JPET #173294 1 Title : Chronic suppression of PDE10A alters striatal expression of genes responsible for neurotransmitter synthesis, neurotransmission and signaling pathways implicated in Huntington’s Disease. Robin J. Kleiman, Lida H. Kimmel, Susan E. Bove, Thomas A. Lanz, John F. Harms, Alison Romegialli, Kenneth S. Miller, Amy Willis, Shelley des Etages, Max Kuhn, and Christopher J. Schmidt Neuroscience Research Unit (RJK, LHK, SEB, TAL, JFH, AR, AW, CJS) Genetically Modified Animals (KSM), Cardiovascular and Metabolic Disease Research Unit (SdE), Biostatistics Unit (MK) , Pfizer Global Research and Development, Pfizer, Inc., Eastern Point Road, Groton CT 06379. JPET Fast Forward. Published on October 5, 2010 as DOI:10.1124/jpet.110.173294 Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics. This article has not been copyedited and formatted. The final version may differ from this version. JPET Fast Forward. Published on October 5, 2010 as DOI: 10.1124/jpet.110.173294 at ASPET Journals on December 26, 2018 jpet.aspetjournals.org Downloaded from
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JPET #173294
1
Title : Chronic suppression of PDE10A alters striatal expression of genes responsible for
neurotransmitter synthesis, neurotransmission and signaling pathways implicated in
Huntington’s Disease.
Robin J. Kleiman, Lida H. Kimmel, Susan E. Bove, Thomas A. Lanz, John F. Harms, Alison
Romegialli, Kenneth S. Miller, Amy Willis, Shelley des Etages, Max Kuhn, and Christopher J.
Schmidt
Neuroscience Research Unit (RJK, LHK, SEB, TAL, JFH, AR, AW, CJS) Genetically Modified
Animals (KSM), Cardiovascular and Metabolic Disease Research Unit (SdE), Biostatistics Unit
(MK) , Pfizer Global Research and Development, Pfizer, Inc., Eastern Point Road, Groton CT
06379.
JPET Fast Forward. Published on October 5, 2010 as DOI:10.1124/jpet.110.173294
Copyright 2010 by the American Society for Pharmacology and Experimental Therapeutics.
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Recommended Section Assignment: Cellular and Molecular
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Inhibition of phosphodiesterase 10A (PDE10A) promotes cyclic nucleotide signaling, increases
striatal activation and decreases behavioral activity. Enhanced cyclic nucleotide signaling is a
well-establish route to producing changes in gene expression. We hypothesized that chronic
suppression of PDE10A activity would have significant effects on gene expression in the
striatum. A comparison of the expression profile of PDE10A knockout mice (KO) and wild-type
(WT) mice following chronic PDE10A inhibition revealed altered expression of 19 overlapping
genes with few significant changes outside the striatum or following administration of a
PDE10A inhibitor to KO animals. Chronic inhibition of PDE10A produced up-regulation of
mRNAs encoding genes that included prodynorphin, synaptotagmin10, phosphodiesterase 1C
(PDE1C), glutamate decarboxylase 1 (GAD67), diacylglycerol O-acyltransferase (DGAT2) and
a down regulation of mRNA encoding choline acetyltransferase (ChAT) and Kv1.6, suggesting
long-term suppression of the PDE10A enzyme is consistent with altered striatal excitability and
potential utility as a antipsychotic therapy. Additionally, upregulation of mRNA encoding
histone H3 and downregulation of histone deacetylase 4, follistatin and claspin mRNAs suggests
activation of molecular cascades capable of neuroprotection. We utilized lentiviral delivery of
CRE-luciferase reporter constructs into the striatum and live animal imaging of TP-10 induced
luciferase activity to further demonstrate PDE10 inhibition results in CRE-mediated
transcription. Consistent with potential neuroprotective cascades, we also demonstrate
phosphorylation of mitogen- and stress-activated kinases 1 (MSK1) and histone H3 in vivo
following TP-10 treatment. The observed changes in signaling and gene expression are predicted
to provide neuroprotective effects in models of Huntington’s Disease.
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Phosphodiesterase 10A (PDE 10A) is one of the eleven families of phosphodiesterases that
serve to limit cyclic nucleotide signaling via enzymatic hydrolysis of these widely utilized
second messengers. Phosphodiesterase (PDE) enzymes are precisely localized within specific
subcellular compartments to regulate discrete pools of cyclic nucleotides sub-serving
functionally distinct signaling events (Baillie et al., 2005). PDE10A is a dual-substrate PDE
expressed at high levels within medium spiny neurons of both the indirect and direct output
pathways of the striatum (Seeger et al., 2003; Coskran et al., 2006; Xie et al., 2006) where it is
primarily associated with membranes (Kotera et al., 2004; Xie et al., 2006). Outside the striatum,
PDE10A appears to be associated with the perinuclear region of neurons throughout the brain
(Seeger et al., 2003; Coskran et al., 2006). In vivo pharmacological inhibition of PDE10A has
been shown to produce a restricted accumulation of cGMP and cAMP within the striatum and to
trigger transient increases in the phosphorylation of CREB (Schmidt et al., 2008). Other studies
have shown enhanced phosphorylation of Extracellular signal-regulated kinase (ERK), protein
kinase A (PKA)-activated epitopes of dopamine- and cAMP-regulated phosphoprotein-32
(DARPP32) and GluR1 subunits following PDE10A inhibition (Siuciak et al., 2006a; Nishi et
al., 2008; Grauer et al., 2009).
Behavioral responses to acute inhibition with PDE10A inhibitors are consistent with striatal
activation and include decreases in spontaneous and amphetamine-stimulated locomotor activity
as well as disruption of conditioned avoidance responding (Siuciak et al., 2006a; Schmidt et al.,
2008). The behavioral consequences of PDE10A inhibition combined with the associated
biochemical indicators of striatal activation suggest that PDE10A inhibition can enhance the
signaling of medium spiny neurons to alter functional responses of the basal ganglia. Chronic
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suppression of PDE10A activity might therefore be expected to drive significant changes in
striatal gene expression. In the current study we use expression profiling following chronic
suppression of PDE10A activity via an inhibitor or gene knockout to identify significant and
overlapping changes in striatal gene expression. The observed changes in gene expression are
indicative of striatal activation and a putative neuroprotective signaling cascade. We also
generated lentiviral constructs to deliver a cAMP Response Element (CRE)-luciferase reporter
into mouse striatum and employed live animal imaging of light generated by the luciferase
reporter to confirm the role of CRE-mediated transcription in response to PDE10A inhibition.
Furthermore, acute pharmacological inhibition of PDE10A activity produced changes in the
phosphorylation state of multiple signaling kinases in the ERK pathway including ERK, MSK1
and the MSK1 substrate histone H3. This cascade has been suggested to provide neuroprotection
in preclinical models of Huntington’s disease (HD)(Roze et al., 2008).
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TP-10 was synthesized at Pfizer Global Research and Development laboratories in Groton, CT.
Chronic dosing of WT and KO animals:
PDE10A knockout animals backcrossed on C57Bl6 background have been previously described
(Siuciak et al., 2006b). WT and PDE10A KO littermate mice (n=6 per group) were dosed daily
for 18 days with TP-10 by oral gavage (25mg/kg 2.5 mg/mL solution; dose volume of 10 mL/kg)
or methylcellulose vehicle 10 mL/kg body weight. Mice were housed singly and provided
routine ad lib feeding. Animals were sacrificed 1 day after the final dose of drug by CO2
euthanasia. Brains were removed and followed by dissection of striatum, hippocampus and
frontal cortex, and snap frozen. All animal treatment protocols were approved by Pfizer’s
Institutional Care and Use Committee and were compliant with Animal Welfare Act regulations.
Affymetrix chip profiling and data analysis:
RNA isolation and hybridizations to mouse 430 2.0 whole genome Affymetrix chips were performed by
Gene Logic. CEL files data were normalized using Robust Multi-array Analysis (RMA) and subjected to
pairwise comparison followed by Benjamini and Hochberg false discovery rate (FDR) correction. Probe
sets with the designation “_x” were removed from the dataset for potential lack of specificity. Probe
translation and pathway analysis was performed in IPA. Probes that could not be translated to genes
within IPA were identified using NetAffx (Affymetrix).
RT-PCR confirmation of changes in gene expression:
Total RNA was isolated from the striatum of PDE10A KO and PDE10A WT male mice (N=8
per group) using the Qiagen RNeasy Lipid Tissue Mini Kit (Qiagen, Valencia, CA) and cDNA
was made with Applied Biosystems High Capacity RNA-to-cDNA Master Mix (Applied
Biosystems, Foster City, CA) using 1 μg total RNA. Quantitative RT-PCR was performed with
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(1:10K) were added before washing with PBS 0.01% Tween20 and imaging on the Odyssey
System (LiCor). Changes in phophoproteins were normalized to total ERK (Cell Signaling
#4696) at 1:5000. Background was removed using the median of 3 pixels from above and below
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the band of interest. Primary phospho-specific antibodies were from the following sources and
used at the indicated dilutions; ERK1/2 pTpY185/187 (Biosource #44-680G) at 1:5000, pCREB
ser133 (UPSTATE#05-807) at 1:2000 MSK pSer376 (Cell Signaling #9591) at 1:5000 and
Histone pH3Ser10(Cell Signaling) at 1:1000.
Construction of CRE-Luciferase reporter lentivirus:
A synthetic DNA (Integrated DNA Technologies, Coralville, Iowa, USA) containing six cyclic
AMP response elements (TGACGTCA) separated by 7 base pair spacers, followed by a 13 bp
minimal promoter sequence and a chimeric intron (pRL-SV40; Promega, Madison, Wisconsin,
USA) was cloned into the Xho/EcoRV sites of pGL4.10 (Promega) upstream of the luciferase
gene. Subsequently, the Xba to EcoRI sites were removed and subcloned into a pLL3.7 lentiviral
vector (Rubinson et al., 2003) which had been modified using Xba and EcoRI to remove the
polymerase III U6 promoter sequence. The final construct contained six copies of the cAMP
response element, followed by the Elb minimal promoter and the Luciferase 2 open reading
frame. High titer lentivirus (109 IU/mL) for injection was produced in 293FT cells (Invitrogen,
Carlsbad , CA, USA). 293FT cells were transfected using Lipofectamine 2000 per
manufacturer’s instructions (Qiagen, Valencia, CA, USA) overnight in two T175 flasks at 70%
confluence with a 3:1 ratio of Virapower Packaging Plasmids (Invitrogen, Carsbad, CA, USA)
and pLL3.7 containing 6x Cre-Luc2. Supernatants containing viral particles were collected after
14 hours and concentrated over a 100kd mw frit (Millipore, Billerica, MA, USA). Supernatants
were then ultracentrifuged for 3 hours, 25rpm, at 4 degrees Celsius. All supernatant was then
removed from the viral pellet and 200uL PBS was added. The pellet was resuspended by gentle
rocking overnight at 4 degrees Celsius. Viral particles were aliquoted in 10uL volumes and
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stored at -80 degrees Celsius until used. Functional viral titers were determined by Flow
cytometry using a BD FACSCalibur (BD Biosciences, San Jose, CA, USA)
Stereotaxic injection:
Fifteen male CD-1 mice, 60-90 days old, were ordered from Charles River and acclimated in-
house for 1 week prior to the start of the experiment. Mice were housed in 12:12 light/dark cycle
and fed standard chow available ad libitum. On the day of the stereotaxic surgery each mouse
was anesthetized with isoflurane (2% in oxygen). The top of the head was shaved and the mouse
was placed in a stereotaxic frame. The eyes were coated with lubricant to prevent drying out and
the shaved portion of the head and surrounding area was disinfected. Using a no. 15 blade, a
midline skin incision was made and a sterile swab was used to dry the surface of the scull.
Measurements were made from bregma and a small burr hole was drilled at the site of the
injection. The coordinates used for the striatal injections were as follows: left striatum =
Anterior/Posterior (A/P) +0.5, Lateral (L) +2.0, Dorsal/Ventral (D/V) (-) 3.0-2.0 mm. Each
mouse received 2 ul of high titer lenti-virus into the left striatum. The virus was delivered using a
10 ul Hamilton syringe with a 30g blunt tip needle. The syringe was attached to a syringe pump
to enable precise flow rate and volume. For the injection, the needle was slowly inserted to the
first D/V coordinate and left in place for 4 minutes to allow the tissue to settle. The virus was
injected at a rate of 0.25 μl/min. After 1 μl was injected (4 min) the needle was slowly raised to
the 2nd D/V coordinate for the second 1 μl injection (remaining 4 min). When the injection was
complete the needle was left in place for an additional 4 minutes to allow for diffusion of the
virus into the surrounding tissue before being slowly withdrawn. The animals were sutured with
6.0 absorbable Vicryl polysorb suture and administered a single 5 mg/kg, s.c. injection of
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Rimadyl (carprofen) and 500 μl saline sc. A topical antibiotic was used on the incision site and
the animals were allowed to recover in a warm cage prior to returning to the holding room.
Imaging of CRE-Luciferase reporter:
Mice were imaged using an IVIS® 200 Bioluminescent Imaging System (Caliper Life Sciences,
Hopkinton, MA). Each mouse was injected i.p. with 150 mg/kg D-luciferin K+ salt (Caliper Life
Sciences, Hopkinton, MA) substrate 10 min prior to imaging. The mice were then anesthetized
with 2.5-3% isoflurane in oxygen, placed in the prone position on the IVIS® platform and
imaged for 1 min. Baseline images were obtained immediately prior to injection of TP-10
compound (3.2 mg/kg, sc in 10% β−cyclodextrin vehicle). Sixteen hours post TP-10
administration, the mice were imaged again. Images obtained from the IVIS® 200 are an overlay
of the bioluminescent signal as a pseudocolor image on a black and white photograph. Data are
presented as total photon flux (photons/second; p/s) from a 1.5 cm2 circular region of interest.
Histology:
Ten weeks post lentiviral injection, 3 mice were selected for histological analysis to assess the
efficiency of lentiviral transduction in the striatum. The mice were deeply anesthetized with
sodium pentobarbital and transcardially perfused with ice-cold saline, followed by ice-cold 4%
paraformaldehyde solution in phosphate buffer. The brains were removed and post-fixed
overnight in 4% paraformaldehyde at 4°C before transferring to a 20% sucrose solution in
phosphate buffer. Brains were sectioned and stained with rabbit polyclonal anti-green fluorescent
protein (A11122; Invitrogen, Carlsbad, CA) at FD Neurotech.
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Chronic genetic and pharmacological suppression of PDE10A enzyme activity produces
significant changes in striatal gene expression.
Vehicle or 2-{4-[-Pyridin-4-yl-1-(2,2,2-trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-
quinoline succinic acid (TP-10), a selective PDE10A inhibitor (IC50= 0.3nM ; (Schmidt et al.,
2008)), was administered once daily for 18 days to both WT and PDE10A knockout mice. Oral
administration of TP-10 at 25 mg/kg results in a free plasma concentration of 7 nM or >10-fold
above the PDE10A IC50 1 h after drug administration (data not shown). Since TP-10 is brain
permeable, this dose should completely inhibit the enzyme and provide a comparator for changes
in gene expression between WT and KO animals. Given the high degree of selectivity (>1000
fold) of TP-10 over all other PDE enzymes, no off-target PDE activity is expected to contribute
to the changes in gene expression identified. This was confirmed by examination of the effect of
TP-10 on differential gene expression in PDE10A KO mice which produced no statistically
significant change in gene expression for any genes in either striatum or hippocampus after
correction for multiple comparisons using a Benjamini and Hochberg False Discovery Rate
(FDR) of p<0.05.
Following chronic dosing with vehicle or TP-10, RNA isolated from striatum and hippocampus
and subjected to microarray hybridization. Following Robust Multi-array Analysis (RMA)
normalization of hybridization intensities, pairwise comparisons were made between vehicle and
TP-10 treatment for WT and PDE10A KO animals as well as between the WT and KO animals
followed by application of Benjamini and Hochberg correction for multiple comparisons.
Changes in striatal gene expression that exhibited statistically significant differences between
vehicle and TP-10 in WT animals (FDR p<0.05) are displayed in Table 1, rank ordered by
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observed fold change in expression. Among the mRNAs significantly affected by TP-10
treatment were several transcripts encoding genes involved in neurotransmitter synthesis or
catabolism, including a 1.38 fold downregulation of the mRNA for the acetylcholine synthetic
enzyme ChAT. Additionally a 1.2 fold upregulation of mRNA for the GABA synthetic enzyme
GAD67 and 4-aminobutyrate aminotransferase, a GABA catabolic enzyme were observed
following chronic TP-10 treatment. Similarly the dynorphin precursor prodynorphin was
significantly upregulated 1.76 fold following chronic TP-10 treatment. ChAT and prodynorphin
were similarly affected in the WT vs. KO comparison. The arginase, type II enzyme, responsible
for limiting the availability of arginine required for production of the neurotransmitter NO was
upregulated by both TP-10 treatment and KO vs. WT comparisons, but only reached statistical
significance in the TP-10 treated WT animals. In addition to neurotransmitter regulation, many
genes involved in neurotransmission and neuronal excitability were altered by chronic exposure
to TP-10 including synaptotagmin X (increased 1.6 fold), the voltage gated potassium channel
protein Kv1.6 (decreased 1.64 fold) and the dihydropyridine-sensitive L-type calcium channel
beta 3 subunit (decreased 1.5 fold) and the synaptic rhoGEF kinase kalirin (decreased 1.6 fold).
The specificity of the TP-10 induced changes is highlighted by the lack of any significant
changes in transcript levels identified in the KO following TP-10 treatment, including any
significant changes among genes identified as significant following TP-10 treatment in the WT
mice (p-values are presented in Table 1).Very few significant changes were observed between
WT and KO comparisons of hippocampus (Rab11b, Lix1, Lnpep and Psmc2, FDR <0.05) and of
these only 2 were larger than a 1.3 fold change (Lix1 and Lnpep). Only 2 probe sets were altered
in hippocampus following TP-10 treatment and they did not map to any known genes, indicating
that the chronic changes in gene expression were largely confined to the striatum, where
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PDE10A expression is most highly concentrated (Seeger et al., 2003;Coskran et al., 2006)). A
complete list of striatal genes that displayed significant differential expression in a comparison of
WT vs. KO gene expression (FDR p<0.05) are displayed in Table 2. A set of 21 probe sets
representing 19 potential genes were significantly altered in both TP-10 treated vs. vehicle as
well as in the PDE 10 KO vs. WT comparisons, and are highlighted in grey in Table 1.
We were able to identify 18 overlapping genes with Taqman assays for RT-qPCR comparisons
between untreated WT and KO striatum utilizing a separate cohort of similarly aged animals to
provide independent confirmation of affected genes (probe set 1460043_at was significant across
both comparisons and mapped to an the unidentified cDNA mM.405423; it was not evaluated as
Taqman assays were not available). Seven additional genes exhibiting a significant change in the
affymetrix chip analysis of vehicle vs. TP-10 comparison and were selected for RT-PCR
confirmation in a replicate cohort of PDE10A WT and KO animals. Transcripts encoding 15 of
the 18 expected overlapping genes, an additional 3 genes with less stringent FDR corrected p-
values for the WT vs. KO comparison (p=0.1 and 0.25) showed experimentally determined
changes in gene expression via RT-PCR and are shown in Table 3. This result suggests that
stringent statistical criteria utilizing a FDR correction of p<0.05 may slightly underestimate the
actual number of genes in the overlapping sets, however we did fail to replicate the significant
effect of chronic PDE10A suppression on ChAT mRNA by RT-PCR, despite observed changes
in both WT vs. KO and TP-10 vs. vehicle comparisons, demonstrating that there is not perfect
correspondence between methods The relative magnitude and direction of changes in gene
expression observed by RT-PCR matched observations from affymetrix chip data.
To facilitate pathway level analysis of changes in gene expression produced by chronic PDE10A
suppression, two different approaches were taken to analyze the data. First, the significantly
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affected genes that survived FDR correction were analyzed using Ingenuity Pathway Analysis
(IPA) software (version 8.0) to identify top scoring canonical pathways. The eIF4 pathway was
identified as the sole overlapping pathway via identification of 2 pathway genes (EIF2C4,
PPP2R5B) present in both TP-10 induced genes and KO-induced gene sets. This was in contrast
to observations that 21 of the 95 probes sets altered by TP-10 were also significantly altered in
the KO vs. WT comparison. The latter comparison yielded 68 probe sets significantly altered out
of 45101 monitored on the microarray. Given the size of these datasets relative to the number of
total probe sets measured, only 0.14 probe sets would be expected to overlap by random chance.
The identification of 21 overlapping significant probe sets is highly significant using a Poisson
approximation of binomial probability (p=2.814 X 10-38). Additionally, a strong correlation
(r2=0.9) was observed between the direction and fold change in the 19 genes that were
significantly altered in both the WT vs. KO and the TP-10 vs. vehicle comparisons (Figure 1).
Identification of a single overlapping pathway between these data sets suggests that the FDR
correction may result in too small a data set for pathway analysis. To explore this possibility, we
repeated pathway analysis using larger gene sets chosen according to their uncorrected p-values.
We selected genes with nominal p value <0.01 and a minimum fold change > 1.2 for analysis in
IPA. Using these criteria we found 364 probe sets following chronic PDE10A inhibition and 289
probe sets altered in PDE10A KO (Supplemental Table 1). Analysis of these probe sets using
IPA software highlighted statistically significant differences in 4 common canonical pathways
(highlighted in bold font in Table 4). These pathways included the polo-like kinases (PLK)
which includes checkpoint kinases, the p38/MAPK (ERK) pathway, the protein kinase A (PKA)
pathway, and beta-adrenergic signaling. In addition, a significant enrichment was observed in a
cAMP-responsive CREB gene list previously described by Zhang and colleagues, which are
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identified in bold font in Table 1 (Zhang et al., 2005). We also identified genes altered following
TP-10 treatment of PDE10A KO striatum for comparison with the same criteria used for IPA
pathway analysis. Although no genes reached statistical significance after correction for multiple
comparisons, 34 genes were identified with a nominal p-value of less than 0.01 and a greater
than 1.2 fold change (Supplementary Table 2). However, none of these genes overlapped with
those identified following drug treatment in the WT animals.
Consistent with effects on 2 of these pathways, acute PDE10A inhibition has been previously
reported to produce changes in cAMP and the phosphorylation state of PKA pathway targets
including pCREB, GluR1 Ser845, DARPP32 Thr34 (Nishi et al., 2008; Grauer et al., 2009).
Similarly, the phosphorylation of ERK has been previously reported in response to PDE10A
inhibition, suggesting the identified overlapping pathways accurately reflect effects of PDE10A
disruption. A common theme among PKA and ERK affected pathways is that they have been
previously proposed as therapeutic strategies for treating Huntington’s disease (Steffan et al.,
2000; Giampa et al., 2006; Roze et al., 2008; )
Acute administration of TP-10 drives CRE-mediated transcription within the striatum in vivo.
To further demonstrate the functional effects of CREB phosphorylation on transcriptional
activation, we created a reporter vector with 6X-CREB Response Elements (CRE) upstream of a
luciferase reporter gene cloned into a lentiviral (pLL3.7) vector. High titer virus was prepared
and stereotaxically administered to the striatum of adult mice. Animals were allowed to recover
for 1 week prior to the administration of TP-10 (3.2 mpk, s.c.) and subsequent imaging. A
separate cohort of animals that received stereotaxic injection of the CRE-luciferase lentiviral
reporter into the striatum showed that the peak transcriptional response in response to PDE10A
inhibition was detected via in vivo imaging of luciferase activity at 16 hours post drug treatment
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(data not shown). This is consistent with a need to translate and accumulate sufficient luciferase
enzyme prior to significant detection of a bioluminescent enzyme derived signal over skulls of
treated animals. All studies were subsequently carried out approximately 16 hours post drug
treatment, when animals were administered luciferase substrate (luciferin) and briefly
anesthetized for imaging on an IVIS bioimager. Baseline imaging of animals showed no
significant luciferase bioluminescence in the brains of injected animals. TP-10 injected animals
exhibited robust bioluminescence over brain regions approximating the striatum, and confirming
a robust transcriptional activation of the striatally-injected reporter construct (Figure 2A).
Repeated injection and imaging of these mice demonstrated the response of the reporter
construct was stable over several weeks (Figure 2B). Animals were sacrificed 10 weeks post
stereotaxic injection and histochemical verification of the striatal location for the reporter
expression of GFP was confirmed (Figure 2A).
Acute inhibition of PDE10A increased phosphorylation of striatal ERK substrates.
To further probe the ERK pathway, we evaluated the phosphorylation state of several potential
ERK pathway components following PDE10A inhibition including ERK, MSK, and H3. Mice
were administered 3.2 mpk of TP-10 s.c., a dose previously shown to produce elevations of
striatal cAMP and cGMP as well as activity in established models of antipsychotic efficacy
(Schmidt et al., 2008). Western blot analysis of samples probed with antibodies to phospho-
epitopes of ERK (ERK1/2 pTpY185/187 ) and MSK1 (Ser 376) found both kinases were
phosphorylated rapidly and transiently following PDE10A inhibition, returning to baseline levels
by 3 hours post drug administration (Figure 3). MSK has been reported to phosphorylate both
CREB and histone H3, both of which can alter gene transcription (Deak et al., 1998; Arthur,
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2008). Consistent with this relationship, we observed an enhanced phosphorylation of Ser10 on
H3 histone, a substrate for MSK1 (Figure 3). It is worth noting that we also observed significant
upregulation of H3 histone mRNA in affy chip studies following chronic PDE10A inhibition (1.4
fold, p=0.01 following FDR correction).
MSK1 is reported to be phosphorylated within striatonigral and striatopallidal MSNs in response
to cocaine and haloperidol, respectively (Heffron and Mandell, 2005; Bertran-Gonzalez et al.,
2008). In our hands, the haloperidol-induced phosphorylation of ERK and MSK1 is significantly
smaller than that observed following inhibition of PDE10A, consistent with the mechanistic
distinction that the D2 antagonist only activates a subset of MSNs versus the simultaneous
activation of striatonigral and striatopallidal neurons by PDE10A inhibition (Figure 4).
Consistent with this, haloperidol did not produce detectable phosphorylation of MSK1 by
western blot analysis. The dose of haloperidol used in this experiment (0.32 mg/kg, s.c.) was 8X
higher than that needed to produce an ED50 response in the conditioned avoidance responding
assay (Schmidt et al., 2008). This assay is thought to be predictive of antipsychotic activity. By
comparison, the 3.2 mg/kg dose of TP-10 used in this experiment is only 3X the dose required to
produce a ED50 response in the conditioned avoidance response assay (Schmidt et al., 2008).
Thus, the smaller changes in phosphorylation produced by haloperidol as compared to TP-10 are
not due to sub-threshold activity of the drug. The enhanced MSK1 phosphorylation during
PDE10A inhibition treatment likely occurs in all MSNs, however the higher expression of MSK1
in D1-containing neurons (Bertran-Gonzalez et al., 2009) may contribute to the larger signal
observed following TP-10 administration. The MSK1 substrate, H3 showed an identical pattern
of phosphorylation.
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The current study utilized microarray profiling to characterize and contrast the effects of genetic
and pharmacological disruption of PDE10A. Changes in gene expression produced by both
approaches implicate PDE10A in the regulation of signaling cascades that impinge on PKA,
ERK and checkpoint kinase-mediated pathways. The lack of any significant changes in gene
expression produced by the PDE10A inhibitor TP-10 in PDE10A KO animal speaks to the high
degree of specificity of the gene expression signature produced by TP-10 in WT animals.
Similarly, the restriction of changes in gene expression identified within this study largely to the
striatum, despite evaluation of microarray data collected from hippocampus, suggests a lack of
circuit-level regulation of gene expression in other structures. These observations illustrate the
utility of using negative microarray data to demonstrate specificity of pathway manipulations and
differentiate PDE10A inhibitors from other antipsychotic approaches that have been previously
reported to alter gene expression in frontal cortex and striatal regions in rodents (MacDonald et
al., 2005) .
Analysis of changes in gene expression provides insight into striatal neurotransmitter systems
regulated by prolonged PDE10A inhibition with TP-10. The downregulation of mRNA encoding
cholinergic synthetic enzyme ChAT and upregulation of mRNA for the L-arginine catabolic
enzyme arginase II, in both PDE10A KO mice and following PDE10A inhibition, would be
expected to decrease availability of acetylcholine and NO, both used as neurotransmitters by
striatal interneurons. PDE10A protein is absent from striatal cholinergic interneurons ( Coskran
et al., 2006), suggesting changes in ChAT mRNA are a response to changes in neuronal activity
within MSNs. The utility of anticholinergics in the treatment of Parkinson’s disease, albeit
limited, is believed to be due to reductions in the exaggerated striatal output following loss of
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dopaminergic inhibition. Thus, the potential for decreased cholinergic signaling may be a
compensatory response to the enhanced striatal activation. Similarly, the breakdown of L-
arginine by arginase II regulates availability of L-arginine for NO production by NOS (Vockley
et al., 1996). Arginase II was recently identified as one of the most abundant transcripts
represented in D2-containing neurons using Translating Ribosome Affinity Purification (TRAP)
to characterize the translational profiles most highly represented in specific cell types (Doyle et
al., 2008). PDE10A inhibition elevates cGMP levels, increases the excitability of MSNs and the
probability that MSNs will fire action potentials in response to cortical stimulation, particularly
within the D2 expressing MSNs of the indirect pathway (West and Grace, 2004; Threlfell et al.,
2009). Thus, increased mRNA for arginase II in the chronic setting could represent a
compensatory response to limit excessive firing of the more excitable indirect pathway neurons.
However, transcriptional changes are not likely to be limited to indirect pathway neurons since
the upregulation of prodynorphin mRNA is often associated with activity-dependent signatures
of the cAMP dependent D1-mediated signaling cascades (Morris et al., 1988) and represents one
of the most abundant transcripts identified in D1 containing neurons by TRAP analysis of D1
expressing MSNs (Doyle et al., 2008).
The primary neurotransmitter of MSNs is GABA and preferential upregulation of mRNA
encoding the synthetic enzyme GAD67 has been previously associated with antagonism of D2
receptors and most clinically effective antipsychotic agents (Laprade and Soghomonian, 1995;
Zink et al., 2004). GAD67 mRNA was preferentially upregulated by the chronic suppression of
PDE10A activity by TP-10 administration but not genetic KO. Likewise, mRNA for 4-
aminobutyrate aminotransferase which encodes the transaminase responsible for GABA
catabolism, decreased in abundance, suggesting the potential for enhanced GABA levels in
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striatal neurons. Additionally, the mRNA encoding shaker family potassium channel Kv1.6 was
downregulated, and might be expected to increase the excitability of striatal neurons as has been
reported following acute inhibition of PDE10A (Threlfell et al., 2009). It is interesting to note
that downregulation of mRNA encoding potassium channel subunits and associated proteins in
brain tissue has been reported as a consequence of chronic exposure to both typical and atypical
antipsychotics and has been proposed as contributing primary mechanistic underpinnings of
antipsychotic efficacy across treatments (Duncan et al., 2008). Taken together, these changes in
gene expression within the striatal neurons are consistent with chronic changes in both direct and
indirect pathways and a prolonged increase in the excitability of striatal neurons that could be
therapeutically relevant in treating psychosis.
PDE10A KO animals exhibited a number of potential compensatory changes in gene expression
that were absent following pharmacological suppression of activity. The Dual Specificity
Phosphatase DUSP14 is a negative regulator of the mitogen-activated protein kinase
(MAPK)/extracellular signal–regulated kinase 1/2 (ERK1/2) pathway (Patterson et al., 2009) and
was upregulated in the striatum of PDE10A knockout mice. This could provide a compensatory
brake on the chronic stimulation of the ERK signaling cascade. Similarly, the PDE10A KO
animals display an upregulation of mRNA for CREM , the cAMP responsive element modulator
protein that contributes negative feedback control of CREB signaling (De Cesare and Sassone-
Corsi, 2000). Thus, several compensatory mechanisms to counteract the enhanced ERK and
CREB signaling have been invoked in the knockout animals that are not obvious following
inhibitor treatment.
Several lines of evidence suggest changes in transcriptional profiles produced by suppression of
PDE10A activity may offer neuroprotection in HD. Transcripts for PDE10A and PDE1B are
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abundant in striatum, but exhibit significant downregulation in HD brain and the R6/2 mouse
model. The decrease in PDE10A mRNA is due to decreases in transcriptional initiation of the
striatally expressed PDE10A2 gene, which initiates at sites 2 and 3 in the exon 1a-specific
promoter (Hu et al., 2004) and loss of the PDE10A enzyme early in disease has been proposed as
a contributing factor in progression of disease (Hebb et al., 2004). However, the loss of striatally
enriched transcripts early in disease or R6/2 model progression could be a consequence of
transcriptional dysfunction due to a direct interaction of soluble huntingtin protein with
transcription factors required to direct expression of particular mRNAs or compensation for
dysfunctional neuronal signaling. Studies of the regulation of the PDE10A promoter have not
revealed any candidate regulatory transcription factors capable of decreasing PDE10A mRNA as
observed in the R6/R2 mouse model (Hu et al., 2004). Given that inhibition of PDE10A is a
powerful inducer of CREB-mediated transcription in striatum, and the propensity of this circuit
to compensate for chronic deficits in signaling cascades, it seems possible that downregulation of
PDE10A early in the disease process may be an adaptive response to compensate for loss of
cAMP signaling. If so, therapeutic inhibition of the enzyme earlier in the disease (prior to loss of
the enzyme) may stall its progression. Consistent with this, Giampa et al (2009) have recently
reported that chronic PDE10A inhibition with TP-10 can provide striatal neuroprotection from
quinolinic acid lesions of the striatum (Giampa et al., 2009). This group has recently also
observed neuroprotection and improved life expectancy in the R6/2 HD model with chronic
administration of the PDE10A inhibitor TP-10 (Giampa et al., 2010). Downregulation of key
transcripts identified in the current study following chronic TP-10 treatment may contribute to
this neuroprotective effect including follistatin, claspin and histone deacetylase (HDAC) 4.
Follistatin is an endogenous antagonist of activin and its mRNA is highly enriched in MSNs
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from the D1 containing neurons in the direct pathway (Doyle et al 2008). Direct striatal
administration of recombinant human activin A has been demonstrated to be neuroprotective in
the quinolinic acid model of striatal neurodegeneration (Hughes et al., 1999). Additionally,
PDE10A inhibition induced a large downregulation of claspin, a checkpoint kinase (Mrc 1)
involved in S-phase checkpoint damage detection. Expanded CAG/CTG repeats can be detected
by checkpoint machinery via changes in their secondary structure. These expansions are prone to
chromosome breakage requiring DNA repair. Detection of DNA damage within neurons has
been associated with the induction of apoptotic cell death. Claspin checkpoint kinase (Mrc1)
inhibitors have been proposed as promising therapeutic target for degenerative trinucleotide
repeat diseases including Huntington’s Disease (Freudenreich and Lahiri, 2004; Lahiri et al.,
2004). HDAC inhibitors have also been proposed as therapeutic treatment for HD. The HDAC
inhibitor 4b, expected to inhibit both HDAC3 and HDAC4, provides significant neuroprotection
in the R6/2 model of HD (Thomas et al., 2008) and the genetic knockdown of HDAC4 has been
recently reported to improve the HD phenotype in the R6/2 mouse (Bates et al., 2009). The
downregulation of HDAC4 following chronic TP-10 treatment may provide an alternative path
to decreasing HDAC activity.
The potential significance of robust activation of the ERK pathway by TP-10 in striatum is
highlighted by studies in Huntington’s Disease animal models and post-mortem HD brain
analysis suggesting deficiencies in MSK1-induced phosphorylation of H3 or its transcription
may contribute to the degeneration of striatal neurons (Roze et al., 2008). Consistent with the
activation of the ERK cascade, we found enhanced phosphorylation of the nuclear ERK substrate
MSK1 at Ser376, an autophosphorylation site that suggests kinase activation (McCoy et al.,
2005). The overexpression of MSK1 has been demonstrated to provide neuroprotection in vitro
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models of polyglutamine expansion, suggesting that MSK1 activation, or downstream
upregulation of histone-mediated modifications, may be a viable approach to treatment of
Huntington’s Disease (Roze et al., 2008).
This is the first study to characterize the consequences of chronic PDE10A suppression on gene
expression. We have identified several novel changes in signaling within the ERK cascade that
support the therapeutic potential of PDE10A inhibitors in the treatment of both psychosis and
HD. Observed changes in gene expression within striatal neurotransmitter systems are consistent
with previous predictions that PDE10A inhibition may be a novel approach to the treatment of
schizophrenia by enhancing striatal output. The specific biochemical and transcriptional pattern
of activity produced by PDE10A inhibition reported here also points to the potential application
of such agents in neurodegenerative conditions such as HD. The current microarray analysis
highlights the power of evaluating therapeutic targets from both genetic and pharmacological
perspectives to gain a broader insight into the biological system impacted and to more fully
evaluate the therapeutic potential of novel biological targets.
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Acknowledgements: The authors would like to acknowledge the technical expertise of Kari
Fonseca, Fred Nelson, Caroline Proulx-LaFrance and the support of Patrick Verhoest and Dan
Morton in conducting these studies. We are grateful to Caroline Benn and Nick Brandon for
critical reading of this manuscript, and to Eric Blalock for helpful discussion and constructive
statistical advice.
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Financial support for this research was provided by Pfizer, Inc.
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Figure 1. Transcripts altered by both TP-10 treatment and genetic knockout (FDR p<0.05) show
significant correlation of magnitude and direction of observed fold change between genetic and
pharmacological suppression of PDE10A. Ratio of TP-10 treated to vehicle treated striatum
(wild-type mice) is shown on the y-axis, and ratio of KO to WT striatum (vehicle treatment) is
shown on the x-axis. Dotted lines demarcate a ratio of 1.0, separating up-regulated genes from
down-regulated genes. Pearson correlation is shown in the upper left, p<0.0001.
Figure 2. Transduction of neurons in striatum with lentiviral CRE-luciferase reporter yields
activation of CREB-mediated transcription of luciferase reporter in vivo in response to PDE10
inhibition. Stereotaxic delivery of lentiviral CRE-luciferase reporter into striatum was completed
1 week prior to first TP-10 treatment. A) Mice were administered luciferin substrate (150 mpk,
i.p.) 10 minutes prior to collection of baseline images using IVIS platform. Subsequently, 3.2
mpk, s.c of TP-10 or vehicle was administered to N=15 animals per treatment group, and
repeated imaging of animals was conducted 16 hours post drug treatment. Confirmation of the
location of stereotaxic delivery was evaluated 10 weeks post lentiviral transduction by
immunohistochemical verification of GFP signal driven by lentiviral construct. Animals were
administered equivalent doses of TP-10 and imaged weekly. B) Quantitation of luciferase signals
collected from all animals 1 week post-lentiviral transduction and 6 weeks post-lentiviral
transduction are shown in panel B.
Figure 3. Western blot analysis of phosphorylation state of ERK cascade kinases following
inhibition of PDE10. TP-10 (3.2 mg/kg, s.c) or vehicle was administered to CD-1 mice ( N=4
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animals per group) for the length of time indicated prior to microwave fixation and collection of
striatal tissue for Western blot analysis. Each lane represents sample from an individual animal.
A) phosphorylation of ERK was detected within 15 minutes of drug treatment and remained high
for 60 minutes, dropping significantly by 3 hours. B) The ERK substrate MSK1 demonstrated
phosphorylation at Ser376 by 15 minutes, peaked at 30 minutes and began to drop off by 60
minutes. MSK1 phosphorylation state was returned to baseline by 3 hours post drug treatment. A
similar profile was observed for the phosphorylation profile of the MSK1 substrate histone H3
which was phosphorylated at Ser10 with a similar time course. Quantitative analysis of the C)
pERK D) pMSK and E) pHistone3 band intensity after normalization to total ERK band or total
NR1 protein (imaged in second channel on same blot) using the LiCOR Odyssey platform.
Figure 4. Western blot analysis of striatal tissues collected 30 minutes after administration of
haloperidol (0.32 mg/kg, s.c) and TP-10 (3.2 mg/kq, s.c.) reveals effects on phosphorylation of
ERK, MSK and H3 phosphorylation. Each lane represents a sample from an individual animal.
(A) Haloperidol induces a small increase in phosphorylation of ERK I as compared to increases
induced with TP-10 (3.2 mpk, s.c). Increases in phosphorylation of MSK1 (B) and Histone H3
(C) was not evident following haloperidol treatment, but was significant following PDE10A
inhibition.
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Tmem49 transmembrane protein 49 0.04 1.26 1423722_at
Znrf1 zinc and ring finger 1 0.03 1.26 1424384_a_at
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