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Large-scale transcriptomic analysis reveals that pridopidine reverses aberrant gene expression and activates… Kusko, Rebecca; Dreymann, Jennifer; Ross, Jermaine; Cha, Yoonjeong; Escalante-Chong, Renan; Garcia-Miralles, Marta; Tan, Liang J; Burczynski, Michael E; Zeskind, Ben; Laifenfeld, Daphna; Pouladi, Mahmoud; Geva, Michal; Grossman, Iris; Hayden, Michael R May 21, 2018

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RESEARCH ARTICLE Open AccessLarge-scale transcriptomic analysis revealsthat pridopidine reverses aberrant geneexpression and activates neuroprotectivepathways in the YAC128 HD mouseRebecca Kusko1†, Jennifer Dreymann2†, Jermaine Ross1, Yoonjeong Cha1, Renan Escalante-Chong1,Marta Garcia-Miralles3, Liang Juin Tan3, Michael E. Burczynski2, Ben Zeskind1, Daphna Laifenfeld2,Mahmoud Pouladi3,5, Michal Geva2, Iris Grossman2 and Michael R. Hayden2,3,4,5*AbstractBackground: Huntington Disease (HD) is an incurable autosomal dominant neurodegenerative disorder driven byan expansion repeat giving rise to the mutant huntingtin protein (mHtt), which is known to disrupt a multitude oftranscriptional pathways. Pridopidine, a small molecule in development for treatment of HD, has been shown toimprove motor symptoms in HD patients. In HD animal models, pridopidine exerts neuroprotective effects andimproves behavioral and motor functions. Pridopidine binds primarily to the sigma-1 receptor, (IC50 ~ 100 nM),which mediates its neuroprotective properties, such as rescue of spine density and aberrant calcium signaling inHD neuronal cultures. Pridopidine enhances brain-derived neurotrophic factor (BDNF) secretion, which is blocked byputative sigma-1 receptor antagonist NE-100, and was shown to upregulate transcription of genes in the BDNF,glucocorticoid receptor (GR), and dopamine D1 receptor (D1R) pathways in the rat striatum. The impact of differentdoses of pridopidine on gene expression and transcript splicing in HD across relevant brain regions was explored,utilizing the YAC128 HD mouse model, which carries the entire human mHtt gene containing 128 CAG repeats.Methods: RNAseq was analyzed from striatum, cortex, and hippocampus of wild-type and YAC128 mice treatedwith vehicle, 10 mg/kg or 30 mg/kg pridopidine from the presymptomatic stage (1.5 months of age) until 11.5 months of age in which mice exhibit progressive disease phenotypes.Results: The most pronounced transcriptional effect of pridopidine at both doses was observed in the striatumwith minimal effects in other regions. In addition, for the first time pridopidine was found to have a dose-dependent impact on alternative exon and junction usage, a regulatory mechanism known to be impaired in HD.In the striatum of YAC128 HD mice, pridopidine treatment initiation prior to symptomatic manifestation rescues theimpaired expression of the BDNF, GR, D1R and cAMP pathways.(Continued on next page)* Correspondence:;†Rebecca Kusko and Jennifer Dreymann contributed equally to this study.2Research and Development, Teva Pharmaceutical Industries Ltd, Netanya,Israel3Translational Laboratory in Genetic Medicine, Agency for Science,Technology and Research, Singapore (A*STAR), Singapore 138648, SingaporeFull list of author information is available at the end of the article© The Author(s). 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0International License (, which permits unrestricted use, distribution, andreproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link tothe Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver( applies to the data made available in this article, unless otherwise stated.Kusko et al. Molecular Neurodegeneration  (2018) 13:25 from previous page)Conclusions: Pridopidine has broad effects on restoring transcriptomic disturbances in the striatum, particularlyinvolving synaptic transmission and activating neuroprotective pathways that are disturbed in HD. Benefits oftreatment initiation at early disease stages track with trends observed in the clinic.Keywords: Huntington disease, Movement disorders, NeurodegenerationBackgroundHuntington Disease (HD) is a progressive and neuro-logical disorder caused by an autosomal dominant CAGtrinucleotide expansion in the Htt gene [1], character-ized by psychiatric, cognitive and motor disturbances,manifesting usually between 40 and 50 years of age andworsening until death [2]. Htt plays a role in facilitatingaxonal transport of brain-derived neurotrophic factor(BDNF) in the corticostriatal pathway of the motorcircuit in wild-type animals (Fig. 1a and b) [3]. Consist-ently, in animal models of HD, mHtt disrupts severalneuronal functions including corticostriatal communi-cation [4] and cortical release of BDNF [5] (Fig. 1c).Breakdown of corticostriatal transmission reducessynaptic activity of striatal neurons [6] and influencesdownstream signal transduction within the striatum.In addition to the deficiencies in BDNF-TrkB signalingpreviously reported in mouse models of HD [7, 8], cyclicAMP (cAMP) signaling is disrupted in the striatum ofpresymptomatic R6/2 HD mice [9].Pridopidine, a small molecule in development for thetreatment of HD, improved motor function in HD patientsin two large, double-blind, placebo-controlled studies(HART and MermaiHD) as exhibited by UHDRS–TotalMotor Score (TMS), but did not meet primary endpoint ofchanges from baseline to week 12 in Modified Motor Score[10, 11]. Pridopidine is a high affinity sigma-1 receptor [12]ligand and exerts low-binding affinity towards additionalCNS receptors, such as Dopamine D2, Adrenergic a2C,Serotonin 5HT-1A and Histamine H3 [13, 14]. Further, anin-vivo PET imaging study in rats confirmed that pridopi-dine occupies the sigma-1 receptor at low doses (3 and15 mg/kg), and the D2R only at higher doses (60 mg/kg).Pridopidine normalizes endoplasmic reticulum (ER)calcium levels in YAC128 corticostriatal co-cultures [15],mediated by the sigma-1 receptor (Fig. 1d). The sigma-1receptor also mediates pridopidine-induced BDNF in ratneuroblastoma cells [15]. In the striatum of R6/2 HD mice[16, 17], pridopidine treatment increases BDNF proteinlevels in the striatum (Fig. 1d). Finally, a gene expressionanalysis in WT rat striatum demonstrates pridopidineinduces differential expression (DE) of genes enriched forthe BDNF, D1R, and glucocorticoid receptor pathways, pre-sumably mediated via sigma-1 receptor activation.The effect of different doses of chronic pridopidinetreatment, initiated at pre-symptomatic stages, on geneexpression and transcript splicing in the context of HDwas evaluated using single nucleotide resolution RNAsequencing in YAC128 mice, examining specificity ofeffects across brain regions.MethodsAnimalsYAC128 HD mice [18] (referred to herein as YAC128),maintained on the FVB/N strain were used. Mice werebred and housed according to Garcia-Miralles 2017 [19].All mouse experiments were performed with theapproval of and in accordance with the Institutional Ani-mal Care and Use Committee at the Biomedical SciencesInstitute at the Agency for Science, Technology and Re-search. Pridopidine synthesized by Teva PharmaceuticalIndustries was dissolved in sterile water for oral adminis-tration. Pridopidine or vehicle was given every day by anoral gavage for 5 days/week for 10 months starting at apresymptomatic stage (1.5 months of age). Mice weresplit into three treatment groups: vehicle (sterile water),10 mg/kg of pridopidine (“low dose”), or 30 mg/kg ofpridopidine (“high dose”).A second group of WT mice (C57BI6) were bred andhoused at the Department of Experimental Medical Sci-ence of Lund University (Sweden), and treated with pri-dopidine 30 mg/kg for 10 days.Sample preparation and RNA extractionMice were anaesthetised and perfused with ice-coldphosphate-buffered saline followed by ice-cold 4% para-formaldehyde in phosphate-buffered saline as describedin Garcia-Miralles 2017 [19]. Brains were removed fromYAC128 and WT, striatum, hippocampus, and cortexwere frozen on dry ice, mounted with Tissue-TEKO.C.T. compound (Sakura, Torrance, CA, USA), andsliced coronally into 25-μm sections on a cryostat(Microm HM 525, Thermo Fisher Scientific, Waltham,Massachusetts, USA). The sections were collected andkept in RNAlater solution (Ambion, AM7021) overnightat 4 °C and then stored at − 80 °C until use. Total RNAwas isolated by EA Genomics from tissue biopsies frommouse brain regions using the miRNeasy mini kit(Qiagen). RNA was also extracted from blood samplesof the same mice using RNeasy Protect Animal BloodKit EA. RNA integrity was assessed using an AgilentBioanaylzer and only RNA samples with RIN scoresKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 2 of 15> 8 were used. RNA samples were quantified byNanoDrop for RNAseq.RNA sequencing and mappingEA Genomics performed the RNA sequencing on bothmouse studies: 1. Striatum, hippocampus, and cortex ofchronic pridopidine or vehicle treated YAC128 or WTmouse and 2. Blood from acute pridopidine treated WTmice. Sequencing was performed using the IlluminaTruSeq Stranded mRNA Kit with HiSeq 2x50nt pairedend sequencing. Star v.2.5.0a was used to align FASTQfiles [20], using the GRCm38 primary assembly annota-tion and standard options. PCA plots of the sampleswere used to select outliers and to adjust for possiblecovariates. Transcripts that had less than 10 reads onaverage were filtered out. CalcNormFactors from theedgeR R package [21] was used to normalize the countsvia the TMM method.Fig. 1 Pridopidine promotes BDNF/TrkB signaling and restores ER calcium levels in the corticostriatal pathway. a Shown is a schematicrepresentation of the motor circuit in mammals. Motor cortical neurons project to the striatum and form excitatory (glutamate, green line)synapses with D1 and D2 receptor-expressing neurons (D1 and D2, blue box). Inhibitory D1 receptor-expressing neurons make GABAergicconnections (GABA, red line) with the pars reticulata of the substantia nigra (SNr). In contrast, D2 receptor-expressing neurons follow an indirectpathway and send GABAergic projections to the external segment of the globus pallidus (GPe). In turn, GABAergic neurons of the GPe project tothe subthalamic nucleus (STN), and excitatory STN neurons send efferents to the SNr GABAergic projections that innervate thalamus, and thethalamus completes the basal ganglia-thalamocortical circuitry by sending excitatory projections to the motor cortex. b In the WT striatum, thehuntingtin (Htt) protein facilitates axonal transport of synaptic vesicles carrying brain-derived neurotrophic factor (BDNF) and glutamate to theactive zone of cortical neurons. Released glutamate and BDNF bind to their targets on the postsynaptic density of striatal neurons, includingN-methyl-D-aspartate (NMDA) receptors and tropomyosin receptor kinase B (TrkB) receptors, respectively. c In Huntington disease, mutant Htt(mHtt) interferes with the axonal transport process, disrupting normal release of BDNF and consequently TrkB signaling in the striatum. Inaddition, endoplasmic reticulum (ER) calcium is also perturbed in the striatum during HD progression. d Shown is a proposed mechanism ofaction for pridopidine in the corticostriatal pathway. Treatment with pridopidine has been previously shown to improve both sigma 1 receptor(σ1r)-dependent BDNF release in neuroblastoma cells, increase striatal BDNF levels in HD mice and restore proper ER levels of Ca2+ via directactivation of σ1r in cortical and striatum co-culturesKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 3 of 15RNAseq analysisFollowing the lead of MAQC [22], the limma v3.28.21[23] R-package was used to transform and model thegene-level quantification data. Limma::voom was used totransform the count data to log2-counts per million andcalculate the mean-variance relationship. Limma::lmFitwas used to fit a linear model for each gene based onthe experimental design matrix. Limma::eBayes was usedto calculate the empirical Bayes moderated t-statistic forcontrast significance. Multiple hypothesis adjustedp-values were calculated using limma::topTable, whichimplemented the Benjamini-Hochberg procedure tocontrol FDR. In order to decrease the chance of findinga differential expression signature by chance, we utilizedpvalue correction to adjust for the number of hypothesis(genes) we were testing Differential expression contrastswere independently calculated for all three tissuesbetween: A. untreated YAC128 and untreated WT sam-ples, B. 30 mg/kg pridopidine YAC128 and untreatedYAC128 samples, C. 10 mg/kg pridopidine YAC128 anduntreated YAC128 samples, D. 30 mg/kg pridopidineWT and untreated WT samples. In order to compare themagnitude of, and concordance between brain transcrip-tional signatures and peripheral blood profiles, indicativeof the potential to develop biomarkers of disease andresponse to therapy, we examined samples obtainedfrom a 10-day treatment study, contrasting 30 mg/kgpridopidine WT and untreated WT blood (E.).To test whether the treatment gene expression signa-ture is enriched for relevant pathways, Gene Set Enrich-ment Analysis (GSEA) [24] was used. All genes testedfor differential expression were ranked by limma gener-ated t-statistic for a given contrast. This was input as the“ranked list” in GSEA pre-ranked analysis. Moreover,gene sets were made from lists of differentially expressedgenes from literature [25–27] in order to assess whethergenes regulated by pridopidine enriched for genes down-stream of Dopamine 1 Receptor, BDNF, and Gluco-corticoid Receptor. In order to further filter beforepathway analysis, we employ a strict version of what isrecommended by MAQC, combining a fold changecutoff with an adjusted pvalue cutoff [28]. Hypothesisfree broad pathway and transcription factor enrichmentwas done using Enrichr [29], selecting striatal differentialexpressed genes combining a fold change with a p-valuecutoff according to MAQC guidance [28] (absolutelinear fold change > 1.25 and adjusted p-value < 0.05).For all differential expression, splicing, pathway, andtranscription factor analyses we consider “significant” tomean Adj. pval < 0.05 unless otherwise stated.A signature of genes modulated in HD patient tissuewas assembled through a meta-analysis of LIMMAresults from two publicly available gene expression data-sets from caudate nucleus of 48 total HD patients and42 controls (GSE26927 and GSE3790). A signature ofgenes modulated in YAC128 striatum was assembledusing LIMMA results from 9 YAC128 mice and 6 WTmice, aged 11.5 months. The HD and YAC128 diseasesignatures were queried against our expression signa-tures for pridopidine in striatum of YAC128 micetreated for 10 months at 10 or 30 mg/kg daily usingcosine similarity of the moderated t-statistic to assessgene expression reversal.Exon and splice junction analysisStar aligned reads were processed using Quality ofRNA-Seq Toolset (QoRTS) with the parameter–stranded. A flat annotation file for GRCm38.p4 wasgenerated using QoRTs and used for subsequentanalysis. Differential usage of exon and splice junction(DUEJ) analysis was performed using the JunctionSeqBioconductor package. JunctionSeq uses a multivariategeneralized linear model using a negative binomialdistribution to detect exons and splice junctions whoseexpression changes between conditions relative to theexpression of their respective genes. To determine differ-ential usage of exons and splice junctions an adjustedp-value cutoff of 0.05 was used.ResultsPridopidine induces striatal gene expression changes inYAC128 HD miceIn a previous study, behavioral and motor effects of pri-dopidine were evaluated longitudinally, demonstratingimprovements in motor coordination, reduced anxietyand depressive like phenotypes, concordant with reversalof specific striatal transcriptional deficits [19]. Here, tocharacterize the underlying mechanisms, the effect ofpridopidine on the YAC128 HD model, gene expressionwas assessed through the comparison of transcriptomicprofiles in YAC128 mice treated with pridopidine (10 or30 kg/mg, p.o.) or vehicle (5 days/week) and WT micetreated with 30 mg/kg of pridopidine or vehicle (5 days/week). In parallel with the previously described behav-ioral study [19], animals were treated starting at1.5 months of postnatal life (presymptomatic) and sacri-ficed at 11.5 months of age (robust HD phenotype).Gene expression from the striatum, hippocampus, andcortex was evaluated using large RNAseq.To further identify disease-specific gene expressionpatterns, vehicle-treated YAC128 mice were comparedto vehicle-treated wild-type (WT) mice, demonstratinggene expression changes largely restricted to the stri-atum. We identified 1346 differentially expressed genes(DEGs) in the striatum (Adj. p-val < 0.05, Table 1) com-pared to 340 DEGs in the hippocampus and 7 DEGs inthe cortex (Adj. p-val < 0.05, Table 1). Fold change andpvalue ranges are in Additional file 1: Table S1.Kusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 4 of 15To test if disease progression and/or pridopidine in-duced gene expression signatures can also be observedoutside of the brain, and thus potentially produce bio-markers useful for therapeutic development and moni-toring, transcriptomic signals were examined in bloodfrom WT mice treated with pridopidine for 10 days.Only one gene was found to be significantly differentiallyexpressed between 30 mg and vehicle treated mice (IL7Radj pval = 0.03, linear FC 2.11).In the YAC128 HD model, a dose-dependent effectof pridopidine (vs vehicle) was observed in striatum(Adj. p-val < 0.05, Table 1): 10 mg/kg of pridopidinetreatment induced significant differential expression of73 genes, with 30 mg/kg pridopidine inducing roughlythree times as many genes as the 10 mg dose (221striatal DEGs, Adj. p-val < 0.05, Table 1, 55 genesoverlap the two lists). No detectible differences ingene expression were observed in the YAC128 hippo-campus or cortex after pridopidine treatment (Adj.p-val < 0.05, Table 1), suggesting that a robust pridopi-dine signature is brain compartment specific and notan off target effect. In WT mice, treatment with30 mg/kg of pridopidine resulted in striatal differen-tial expression of only 17 genes (Adj. p-val < 0.05,Table 1). Among these genes, four were alsodescribed in a recent microarray study that identified16 DEGs in the striatum of wild-type rats after treat-ment with pridopidine (60 mg/kg) [30]. The fouroverlapping genes are Junb, Egr2, Nr4a1, and Per1.With the exception of Junb, these genes are alsodownregulated in the YAC128 mouse model of HD.Taken together, the data demonstrate that the effectof pridopidine on gene expression is more pro-nounced in a disease model than in WT animals, andis primarily limited to the striatum.Pridopidine reverses YAC128 HD mouse model andhuman HD disease gene expression signaturesWe next quantified the extent to which the genes withexpression modulated by 10 and 30 mg/kg of pridopi-dine reverse: 1) genes with expression modulated in HDpatient tissue relative to healthy controls (fromGSE26927 and GSE3790, described in methods), and 2)genes with expression modulated in YAC128 striatumcompared to WT controls. In agreement withGarcia-Miralles 2017 [19], we observed that treatmentwith 10 and 30 mg/kg of pridopidine significantly re-versed the YAC128 disease signature (Fig. 2, Table 2).Moreover, in this study, we additionally observe reversalof our HD patient signature (derived from GSE26927and GSE3790). The results demonstrate the effectivenessof pridopidine to reverse genes modulated in human HDand the YAC128 mouse model of HD.BDNF, GR, and D1R pathways are downregulated inHD [5, 9, 31], while pridopidine upregulates these path-ways in WT rat striatum [30]. We investigated whetherpridopidine upregulation of these pathways is recapitu-lated in WT and/or YAC128 mice. We performed GeneSet Enrichment Analysis (GSEA) using manually-curatedBDNF, GR, and D1R gene sets. In YAC128 mice, we ob-served the expected reduction of the BDNF pathway vianegative GSEA enrichment in the cortex and striatum,along with downregulation of the D1R pathway in thecortex (Adj. p-val < 0.05, Fig. 3). Consistent with previ-ous reports, GSEA pathway analysis revealed positiveenrichment of BDNF, GR, and D1R pathway genes inWT mouse striatum after 30 mg/kg pridopidine treat-ment (Adj. p-val < 0.05, Fig. 3). Enrichment analysis alsoconfirmed upregulation of the BDNF pathways in theTable 1 Summary of genes with differential expression oralternative junction/exon usageNumber of Differentially Expressed GenesContrast Striatum Hippocampus CortexYAC128 Veh-WT Veh 1346 340 7YAC128 10 mg/kg-YAC128 Veh 73 0 0YAC128 30 mg/kg-YAC128 Veh 221 0 0WT 30 mg/kg-WT Veh 17 0 0Number of Genes with Alternative Junction/Exon usageYAC128 Veh-WT Veh 39 14 4YAC128 10 mg/kg-YAC128 Veh 1 0 0YAC128 30 mg/kg-YAC128 Veh 565 1 4WT 30 mg/kg-WT Veh 0 4 2An Adj. p-val cutoff of 0.05 was used as pre-requisite criterion to identifydifferentially expressed genes (top) and alternative exon/junction usagecases (bottom) for each contrast and each tissueTable 2 Pridopidine treatment signal significantly reverses YAC128 HD signal. Numbers shown are adjusted p-values from Gene SetEnrichment AnalysisDose Direction Striatum Hippocampus Cortex10 mg/kg Up in YAC128, Down with pridopidine 3.58E-04 3.78E-04 2.40E-04Down in YAC128, Up with pridopidine 3.64E-04 4.99E-02 2.40E-0430 mg/kg Up in YAC128, Down with pridopidine 3.15E-04 3.46E-04 2.35E-04Down in YAC128, Up with pridopidine 3.37E-04 3.53E-03 2.35E-04Reversal was significant for all doses of pridopidine across the striatum, hippocampus, and cortex of YAC128 miceKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 5 of 15striatum, hippocampus, and cortex of YAC128 animalsafter treatment with either the 10 or 30 mg/kg dose ofpridopidine (Adj. p-val < 0.05, Fig. 3). In addition, D1Rand GR pathways were positively enriched across allthree tissues after either 10 or 30 mg/kg treatment ofpridopidine, with two exceptions: no significant enrich-ments were observed 1) for the GR pathway in the cor-tex after 10 mg/kg treatment; and 2) for the D1Rpathway in the hippocampus after 30 mg/kg treatment.Database-driven enrichment analysis reveals thatpridopidine enhances relevant biological pathwaysaltered in the YAC128 HD striatumWhile GSEA provides a robust approach for pathwayanalysis, the method is limited to the manual curationof gene sets. To expand systematically our search forbiological processes modulated by pridopidine, wenext employed the enrichment tool Enrichr, whichutilizes several comprehensive datasets including theGene Ontology (GO) database. Our analysis focusedon DEGs identified in the striatum, where the effectof pridopidine was most pronounced. Enrichmentanalysis was performed to identify pathways that aredownregulated in vehicle-treated YAC128 and upregu-lated in pridopidine-treated YAC128 mice. Top 10 (of63) downregulated pathways in the YAC128-vehiclemice (Adj. p-val < 0.05, Additional file 2: Table S2),include impaired synaptic transmission processes,MAP kinase activity, cAMP metabolism, and adenyl-ate cyclase signaling, as well as response to amphet-amine and cocaine (Adj. p-val < 0.05, Fig. 4).Pridopidine treatment upregulated cAMP and kinaseactivity pathways impaired in the YAC128 striatum. En-richment analysis revealed upregulation of enhanced bio-logical pathways in the YAC128 striatum, with the cAMPresponse biological process showing a robust, statisticallysignificant signal, ranking 1st after treatment with eitherthe 10 or 30 mg/kg dose of pridopidine (Adj. p-val =2.49E-09 and 2.53E-09 respectively, Fig. 4). For both dosesof pridopidine, other highly ranked biological processesinclude negative regulation of kinase and phosphorylation(Adj. p-val < 0.05, Fig. 4 and Additional file 2: Table S2). Inthe YAC128 striatum, there were no significant GO en-richments using DEGs 1) upregulated after vehicle treat-ment, or 2) downregulated after pridopidine treatment. Inwild-type mice, pridopidine induced enrichment of cAMPand p38-MAPK regulation pathways (Adj. p-val < 0.05,Fig. 4, Additional file 2: Table S2).To identify enriched transcriptional factors (TFs) consti-tuting upstream regulatory mechanisms, ENCODE andChIP Enrichment Analysis (ChEA) databases were queriedvia Enrichr using DEGs that were: 1) downregulated inthe YAC128 versus WT striatum, or 2) upregulated in thepridopidine treatment group. TF analysis of genes down-regulated in YAC128 striatum revealed enrichment forgene targets of the transcription factor SUZ12 (Adj. p-val< 0.05, Additional file 3: Table S3). These enrichments areconsistent with previous studies showing that SUZ12 isperturbed due to epigenetic dysregulation in HD [32].TF analysis of DEGs upregulated in the pridopidinetreatment group revealed CREB1 transcription factor asa highly significantly enriched gene set (10 and 30 mg/Fig. 2 Pridopidine reverses mouse YAC128 and human Huntington disease signatures. Pridopidine reversal of genes modulated in the YAC128mouse model of Huntington disease (y-axis) as a function of human Huntington disease (x-axis). The blue dots represent 10 and 30 mg/kg doseof pridopidineKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 6 of 15Fig. 3 Pathway enrichments for the BDNF, dopamine receptor, glucocorticoid receptor pathways after pridopidine treatment. Each box showsthe Adj. p-val for either the BDNF, dopamine D1 receptor (D1R), or glucocorticoid (GR) pathway in a given treatment, genotype, and tissue.Consistent with the gene level results from the RNAseq data, the pathway signal is most consistent in the WT and YAC128 striatum aftertreatment with pridopidine. “High” = 30 mg/kg of pridopidine; “Low” = 10 mg/kg of pridopidine; “Veh” = vehicle. Grey = no enrichment.Red = significant positive enrichment. Blue = significant negative enrichmentKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 7 of 15kg) in the YAC128 striatum (Adj. p-val < 0.05, Additionalfile 3: Table S3). This is consistent with both disruptedCREB activity in HD [33] and the identification of up-regulation of cAMP-response genes after pridopidinetreatment (Adj. p-val < 0.05, see Additional file 2: TableS2). CREB1 gene set was also enriched after 30 mg/kgpridopidine treatment in WT striatum (Adj. p-val < 0.05,Additional file 3: Table S3). Taken together, theseobservations suggest that pridopidine treatment may in-duce gene expression regulated via the CREB transcrip-tional pathway, known to be disrupted in HD.Pridopidine reverses compromised cAMP response geneactivity in the YAC128 HD striatumIn YAC128 striatum, pridopidine enrichment of cAMPresponse is composed of 8 upregulated genes (Dusp1,Fig. 4 Top 10 enriched Gene Ontology (GO) biological process pathways. Pathway enrichment was performed on striatal DEGs downregulated invehicle-treated YAC128 mice (top panel), striatal DEGs upregulated after treatment with 10 mg/kg pridopidine in YAC128 (second panel), striatalDEGs upregulated after treatment with 30 mg/kg pridopidine in YAC128 (third panel), and striatal DEGs upregulated after treatment with 10 mg/kg pridopidine in WT (second panel)Kusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 8 of 15Egr1, Egr2, Egr4, Fos, Fosb, Fosl2, and Junb) after treat-ment with 10 mg/kg of pridopidine, and 9 upregulatedgenes (Dusp1, Egr1, Egr2, Egr3, Egr4, Fos, Fosb, Fosl2,and Junb) after treatment with 30 mg/kg of pridopidine(Adj. p-val < 0.05, Figs. 5 and 6). In addition, we alsoidentified another cAMP-regulated gene, Rgs2 [34], up-regulated after pridopidine treatment (Fig. 6). Five ofthese genes (Dusp1, Egr1, Egr2, Fosl2, and Rgs2) aredownregulated in the striatum of vehicle-treatedYAC128 mice (Adj. p-val < 0.05, Fig. 5, Additional file 4:Table S4). qPCR confirmed pridopidine reversed the ex-pression of Dusp1, and Egr2, and Fosl2 (Adj. p-val < 0.05,Fig. 5 and Additional file 4: Table S4).Pridopidine modulates exon and transcript junction in thestriatum of HD miceAlternative splicing represents a key transcriptomicregulatory mechanism required for many basic cellularfunctions. Recent evidence suggests alternative splicingmay be perturbed in HD. To determine if alternativesplicing also occurs in YAC128 mice, we performeddifferential usage of exon and splice junction (DUEJ)analysis in the striatum, hippocampus, and cortex. InYAC128 mice compared to WT controls, we observed39, 14, and 4 DUEJs in striatal, hippocampal, and cor-tical genes, respectively (all Adj. p-val < 0.05, Table 1). Inconcordance with gene level differential expression, themajority of DUEJ occurred in the striatum.We then examined whether treatment with pridopi-dine compared to vehicle induces DUEJ in the brains ofWT and YAC128 mice. In the YAC128 striatum, pridopi-dine induced dose-dependent DUEJ, with 565 genessignificant in the 30 mg/kg group compared to only asingle gene in 10 mg/kg pridopidine-group (both Adj.p-val < 0.05, Table 1). In WT mice, treatment with prido-pidine did not lead to any DUEJ differences in striatum(all Adj. p-val < 0.05, Table 1). Eleven genes (Kifap3,Zwint, Cltc, Rtn1, Acin1, Ano3, Dclk1, Ppp3ca, Atp2b2,Arpp19, and Arpp21) demonstrated significant DUEJand reversal after 30 mg/kg pridopidine treatment in theYAC128 striatum (Adj. p-val < 0.05). Pridopidine in-duced minimal to no DUEJ changes in hippocampus andcortex (Table 1). These results are consistent with thedose-dependent effect of pridopidine on gene expressionrestricted to the YAC128 striatum.Pathway analysis on the 565 genes demonstratingDUEJ after 30 mg/kg pridopidine treatment in YAC128mice showed enrichments for pathways previously de-scribed to be involved in pridopidine’s mechanism of ac-tion such as calcium regulation (adj.pval = 3.6E-06), andSynaptic Vesicle (3.4E-06). Additional pathways ofinterest previously reported as part of pridopidine’smechanism of action include: BDNF signaling and Gprotein signaling, (Adj. p-val < 0.05, Fig. 7a, b andAdditional file 5: Table S5). Taken together, these resultsdemonstrate that the 30 mg/kg of pridopidine inducesexon and junction level changes in pathways that arerelevant to HD pathology.DiscussionIt has recently been demonstrated that 30 mg/kg of pri-dopidine rescues motor behavioral deficits in YAC128mouse model of HD [19]. To identify potential mecha-nisms by which pridopidine confers motor benefits, thisstudy focuses on pridopidine induced changes intranscription across multiple brain regions and doseregimens. To characterize the functional relevance oftranscriptomic changes, RNAseq data was analyzed forexpression signaling, as well as splice variant modifica-tions in pre-specified pathways, as well as across thegenome unbiasedly. Testing was performed on WT andYAC128 striatum, cortex, and hippocampus after treat-ment with vehicle, 10 or 30 mg/kg pridopidine from apresymptomatic stage through disease progression. Bothdoses of pridopidine had a significant effect on gene andtranscript levels in the striatum, with modest to unob-served effects in the cortex and hippocampus. However,the transcriptional effect of pridopidine in YAC128striatum is dose-dependent. The two doses tested hereinsuggest linearity of the effect, which future studiesemploying additional doses will serve to shed furtherlight on. While this study cannot directly query whetherpridopidine’s behavioral benefits are transcriptionallymediated, the fact that pridopidine’s main transcriptomiceffect is detected in the striatum supports this hypoth-esis. Moreover, the dose dependent functional expressionsignals induced by pridopidine track well with thedose-dependent behavioral benefits induced it inducesat parallel experimental conditions (Garcia-Miralleset al., 2017). Lastly, genes with perturbed expressionin YAC128 pathology are oppositely modulated bypridopidine in the striatum, far more so thanexpected by chance.The transcriptional footprint of pridopidine demon-strates a reversal of the disease-specific gene expressionand alternative splicing. The disease mechanismsreversed by pridopidine include critical neuroprotectivepathways such as BDNF, D1R and glucocorticoid path-ways previously reported. qPCR confirmed differentialexpression of many genes in these pathways. In addition,pridopidine induced gene expression triggered by cAMPtransduction, also supported by modulation of down-stream transcription factors (e.g. CREB1). Together,these findings provide robust data to demonstrate prido-pidine restores mechanisms impaired in HD, specificallyin the striatum.Previous studies reported rescue of several aspects of HD,including phenotype and behavior, in the YAC128 mouseKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 9 of 15through BDNF overexpression [35]. Dexamethasone, aglucocorticoid that activates the GR pathway, also dampensdisease progression in a HD animal model [36]. Both 10and 30 mg/kg pridopidine treatment in YAC128 mice sig-nificantly induced the BDNF, GR and D1R pathways in thestriatum, hippocampus, and cortex, consistent with priorreports in WT rat [30]. As pridopidine does not directlybind GR (internal data, not shown), it suggests that the up-regulation of the GR pathway may be indirect. Pridopidineincreases dopamine efflux in the striatum [37], which mayexplain the observed upregulation of expression for D1Rpathway genes after pridopidine treatment.Fig. 5 qPCR validation of differential expression of striatal genes in YAC128 mice. Shown are RNAseq and qPCR results for genes differentiallyexpressed in the striatum of YAC128 mice after pridopidine treatment. “**” and “##” represent significant (Adj. p-val < 0.05) differential expressionin YAC128 Veh-WT Veh and YAC128 Low/High-YAC128 Veh contrasts, respectively. “High” = 30 mg/kg of pridopidine; “Low” = 10 mg/kg ofpridopidine; “Veh” = vehicleKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 10 of 15Fig. 6 RNAseq differential expression analysis of cAMP-related genes in the YAC128 striatum. Shown are RNAseq results for differentiallyexpressed genes in the YAC128 striatum after pridopidine treatment. “**” and “##” represent significant (Adj. p-val < 0.05) differential expression inYAC128 Veh-WT Veh and YAC128 Low/High-YAC128 Veh contrasts, respectively. “High” = 30 mg/kg of pridopidine; “Low” = 10 mg/kg ofpridopidine; “Veh” = vehicleFig. 7 Pathway and differential exon/splice junction analysis in the YAC128 striatum after pridopidine treatment. Pathway analysis was performed ongenes that demonstrated pridopidine induced alternative exon/junction usage at a high dose (30 mg/kg). a Top 10 significant (Adj. p-val < 0.05)pathways from the WikiPathway database. b Top 10 significant (Adj. p-val < 0.05) pathways from the Gene Ontology (GO) pathway databaseKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 11 of 15In the WT striatum, dopamine is a central regulator ofcAMP activity in both D1 and D2 receptor-expressingneurons, namely, medium spiny neurons, where D1Rsand D2Rs have opposing effects on cAMP levels [38].Previous studies of HD postmortem brain tissue and ani-mal models have shown that cAMP signaling becomesderegulated in the striatum of humans and animal HDmodels [9, 39, 40]. Restoration of cAMP levelsreduced mHtt aggregates in the striatum of R6/2 HDmice [40], underscoring the importance of rescuingstriatal cAMP signaling. In the YAC128 striatum, weobserved downregulation of cAMP pathway genes,which are upregulated after treatment with pridopi-dine (Figs. 5, 6, and 8).Fig. 8 Pridopidine enhances cAMP/PKA and TrkB pathway genes in the YAC128 striatum. Shown is a model of gene regulation after treatmentwith pridopidine in the YAC128 striatum. Dopamine transmission directs the activation of dopamine D1 and D2 receptors (D1R and D2R,respectively) in medium spiny neurons (MSNs) of the striatum. On the WT postsynaptic density of a D1 synapse, D1Rs activate adenylyl cyclase(AC) in MSNs, whereas muscarinic acetylcholine receptor M4 (M4R) inhibits AC activity. In contrast, D2Rs negatively regulate AC in MSNs, whileA2ARs are AC agonists. GPR3 activates AC in both D1R and D2R-expressing MSNs, where RGS2 is a target of cAMP signaling. Activation of AC isupstream of cAMP and PKA, which augments NMDAR activity. Dopamine D1 and D2 receptor genes (Drd1 and Drd2) and A2AR gene (Adora2a)are differentially expressed (DE) and downregulated in the YAC128 striatum, but unchanged after pridopidine treatment (black highlighting). BothRgs2 and Gpr3 are downregulated in YAC128 striatum (Adj. p-val < 0.05) and upregulated after treatment of pridopidine. NMDARs and TrkBreceptors are expressed in both D1R and D2R-expression MSNs. NMDAR and TrkB receptors both indirectly activate the transcription factor (TF)CREB in the nucleus via downstream pathways. In turn, CREB activates gene expression of several targets. Genes Arc, Dusp1, Egr1 and Egr2 aredownregulated in the YAC128 striatum, but upregulated after pridopidine treatment (Adj. p-val < 0.05). Fos, Fosb, Fosl2, Egr3, and Egr4 areunperturbed in the striatum of YAC128 mice, but expression of these genes is restored after treatment with pridopidine (Adj. p-val < 0.05).Dopamine pathway genes Nr4a1 and Nr4a3 and TF gene Npas4 are downregulated in YAC128 and upregulated after pridopidine treatment (Adj.p-val < 0.05). NPAS4 regulates Gpr3 gene expressionKusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 12 of 15In agreement with Garcia-Miralles et al. [19], we notedreversal of compromised expression of dopamine recep-tor genes (Drd1 and Drd2) after pridopidine treatmentin the YAC128 striatum. However, reversal of Drd1 andDrd2 expression was only nominally significant aftertreatment with either the 10 or 30 mg/kg dose ofpridopidine [19]. Therefore, pridopidine could partlyrestore dopamine-cAMP signaling via compensatorymechanisms. One possibility is that pridopidine inducespost-translational regulation of the D1R protein. PSD-95has been shown to increase D1R surface level expression[41], regulate D1R internalization, and D1R-cAMPsignaling [42, 43]. In the striatum, wild-type HTT bindsto postsynaptic density protein 95 and promotes its clus-tering (PSD-95) [42, 44], whereas mHTT lacks bindingaffinity to PSD-95 [44]. In agreement with increasedD1R activity, compromised expression of D1R-regulatedgenes Nr4a1 and Nr4a3 is rescued after treatment witheither 10 or 30 mg/kg pridopidine in the YAC128 stri-atum (Adj. p-val < 0.05, Fig. 5).In addition to dopamine signaling, other signal trans-duction pathways regulate cAMP response targets,which may also explain the putative effect of pridopidineon cAMP signaling. For example, we identified twoadditional cAMP-related DEGs (Npas4 and Gpr3)upregulated after treatment with pridopidine in theYAC128 striatum (Additional file 6: Figure S1).Activity-dependent NPAS4 has been shown to upregu-late both BDNF and Gpr3 expression in cultured excita-tory and inhibitory neurons, respectively [45]. GPR3 is aconstitutive activator of cAMP signaling via adenylylcyclase (AC) [46, 47]. Interestingly, Npas4 gene expres-sion is compromised in the YAC128 striatum, butrescued after pridopidine treatment with either dose (10or 30 mg/kg, Adj. p-val < 0.05, Fig. 5). In addition, Gpr3gene expression is downregulated in the YAC128striatum, whereas striatal expression of Gpr3 is upreg-ulated after either 10 or 30 mg/kg pridopidine treat-ment in YAC128 mice (Additional file 5: Figure S5,Adj. p-val < 0.05). Taken together, this suggests thatpridopidine could partly rescue dysregulated cAMPsignaling by modulating Npas4 and Gpr3 gene expres-sion. In addition to the GPR3-cAMP pathway,BDNF-TrkB signaling has also been shown to activatecAMP response element binding (CREB) proteinactivity and thus facilitate gene expression in culturedstriatal neurons [26]. In other words, pridopidine mayinduce transcription by binding to S1R, which leadsto enhanced BDNF activity, in turn activating geneand splice-variant expression. This alternative mech-anism is also supported by the fact that pridopidineinduces BDNF release in neuroblastoma cells [30].Recently, it was demonstrated that treatment with30 mg/kg of pridopidine rescues motor deficiencies inYAC128 mice, whereas no effect was detected inYAC128 animals treated with 10 mg/kg of pridopidine[19]. In agreement with this observation, we report abroader effect of 30 mg/kg pridopidine on gene expres-sion in the YAC128 striatum compared to the 10 mg/kgdose, but also report that either dose reverses disease as-sociated gene expression. The effect of 30 mg/kg prido-pidine on motor function diminishes during theprogression of the disease [19], and the lowest loco-motor performance is observed between 10 and12 months of age when mice are very ill. Given that RNAsamples for this study were collected during the decline ofmotor activity in 30 mg/kg pridopidine-treated YAC128mice, it is difficult to correlate improvement in motor def-icit and pridopidine-induced gene expression in the stri-atum of YAC128 animals. Moreover, our study showedthat pridopidine induces a robust gene expression signalwhen treatment begins early in disease course. Thismay suggest that in humans, pridopidine may bemore effective if started in early disease stages. Forboth of these reasons, a longitudinal study withearlier time points would better illuminate the linkbetween gene expression and motor behavior aftertreatment with pridopidine in YAC128 mice.ConclusionsIn conclusion, pridopidine reverses HD associatedchanges in transcription at the pathway, gene andsplice-variant level. Pathways with transcriptomicaberrations in the YAC128 mouse that are restored toWT levels by pridopidine treatment include BDNF, D1R,GR, cAMP, and calcium signaling. These pathwaystogether are known to interact, and likely positively feedinto each other downstream of pridopidine treatment, torelieve HD associated motor symptoms. Beneficialeffects when treatment is initiated early, before symp-toms are manifest, tracks with trends observed in clinicaltrials. Studying the effect of pridopidine at multiple timepoints over the course of treatment against transcrip-tomic aberrations in YAC128 will reveal additional regu-latory dynamics. The results in this study, all takentogether, support exploring pridopidine’s role as a thera-peutic for neuroprotection in HD and similar neuro-logical movement disorders.Additional filesAdditional file 1: Table S1. Adjusted p-val range and fold changerange for differential expression and DUEJ genes meeting adj p-val < 0.05cutoff. (XLSX 11 kb)Additional file 2: Table S2. Gene Ontology pathway analysis of genesdifferentially expressed in the mouse striatum. (XLSX 27 kb)Additional file 3: Table S3. Transcription factor enrichment analysis ofgenes differentially expressed in the mouse striatum. (XLSX 18 kb)Kusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 13 of 15Additional file 4: Table S4. qPCR validation of striatal gene expressionidentified in RNAseq and pathway analysis. (XLSX 10 kb)Additional file 5: Table S5. Pathway analysis of alternatively splicedgenes identified after high dose treatment with pridopidine. (XLSX 31 kb)Additional file 6: Figure S1. Pridopidine reverses downregulation of GProtein-Coupled Receptor 3 (Gpr3) gene expression in the striatum ofYAC128 mice. Shown are RNAseq results for Gpr3 in the YAC128 striatumafter pridopidine treatment. “**” and “##” represent significant (Adj. p-val< 0.05) differential expression in YAC128 Veh-WT Veh and YAC128 Low/High-YAC128 Veh contrasts, respectively. “High” = 30 mg/kg of pridopi-dine; “Low” = 10 mg/kg of pridopidine; “Veh” = vehicle. (PDF 151 kb)AbbreviationsAC: Adenylyl cyclase; BDNF: Brain-derived neurotrophic factor; cAMP: CyclicAMP; ChEA: ChIP Enrichment Analysis; CREB: cAMP response elementbinding; D1R: Dopamine D1 receptor; DE: Differential expression;DUEJ: Differential usage of exon and splice junction; ER: Endoplasmicreticulum; GO: Gene ontology; GPe: External segment of the globus pallidus;Gpr3: G protein-coupled receptor 3; GR: Glucocorticoid receptor; GSEA: Geneset enrichment analysis; HD: Huntington disease; Htt: Huntingtin;M4R: Muscarinic acetylcholine receptor M4; MAQC: MicroArray qualitycontrol; mHtt: Mutant Htt; MSNs: Medium spiny neurons; NMDA: N-methyl-D-aspartate; QoRTs: Quality of RNA-Seq toolset; SNr: Pars reticulata of thesubstantia nigra; STN: Subthalamic nucleus; TF: Transcriptional factors;TMS: Total Motor Score; TrkB: Tropomyosin receptor kinase B; WT: Wild-type;σ1r: Sigma-1 receptorFundingTeva Pharmaceuticals provided funding for the study.Availability of data and materialsThe datasets used and/or analysed during the current study are availablefrom the corresponding author on reasonable request.Authors’ contributionsAll authors discussed the results and contributed to the manuscript. RK, JD,MG, MP, DL, MGM, LJT, MEB, and MRH designed the study and participatedin its design and coordination. RK, JR, YC, and REC processed and analyzedgene expression data. RK, JD, JR, MG, DL, IG, MEB, MP, BZ, and MRH draftedand revised the manuscript. All authors read and approved the finalmanuscript.Authors’ informationInformation for all the co-authors is listed in the title page.Ethics approvalAll mouse experiments were performed with the approval of and inaccordance with the Institutional Animal Care and Use Committee at theBiomedical Sciences Institute at the Agency for Science, Technology andResearch.Competing interestsRK, JR, YC, REC, BZ are employees of Immuneering Corporation. JD, MEB, DL,MG, IG, and MRH are employees of Teva Pharmaceutical. TevaPharmaceuticals played no role in the treatment or testing of animals, or thecollection, or analysis of the results.Publisher’s NoteSpringer Nature remains neutral with regard to jurisdictional claims inpublished maps and institutional affiliations.Author details1Immuneering Corporation, Cambridge, MA 02142, USA. 2Research andDevelopment, Teva Pharmaceutical Industries Ltd, Netanya, Israel.3Translational Laboratory in Genetic Medicine, Agency for Science,Technology and Research, Singapore (A*STAR), Singapore 138648, Singapore.4Centre for Molecular Medicine and Therapeutics, Child and Family ResearchInstitute, University of British Columbia, Vancouver, BC V5Z 4H4, Canada.5Department of Medicine, Yong Loo Lin School of Medicine, NationalUniversity of Singapore, Singapore 117597, Singapore.Received: 9 April 2018 Accepted: 13 May 2018References1. Macdonald M. A novel gene containing a trinucleotide repeat that isexpanded and unstable on Huntington’s disease chromosomes. Cell. 1993;72:971–83.2. Foroud T, Gray J, Ivashina J, Conneally PM. Differences in duration ofHuntington’s disease based on age at onset. J Neurol Neurosurg Psychiatry.1999;66:52–6.3. Colin E, Zala D, Liot G, Rangone H, Borrell-Pagès M, Li X-J, et al. Huntingtinphosphorylation acts as a molecular switch for anterograde/retrogradetransport in neurons. EMBO J. 2008;27:2124–34.4. Miller BR, Bezprozvanny I. Corticostriatal circuit dysfunction in Huntington’sdisease: intersection of glutamate, dopamine and calcium. Future Neurol.2010;5:735–56.5. Gauthier LR, Charrin BC, Borrell-Pagès M, Dompierre JP, Rangone H,Cordelières FP, et al. Huntingtin controls neurotrophic support and survivalof neurons by enhancing BDNF vesicular transport along microtubules. Cell.2004;118:127–38.6. André VM, Fisher YE, Levine MS. Altered balance of activity in the striataldirect and indirect pathways in mouse models of Huntington’s disease.Front Syst Neurosci. 2011;5:46.7. Plotkin JL, Day M, Peterson JD, Xie Z, Kress GJ, Rafalovich I, et al. ImpairedTrkB receptor signaling underlies corticostriatal dysfunction in Huntington’sdisease. Neuron. 2014;83:178–88.8. Nguyen KQ, Rymar VV, Sadikot AF. Impaired TrkB signaling underliesreduced BDNF-mediated trophic support of striatal neurons in the R6/2mouse model of Huntington’s disease. Front Cell Neurosci. 2016;10:37.9. Bibb JA, Yan Z, Svenningsson P, Snyder GL, Pieribone VA, Horiuchi A, et al.Severe deficiencies in dopamine signaling in presymptomatic Huntington’sdisease mice. Proc Natl Acad Sci U S A. 2000;97:6809–14.10. de Yebenes JG, Landwehrmeyer B, Squitieri F, Reilmann R, Rosser A, BarkerRA, et al. Pridopidine for the treatment of motor function in patients withHuntington’s disease (MermaiHD): a phase 3, randomised, double-blind,placebo-controlled trial. Lancet Neurol. 2011;10:1049–57.11. The Huntington Study Group HART Investigators. A randomized, double-blind, placebo-controlled trial of pridopidine in Huntington’s disease. MovDisord. 2013;28:1407–15.12. Sahlholm K, Sijbesma JWA, Maas B, Kwizera C, Marcellino D, Ramakrishnan NK,et al. Pridopidine selectively occupies sigma-1 rather than dopamine D2receptors at behaviorally active doses. Psychopharmacology. 2015;232:3443–53.13. Ponten H, Kullingsjö J, Sonesson C, Waters S, Waters N, Tedroff J. Thedopaminergic stabilizer pridopidine decreases expression of L-DOPA-induced locomotor sensitisation in the rat unilateral 6-OHDA model. Eur JPharmacol. 2013;698:278–85.14. Dyhring T, Nielsen EØ, Sonesson C, Pettersson F, Karlsson J, Svensson P,et al. The dopaminergic stabilizers pridopidine (ACR16) and (−)-OSU6162display dopamine D2 receptor antagonism and fast receptor dissociationproperties. Eur J Pharmacol. 2010;628:19–26.15. Ryskamp D, Wu J, Geva M, Kusko R, Grossman I, Hayden M, et al. The sigma-1 receptor mediates the beneficial effects of pridopidine in a mouse modelof Huntington disease. Neurobiol Dis. 2017;97:46–59.16. Squitieri F, Di Pardo A, Favellato M, Amico E, Maglione V, Frati L.Pridopidine, a dopamine stabilizer, improves motor performance and showsneuroprotective effects in Huntington disease R6/2 mouse model. J CellMol Med. 2015;19(11):2540–548.17. Altar CA, Cai N, Bliven T, Juhasz M, Conner JM, Acheson AL, et al.Anterograde transport of brain-derived neurotrophic factor and its role inthe brain. Nature. 1997;389:856–60.18. Slow EJ, van Raamsdonk J, Rogers D, Coleman SH, Graham RK, Deng Y,et al. Selective striatal neuronal loss in a YAC128 mouse model ofHuntington disease. Hum Mol Genet. 2003;12:1555–67.19. Garcia-Miralles M, Geva M, Tan JY, et al. Early pridopidine treatment improvesbehavioral and transcriptional deficits in YAC128 Huntington disease mice. JCIInsight. 2017;2(23):e95665. Dobin A, Davis CA, Schlesinger F, Drenkow J, Zaleski C, Jha S, et al. STAR:ultrafast universal RNA-seq aligner. Bioinformatics. 2013;29:15–21.Kusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 14 of 1521. Robinson MD, McCarthy DJ, Smyth GK. edgeR: a Bioconductor package fordifferential expression analysis of digital gene expression data.Bioinformatics. 2010;26:139–40.22. Su Z, Łabaj PP, Li S, Thierry-Mieg J, Thierry-Mieg D, et al. Seqc/Maqc-IiiConsortium. A comprehensive assessment of RNA-seq accuracy,reproducibility and information content by the sequencing quality controlconsortium. Nat Biotechnol 2014;32:903–914.23. Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, et al. Limma powersdifferential expression analyses for RNA-sequencing and microarray studies.Nucleic Acids Res. 2015;43:e47.24. Subramanian A, Tamayo P, Mootha VK, Mukherjee S, Ebert BL, Gillette MA,et al. Gene set enrichment analysis: a knowledge-based approach forinterpreting genome-wide expression profiles. Proc Natl Acad Sci. 2005;102:15545–50.25. Sato H, Horikawa Y, Iizuka K, Sakurai N, Tanaka T, Shihara N, et al. Large-scaleanalysis of glucocorticoid target genes in rat hypothalamus. J Neurochem.2008;106:805–14.26. Gokce O, Runne H, Kuhn A, Luthi-Carter R. Short-term striatal geneexpression responses to brain-derived neurotrophic factor are dependenton MEK and ERK activation. PLoS One. 2009;4:e5292.27. Cadet JL, Jayanthi S, McCoy MT, Beauvais G, Cai NS. Dopamine D1receptors, regulation of gene expression in the brain, andneurodegeneration. CNS Neurol Disord Drug Targets. 2010;9:526–38.28. Maqc Consortium SL, Shi L, Reid LH, Jones WD, Shippy R, et al. The MicroArrayquality control (MAQC) project shows inter- and intraplatform reproducibilityof gene expression measurements. Nat Biotechnol. 2006;24:1151–61.29. Chen EY, Tan CM, Kou Y, Duan Q, Wang Z, Meirelles GV, et al. Enrichr:interactive and collaborative HTML5 gene list enrichment analysis tool. BMCBioinformatics. 2013;14:1–14.30. Geva M, Kusko R, Soares H, Fowler KD, Birnberg T, Barash S, et al.Pridopidine activates neuroprotective pathways impaired in Huntingtondisease. Hum Mol Genet. 2016;25(18):3975–987.31. Aziz NA, Pijl H, Frölich M, van der Graaf AWM, Roelfsema F, Roos RAC.Increased hypothalamic-pituitary-adrenal axis activity in Huntington’sdisease. J Clin Endocrinol Metab. 2009;94:1223–8.32. Dong X, Tsuji J, Labadorf A, Roussos P, Chen J-F, Myers RH, et al. The role ofH3K4me3 in transcriptional regulation is altered in Huntington’s disease.PLoS One. 2015;10:e0144398.33. Choi Y-S, Lee B, Cho H-Y, Reyes IB, Pu X-A, Saido TC, et al. CREB is a keyregulator of striatal vulnerability in chemical and genetic models ofHuntington’s disease. Neurobiol Dis. 2009;36:259–68.34. Taymans J-M, Leysen JE, Langlois X. Striatal gene expression of RGS2 andRGS4 is specifically mediated by dopamine D1 and D2 receptors: clues forRGS2 and RGS4 functions. J Neurochem. 2003;84:1118–27.35. Xie Y, Hayden MR, Xu B. BDNF overexpression in the forebrain rescuesHuntington’s disease phenotypes in YAC128 mice. J Neurosci. 2010;30:14708–18.36. Maheshwari M, Bhutani S, Das A, Mukherjee R, Sharma A, Kino Y, et al.Dexamethasone induces heat shock response and slows down diseaseprogression in mouse and fly models of Huntington’s disease. Hum MolGenet. 2014;23:2737–51.37. Ponten H, Kullingsjö J, Lagerkvist S, Martin P, Pettersson F, Sonesson C, et al.In vivo pharmacology of the dopaminergic stabilizer pridopidine. Eur JPharmacol. 2010;644:88–95.38. Nagai T, Yoshimoto J, Kannon T, Kuroda K, Kaibuchi K. Phosphorylation signalsin striatal medium spiny neurons. Trends Pharmacol Sci. 2016;37:858–71.39. Gines S, Seong IS, Fossale E, Ivanova E, Trettel F, Gusella JF, et al. Specificprogressive cAMP reduction implicates energy deficit in presymptomaticHuntington’s disease knock-in mice. Hum Mol Genet. 2003;12:497–508.40. Lin J-T, Chang W-C, Chen H-M, Lai H-L, Chen C-Y, Tao M-H, et al. Regulationof feedback between protein kinase a and the proteasome system worsensHuntington’s disease. Mol Cell Biol. 2013;33:1073–84.41. Porras G, Berthet A, Dehay B, Li Q, Ladepeche L, Normand E, et al. PSD-95expression controls l-DOPA dyskinesia through dopamine D1 receptortrafficking. J Clin Invest. 2012;122:3977–89.42. Parsons MP, Kang R, Buren C, Dau A, Southwell AL, Doty CN, et al.Bidirectional control of postsynaptic density-95 (PSD-95) clustering byhuntingtin. J Biol Chem. 2014;289:3518–28.43. Zhang J, Vinuela A, Neely MH, Hallett PJ, Grant SGN, Miller GM, et al.Inhibition of the dopamine D1 receptor signaling by PSD-95. J Biol Chem.2007;282:15778–89.44. Sun Y, Savanenin A, Reddy PH, Liu YF. Polyglutamine-expanded huntingtinpromotes sensitization of N-methyl-D-aspartate receptors via post-synapticdensity 95. J Biol Chem. 2001;276:24713–8.45. Spiegel I, Mardinly A, Gabel H, Bazinet J, Couch C, Tzeng C, et al. Npas4regulates excitatory-inhibitory balance within neural circuits through celltype-specific gene programs. Cell. 2014;157:1216–29.46. Eggerickx D, Denef JF, Labbe O, Hayashi Y, Refetoff S, Vassart G, et al.Molecular cloning of an orphan G-protein-coupled receptor thatconstitutively activates adenylate cyclase. Biochem J. 1995;309(Pt 3):837–43.47. Valverde O, Célérier E, Baranyi M, Vanderhaeghen P, Maldonado R, SperlaghB, et al. GPR3 receptor, a novel actor in the emotional-like responses. PLoSOne. 2009;4:e4704.Kusko et al. Molecular Neurodegeneration  (2018) 13:25 Page 15 of 15


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