Title : Chronic suppression of PDE10A alters striatal

JPET #173294

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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

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Running title: PDE10 suppression affects transmitter and signaling genes

Corresponding Author: Robin J Kleiman, Ph.D.

Neuroscience Research Unit

Eastern Point Road

Pfizer Inc, Global Research and Development

Groton, CT

Email: [email protected]

44 Text pages; 4 Tables; 4 Figures; 2 Supplementary Tables; 40 References

Abstract -243 words

Introduction-436 words

Discussion-1606 words

Nonstandard Abbreviations:

PDE10A, Phosphodiesterase 10A, Phosphodiesterase, PDE; TP-10: 2-{4-[-Pyridin-4-yl-1-(2,2,2-

trifluoro-ethyl)-1H-pyrazol-3-yl]-phenoxymethyl}-quinoline succinic acid; CREB, cAMP

responsive element binding protein; CRE, cAMP Response Element, RT-PCR, Real Time-

Polymerase Chain Reaction;WT, wild type; KO, knockout; PKA, Protein kinase A; ERK,

Extracellular signal-regulated kinase; MSK1, mitogen- and stress-activated kinases 1; FDR,

False Discovery Rate;DARPP32, dopamine- and cAMP-regulated phosphoprotein-32;

H3,Histone 3; HD, Huntington’s Disease; ChAT, Choline acetyltransferase; GAD67, Glutamate

decarboxylase 1,; DGAT2, Diacylglycerol O-acyltransferase ; PDE1C, Phosphodiesterase 1C,;

HDAC, Histone deacetylase

Recommended Section Assignment: Cellular and Molecular


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Abstract:

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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Introduction:

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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Methods:

Compounds:

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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TaqMan® assays from Applied Biosystems using primer sets detailed in Table III. All qRT-PCR

assays were performed using TaqMan® Gene Expression Master Mix (Applied Biosystems,

Foster City, CA) and the following cycling conditions: (1) 50 °C hold for 2 min, (2) 95 °C hold

for 10 min, (3) 40 cycles of 3 sec at 95 °C, 30 sec at 60 °C. Ribophorin (Rpn1) was used as the

endogenous control gene and relative quantities for gene expression were calculated using RQ

manager software (Applied Biosystems, Foster City, CA).

Phosphoprotein Profiling:

CD-1 mice dosed with vehicle, haloperidol (0.32mg/kg) or TP-10 (3.2mg/kg), s.c in a vehicle

consisting of 30% b-cyclodextrin for the indicated times were sacrificed by 3 second focused

microwave irradiation to optimally preserve phosphorylation state of selected epitopes. Dissected

striatum were placed on dry ice and stored at -70 C. Homogenates prepared on a wt/vol basis

(100ug/uL) in 25mM Tris-HCL pH 7.4, 150mM NaCl, 0.1% NP-40, and one complete protease

inhibitor tablet (Roche). Samples homogenized on Mixer-Mill 300 (Retsch) at 25 shakes/sec for

4 min were placed on wet ice with 30ul of homogenate mixed with 50uL 4x LDS sample buffer

(Invitrogen) including 1mM DTT and 90uL water and warmed to 70oC for 10 minutes. Samples

(10 ul) were loaded onto EPAGE gels (Invitrogen) with seeBlueplus2 makers and transferred to

nitrocellulose using the I-blot system (Invitrogen). Membranes were blocked with Rockland

Near Infrared blocking solution for 1 hour and primary antibodies added overnight at 4 oC in

50/50 Rockland Block/PBS with 0.01% Tween 20. Blots were washed in PBS 0.01% Tween 20.

Incubation in secondary antibodies, goat anti-rabbit Alexa-680 and/or goat anti-mouse Alexa 800

(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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Results:

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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Discussion:

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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Authorship Contributions:

Participated in research design: Kleiman, Schmidt

Conducted experiments: Bove, Harms, Kimmel, Romegialli

Contributed new reagents: Miller, Willis

Performed data analysis: Kuhn, Des Etages, Lanz

Wrote the manuscripts: Kleiman, Schmidt


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Footnotes:

Financial support for this research was provided by Pfizer, Inc.


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Legends for Figures:

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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Table 1. Genes encoded by transcripts that were significantly altered in mouse striatum following chronic pharmacological inhibition

of PDE10A activity, following correction for multiple comparisons. A comparison between PDE10A KO and WT striatum for each

pharmacologically regulated gene is also shown with the associated FDR corrected p-value and fold change. Significant changes

across treatments are highlighted in grey. No significant pharmacologically-induced differences were observed following TP-10

treatment in the PDE10A knockout animals, consistent with the specificity of these effects via PDE10A inhibition. Genes represented

in bolded text were reported to be cAMP responsive genes by Zhang et al. (Zhang et al 2005).

Gene Symbol Gene Name Veh vs. TP-10 Veh vs. TP-

10 KO vs.

WT KO vs. WT KO+TP-10

vs. Veh KO+TP-10

vs. Veh AffyID

FDR p-value Fold change FDR p-value

Fold change FDR p-value

Fold change

Csda cold shock domain protein A 0.01 7.86 0.89 1.76 0.83 -1.22 1453238_s_at

1200016E24Rik Mus musculus RIKEN cDNA 1200016E24 gene 0.02 7.65 0.89 1.76 1.00 1.01 1452418_at

4631426E05Rik hypothetical protein MGC39715 0.04 3.58 0.89 1.48 0.99 -1.06 1453177_at

Mm.405423

Mus musculus adult male corpus striatum cDNA, RIKEN clone:C030023B07 0.00 3.36 0.00 2.95 1.00 -1.02 1460043_at

Nnat neuronatin 0.00 2.69 0.00 2.36 0.99 1.03 1423506_a_at

Irak3 Interleukin-1 receptor-associated kinase M 0.01 2.34 0.69 1.38 0.98 -1.05 1435040_at

Myo3b myosin IIIB 0.01 2.20 0.26 1.64 0.98 1.05 1459299_at

Dlk1 delta-like 1 homolog (Drosophila) 0.03 1.94 0.49 1.45 0.96 -1.08 1449939_s_at

Pde1c

PDE1C (Calmodulin 3',5'cyclic nucleotide phosphodiesterase 1C / HSPDE1C) 0.02 1.94 0.10 1.69 0.88 1.18 1440374_at

Gpr149 G protein-coupled receptor 149 0.02 1.92 0.93 1.16 0.98 -1.05 1438210_at

Arg2 arginase, type II 0.04 1.91 0.58 1.40 0.99 1.01 1418847_at

Smpdl3b sphingomyelin phosphodiesterase, acid-like 3B 0.03 1.86 0.43 1.45 1.00 -1.02 1417300_at

This article has not been copyedited and form

atted. The final version m

ay differ from this version.

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BC022623 family with sequence similarity 43, member A 0.00 1.84 0.00 1.83 0.95 1.05 1426734_at

Ppp1r2 protein phosphatase 1, regulatory (inhibitor) subunit 2 0.02 1.78 0.86 1.20 0.99 -1.02 1456830_at

Pdyn prodynorphin 0.03 1.76 0.00 2.61 0.87 -1.07 1416266_at

Rgs5 regulator of G-protein signalling 5 0.04 1.71 0.65 1.30 0.93 -1.09 1420941_at

Pde1c

PDE1C (Calmodulin 3',5'cyclic nucleotide phosphodiesterase 1C / HSPDE1C) 0.02 1.70 0.71 1.24 0.95 -1.04 1436251_at

Syt10 synaptotagmin X 0.02 1.67 0.25 1.41 1.00 -1.01 1450347_at

Filip1 filamin A interacting protein 1 0.03 1.63 0.82 1.19 0.97 -1.03 1436650_at

Rgs5 regulator of G-protein signalling 5 0.03 1.62 0.62 1.26 0.91 -1.07 1417466_at

Ppp1r2 protein phosphatase 1, regulatory (inhibitor) subunit 2 0.00 1.59 0.35 1.25 0.73 -1.39 1448684_at

Hs6st2 Heparan-sulfate 6-sulfotransferase 2 0.03 1.56 0.68 1.22 0.94 1.09 1450047_at

1700012A16Rik RIKEN cDNA 1700012A16 gene 0.03 1.55 0.64 1.24 0.98 -1.03 1429324_at

Frem2 FRAS1 related extracellular matrix protein 2 0.03 1.54 0.24 1.37 0.96 -1.06 1457038_at

Dgat2 diacylglycerol O-acyltransferase homolog 2 (mouse) 0.01 1.54 0.03 1.46 1.00 -1.03 1422678_at

D10Ertd438e chromosome 6 open reading frame 68 0.03 1.54 0.29 1.34 1.00 -1.02 1419915_at

Lor loricrin 0.02 1.53 0.11 1.42 0.92 1.11 1448745_s_at

Dgat2 diacylglycerol O-acyltransferase homolog 2 (mouse) 0.04 1.53 0.06 1.51 0.75 -1.14 1422677_at

Slc7a8

solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 (SLC7A8) 0.00 1.53 0.00 1.63 0.85 -1.29 1417929_at

Itm2a integral membrane protein 2A 0.02 1.51 0.53 1.23 0.22 1.42 1423608_at

Wdr17 WD repeat domain 17 0.00 1.48 0.11 1.28 0.92 1.14 1435392_at




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Dcbld1 Homo sapiens cDNA FLJ30900 fis, clone FEBRA2005752. 0.03 1.46 0.27 1.29 0.92 -1.71 1418966_a_at

Rasl10b RAS-like, family 10, member B 0.03 1.45 0.48 1.23 0.89 -1.10 1433566_at

Avpi1 arginine vasopressin-induced 1 0.01 1.41 0.03 1.36 0.95 -1.11 1423122_at Hist3h2ba histone 3, H2bb 0.01 1.41 0.20 1.23 0.95 -1.14 1449482_at

Lor loricrin 0.02 1.40 0.08 1.34 0.77 -1.11 1420183_at

1110018G07Rik KIAA0317 0.01 1.39 0.41 1.20 1.00 -1.04 1433767_at

0610012D17Rik hypothetical protein MGC33212 0.04 1.39 0.79 1.14 0.91 -1.73 1428972_at

Esm1 endothelial cell-specific molecule 1 0.05 1.36 0.88 1.11 0.90 -1.06 1449280_at

Chmp1b chromatin modifying protein 1B 0.02 1.35 0.00 1.49 0.99 1.02 1418817_at

Ninj1 ninjurin 1 0.03 1.33 0.44 1.18 0.99 -1.05 1441281_s_at

Tmem35 transmembrane protein 35 0.05 1.31 0.37 1.20 1.00 -1.01 1416710_at

Tmem41a transmembrane protein 41A 0.03 1.31 0.00 1.42 0.96 -1.08 1424191_a_at

Erlin1 ER lipid raft associated 1 0.04 1.30 0.87 1.09 0.98 -1.05 1424210_at

1110059G10Rik KIAA1143 0.04 1.30 0.87 1.09 1.00 -1.02 1418048_at

D10Ertd438e chromosome 6 open reading frame 68 0.02 1.30 0.38 1.16 0.95 -1.05 1419914_s_at

Pigx phosphatidylinositol glycan, class X 0.03 1.29 0.14 1.23 0.64 -1.05 1425134_a_at

Abat 4-aminobutyrate aminotransferase 0.01 1.29 0.07 1.23 1.00 -1.01 1433855_at

Znf346 zinc finger protein 346 0.04 1.28 0.88 1.08 0.91 1.13 1417088_at

Nudt11 nudix (nucleoside diphosphate linked moiety X)-type motif 11 0.01 1.28 0.12 1.19 0.92 -1.07 1426887_at

Ppp2r5b

protein phosphatase 2, regulatory subunit B (B56), beta isoform 0.01 1.27 0.02 1.24 0.95 -1.07 1426811_at

Tmem68 transmembrane protein 68 0.01 1.26 0.06 1.20 0.93 1.10 1423649_at

Gpr176 G protein-coupled receptor 176 0.02 1.25 0.29 1.15 0.94 -1.05 1442116_at

Fkbp1a FKBP (12 FK-506) 0.03 1.25 0.84 1.08 0.87 -1.16 1448184_at

Ppp1r7 protein phosphatase 1, regulatory subunit 7 0.04 1.24 0.05 1.25 0.98 -1.06 1417919_at




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Cbx4 chromobox homolog 4 (Pc class homolog, Drosophila) 0.01 1.23 0.01 1.24 0.89 1.11 1419583_at

Znrf1 zinc and ring finger 1 0.04 1.22 0.03 1.26 0.95 -1.09 1424384_a_at

Bzw2 basic leucine zipper and W2 domains 2 0.03 1.22 0.63 1.10 0.80 -1.16 1423456_at

Rnf103 ring finger protein 103 0.02 1.22 0.45 1.11 0.90 -1.23 1448434_at

Gad1 glutamate decarboxylase 1 (brain, 67kDa) (GAD1) 0.05 1.21 0.87 1.07 0.96 -1.03 1416561_at

Hif1a

hypoxia-inducible factor 1, alpha subunit (basic helix-loop-helix transcription factor) 0.03 1.19 0.55 1.10 0.98 1.07 1416035_at

Rwdd4a RWD domain containing 4A 0.04 1.18 0.54 1.10 1.00 -1.02 1424243_at

Rps2 ribosomal protein S2 0.04 1.17 0.33 1.11 0.91 -1.16 1431765_a_at

KIAA1109 KIAA1109 0.01 1.17 0.65 1.06 0.93 -1.12 1427016_at

Tsc22d1 TSC22 domain family 1 0.03 1.16 0.25 1.11 0.95 -1.05 1425742_a_at

Cox7a2l cytochrome c oxidase subunit VIIa polypeptide 2 like 0.04 1.15 0.98 -1.02 0.97 -1.03 1421772_a_at

Phb2 prohibitin 2 0.05 1.15 0.99 1.02 0.95 -1.15 1416202_at

Ngfrap1

nerve growth factor receptor (TNFRSF16) associated protein 1 0.04 1.13 0.18 1.10 0.98 -1.08 1428842_a_at

Usmg5 upregulated during skeletal muscle growth 5 0.02 1.11 0.58 1.05 0.98 -1.07 1448179_at

Slc25a3

solute carrier family 25 (mitochondrial carrier; phosphate carrier), member 3 0.03 1.10 0.43 1.06 0.74 1.15 1416300_a_at

Pttg1 pituitary tumor-transforming 1 0.02 -1.21 0.11 -1.16 0.91 -1.03 1438390_s_at

Snrpb small nuclear ribonucleoprotein polypeptide N 0.01 -1.23 0.46 -1.11 0.97 -1.04 1437193_s_at

D630004N19Rik RIKEN cDNA D630004N19 gene 0.03 -1.26 0.94 -1.06 1.00 1.03 1442016_at

Mm.326577 Mus musculus similar to cadherin 11 0.04 -1.26 0.65 -1.12 0.99 -1.02 1441565_at




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Rnf144a ring finger protein 144A 0.01 -1.29 0.00 -1.37 0.99 1.04 1438404_at

Chat Choline O-acetyltransferase 0.02 -1.38 0.03 -1.37 0.99 -1.04 1443372_at

Ankrd35 ankyrin repeat domain 35 0.03 -1.38 0.23 -1.27 0.87 -1.10 1438474_at

Hdac4 histone deacetylase 4 0.03 -1.38 0.85 -1.11 0.53 -1.14 1436758_at

Camkk2

Ca2+/calmodulin-dependent protein kinase kinase beta (CAMKKB) 0.03 -1.45 0.55 -1.21 1.00 -1.01 1455401_at

Il17rc interleukin 17 receptor C 0.01 -1.45 0.05 -1.36 0.85 -1.60 1419671_a_at

Eif2c4 eukaryotic translation initiation factor 2C, 4 0.00 -1.46 0.01 -1.39 0.92 -1.20 1453289_at

Nfix nuclear factor I/X (CCAAT-binding transcription factor) 0.01 -1.47 0.76 -1.15 0.84 1.16 1436363_a_at

Fst follistatin 0.04 -1.48 0.08 -1.44 0.95 -1.08 1421365_at

Eif2c4 eukaryotic translation initiation factor 2C, 4 0.00 -1.48 0.03 -1.39 0.94 -1.04 1429779_at

Cacnb3 Dihydropyridine-sensitive L-type calcium channel beta-3 subunit 0.01 -1.50 0.57 -1.20 0.98 -1.02 1448656_at

Camkk2

Ca2+/calmodulin-dependent protein kinase kinase beta (CAMKKB) 0.02 -1.53 0.31 -1.32 0.88 -1.09 1424474_a_at

Ptprv protein tyrosine phosphatase, receptor type, V (pseudogene) 0.01 -1.58 0.00 -1.74 1.00 1.01 1449957_at

1110032E23Rik hypothetical protein DKFZp434L142 0.03 -1.60 0.01 -1.72 0.97 -1.04 1416805_at

Kcna6 Voltage-gated potassium channel protein KV1.6 0.03 -1.64 0.57 -1.29 0.93 -1.16 1441049_at

EG625121 predicted gene, EG625121 0.03 -1.65 0.33 -1.38 0.99 -1.01 1457143_at

Fst follistatin 0.01 -1.65 0.03 -1.62 1.00 -1.01 1434458_at

Kalrn kalirin, RhoGEF kinase 0.01 -1.66 0.99 -1.06 1.00 1.00 1436066_at

1110032E23Rik hypothetical protein DKFZp434L142 0.00 -1.91 0.00 -2.24 0.96 -1.04 1429637_at

2210419I08Rik RIKEN cDNA 2210419I08 gene 0.01 -1.93 0.25 -1.52 1.00 -1.02 1441368_at

Clspn claspin homolog (Xenopus laevis) 0.00 -2.78 0.00 -2.55 1.00 -1.01 1456280_at




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Table 2. Gene list of mRNAs significantly altered in mouse striatum following genetic

disruption of PDE10A activity, following correction for multiple comparisons.

Gene Symbol Gene Name KO vs. WT KO vs. WT AffyID

FDR p-value Fold change

Mm.405423 Mus musculus adult male corpus striatum cDNA, RIKEN clone:C030023B07 0.00 2.95 1460043_at

Pdyn prodynorphin 0.00 2.61 1416266_at

Gnb4 guanine nucleotide binding protein, beta 4 0.02 2.37 1439632_at

Nnat neuronatin 0.00 2.36 1423506_a_at

Hist2h3c2 histone 2, H3c2 0.03 2.31 1422155_at

Hist1h4e histone 1, H4e 0.00 2.16 1422948_s_at

Hist1h4j histone 1, H4j 0.02 2.12 1428014_at


Histh3h3 histone 3, H3 0.01 2.06 1460314_s_at

Hist1h4b histone 1, H4b 0.01 1.94 1424854_at


Crem cAMP responsive element modulator 0.03 1.90 1449037_at

BC022623 cDNA sequence BC022623 0.00 1.83 1426734_at

Rab3c RAB3C, member RAS oncogene family 0.01 1.83 1449494_at

Dusp14 dual specificity phosphatase 14 0.04 1.75 1431422_a_at

Hist1h2bd histone 1, H2b5 0.02 1.70 1452540_a_at

Slc7a8 solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 0.00 1.63 1417929_at

Hist1h2bc solute carrier family 7 (cationic amino acid transporter, y+ system), member 8 0.05 1.57 1418072_at

Sema3e

sema domain, immunoglobulin domain (Ig), short basic domain, secreted, (semaphorin) 3E 0.03 1.55 1427673_a_at

Trim17 tripartite motif-containing 17 0.05 1.50 1440932_at

Chmp1b chromatin modifying protein 1B 0.00 1.49 1418817_at

Zdhhc14 zinc finger, DHHC domain containing 14 0.05 1.48 1423668_at

Dgat2 diacylglycerol O-acyltransferase 2 0.03 1.46 1422678_at

Rab3c RAB3C, member RAS oncogene family 0.01 1.46 1435047_at

Tmem41a transmembrane protein 41a 0.00 1.42 1424191_a_at

2610002M06Rik RIKEN cDNA 2610002M06 gene 0.03 1.42 1418816_at

Esd esterase D/formylglutathione hydrolase 0.00 1.38 1417825_at

Abca3 ATP-binding cassette, sub-family A (ABC1), member 3 0.04 1.37 1451731_at

Slc3a1 solute carrier family 3, member 1 0.05 1.37 1448741_at

Avpi1 arginine vasopressin-induced 1 0.03 1.36 1423122_at

Mef2d myocyte enhancer factor 2D 0.03 1.29 1437300_at

Dctn3 dynactin 3 0.05 1.28 1416247_at

Asph aspartate-beta-hydroxylase 0.05 1.27 1433906_at

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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Cbx4 chromobox homolog 4 (Drosophila Pc class) 0.01 1.24 1419583_at

Slc3a2 solute carrier family 3 member 2 0.01 1.24 1425364_a_at

Ppp2r5b protein phosphatase 2, regulatory subunit B (B56), beta isoform 0.02 1.24 1426811_at

Trak1 trafficking protein, kinesin binding 1 0.04 1.19 1428327_at

Reps2 RALBP1 associated Eps domain containing 2 0.04 1.19 1435389_at

Tmem33 transmembrane protein 33 0.02 1.16 1436028_at

Atp5d ATP synthase, H+ transporting, mitochondrial F1 complex, delta subunit 0.03 1.15 1423716_s_at

Ckap5 cytoskeleton associated protein 5 0.04 -1.19 1442271_at

Ubxn2b UBX domain protein 2B 0.04 -1.24 1430816_at

2310069P03Rik RIKEN cDNA 2310069P03 gene 0.04 -1.25 1441006_at

Pard6g par-6 partitioning defective 6 homolog gamma (C. elegans) 0.04 -1.28 1420851_at

Taf1d

TATA box binding protein (TBP)-associated factor, RNA polymerase I, D, 41kDa 0.05 -1.30 1457292_at

Hla-dma major histocompatibility complex, class II, DM alpha 0.03 -1.36 1422527_at

Rnf144a ring finger protein 144A 0.00 -1.37 1438404_at

Chat choline acetyltransferase 0.03 -1.37 1443372_at

Cgref1 cell growth regulator with EF hand domain1 0.02 -1.38 1424529_s_at

Eif2c4 eukaryotic translation initiation factor 2C, 4 0.01 -1.39 1453289_at

BM119687.2

Mouse Newborn Kidney cDNA Library (Long) Mus musculus cDNA clone NIA:L0929D02 0.04 -1.39 1447060_at

Eif2c4 eukaryotic translation initiation factor 2C, 4 0.03 -1.39 1429779_at

Clca6 chloride channel calcium activated 6 0.01 -1.42 1443256_at

Gm715 gene model 715 0.01 -1.45 1445503_at

Kcns2 K+ voltage-gated channel, subfamily S, 2 0.02 -1.46 1457325_at

Mm.402736 transcribed locus 0.05 -1.48 1458052_at

Id4 inhibitor of DNA binding 4 0.04 -1.48 1423260_at

Id4 inhibitor of DNA binding 4 0.02 -1.59 1450928_at

Rps6ka5 ribosomal protein S6 kinase, polypeptide 5 0.01 -1.62 1440343_at

Fst follistatin 0.03 -1.62 1434458_at

C030009J22Rik RIKEN cDNA C030009J22 gene 0.02 -1.64 1458386_at

1110032E23Rik RIKEN cDNA 1110032E23 gene 0.01 -1.72 1416805_at

Ptprv protein tyrosine phosphatase, receptor type, V 0.00 -1.74 1449957_at

Neu2 neuraminidase 2 0.04 -1.83 1431936_a_at

1110032E23Rik RIKEN cDNA 1110032E23 gene 0.00 -2.24 1429637_at

Clspn claspin homolog (Xenopus laevis) 0.00 -2.55 1456280_at


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Table 3. RT-PCR confirmation of genes significantly regulated by inactivation of PDE10A activity. Significant changes in gene

expression between WT and KO striatum mRNA samples as determined by affy chip analysis were expected for the 18 genes

highlighted in grey. The directional fold change observed between KO and WT are listed next to p-values. Positive number indicate

more mRNA detected in KO animals relative to WT. Individual TaqMan assays used to carry out PCR are listed next to each result.

Gene Symbol Gene Name Veh vs. TP-10 affy KO vs. WT affy KO vs. WT qRT-PCR

TaqMan Assay AffyID FDR p-value Fold change FDR p-

value Fold

change p-value Fold change

Nnat neuronatin 0.0003 2.7 0.0031 2.4 0.0000 2.3 Mm00731416_s1 1423506_a_at

Pdyn prodynorphin 0.0328 1.8 0.0002 2.6 0.0000 3.3 Mm00457573_m1 1416266_at

Fam43a family with sequence similarity 43, member A 0.0004 1.8 0.0007 1.8 0.0000 1.8 Mm00617309_s1 1426734_at

Dgat2 diacylglycerol O-acyltransferase 2 0.0074 1.5 0.0297 1.5 0.0000 1.4 Mm00499536_m1 1422678_at

Slc7a8 solute carrier family 7, member 8 0.0017 1.5 0.0004 1.6 0.0000 1.9 Mm00444250_m1 1417929_at

Avpi1 Avpi1 arginine vasopressin-induced 1 0.0095 1.4 0.0329 1.4 0.0000 1.5 Mm00503550_m1 1423122_at

Chmp1b Chmp1b chromatin modifying protein 1B 0.0245 1.3 0.0023 1.5 0.0137 1.1 Mm04179599_s1 1418817_at

Tmem41a

Tmem41a transmembrane protein 41a 0.0333 1.3 0.0047 1.4 0.0000 1.4 Mm00508557_m1 1424191_a_at

Ppp2r5b

Ppp2r5b protein phosphatase 2, regulatory subunit B 0.0067 1.3 0.0228 1.2 0.2794 1.1 Mm01233429_m1 1426811_at

Cbx4

Cbx4 chromobox homolog 4 (Drosophila Pc class) 0.0089 1.2 0.0081 1.2 0.1303 1.1 Mm00483089_m1 1419583_at

Znrf1 Znrf1 zinc and ring finger 1 0.0447 1.2 0.0258 1.3 0.0237 1.1 Mm00460000_m1 1424384_a_at

Rnf144a Rnf144a ring finger protein 144A 0.0062 -1.3 0.0007 -1.4 0.0001 -1.4 Mm00475638_m1 1438404_at




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Chat choline acetyltransferase 0.0156 -1.4 0.0254 -1.4 0.1449 -1.2 Mm01221880_m1 1443372_at

Eif2c4 eukaryotic translation initiation factor 2C, 4

0.0011 -1.5 0.0088 -1.4

0.0000 -1.7 Mm01188521_m1

1453289_at

0.0036 -1.5 0.0267 -1.4 1429779_at

Ptprv

Ptprv protein tyrosine phosphatase, receptor type, V 0.0119 -1.6 0.0030 -1.7 0.0000 -3.9 Mm00468369_m1 1449957_at

1110032E23Rik (Fam198b)

Fam198b family with sequence similarity 198, member B 0.0303 -1.6 0.0130 -1.7 0.0000 -3.2 Mm00500026_m1 1416805_at

Fst follistatin 0.0139 -1.7 0.0272 -1.6 0.0000 -2.0 Mm03023987_m1 1434458_at

Clspn claspin homolog (Xenopus laevis) 0.0001 -2.8 0.0007 -2.5 0.0000 -3.3 Mm01181258_m1 1456280_at

Pde1c phosphodiesterase 1C 0.0156 1.9 0.1043 1.7 0.0001 1.5 Mm00478049_m1 1440374_at

Rgs5 regulator of G-protein signaling 5 0.0435 1.7 0.6516 1.3 0.3174 -1.1 Mm00501393_m1 1420941_at

Syt10 synaptotagmin 10 0.0192 1.7 0.2507 1.4 0.0000 2.8 Mm00444516_m1 1450347_at

Lor loricrin 0.0227 1.5 0.1051 1.4 0.0007 1.5 Mm01962650_s1 1448745_s_at

Ninj1 ninjurin 1 0.0259 1.3 0.4371 1.2 0.9999 1.0 Mm00479015_m1 1441281_s_at

Gad1 glutamic acid decarboxylase 1 0.0456 1.2 0.8697 1.1 0.9934 1.0 Mm00725661_s1 1416561_at

Kcna6

potassium voltage-gated channel, shaker-related, subfamily, member 6 0.0333 -1.6 0.5681 -1.3 0.0000 -1.6 Mm00496625_s1 1441049_at




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Table 4. Canonical Pathways identified by Ingenuity Pathway Analysis of statistically significant

genes (p<0.01, without FDR correction) and a greater than 1.2 fold difference between TP-10

and vehicle treated mouse striatum, or between PDE10 KO and WT mouse striatum. Genes

utilized for pathway analysis are listed in supplementary Table 1. Pathways identified in bold

font were determined to be significantly affected in both comparisons.

TP-10 vs. vehicle treated mouse striatum p-value PDE10 knockout vs. WT mouse striatum p-value

Alanine and Aspartate Metabolism 0.003 Protein Kinase A Signaling 0.003

GABA Receptor Signaling 0.003 p38 MAPK Signaling 0.003

Mitotic Roles of Polo-Like Kinase 0.007 ERK/MAPK Signaling 0.009

Aryl Hydrocarbon Receptor Signaling 0.009 Calcium-induced T Lymphocyte Apoptosis 0.015

Axonal Guidance Signaling 0.009 Mitotic Roles of Polo-Like Kinase 0.020

Taurine and Hypotaurine Metabolism 0.010 Growth Hormone Signaling 0.026

Glutamate Metabolism 0.012 ERK5 Signaling 0.026

Cyanoamino Acid Metabolism 0.020 Cardiac beta-adrenergic Signaling 0.035

Nitrogen Metabolism 0.022 Reelin Signaling in Neurons 0.042

Cardiac beta-adrenergic Signaling 0.022

Calcium Signaling 0.023

Hepatic Fibrosis / Hepatic Stellate Cell Activation 0.026

Ephrin Receptor Signaling 0.028

beta-alanine Metabolism 0.031

G Beta Gamma Signaling 0.032

Synaptic Long Term Depression 0.035

Protein Kinase A Signaling 0.035

p38 MAPK Signaling 0.038

Type I Diabetes Mellitus Signaling 0.039

Butanoate Metabolism 0.047


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Figure 1. JPET #173294


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Baseline

B

16 hrPost TP-10 treatment

A

Figure 2. JPET#179294


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vehicle 15min 30min 60min 3hrs 6hrs

pMSKs376

pHistoneS10

NR1

pERK

Total ERK

vehicle 15min 30min 60min 3hrs 6hrsA

B

DC E

Figure 3. JPET #173294




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B

Vehicle Haloperidol TP-10Vehicle Haloperidol TP-10Vehicle Haloperidol TP-10Vehicle Haloperidol TP-10

C

Vehicle Haloperidol TP-10

A Vehicle Haloperidol TP-10

Vehicle Haloperidol TP-10

pERK

tubulin

pMSK

tubulin

pHistone H3

tubulin

Figure 4. JPET 173294


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Title : Chronic suppression of PDE10A alters striatal

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