphorylated
CREB and BDNF in the striatum and cortex in samples taken from R6/2 mice
treated with TP-10 or vehicle. We will use immunohistochemical methods
established and validated in our previous studies [DeMarch et al. 2007;
DeMarch et al. 2008; Giampà et al. 2009].
Specific Aim 2: Cortical neuron pathology. In the previous study in our rat
QA lesion model, we observed a significant effect of TP-10 treatment on cortical
neuron survival in addition to an effect in striatum. We propose to determine
whether TP-10 treatment had an effect on cortical neuron loss in the
R6/2 mice in samples from the current study. We will use the cortical neuronal
counting methodology established and validated in our previous studies
[DeMarch et al. 2007; DeMarch et al. 2008; Giampà et al. 2009]. Specifically,
cortical neurons in layers 3, 5 and 6 are counted in both hemispheres in each
of three 1.0-mm-square confocal microscope fields in each of two sections
from six mice. The apparent density of immunoreactive neurons per mm 2 is
thus obtained [Fusco et al. 2003].
Specific Aim 3: PDE10A levels in the R6/2 mice. Hebb and coworkers
reported that PDE10A mRNA and protein levels decline very early in
the course of disease progression in the R6/2 mice. PDE10A protein is
also decreased in striata obtained post mortem from HD patients [Hebb et
al. 2004]. These findings raise several issues that bear further investigation.
The first is whether following PDE10A loss may be an early and
quantifiable biomarker of HD progression? The second is, if PDE10A is
lost early in the disease, does that impact the timing for initiating PDE10A
668 2009