Conference Abstract

Conference posters abstract

Cortico-striatal-thalamo-cortical white matter structure in Tourette syndrome and ADHD (OHBM 2017)

Dysregulation within cortico-striatal-thalamo-cortical (CSTC) networks is hypothesized to underlie multiple disorders including Tourette syndrome (TS), attention-deficit/hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD; Leisman & Melillo, 2013). Dysfunction of individual loops is supposedly responsible for specific symptoms such as tics, obsessions and compulsions (Mink, 2006) with the interplay between the loops accounting for the high rate of comorbidity between the disorders. However, evidence from neuroimaging studies of the respective disorders implicating the structural connectivity of the CSTC networks has been inconsistent (van Ewijk et al, 2012; Makki et al, 2009) possibly due to methodological variation across studies, small sample sizes and heterogeneity of participants, especially regarding age. Here, we investigated CSTC networks associated with tics, ADHD and obsessive compulsive symptoms in a large group of children with TS and/or ADHD and healthy controls. We focussed on a restricted age range (8-12) where both TS and ADHD are highly prevalent. Eighty-two diffusion-weighted (DW, B0=2, diffusion gradients=64 [b=1500s/mm2]) and T1-weighted (MPRAGE) magnetic resonance datasets that survived quality assessment were included. Children with TS (n=22, age 10.7[1.2], n=8 of which also had ADHD), 19 with ADHD (age 11.1[1.3]) and 41 healthy controls (age 11.0[1.1]) were included. DW images were pre-processed to correct for motion, cardiac and other imaging artefacts (PATCH (Zwiers, 2010)). The constrained spherical deconvolution (CSD) algorithm was applied followed by EuDX tractography within Dipy (Garyfallidis et al, 2014). Tractography seeds (striatum and thalamus) and targets (superior frontal, rostral middle frontal, precentral, lateral orbito frontal and caudalmiddle frontal) were derived from subject specific Freesurfer reconstructions (27 tracks were propagated per seed voxel, step size = 0.5, stop threshold fractional anisotropy [FA]=0.15). Targets were selected based on previous literature. In total 20 connections were isolated; 2 seeds x 5 targets x 2 hemispheres. FA was extracted from each isolated tract with TrackVis (Wang et al, 2007). FA was modelled in relation to TS (categorical), ADHD-severity (continuous across all subjects) and their interaction (as an indicator of comorbidity) for each connection of interest, with age and sex as covariates. Analysis revealed no association between FA and TS, ADHD severity or their interaction (p>.05 in all cases following multiple comparison correction) in any white matter tracts between the striatum or thalamus and frontal targets. Furthermore, FA was not related to obsessive-compulsive symptoms, tic severity, duration since tic onset or age at tic onset within participants with TS in any of the tracts (p>.05 in all cases following multiple comparison correction). This is the first study to investigate CSTC white matter microstructure of children with TS and/or ADHD together. In keeping with some (Jackson et al, 2011; Thomalla et al, 2009) but not all (van Ewijk et al, 2012; Makki et al, 2009) of the previous literature investigating these disorders separately we see no white matter differences associated with the disorders. However, methodological limitations remain, most notably due to the size of the seed and target regions. In some cases multiple fibre bundles were reconstructed between a seed (thalamus or striatum) and target. As a result mean FA was sometimes extracted from a composite of multiple bundles rather than one individual fibre bundle and thus potentially influenced by the quality of reconstruction of the respective bundles. Individual bundles should be isolated and compared in future. Variation in CSTC white matter was not associated with TS, ADHD-severity or their interaction in this study. However, further methodological refinements should be implemented to ensure appropriate comparison of tracts before final conclusions can be made.

Cortical development through adolescence and early adulthood (OHBM 2016)

Introduction Despite many years of neuroimaging, the precise developmental changes that occur in the brain during development are yet to be fully characterised. Here we utilised the novel application of intrinsic curvature (IC) analysis of the cortical surface, along with more traditional measures such as cortical thickness (CT), local gyrification index (LGI) and surface area (SA), to help further understand the neurodevelopmental changes that occur in healthy development. IC is predictive of the degree and pattern of gyrification (Ronan et al., 2012) and reflects the differential expansion of the cortex. It is hypothesised to reflect both cortical cell density and cortical connectivity (Ronan et al., 2012). Methods T1 magnetic resonance images from 218 healthy controls (age in years; mean(SD) 16.5(3.37), range 8.3-29.2) collected at two sites in the Netherlands (as part of the NeuroIMAGE study) were used. Data sets were processed with Freesurfer software. IC was calculated with Caret software from the Freesurfer reconstructions. IC, LGI, CT and SA were extracted per region (frontal, parietal, occipital, temporal, insula and cingulate), using regions generated by combining labels from the Desikan-Killiany Atlas (Desikan et al., 2006). Developmental profiles for each parameter per region were modelled using generalised additive mixed-effect models (GAM; Wood, 2006) with the gamm4 (Wood and Scheipl, 2014) and mgcv (Wood, 2011) packages in R. Each model also included scanner location and sex and accounted for the non-independence of subjects (sibling pairs) by modelling family as a random factor. IQ and the interaction of age and sex were additionally investigated. Results were adjusted for multiple comparisons at the p=0.002 level, according to a Bonferroni correction. Results IC was significantly inversely associated with age in the frontal, parietal, temporal, cingulate and insula regions, indicating lower IC with increasing age. LGI and CT were also significantly inversely associated with age in all regions, with both LGI and CT declining steadily throughout the age range. SA was significantly associated with sex in all regions, with male SA consistently larger than that of female SA, while there was no significant effect of age. A significant effect of sex was also seen with males consistently having a larger LGI than females in all regions and also in IC of the frontal lobe, however these sex effects did not persist when brain size was accounted for by including total brain SA in the models. There were no age-by-sex interactions or effects of IQ seen in any of the measures. Discussion In keeping with the previous literature, and now reporting regionally instead of globally, we observed diverse effects of age and sex on the different cortical measures, including IC. We could not confirm previous findings (Raznahan et al., 2011) showing a difference between males and females in CT; furthermore, we did not detect an effect of age on SA within the age range under investigation. This may be due to the cross-sectional character of the study and/or the restricted age range which had a particular focus on adolescence. This unfortunately resulted in the study being underpowered to detect cortical changes in early adolescence and early adulthood. This study adds an innovative cortical measure to the study of neurodevelopment, IC, which appears to decrease with age. This may reflect a higher neuronal density and/or a decrease in cortico-cortical connectivity. 

Basal ganglia structure in Tourette’s Disorder and Attention-Deficit/Hyperactivity Disorder (Eunethydis 2016)

Tourette’s disorder (TD) and Attention-Deficit/Hyperactivity disorder (ADHD) which often co-occur have both been associated with structural variation of the basal ganglia (BG)1,2, although not consistently and many studies have failed to account for comorbidity, medication use and other possible confounders. Herein, we aimed to determine the relationship between TD, ADHD and the combination thereof and BG structure. T1-weighted magnetic resonance (MR) images and phenotypic information from 141 children (age 8-12 years) were used; TD (n=47, age=10.5[1.4], 47% with comorbid ADHD), ADHD (n=39, age=10.7[1.3]) and controls (n=55, age=11.0[1.0]). Caudate nucleus (CN), putamen (Pu) and globus pallidus (GP) volumes were determined from MR images following processing with the FMRIB integrated registration and segmentation tool (FIRST)3. Structure surfaces were reconstructed and used to determine shape. Across all participants, BG nuclei volume and shape were estimated in relation to TD (categorical), ADHD severity (continuous measure across all participants) and TD-by-ADHD severity interaction. Sex, age and IQ were included as covariates with total brain volume additionally included in the volume analysis. Analysis revealed no difference in CN, Pu or GP volumes between children with and without tics (F(1,134)=0.29, p=0.59; F(1,134)=0.27, p=0.60; F(1,134)=3.60, p=0.06), no association with ADHD severity (F(1,134)=0.001, p=0.98; F(1,134)=0.04, p=0.85; F(1,134)=0.06, p=0.81) and no interaction between TD and ADHD severity (F(1,133)=2.55, p=0.11; F(1,133)=0.14, p=0.71; F(1,133)=0.80, p=0.37). Similarly shape analysis showed no significant association with either TD, ADHD severity or their interaction. In conclusion, we found no evidence that TD, ADHD or the combination thereof are associated with structural variation of the BG.

Glutamate levels in the Fronto-striatal circuit in Tourette’s Disorder and Attention-deficit/ Hyperactivity Disorder: A proton Magnetic Resonance Spectroscopy study (ESSTS 2016)

Both Tourette’s Disorder (TD) and Attention-Deficit/Hyperactivity Disorder (ADHD) have been related to abnormalities in glutamatergic neurochemistry in the fronto-striatal circuitry. TD and ADHD often co-occur and this co-occurrence has been insufficiently taken into account in prior studies. Here, we used proton magnetic resonance spectroscopy (1H-MRS) in children between 8-12 years of age (TD n=15, ADHD n=39, TD+ADHD n=29 and control n=53) as an in vivo method of evaluating glutamate concentrations in the fronto-striatal circuit. Spectra were collected on a 3 Tesla Siemens scanner from two 8 cm3 voxels in each participant: the anterior cingulate cortex (ACC) and the left dorsal striatum. LC-model was used to process spectra and generate absolute glutamate concentrations. To determine if there was a significant effect of group on concentrations, a one-way analysis of variance was performed in R. Neither TD or ADHD or their combination was associated with differences in glutamate levels in either the ACC (F(3,132)=0.97, p=0.41) or striatum (F(3,121)=0.59, p=0.62). Variation in glutamate concentration was unrelated to age, sex, medication use, IQ, tic or ADHD severity. Obsessive-compulsive (OC) traits were positively correlated with ACC glutamate concentration within the participants with TD (r=0.45, pcorrected=0.02). We found no evidence that TD or ADHD are directly associated with alterations in glutamatergic transmission within the fronto-striatal circuits. However, OC-traits correlating with ACC glutamate concentration supports a role for involvement of glutamatergic transmission in OC-symptoms within TD. As previous literature is inconclusive, further investigation is warranted to determine if our finding  extends to OC-symptoms outside of TD.

Basal ganglia structure in Tourette’s Disorder and Attention-Deficit/Hyperactivity Disorder: Influence of comorbidity and medication use (ECNP 2016)

Structural variation of the basal ganglia has been associated with both Tourette’s disorder (TD)[1] and Attention-Deficit/Hyperactivity disorder (ADHD)[2]. However, previous studies have been inconsistent in their findings and often failed to account for comorbidity, medication use and other possible confounders. This is of particular importance considering the high rate of comorbidity of ADHD in TD (~40%) [3] and the common use of antipsychotic medication in the treatment of tics. Antipsychotics have previously been associated with grey matter volume reductions in other patient groups (i.e. schizophrenia) and animal models [4]. Herein, we aimed to determine the relationship of TD, ADHD and the combination thereof with basal ganglia volumes, taking medication into account. A total of 141 participants (age 8-12 years) were included in this analysis from whom good quality T1-weighted magnetic resonance (MR) datasets and full phenotypic information were available; Conners’ Parent Rating Scale, Yale Global Tic Severity Scale, Children’s Yale-Brown Obsessive Compulsive Scale, Children’s Social Behaviour Questionnaire, medication history and estimated full-scale IQ. These included participants with TD (n=47, age=10.5[1.4], 47% with comorbid ADHD), ADHD (n=39, age=10.7[1.3]) and controls (n=55, age=11.0[1.0]). T1-weighted datasets were processed within the FMRIB Software Library (FSL). This included registration to standard space and segmentation to isolate subcortical structures with the FMRIB integrated registration and segmentation tool (FIRST)[5]. Volumes for the caudate nucleus and putamen were then extracted for both the left and right hemisphere. Across all participants, basal ganglia volumes were estimated as a function of TD (categorical), ADHD severity (continuous measure across all participants) and TD-by-ADHD severity interaction. Total brain volume, sex, age and IQ were included as covariates. To assess left-right asymmetry, hemisphere was added as a repeated measures variable interacting with TD. Furthermore, a left-right asymmetry index (AI=(R-L)/(R+L)) was calculated to investigate associations between asymmetry and phenotype/medication within the TD group. Analysis revealed no difference in absolute caudate nucleus or putamen volumes between those with and without tics (F(1,134)=0.29, p=0.59; F(1,134)=0.27, p=0.60), no association with ADHD severity (F(1,134)=0.001, p=0.98; F(1,134)=0.04, p=0.85) or interaction between ADHD severity and TD (F(1,133)=2.55, p=0.11; F(1,133)=0.14, p=0.71). A trend towards a hemisphere-by-TD interaction in the caudate nucleus (F(1,139)=3.13, p=0.08) was seen. There was no association between caudate nucleus AI and tic severity, obsessive-compulsive symptoms, age of tics onset or duration since tic onset. The degree of AI did not differ with medication status regarding current antipsychotic use versus no-antipsychotic use (F(1,42)=0.53, p=0.47) or current stimulant versus no-stimulant use (F(1,42)=-0.22, p=0.64). In conclusion, we found no evidence that TD, ADHD or the combination thereof are associated with volume differences in the basal ganglia. A trend finding tentatively supports previous literature indicating alterations in left-right asymmetry associated with TD. However, the degree of asymmetry was unrelated to phenotypic measures or medication status. Further research is required to confirm this result regarding medication as the current study may have been underpowered to detect their effects.

Cortical connectivity in adolescents and young adults with attention-deficit/hyperactivity disorder (ECNP 2015)

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder affecting approximately 5% of the school age population and characterised by a pattern of pervasive inattention and/or hyperactivity and impulsivity that is associated with impairments of functioning. Neuroimaging studies have highlighted structural and functional abnormalities associated with ADHD; including cortical and connectivity differences based both on structural (diffusion magnetic resonance imaging [MRI]) and functional MRI data. The innovative approach of applying intrinsic curvature analysis to the cortical surface applied herein allowed us determine whether the structural connectivity abnormalities thus far identified at a centimeter scale also occur at a millimeter scale within the cortical surface. Differential expansion of the surface during development underlies both intrinsic curvature and also results in a higher proportion of shorter connections, from which we infer more efficient connectivity [1, 2]. Thus, we can use intrinsic curvature as a quantifiable measure of connectivity within the cortex. Furthermore, intrinsic curvature is related to gyrification [2]; however, it holds greater power to detect subtle shape differences in the cortex indicative of abnormal neurodevelopment [2]. We hypothesised that previous gyrification findings may have been obfuscated by the scale of morphological parameters employed; we therefore proposed that the intrinsic curvature may have increased power to detect subtle differences in cortical connectivity of ADHD, but that these would not be detectable with the related but larger-scale gyrification index. Structural MR images from NeuroIMAGE (www.neuroimage.nl) participants, whose scans were acquired at one of two test sites (Amsterdam and Nijmegen), were utilised in this study. 618 full, good quality datasets from 374 families were available. Of these, 164 were healthy controls [mean(SD) age 16.8 (3.2)], 306 had ADHD [17.2 (3.4)] and 148 were healthy siblings of a person with ADHD [17.7 (3.8)]. FreeSurfer was used to reconstruct the cortical surfaces and compute the local gyrification index (lGI) for each point on the surface (vertex) [3]. Intrinsic curvature was calculated from the FreeSurfer reconstructions per vertex using Caret software. The skew of the intrinsic curvature distribution was calculated per hemisphere. Because we were interested in the degree of intrinsic curvature independent of brain size, we used the skew of the distribution rather than the mean for analysis [2, 4]. A vertex-wise general linear model analysis of lGI was performed using glmfit in FreeSurfer with age, gender, scanner site and total brain volume as covariates and a monte carlo simulation for multiple comparison correction. An univariate analysis of co-variance (ANCOVA) was used to assess group differences in intrinsic curvature skew per hemisphere with age, gender, surface area of hemisphere and scanner site as covariates. No group differences were found in lGI or intrinsic curvature skew.

Despite the increased sensitivity of intrinsic curvature to subtle morphological abnormalities of the cortical surface we found no differences between groups. This suggests that there are no milimeter scale connectivity abnormalities associated with ADHD. This highlights the diffference between ADHD and other neurodevelopmental disorders like schizophrenia and autism spectrum disorder that have shown differences in intrinsic curvature or related measures [2, 5].

Neural Structure and Function in Tourette syndrome: distinguishing neural structure and function in Tourette syndrome from healthy controls and ADHD (ESSTS 2014)

Rationale: Attention-Deficit/Hyperactivity Disorder (ADHD) is often found comorbid with Tourette syndrome (TS), similarly tic disorders often comprise secondary diagnoses in ADHD, making it difficult to eliminate the potential confounds of comorbidity on research findings in TS. Although the frontostriatal circuits and its main regions, the prefrontal cortex and the basal ganglia have been implicated in TS, there are multiple inconsistent findings in imaging measures and executive functioning in TS. Furthermore glutamate is a major neurotransmitter modulating the activity of the frontostriatal circuits, but its role in TS in unclear. Objectives: The aim of the study is to identify brain (1) structural, (2) functional and (3) connectivity abnormality differences, and (4) neuropsychological differences between TS and ADHD, to elucidate which neural correlates correspond to each condition, which are common and which unique. Similarly (5) glutamate concentrations from the frontostriatal region, acquired with magnetic resonance spectroscopy (MRS), will be compared between groups. Methods: 180 children (n=60 per group, 8-12 years old); (1) TS, (2) ADHD and (3) healthy controls will be recruited and undergo imaging and neuropsychological assessment. Structural, diffusion and functional (resting state and task specific) magnetic resonance images will be obtained as well as MRS glutamate and neuropsychological data. Advanced neuroimaging analysis techniques will be applied, including the use of FreeSurfer. Results from the three groups will be analysed in a multiple regression model. Outcome: This will be the first study to explicitly investigate the neural correlates of TS and ADHD in children.

Gene expression analysis of Tourette Syndrome cohort. European Society for the study of Tourette Syndrome (ESSTS) 2014 Annual Meeting, April 25-26 2014, Paris, France.

 

The aetiology Tourette Syndrome (TS) is complex with multiple genes interacting with environmental factors. Given this complexity, the investigation of gene expression patterns in TS may provide useful insights on gene and environment interactions. Changes in gene expression in the brain can be observed in peripheral blood as expected through its interaction with the brain. Studies have shown distinct gene expression profiles in peripheral blood for many neuropsychiatric disorders. Here, we are performing meta-analyses of gene expression datasets for TS (made available to us by our collaborators at the University of California Davis). The gene expressions arrays that were used are the Affymetrix U133 Plus 2.0 arrays and the Affymetrix HuExon arrays for 26 TS cases and 23 Healthy Control samples. We are exploring various algorithms for clustering and classification of the gene expression data, ranging from Principal Component Analysis (PCA) and other dimensionality reduction techniques to clustering and classification tools such as k-means and Support Vector Machines to predict tic severity (which is scored through standard measures) using gene expression profiles. The algorithmic methods and pipeline established here will be transferred to the study of a much larger dataset that is currently being collected under the auspices of the EU-funded EMTICS project.

 

Genomewide investigation of Copy Number Variants in relation to Tourette Syndrome in a Danish cohort. 12th World Congress of Biological Psychiatry. 14-18 June 2015, Athens, Greece.

Tourette Syndrome is a neuropsychiatric disorder characterized by multiple motor and phonic tics. The etiology of TS involves complex interaction of genetic variants and environmental factors. The role of Copy Number Variants (CNVs) in relation to TS susceptibility has not yet been studied in detail at a genomewide level. At the same time, recent studies have shown an implication of Copy Number Variants (CNVs) in other neurodevelopmental disorders such as Autism Spectrum Disorders (ASD). Here, we explore the impact that CNVs may have on the aetiology of TS analysing a well characterized cohort of individuals with TS from Denmark. 261 Danish individuals with TS were genotyped using the Affymetrix CytoScan High-Density (HD) array which covers 2.6 Million copy number markers with 500-600 bases median marker distance. CytoScan HD array also includes ~750,000 SNP marker allelic information to give additional confidence for the CNV calls. CNVs were called using PennCNV and  BirdSuite. These softwares give QC metrics on CNV analysis where bad calls and outlier samples can be ignored before proceeding to further analysis. Called CNVs were subsequently tested for association with TS.  Results were compared to current literature for the role of CNVs in neurodevelopmental and other neuropsychiatric disorders. Our study sheds further light into the possible role of CNVs in the etiology of neurodevelopmental disorders.

Genome-wide Association of SNPs in Danish cohort for Gilles de la Tourette Syndrome (GTS). 1st World Congress on Tourette Syndrome and Tic Disorders. 24-26 June 2015, London, UK.

Gilles de la Tourette Syndrome (GTS) is a neuropsychiatric disorder characterized by multiple motor and phonic tics. The aetiology of TS involves complex interaction of different genetic variants and environmental factors. The first Genome-wide Association Study (GWAS), investigated the role of genetic variant Single Nucleotide Polymorphisms (SNPs) in GTS, identified SNPs that did not meet the genome-wide significance level, but they serve as indicators of possible association with GTS. Here we explore the role of SNPs in aetiology of GTS using well characterised cohort of individuals with GTS from Denmark. 261 Danish individuals with GTS were genotyped in four different batches for around ~ 750,000 SNPs from Affymetrix CytoScan High-Density (HD) array and 800 Healthy Controls with European ancestry were used to find significant SNPs associated with Tourette Syndrome (TS) in the Danish cohort. Quality Control (QC) was applied at SNP level to remove SNPs with lower genotyping rate, ambiguous strand orientation, deviations from Hardy-Weinberg Equilibrium and batch effects. Quality control at sample level checked for genotype call rate, heterozygosity, discordance in sex, removal of closely related samples and population stratification. European ancestry check of both cases and controls is performed using the 500 European samples from 1000genomes. Multiple models of association tests were performed on QC passed samples. The whole analysis was performed using software PLINK and GCTA software. The scripts for whole analysis were written in R and Python scripting languages. Results from quality control at SNP level left with 654717 SNPs and at sample level left with 968 samples of which 228 cases and 740 controls. Association analysis was performed using logistic regression on 967 samples on PLINK and GCTA. Our study sheds further light into possible role of SNPs in aetiology of Gilles de la Tourette Syndrome.

Investigation of NTN4, variant rs2060546, in a Danish Gilles de la Tourette syndrome cohort. European Society for the study of Tourette Syndrome (ESSTS) 2016 Annual Meeting, June 8-11 2016, Warsaw, Poland.

Gilles de la Tourette syndrome (GTS) is a neuropsychiatric disorder characterized by repetitive multiple motor and vocal tics. GTS is a complex disorder, where environmental factors and several genes are thought to be involved in the aetiology. Although few genes such as AADAC, NRXN1, SLITRK1, and IMMP2L have been associated with GTS, the causative genes underlying GTS pathophysiology remain unknown. In a previous genome-wide association study a single nucleotide polymorphism (SNP, rs2060546) near the Netrin-4 (NTN4 - MIM 610401) gene was shown to be associated with GTS [odds ratio (OR) = 1.7 with a p-value (p) = 5.8x10-7] and merit further investigations. As NTN4 is one of the axon guidance molecules expressed in the central nervous system and it interacts with SLIT and WNT genes guiding the growth cone towards its target, it is an attractive candidate susceptibility gene for GTS. In this study we attempted to replicate the association of rs2060546 with GTS by genotyping a cohort of 240 Danish GTS patients and 1007 healthy controls (HC), but our results did not reveal an association (OR= 1.363; p = 0.3329). However, a meta-analysis of association results from Danish cohort along with Gilles de la Tourette syndrome Genome-wide Association Study Replication Initiative (GGRI) and first GTS Genome Wide Association study (GTS-GWAS) cohorts yielded a significant signal OR= 3.74; p = 0.00018.   Our study strengthens the evidence for the involvement of NTNT4 gene with GTS suggesting for functional studies to investigate the role of this regions in pathogenesis of GTS.

MicroRNA regulation of candidate genes for Tourette Syndrome(WCPG 2016)

Tourette Syndrome (TS) is a neurodevelopmental disorder that presents early in childhood and is marked by the appearance of multiple involuntary motor tics and at least one vocal tic. It presents high comorbidity rates with other disorders such as attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD). Despite a strong genetic contribution, the molecular mechanisms behind TS are still uncertain, although multiple lines of evidence suggest involvement of specific candidate genes and corresponding epigenetic mechanisms via miRNA regulators. To date only a few genetic findings have been replicated in TS. Among these the nicotinic acetylcholine receptor alpha 7 subunit (CHRNA7) gene has been recently suggested as candidate susceptibility gene. CHRNA7 is known to regulate a wide variety of developmental and secretory functions, however, the mechanism of its transcriptional regulation is still unclear. Another recent promising finding in TS genetic research is Netrin 4 (NTN4), which belongs to a family of extracellular proteins that direct axon outgrowth and guidance. Several microRNAs have already been associated with neuropsychiatric disorders, but most importantly a few miRNA have also been associated with Tourette Syndrome. Our goal was to investigate the role of selected candidate genes in a case-control setup along with functional validation involving miRNA regulation in the possible involvement in TS pathogenesis through analysis of 3’UTR regions. An OpenArray platform (TaqMan® OpenArray® Genotyping System) was used for the case-control analysis of TS patients (N=564) and healthy controls. Starting from a list of candidate genes previously indicated as possible risk factors for TS, we screened for presence of SNPs in the 3’UTR regulatory regions. This analysis (data retrieved by mirSNP and polymiRTS databases) led to the identification of 32 SNPs which were predicted to change the seed sequence of in silico proposed miRNAs. To proof this concept, we performed functional validation study using a luciferase assay reporter containing 3’ UTR regions of CHRNA7 and NTN4 transfected with their predicted miRNA. Specifically, miRNA-106b and miRNA-198b were identified as the most potent miRNA candidates for regulation of CHRNA7 and NTN4 gene expression. SKNF1 and HEK human cell lines were co-transfected using lipofectamine with the luciferase reporter-3’ UTR construct of CHRNA7 and NTN4 (SwitchGear Genomics) and the corresponding putative miRNAs (miR-106b, miR-198b) along with non-targeting control miRNAs (miR-196b, miR-641b) in the functional validation studies. OpenArray analysis identified significant differences among the selected candidates genes (GDNF, LHX6, DRD2, CNTNAP2). In the luciferase assays we characterized the regulatory effect of the predicted miRNAs on the expression of the studied genes in a concentration-dependent manner, which showed a dramatic, up to 5-fold change in the relative gene expression levels in case of CHRNA7 and NTN4. In order to increase our understanding of the underlying genetic and epigenetic mechanisms of TS, we aimed to study the possible miRNA regulation processes in TS-related genes, which would help not only to better understand the full genetic architecture of this disorder but also to determine how miRNAs contribute to the complexity of gene regulation in the development of disease.

Effect of chronic treatment with psychiatric medications aripiprazole and riluzole on DNA methylation profiles in the rat striatum and prefrontal cortex (WCPG 2016)

One of the main epigenetic mechanisms is DNA methylation. It mostly occurs in CpG islands in the promoters by addition of a methyl group to a cytosine that may inhibit gene expression. It is known that many developmental disorders are characterized by alterations in epigenetic mechanisms. Moreover, studies in animal models of neuropsychiatric disorders have shown that epigenetics may play an important role in pathogenesis and therapy. The aim of this study is to investigate the effect of drugs used in different psychiatric diseases including Tourette Syndrome (TS) on DNA methylation to find epigenetic targets that may influence the efficacy of treatment.Wistar Kyoto rats were chronically treated from post-natal day (PND) 35 to PND50 by intraperitoneal injection of either aripiprazole or riluzole or vehicle. Metabolite changes during the treatment were quantified by magnetic resonance spectroscopy (MRS) in the striatum and prefrontal cortex. At PND50 rats were sacrificed and plasma, striatum, prefrontal cortex, hippocampus and cerebellum were stored for further analyses.DNA isolated from striatum and prefrontal cortex was used for methylation studies.RRBS is a high-throughput technique used to analyze genome-wide methylation profiles at the single nucleotide level. It provides information of the most relevant genomic regions (including CpG islands), which are likely to influence gene expression. We analyzed striatum and prefrontal cortex of 2 rats in each group treated with aripiprazole (a dopaminergic neuroleptic) or riluzole (a glutamatergic anxiolytic) or vehicle in order to identify DNA methylation changes due to these drug treatments. The analysis was performed by LC-MS/MS on striatum and prefrontal cortex in order to quantify the amount of 5-methyl-Cytosine (mC) and 5-HydroxyMethylCytosine (5hmC) after drug treatment.LC-MS/MS showed significant differences in 5hmC levels in prefrontal cortex after treatment with aripiprazole and riluzole. The same trend was also observed for mC levels. We found no differences in methylation or hydroxy-methylation levels in striatal samples. We observed differences in overall methylation levels between treatment using the two drugs and the vehicle. We identified differentially methylated CpG sites (DMS) and characterized their distribution across the genome (eg. promoter, intron, exon, UTR, etc.). Treatment with riluzole led to identification of 156 DMS in striatum and 346 DMS in prefrontal cortex. Treatment with aripiprazole yielded 117 DMS in striatum and 466 DMS in prefrontal cortex. The identified CpG sites were annotated, a candidate gene list was created and pathway analysis was also performed on these genes. Interestingly, most of the changes occurred in intergenic regions far from TSS suggesting that enhancers are the regions most prone to undergo methylation changes. Moreover, several genes (eg. OXTR, DRD2, etc.) have been associated with brain disorders or functions.The known functions of implicated genes suggest that some of the observed epigenetic changes might underlie the amelioration of symptoms by these drugs and some may account for certain adverse effects. Data on differential DNA methylation will be compared with imaging data on the respective brain regions. The results give insights into the mechanism of action of aripiprazole and riluzole as well as the side effects, not just in TS, but also in a broader context regarding these psychiatric medications.

Changes in DNA methylation profiles followed by sub-chronic treatment with psychiatric drugs aripiprazole and riluzole in the rat striatum and prefrontal cortex (Danube Epigenetic 2016)

Studies in neuropsychiatric disorders have shown that epigenetics may play an important role in pathogenesis and therapy. The aim of this study is to investigate the effect of drugs used in Tourette Syndrome (TS) and other psychiatric disorders on DNA methylation in the CNS to find epigenetic targets that may influence efficacy of treatment. MaleWistar Kyoto rats were treated from post-natal day 35 to 50 by intraperitoneal injection of either aripiprazole (neuroleptic), or riluzole (glutamatergic) or vehicle. Then rats were sacrificed and DNA was isolated from brain regions for methylation studies. Reduced Representation Bisulfite Sequencing (RRBS) is a high-throughput technique used to analyze genome-wide methylation profiles at the single nucleotide level. We analyzed striatum and prefrontal cortex of 2 rats per drug group in order to identify DNA methylation changes due to these medications. We observed differences in overall methylation levels between the two drugs and controls. We identified hundreds of differentially methylated CpG sites and characterized their distribution across the genome (eg. promoter, intron, exon, UTR, etc.). The recognized CpG sites were annotated, a candidate gene list was created and pathway analysis was also performed on these genes. The known functions of implicated genes suggest that some of the observed epigenetic changes might underlie the amelioration of symptoms by these drugs and account for certain adverse effects. The results give insights into the mechanism of action of aripiprazole and riluzole, as well as the side effects regarding these psychiatric medications.

MicroRNA regulation of candidate genes for Tourette Syndrome (ECNP 2016)

Tourette Syndrome (TS) is a neurodevelopmental disorder that presents early in childhood and is marked by the appearance of multiple involuntary motor tics and at least one vocal tic. It presents high comorbidity rates with other disorders such as attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD. Despite a strong genetic contribution, the molecular mechanisms behind TS are still uncertain, although multiple lines of evidence suggest involvement of specific candidate genes and corresponding epigenetic mechanisms via miRNA regulators. To date only a few genetic findings have been replicated in TS. Among these the nicotinic acetylcholine receptor alpha 7 subunit (CHRNA7) gene has been recently suggested as candidate susceptibility gene. CHRNA7 is known to regulate a wide variety of developmental and secretory functions, however the mechanism of its transcriptional regulation is still unclear. Another promising finding of the last years in TS genetic research is Netrin 4 (NTN4), which belongs to a family of extracellular proteins that direct axon outgrowth and guidance. Several microRNAs have already been associated with neuropsychiatric disorders, but most importantly a few have also been associated with Tourette Syndrome. Our goal was to investigate the role of selected candidate genes in a case-control setup along with functional validation involving miRNA regulation in the possible involvement in TS pathogenesis through analysis of 3’UTR regions. An OpenArray platform (TaqMan® OpenArray® Genotyping System) was used for the case-control analysis of TS patients (N=564) and healthy controls. Starting from a list of candidate genes previously indicated as possible risk factors for TS, we screened for presence of SNPs in the 3’UTR regulatory regions. This analysis (data retrieved by mirSNP and polymiRTS databases) led to the identification of 32 SNPs predicted to change the seed sequence of in silico proposed miRNAs. In the functional validation study the applied plasmids containing the 3’ UTR regions of CHRNA7 and NTN4 genes were used in transfection assays. Using databases, miRNA-106b and miRNA-198b were identified as the most potent miRNA candidates for regulation of CHRNA7 and NTN4 gene expression, respectively. SKNF1 and HEK human cell lines were co-transfected using lipofectamine with the luciferase reporter-3’ UTR construct of CHRNA7 and NTN4 (SwitchGear Genomics) and the corresponding putative miRNAs (miR-106b, miR-198b) along with non-targeting control miRNAs (miR-196b, miR-641b) in the functional validation studies. OpenArray analysis identified nominally significant differences among the selected candidates, however, follow-up studies are still required. In the luciferase assays we characterized the regulatory effect of the predicted miRNAs on the expression of the studied genes in a concentration-dependent manner, which showed a dramatic, up to 5-fold change in the relative gene expression levels in case of CHRNA7 and NTN4. In order to increase our understanding of the underlying genetic and epigenetic mechanisms of TS, we aimed to study the possible miRNA regulation processes in TS-related genes, which would help not only to better understand the full genetic architecture of this disorder but also to determine how miRNAs contribute to the complexity of gene regulation in the development of disease.

Epigenetic changes following striatal 6-OH-dopamine lesion and L-DOPA treatment in a rodent tic model (WARSAW 2016)

Tourette Syndrome (TS) is a neurodevelopmental disorder characterized by multiple repetitive involuntary movements, motor tics and at least one vocal tic, lasting longer than a year. The cause of TS remains elusive but dopamine (DA) appears to have a central role through the nigrostriatal pathway. TS has a complex genetic background, but environmental factors also play an important role in the development of the disease. These factors may operate through epigenetic mechanisms (covalent modifications and accessibility of chromatin). The aim of this project is to investigate DNA methylation changes in a juvenile animal tic model. Juvenile male Wistar Kyoto rats received stereotaxic injections of 6-hydroxydopamine (6-OH-DA) or vehicle in the left medial forebrain bundle. This results in degeneration of nigrostriatal neurons. Chronic application of L-DOPA after the lesion leads to the development of motor tics as a consequence of the striatal hypersensitivity to DA. This is a putative pathological mechanism of TS and the model is obtained by prior deprivation of the neurotransmitter. 3 groups were investigated by methylome and transcriptome analysis. Two weeks after 6-OH-DA injection, rats underwent different treatments. Group 1 was chronically administered (ten times in 2 weeks) with a solution of L-DOPA. Group 2 did not receive treatment. Group 3 was chronically administered (ten times in 2 weeks) with riluzole and L-DOPA. At post natal day 53 rats were administered one more time based on their respective treatments and sacrificed. Striatum, prefrontal cortex, hippocampus and cerebellum were stored for further analyses. DNA/RNA isolated from striatum of lesioned and control side were used for methylation and transcriptome studies. DNA methylation was investigated by Reduced representation bisulfite sequencing (RRBS). This technique provides methylation profile at the single nucleotide level of the most informative CpG regions of the genome (eg. promoter, intron, exon, UTR etc.) We analyzed striatum of 3 rats for each group (lesion and control sides) in order to identify methylation changes due to levodopa treatment as well as due to  treatment response. The results showed reduced overall methylation levels in lesioned sides compared to control in the striatum. We identified approximately 35,000 differentially methylated sites (DMS)/animal. The comparison between rats of the same group lead to the identification of approximately 3,000 DMS. The majority (90%) of those sites were hypo-methylated. Interestingly, most of those changes happened in intergenic regions and outside CpG islands. The annotation of these sites resulted in a list of candidate gene that will be compared to gene expression data (RNA-Sequencing).We observed 1) an important fraction of random changes, 2) presence of regions which are more prone to be methylated, 3) very few CpG islands were involved in those changes. This project is a combined methylome/transcriptome study on Tourette Syndrome animal model that may provide candidate genes as well as  a better understanding of the molecular mechanism behind pathophysiology of TS.

MicroRNA regulation in 3UTR regions of candidate genes for Tourette Syndrome

Tourette Syndrome (TS) is a common developmental neuropsychiatric disorder with one of the highest familial recurrence rates among such diseases. Despite a strong genetic contribution, the molecular underpinnings of the disorder remain uncertain, although multiple lines of evidence suggest involvement of specific candidate genes and corresponding miRNA regulators. To date only a few findings have been replicated. Among these the nicotinic acetylcholine receptor alpha 7 subunit (CHRNA7) gene has been recently suggested as candidate susceptibility gene in TS. CHRNA7 is known to regulate a wide variety of developmental and secretory functions, however the mechanism of its transcriptional regulation is still unclear. Another promising finding of the last years is Netrin 4 (NTN4), which belongs to a family of extracellular proteins that direct axon outgrowth and guidance. MicroRNAs are also implicated in the regulation of expression of these genes based on in silico search. Our goal was to investigate the role of the selected candidate genes in a case-control setup along with functional validation involving miRNA regulation in the possible involvement in TS pathogenesis through analysis of 3’UTR regions. OpenArray platform (TaqMan® OpenArray® Genotyping System) was used for the case-control analysis of TS patients (N=564) and healthy controls. Starting from a list of candidate genes previously indicated as possible risk factors for TS, we screened for presence of SNPs in the 3’UTR regulatory regions. This analysis (data retrieved by mirSNP and polymiRTS databases) led to the identification of 32 SNPs predicted to change the seed sequence of in silico proposed miRNAs. In the functional validation study the applied plasmids containing the 3’ UTR regions of CHRNA7 and NTN4 genes were used in transfection assays. Using databases miRNA-106b and miRNA-198b were identified as the most potent miRNA candidates for regulation of CHRNA7 and NTN4 gene expression, respectively. SKNF1 and HEK human cell lines were co-transfected using lipofectamine with the luciferase reporter-3’ UTR construct of CHRNA7 and NTN4 (SwitchGear Genomics) and the corresponding putative miRNAs (miR-106b, miR-198b) along with non-targeting control miRNAs (miR-196b, miR-641b) in the functional validation studies. OpenArray analysis identified nominally significant differences among the selected candidates, however, follow-up studies are still required. In the luciferase assays we characterized the regulatory effect of the predicted miRNAs on the expression of the studied genes in a concentration-dependent manner, which showed a dramatic, up to 5-fold change in the relative gene expression levels in case of CHRNA7. In order to increase our understanding of the underlying genetic and epigenetic mechanisms of TS, we aimed to study the possible miRNA regulation processes in TS-related genes, which would help not only to better understand the full genetic architecture of this disorder but also to determine how miRNAs contribute to the complexity of gene regulation in the development of disease.

Epigenetic changes following striatal 6-OH-dopamine lesion and L-DOPA treatment in a rodent tic model (WCPG 2015)

Tourette Syndrome (TS) is a neurodevelopmental disorder characterized by multiple repetitive involuntary movements ie. motor tics and at least one vocal tic, lasting longer than a year. It presents high comorbidity rates with attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD). The cause of TS remains elusive but dopamine (DA) appears to have a central role through the nigrostriatal pathway. TS has a complex genetic background but environmental factors also play an important role in the development of the disease. These factors operate through epigenetic mechanisms (covalent modifications and accessibility of chromatin). The aim of this project is to investigate these mechanisms (such as DNA methylation at CpG dinucleotides and histone modifications) in a juvenile animal model of tic.Juvenile male Wistar rats received stereotaxic injections of 6-hydroxydopamine (6-OHDA) or vehicle in the left medial forebrain bundle. This results in degeneration of nigrostriatal neurons. Chronic application of L-dopa after the lesion leads to the development of motor tics as a consequence of the striatal hypersensitivity to DA. This is a putative pathological mechanism of TS and the model is obtained by prior deprivation of the neurotransmitter.Lesioned and unlesioned sides of striatum, cerebellum, prefrontal cortex and hippocampus have been collected for epigenetic analysis. Global methylation quantification: The analysis was performed by LC-MS/MS on several brain regions in order to quantify the amount of 5methyl-Cytosine (mC) and 5-HydroxyMethylCytosine (5hmC) in lesioned and control sides. Reduced representation bisulfite sequencing (RRBS): we pooled 2 lesioned striata and 2 contralateral striata as controls to analyze the methylation pattern by NGS after bisulfite treatment of the most CpG rich regions of the genome. Chromatin immunoprecipitation (ChIP) has been performed to unravel chromatin activity by the following antibodies H3K36me3 (elongation), H3K4me3 (promoter), H3K27me3 (heterochromatin) and H3K27ac (enhancer). MeC and 5hmC levels were quantified with no significant differences between the lesioned and control sides indicating that two sides remain comparable for regional analysis after the lesion. Differenially methylated regions across the genome between lesioned and control side of striata have been identified by RRBS. The ChIP conditions were set up and calibrated for the number of striata to be used for the experiments. Bioinformatic analyses identified active and inactive genomic regions in the lesioned and contralateral sides. This project is a combined epigenetic study on Tourette Syndrome. It is a new field of research and will provide a possible link that may help to better explain the pathophysiology of TS.

Studies on epigenetic mechanisms in Tourette Syndrome (LONDON 2015)

Tourette Syndrome (TS) is a neurodevelopmental disorder characterized by motor and vocal tics. It presents high comorbidity rates with other disorders such as attention deficit hyperactivity disorder (ADHD)  and obsessive compulsive disorder (OCD). Epigenetics represents all the phenotype alterations which are not due to changes in DNA sequence. It is known that many developmental disorders are characterized by alterations in genes linked to  epigenetic mechanisms. Moreover, environmental factors may represent a risk for TS and they might act via epigenetic alterations. However, very few studies on epigenetics of TS have been done so far. The aim of this project, within the TS-EUROTRAIN network, is to investigate all the main epigenetic mechanisms (such as miRNA regulation, DNA methylation and histone modifications) and their possible involvement in the pathogenesis of TS.Starting from a list of all candidate genes (n=74) for TS available from the literature, we screened for presence of SNPs in the 3’UTR regulatory regions in our susceptibility gene list. This analysis (data retrieved by mirSNP1 and polymiRTS2) led to the identification of those SNPs (n=32) which are predicted to change the seed sequence of in silico proposed miRNAs. Genotyping of a sample of n=564 TS patients and controls was performed by TaqMan method on an OpenArray platform. Furthermore, other polymorphisms in TS candidate genes (GDNF and WFS1) were investigated by real-time PCR. Different techniques were used to analyze DNA methylation patterns (such as targeted bisulfite sequencing, RRBS, HRM) both on human TS and control samples as well as on a rodent model of TS where the striatal dopaminergic neurons were lesioned unilaterally by 6-OHDA (Nespoli et al. WorkPackage 3 in TS-EUROTRAIN). Chromatin immunoprecipitation from striatum of the rodent model was set up using H3K27ac, H3K27me3, H3K4me3 and H3K36me3 antibodies. Chip-Seq from these samples is underway. We identified SNPs in 3’UTR of TS candidate genes, which might affect the binding of some miRNAs and thus have an impact on the gene expression. Some of these were followed up by functional validation studies using luciferase assay (see poster by Vereczkei et al. for more details). Analyses of methylation and chromatin structure also identified differences in a number of gene regions. This project is a combined epigenetic investigation on Tourette Syndrome. Further analyses such as genome-wide Illumina-450K methylation array, targeted bisulfite sequencing and ChIP-Seq will provide more information about the role of epigenetic mechanisms in the pathophysiology of TS.

MicroRNA regulation of candidate genes in Tourette Syndrome(WCPG 2014)

Gilles de la Tourette Syndrome (TS) is a neurodevelopmental disorder that presents early in childhood and is marked by the appearance of multiple involuntary motor tics and at least one vocal tic. It presents high comorbidity rates with other disorders such as attention deficit hyperactivity disorder (ADHD) and obsessive compulsive disorder (OCD). Compared to other psychiatric disorders very little is known about the genetic background of TS. MicroRNAs are single stranded RNAs of ~22 nucleotides which regulate gene expression and their importance seems to be emerging in the last few years. The aim of this work is the identification of candidate genes involved in the pathogenesis of TS and possibly also implicated in OCD and ADHD; the selection of miRNAs regulating the expression of those genes and the study of genetic variants in the candidate genes involved in this regulation, thus identifying possible targets in the pathomechanism of TS. The list of candidate genes was obtained through data available in the literature, while the list of the corresponding miRNAs was acquired through the interpolation of data from miRNA databases such as miRDB, DIANA, TargetHumanScan, microRNA.org and miRTarBase. Subsequently, a ranking was made based on the number of publications implicating a genetic link to TS and the other co-morbidities, as well as some other factors. Starting from the list of all candidate genes for TS (n=74) we cross-referenced between the set of genes that were also implicated in OCD and ADHD (n=30). Then, we searched miRNAs targeting these candidate genes seeking for a correlation between data form different databases and genes as well. We were able to track down a list of candidate miRNAs (n=27). First, we compared and contrasted the target miRNAs from the different databases to identify a short-list for each gene. Second, we looked for a connection between miRNAs targeting different genes within our list. Third, we identified some miRNAs (eg. mir-200, mir-374, mir-429) in our top ranking gene set (for example SLITRK1, DRD4, IMMP2L), as well as some others (such as mir-520, mir-302) with the capacity to regulate the highest number of genes in our total gene set. The sets were also validated for evidence of expression in the CNS. Finally, we searched for genetic variation in regulatory sequences of these genes. The lack of information and the dissonance between the available databases required certain adjustments and a personal elaboration of the data in order to establish a credible list of genes and miRNAs for subsequent functional assays. So far there is only one GWAS and only a few miRNA studies focusing on TS. This project implemented a combined and systematic approach to identify plausible candidate gene-miRNA pairs and genetic variants in 3’-UTR regulatory regions for further functional studies in order to shed some light on the pathophysiology of TS.

Genetic and Epigenetic risk factor in Tourette Syndrome (Danube Epigenetics 2014)

The aim of this work is the identification of candidate genes involved in the pathogenesis of Tourette Syndrome (TS) and possibly also implicated in obsessive compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD); the selection of miRNAs regulating the expression of those genes; the study of genetic variants in the candidate genes involved in this regulation, thus identifying possible targets in the pathomechanism of TS. Starting from the list of all candidate genes for TS we cross-referenced between the set of genes that were also implicated in OCD and ADHD. Then, we searched miRNAs targeting these candidate genes seeking for a correlation between data form different databases and genes as well. We were able to track down a list of candidate miRNAs. First, we compared and contrasted the target miRNAs from the different databases to identify a short-list for each gene. Second, we looked for a connection between miRNAs targeting different genes within our list. Third, we identified some miRNAs in our top ranking gene set as well as some others with the capacity to regulate the highest number of genes in our total gene set. Finally, we searched for genetic variation in regulatory sequences of these genes. The lack of information and the dissonance between the available databases required certain adjustments and a personal elaboration of the data in order to establish a credible list of genes and miRNAs for subsequent functional assays. This project implemented a combined and systematic approach to identify plausible candidate gene-miRNA pairs and genetic variants in 3’-UTR regulatory regions for further functional studies in order to shed some light on the pathophysiology of TS.

An Integrated Molecular Landscape for Tourette syndrome Yields Novel Clues for Diagnosis and Treatment.

Tourette syndrome (TS) is a childhood-onset neurodevelopmental disorder characterized by multiple and involuntarily motor tics and at least one vocal tic that persist for more than one year. TS has a prevalence of 0,3-0,8 % and occurs more frequently in boys than girls. In addition, the disorder is often accompanied by other, comorbid neurodevelopmental disorders, especially obsessive-compulsive disorder (OCD) and attention deficit hyperactivity disorder (ADHD). TS is caused by a complex interplay between genetic and environmental factors and although it is highly heritable, a thorough understanding of the biological processes underlying its etiology is essentially lacking. Therefore, we built a molecular landscape for TS, through applying an approach that we have used before for other neurodevelopmental disorders such as ADHD (Poelmans et al., 2011) and autism (Poelmans et al., 2013) and based on all available genetic and expression data (Paschou et al., 2013; Scharf et al., 2013; Lennington et al., 2014; Tian et al., 2011; Tian et al., 2012). To build a molecular landscape of the (dysregulated) biological processes underlying TS, we combined a genetic network analysis of the top-ranked genes from the only thus far published genome-wide association study (GWAS) of TS (Scharf et al., 2013)  with elaborate literature searches for these GWAS genes. We also integrated genes implicated in TS through other evidence, including functional and animal studies, into the landscape. In addition, we corroborated our findings and refined the landscape through an upstream regulator analysis of the published genome-wide expression data from (postmortem) brain (Lennington et al., 2014) and blood of TS patients (Tian et al., 2011; Tian et al., 2012). We also applied our customized individual SNP-weighted P-value (ISWP) calculation method to the summary statistics data from the published TS GWAS to identify (additional) genes implicated in TS. The molecular TS landscape is located in neurons and microglia cells and regulates a number of distinct biological processes and signaling cascades that contribute to key neuronal functions such as neuronal growth and myelination. Moreover, cascades within the landscape are directly linked to some of the etiological hypotheses about TS, such as disturbances in GABA and histamine metabolism. Lastly and importantly, the landscape contains putative biomarkers and drug targets for TS that could be "leads" for the further development of novel diagnostic and therapeutic strategies. This approach provides novel insights into the genetic and molecular underpinnings of TS, and highlights putative biomarkers and drug targets as worthy of further investigation as diagnostic and therapeutic tools.

 

ESSTS Annual Meeting COST International Conference for Tourette Syndrome (2014)

Obsessive-Compulsive Disorder (OCD) is a neuropsychiatric illness with a complex genetic etiology. It is often associated with comorbid behavioral diseases such as Tourrette’s Syndrome, Attention deficit hyperactivity disorder (ADHD), and tics disorder. Several studies have sought candidate genes for this disorder with largely inconsistent results. Therefore, recent collaborative efforts have aimed at increasing sample sizes and aimed at replication of results in discovery cohorts. Here we report results from a Genome-Wide Association Analysis (GWAS), to search for common Single Nucleotide Polymorphisms (SNPs) responsible for predisposition for OC symptoms, and a set-based test (PLINK) for the association between sets of markers (genes) and OC symptoms; this combines the effects of all SNPs in a gene into a test statistic and considers the association between the trait and the genes instead of all markers individually. In a primary analysis we performed a GWAS on 6931 subjects registered at the Netherlands Twin Register; secondly, for the identification of genes associated with OC symptoms, we performed a set-based test based on permutations at the gene level. By complementing GWAS with a gene-based test, a gene centric result is obtained with the relative importance of each gene. Furthermore, confounding factors such as Linkage Disequilibrium (LD) structure and gene size are corrected for. Combining these two approaches is ideally suited for pathway analysis to better interpret the findings from GWAS. We hope with this work to set the ground for a more clear understanding of the genetic susceptibility variants underlying the genetic architecture of OCD.

World Congress of Psychiatric Genetics (WCPG 2014)

Obsessive-Compulsive Disorder (OCD) is a neuropsychiatric illness with a complex genetic etiology. It is often associated with comorbid behavioral diseases such as Tourrette’s Syndrome, Attention deficit hyperactivity disorder (ADHD), and tics disorder. Several studies have sought candidate genes for this disorder with largely inconsistent results. We measured Obsessive Compulsive (OC) symptoms using 12-item Padua Inventory Revised Abbreviated in a longitudinal sample of 20449 individuals (twins and siblings) registered at the Netherlands Twin Register, to estimate the relative genetic and environmental component of OC symptoms and the stability of OC symptoms across time. Our results, for comparing the observed patterns in familial correlations between monozygotic twins and other first-degree relatives, suggest that genetic factors play a role in individual differences for OC symptoms and that the OC scale is stable (after an average period of about 2.5 years). Next, we performed Genome-Wide Association Analysis (GWAS) in 6931 individuals to search for common Single Nucleotide Polymorphisms (SNPs) predicting level of OC symptoms. We obtained one significant SNP located on Chromosome 19, exceeding the genome-wide threshold for significance. In addition, using the GWAS results we performed a gene-based test for the association between sets of markers clustered in and around genes, and OCD symptoms; this combines the effects of all SNPs in a gene into a test statistic per gene and considers the association between the trait and the genes instead of all markers individually. No statistically significant result was obtained for the tested genes. By complementing GWAS with a gene-based test, a gene centric result is obtained with the relative importance of each gene. Furthermore, confounding factors such as Linkage Disequilibrium (LD) structure and gene size are corrected for. Combining these two approaches is ideally suited for pathway analysis to better interpret the findings from GWAS. We hope with this work to set the ground for a more clear understanding of the genetic epidemiology and the genetic susceptibility variants underlying the genetic architecture of OCD.

1st World Congtress on Tourette Syndrome and tic disorders

Hoarding, Obsessive-Compulsive Disorder and Tourette Syndrome are common psychiatric disorders that share symptom overlap, which might partly be a result of shared genetic variation. Population-based twin studies have found significant genetic correlations between hoarding and OCD symptoms, with correlations varying between 0.1 and 0.45. Other lines of research including clinical samples and GWAS or CNV data to explore relationships between tics and OCD have failed to detect shared variation. Here our aim was to extend current knowledge on the genetic structure underlying hoarding, OC symptoms and tics and, in a trivariate analysis, assess the degree of common and unique genetic factors contributing to the etiology of these disorders. METHODS. Data have been gathered from participants in the Netherlands Twin Register 4 comprising a total of 5293 individuals from a sample of adult monozygotic (n=2460) and dizygotic (n=2833) twin pairs (mean age 33.61 years). The data on Hoarding, Obsessive-Compulsive Disorder and Tourrette were simultaneously analyzed in Mplus. A genetic a liability threshold model was fitted to the twin data, analyzing heritability of phenotypes and of their co-morbidity. RESULTS AND DISCUSSION. Following the criteria for clinical diagnosis in all phenotypes, 6.8% of participants had a diagnose for Hoarding, 6.3% for OCD, and 12.8% for TS. p { margin-bottom: 0.1in; direction: ltr; line-height: 120%; text-align: left; widows: 2; orphans: 2; } Genetic factors explained 52.7%, 69.8% and 74.2% of the phenotypic covariance between Hoarding-OCS, Hoarding-TS and OCS-TS, respectively. Substantial genetic correlations were observed between Hoarding and OCS (0.46), Hoarding and TS (0.39) and between OCS and TS (0.53). CONCLUSIONS. These results support the contribution of genetic factors in the development of these disorders, as well as for their co-morbidity. Furthermore, the values for the genetic correlations indicate that there are shared components in the genetic architecture of these disorders.

Heritability of Tic Disorders: a Twin-Family Study. European Society for the study of Tourette Syndrome (ESSTS)

Genetic-epidemiological twin family studies to estimate the relative contributions of genetic and environmental factors to tic symptoms are extremely scarce. Therefore we explored the extent to which genetic and environmental factors contribute to tics, using various phenotypic definitions ranging between mild and severe symptomatology, in a large population-based adult twin family sample. In an extended twin-family design, we analyzed lifetime tic data reported by adult mono- and dizygotic twins (n= 8,323) and their family members (n=7,164; parents and siblings) from 7,311 families of the Netherlands Twin Register (NTR), using  tic self-reports based on the Schedule for Tourette and Other Behavioral Syndromes (STOBS) (TSAICG, 2007). We used genetic Structural Equation Modeling (SEM) to analyze four probable tic disorder definitions: three dichotomous and one trichotomous phenotype, characterized by increasingly strict criteria to define tic status. Prevalence rates for the different tic definitions varied between 0.3-4.5%. Tic frequencies decreased with increasing age. Heritability estimates varied between .25 and .37, depending on phenotypic definitions, with the more broadly defined phenotypes showing lower heritability and narrowest confidence intervals (0.31, 95% CI [0.23, 0.40]). There was no evidence for assortative mating, influence of common environment, or for non-additive genetic effects on any of the tic phenotypes. Heritabilities of mild and severe tic phenotypes were found to be moderate in effect size.

Exploration of the role of the histamine metabolic and signaling pathways in the pathogenesis of Gilles de la Tourette Syndrome. 65Th congress of HSBMB, November 28-30 2014, Thessaloniki Greece.

Gilles de la Tourette Syndrome (TS) is a neurodevelopmental disorder of childhood onset, marked by the appearance of multiple involuntary movements and vocalizations (tics), likely caused by a complex genetic basis, which remains largely elusive. The implication of the L-histidine decarboxylase (HDC) gene recently raised the intriguing hypothesis of the involvement of the histaminergic pathway in disease onset1. In the present study we report on a targeted re-sequencing approach with NGS technology, aiming to identify disease-causing variants of HDC, following up on our recent work that certain HDC haplotypes are significantly associated with TS in Caucasians2. In addition, we present an investigation of potential associations of variation across genes encoding for proteins involved in the histamine metabolic and upstream signaling pathway, in TS and controls, by:

  • Exploring possible genetic associations of TS to polymorphic variants in genes encoding: a) Proteins of the histamine metabolic pathway, including D-amino-oxidase and histamine N-methyltransferase, and b) Histamine’s G-protein-coupled receptors HRH1-HRH4. Information will be mined upon genotyping family trios of French origin, with one or more affected individuals, using genome-wide SNP arrays, and by genotyping of selected tagging SNPs on independent family trios of Caucasian origin.

  • Investigating altered expression patterns of the aforementioned genes in: a) TS cases versus controls, and b) Subgroups of TS patients (i.e. according to tic severity or related comorbidities). Custom-made array-based assays are being developed, in order to examine differential gene expression across genes representing the two pathways, in ~50 TS cases versus an equal number of controls. Selection of the most stably expressed housekeeping genes (HKGs) in peripheral blood is based on analyzing an array of 12 HKG candidates in cases and controls. Whole-genome microarray transcriptome data from a similar project are being analyzed in order to further inform our selection of targeted genes and to compare microarray-generated data versus real-time PCR-generated gene expression data from the same tissue.  

 

Targeted re-sequencing approach of TS candidate genes implicates potentially functional variants in TS etiology. European Society for the study of Tourette Syndrome (ESSTS) 2014 Annual Meeting, April 25-26 2014, Paris, France.

Although the genetic basis of Tourette Syndrome remains unclear, several candidate genes have been implicated. Here, using a set of 382 TS individuals we investigate four of the most prominent candidate genes for TS (HDC, SLITRK1, BTBD9 and SLC6A4) and attempt to identify possibly causal variants using a targeted re- sequencing approach and next generation sequencing technology. Identification of possible disease causing variants under different modes of inheritance was performed using the algorithms implemented in VAAST. Furthermore, we propose a novel method to prioritize and rank variants from exome sequencing capture by integrating information from open-source annotation databases. We identified several novel candidate variants that could be implicated in TS etiology and are yet to be further validated using Sanger sequencing. Our method can be used with supplementary bioinformatics filters to prioritize variants in future sequencing projects.

Investigating gene pathways involved in the aetiology of Gilles de la Tourette syndrome. 12th World Congress of Biological Psychiatry, 14-18 June 2015, Athens, Greece.

Tourette syndrome (TS) is a neurodevelopmental disorder with a complex genetic background characterised by vocal and motor tics (Paschou P. 2013. Neuroscience & Behavioral Reviews).Using meta analysis data comprising of 1285 TS cases and 4964 TS ancestry-matched controls of European ancestry from the first genome-wide association study (GWAS) of TS (Scharf et al 2013,Molecular psychiatry), we performed functional analysis in order to dissect the molecular mechanisms underlying TS. Using various bioinformatics tools for single nucleotide polymorphism (SNP) based and gene based functional analysis, we perform candidate gene prioritization, geneset enrichment and tissue enrichment. Using combined information from the enriched functional and pathway results we also construct functional interaction networks. Our results provide additional insights into the possible genetic mechanisms causing TS. We conclude that performing pathway analysis on GWAS data can help prioritize SNPs and genes operating together in the etiology of TS.

Network Analysis on Tourette Syndrome associated genes using genome wide. 1st World Congress on Tourette Syndrome & Tic Disorders, London, United Kingdom, 24-26 June 2015

Tourette syndrome (TS) is a neurodevelopmental disorder with a complex genetic background characterized by vocal and motor tics. Analyzing 4220 TS cases of European ancestry and 8994 ancestry-matched controls from the first and second genome-wide association study (GWAS) of TS, we investigate the molecular mechanisms underlying TS by performing protein-protein interaction analysis and constructing networks of genes that harbor the top TS-associated and potentially functional SNPs. Using DAPPLE, we construct networks of the top TS associated genes, looking for significant physical connectivity among proteins encoded for genes in loci associated to disease according to protein-protein interactions reported in the literature. Using a novel ranking method developed for prioritizing genes and variants according to functional annotations, we rank variants by functional importance, exploiting information from open-source annotation databases. Using Cytoscape and combined information from our protein-protein interaction and prioritization analysis we build networks of genes that are implicated in TS etiology. Our ranking method prioritizes various genes and single nucleotide polymorphisms (SNPs) playing a role in neurogenesis (PNMA1, EPHA1) and our network analysis reveals pathways related to nervous system and signaling pathways. Protein interaction networks from DAPPLE reveal genes involved with cell adhesion, neurotransmission, and neuronal cell development (including NRG1, GABRA2, NEUROG1, CSDC2). Our analysis reveals novel genes operating together in the aetiology of TS.

TS-EUROTRAIN: European wide investigation of the etiology and pathophysiology of Gilles de la Tourette syndrome and related disorders, 12th World Congress of Biological Psychiatry, 14-18 June 2015, Athens, Greece.

Common psychiatric disorders are rarely caused by single gene effects that exhibit classic mendelian patterns of inheritance.  The majority of psychiatric disorders fall into the category of complex genetic disorders, which are likely caused by genetic susceptibility factors that interact with the environment to confer the total risk of acquiring complex phenotypes. Such complex traits do not usually exhibit clear-cut boundaries between affected and non-affected states, but exhibit a phenotype that extends over a range of severity and comorbidity. Over the past decade, it has become increasingly evident that a wide range of clinically distinct, developmental and neuropsychiatric disorder exhibit a significant degree of etiological overlap. As a result, there is an increased need for the identification and investigation of good models of developmental and psychiatric disorders in a bid to (a) explore the shared etiology across such disorders and to (b) develop new treatment strategies. Gilles de la Tourette Syndrome  (GTS) provides such a model, as it is a complex developmental disorder that wavers along the fine margin between psychiatry and neurology with an enshrouding set of motor and behavioral symptoms. Clinically, GTS is characterized by the presence of multiple motor tics and one or more vocal tics, and also presents with other comorbid conditions that most commonly include Attention Deficit Hyperactivity Disorder (ADHD) and Obsessive Compulsive Disorder (OCD). Currently, a complete picture of the etiology and the pathogenesis of motor and behavioral symptoms of GTS is an open question in the literature. TS-EUROTRAIN is a European FP7 funded consortium that aims to push the build on the current knowledge in the field by tackling several major challenges that include: (i) assembling a large genetic database for the evaluation of the complex etiology of GTS and related disorders with high statistical power; (ii) exploring the role of gene-environment interaction as the role of epigenetic phenomena in GTS have not even begun to be evaluated; (iii) employing endophenotypic based approaches to dissect the basis of  shared etiology between TS, OCD and ADHD; (iv) establish new development animal models for GTS as such models are important and severely lacking; (v) gain new insights into the neurobiological mechanisms of GTS via transverse and longitudinal imaging based studies to pave the way for new treatment strategies.

 

Pathway Analysis on Genome-wide data for Tourette Syndrome Shows Enrichment in Genes expressed in Nervous System Tissues. American Society of Human Genetics Annual Meeting. October 6-10 2015,Baltimore,USA.

Gilles de la Tourette Syndrome is a childhood-onset neuropsychiatric disorder that is characterised by a multitude of vocal and motor tics. Recent advances in the genetics of Gilles de la Tourette Syndrome are starting to shed new light in its genetic aetiology. Yu et al. in 2015 report a second Genome Wide Association Study on Gilles de la Tourette Syndrome on 2,859 cases/3,855 controls. Following meta-analysis with the first Genome Wide Association Study (1,117 cases /4,955 controls) and the TIC Genetics Trios (184 cases/184 controls), they report multiple LD-independent SNPs with p-value less than 10-5. These SNPs implicate novel loci on Tourette Syndrome pathogenesis. We focused on the top scoring SNPs from the second Genome Wide Association Study on Gilles de la Tourette Syndrome for downstream pathway analysis. For the pathway analysis we used  DEPICT, a novel tool for the interpretation of GWAS results, and additional pathway analysis software, including MAGENTA and INRICH. Furthermore, we also applied a set-based association approach on the Second Genome Wide Association Study case and control data. In order to perform the set-based association, we collected pathway gene sets from KEGG, REACTOME, Molecular Signatures Database, OMIM and we constructed sets of SNPs located at genes present on the pathway gene sets. DEPICT analysis showed significant enrichment among top TS associated SNPs in genes expressed in nervous system tissues, including the parietal lobe and the basal ganglia. We also report the results from the set based association analysis, which further illuminate candidate pathways for investigation. Our study aims to untangle the complex interaction networks of genes implicated in TS and points to candidate etiological pathways for further investigation in future studies.

Pathway Analysis on Genome-wide data for Tourette Syndrome Shows Enrichment in Genes expressed in Nervous System Tissues. 1st Tourette World Congress. June 24-26 2015, London, UK.

 

Gilles de la Tourette Syndrome (GTS) is a common neurodevelopmental disorder characterized by the occurrence of multiple chronic motor and vocal tics. Recent advances in the genetics of GTS are starting to illuminate its genetic aetiology. We have recently completed a second Genome Wide Association Study (GWAS) of GTS on 2,859 cases/3,855 controls. A subsequent meta-analysis with existing GTS GWAS data (final sample size of 4645 cases and 9,750 controls) generated multiple LD-independent SNPs with p-values less than 10-5. These SNPs implicate novel loci on Tourette Syndrome pathogenesis. Our study uses downstream pathway analysis to functionally investigate these results. In this study, we performed pathway analysis using a multitude of different methods on the GTS GWAS data. We focused on the top scoring LD-independent SNPs from the GTS GWAS meta-analysis by using DEPICT, a novel method for interpreting GWAS results, INRICH, FORGE and DAPPLE. We also utilized a set-based association approach on the second GWAS genomic data. In order to perform the set-based association, we collected pathway gene-sets from the Molecular Signatures Database, an in-depth library of collections of annotated gene sets, and constructed sets of SNPs located at genes present on the pathway gene-sets. The set-based association was run on PLINK using 1 million permutations. INRICH analysis reported a significant enrichment p-value of 10-5 that after correction for multiple testing was significant at 6x10-3 in the TCF3 binding motif gene set, hinting towards the involvement of neurodevelopmental transcription factor binding sites in Tourette Syndrome aetiology. This finding was further reinforced by the results of the set-based association, where the same gene set was the top hit, achieving an empirical p-value of 4x10-5, which still remained significant after correcting for multiple testing. DEPICT analysis showed significant enrichment among top GTS associated SNPs in genes expressed in nervous system tissues, including the parietal lobe and the basal ganglia. Our study has resulted in the enrichment of a neurodevelopmental transcription factor, TCF3, binding sites. TCF3 competes against the Wnt/β-catenin pathways, by a complex interplay. Involvement of neurodevelopment in the genetic aetiology of GTS is an analytical direction that could potentially lead to interesting findings. Our results aim to untangle the complex interaction networks of genes implicated in GTS and points to candidate etiological pathways for further investigation in future studies

Cortico-striatal-thalamo-cortical white matter structure in Tourette syndrome and ADHD (OHBM 2017)

Dysregulation within cortico-striatal-thalamo-cortical (CSTC) networks is hypothesized to underlie multiple disorders including Tourette syndrome (TS), attention-deficit/hyperactivity disorder (ADHD) and obsessive-compulsive disorder (OCD; Leisman & Melillo, 2013). Dysfunction of individual loops is supposedly responsible for specific symptoms such as tics, obsessions and compulsions (Mink, 2006) with the interplay between the loops accounting for the high rate of comorbidity between the disorders. However, evidence from neuroimaging studies of the respective disorders implicating the structural connectivity of the CSTC networks has been inconsistent (van Ewijk et al, 2012; Makki et al, 2009) possibly due to methodological variation across studies, small sample sizes and heterogeneity of participants, especially regarding age. Here, we investigated CSTC networks associated with tics, ADHD and obsessive compulsive symptoms in a large group of children with TS and/or ADHD and healthy controls. We focussed on a restricted age range (8-12) where both TS and ADHD are highly prevalent. Eighty-two diffusion-weighted (DW, B0=2, diffusion gradients=64 [b=1500s/mm2]) and T1-weighted (MPRAGE) magnetic resonance datasets that survived quality assessment were included. Children with TS (n=22, age 10.7[1.2], n=8 of which also had ADHD), 19 with ADHD (age 11.1[1.3]) and 41 healthy controls (age 11.0[1.1]) were included. DW images were pre-processed to correct for motion, cardiac and other imaging artefacts (PATCH (Zwiers, 2010)). The constrained spherical deconvolution (CSD) algorithm was applied followed by EuDX tractography within Dipy (Garyfallidis et al, 2014). Tractography seeds (striatum and thalamus) and targets (superior frontal, rostral middle frontal, precentral, lateral orbito frontal and caudalmiddle frontal) were derived from subject specific Freesurfer reconstructions (27 tracks were propagated per seed voxel, step size = 0.5, stop threshold fractional anisotropy [FA]=0.15). Targets were selected based on previous literature. In total 20 connections were isolated; 2 seeds x 5 targets x 2 hemispheres. FA was extracted from each isolated tract with TrackVis (Wang et al, 2007). FA was modelled in relation to TS (categorical), ADHD-severity (continuous across all subjects) and their interaction (as an indicator of comorbidity) for each connection of interest, with age and sex as covariates. Analysis revealed no association between FA and TS, ADHD severity or their interaction (p>.05 in all cases following multiple comparison correction) in any white matter tracts between the striatum or thalamus and frontal targets. Furthermore, FA was not related to obsessive-compulsive symptoms, tic severity, duration since tic onset or age at tic onset within participants with TS in any of the tracts (p>.05 in all cases following multiple comparison correction). This is the first study to investigate CSTC white matter microstructure of children with TS and/or ADHD together. In keeping with some (Jackson et al, 2011; Thomalla et al, 2009) but not all (van Ewijk et al, 2012; Makki et al, 2009) of the previous literature investigating these disorders separately we see no white matter differences associated with the disorders. However, methodological limitations remain, most notably due to the size of the seed and target regions. In some cases multiple fibre bundles were reconstructed between a seed (thalamus or striatum) and target. As a result mean FA was sometimes extracted from a composite of multiple bundles rather than one individual fibre bundle and thus potentially influenced by the quality of reconstruction of the respective bundles. Individual bundles should be isolated and compared in future. Variation in CSTC white matter was not associated with TS, ADHD-severity or their interaction in this study. However, further methodological refinements should be implemented to ensure appropriate comparison of tracts before final conclusions can be made.

Cortical development through adolescence and early adulthood (OHBM 2016)

Introduction Despite many years of neuroimaging, the precise developmental changes that occur in the brain during development are yet to be fully characterised. Here we utilised the novel application of intrinsic curvature (IC) analysis of the cortical surface, along with more traditional measures such as cortical thickness (CT), local gyrification index (LGI) and surface area (SA), to help further understand the neurodevelopmental changes that occur in healthy development. IC is predictive of the degree and pattern of gyrification (Ronan et al., 2012) and reflects the differential expansion of the cortex. It is hypothesised to reflect both cortical cell density and cortical connectivity (Ronan et al., 2012). T1 magnetic resonance images from 218 healthy controls (age in years; mean(SD) 16.5(3.37), range 8.3-29.2) collected at two sites in the Netherlands (as part of the NeuroIMAGE study) were used. Data sets were processed with Freesurfer software. IC was calculated with Caret software from the Freesurfer reconstructions. IC, LGI, CT and SA were extracted per region (frontal, parietal, occipital, temporal, insula and cingulate), using regions generated by combining labels from the Desikan-Killiany Atlas (Desikan et al., 2006). Developmental profiles for each parameter per region were modelled using generalised additive mixed-effect models (GAM; Wood, 2006) with the gamm4 (Wood and Scheipl, 2014) and mgcv (Wood, 2011) packages in R. Each model also included scanner location and sex and accounted for the non-independence of subjects (sibling pairs) by modelling family as a random factor. IQ and the interaction of age and sex were additionally investigated. Results were adjusted for multiple comparisons at the p=0.002 level, according to a Bonferroni correction. IC was significantly inversely associated with age in the frontal, parietal, temporal, cingulate and insula regions, indicating lower IC with increasing age. LGI and CT were also significantly inversely associated with age in all regions, with both LGI and CT declining steadily throughout the age range. SA was significantly associated with sex in all regions, with male SA consistently larger than that of female SA, while there was no significant effect of age. A significant effect of sex was also seen with males consistently having a larger LGI than females in all regions and also in IC of the frontal lobe, however these sex effects did not persist when brain size was accounted for by including total brain SA in the models. There were no age-by-sex interactions or effects of IQ seen in any of the measures. In keeping with the previous literature, and now reporting regionally instead of globally, we observed diverse effects of age and sex on the different cortical measures, including IC. We could not confirm previous findings (Raznahan et al., 2011) showing a difference between males and females in CT; furthermore, we did not detect an effect of age on SA within the age range under investigation. This may be due to the cross-sectional character of the study and/or the restricted age range which had a particular focus on adolescence. This unfortunately resulted in the study being underpowered to detect cortical changes in early adolescence and early adulthood. This study adds an innovative cortical measure to the study of neurodevelopment, IC, which appears to decrease with age. This may reflect a higher neuronal density and/or a decrease in cortico-cortical connectivity.

Basal ganglia structure in Tourette’s Disorder and Attention-Deficit/Hyperactivity Disorder (Eunethydis 2016)

Tourette’s disorder (TD) and Attention-Deficit/Hyperactivity disorder (ADHD) which often co-occur have both been associated with structural variation of the basal ganglia (BG)1,2, although not consistently and many studies have failed to account for comorbidity, medication use and other possible confounders. Herein, we aimed to determine the relationship between TD, ADHD and the combination thereof and BG structure. T1-weighted magnetic resonance (MR) images and phenotypic information from 141 children (age 8-12 years) were used; TD (n=47, age=10.5[1.4], 47% with comorbid ADHD), ADHD (n=39, age=10.7[1.3]) and controls (n=55, age=11.0[1.0]). Caudate nucleus (CN), putamen (Pu) and globus pallidus (GP) volumes were determined from MR images following processing with the FMRIB integrated registration and segmentation tool (FIRST)3. Structure surfaces were reconstructed and used to determine shape. Across all participants, BG nuclei volume and shape were estimated in relation to TD (categorical), ADHD severity (continuous measure across all participants) and TD-by-ADHD severity interaction. Sex, age and IQ were included as covariates with total brain volume additionally included in the volume analysis. Analysis revealed no difference in CN, Pu or GP volumes between children with and without tics (F(1,134)=0.29, p=0.59; F(1,134)=0.27, p=0.60; F(1,134)=3.60, p=0.06), no association with ADHD severity (F(1,134)=0.001, p=0.98; F(1,134)=0.04, p=0.85; F(1,134)=0.06, p=0.81) and no interaction between TD and ADHD severity (F(1,133)=2.55, p=0.11; F(1,133)=0.14, p=0.71; F(1,133)=0.80, p=0.37). Similarly shape analysis showed no significant association with either TD, ADHD severity or their interaction. In conclusion, we found no evidence that TD, ADHD or the combination thereof are associated with structural variation of the BG.

Glutamate levels in the Fronto-striatal circuit in Tourette’s Disorder and Attention-deficit/ Hyperactivity Disorder: A proton Magnetic Resonance Spectroscopy study (ESSTS 2016)

Both Tourette’s Disorder (TD) and Attention-Deficit/Hyperactivity Disorder (ADHD) have been related to abnormalities in glutamatergic neurochemistry in the fronto-striatal circuitry. TD and ADHD often co-occur and this co-occurrence has been insufficiently taken into account in prior studies. Here, we used proton magnetic resonance spectroscopy (1H-MRS) in children between 8-12 years of age (TD n=15, ADHD n=39, TD+ADHD n=29 and control n=53) as an in vivo method of evaluating glutamate concentrations in the fronto-striatal circuit. Spectra were collected on a 3 Tesla Siemens scanner from two 8 cm3 voxels in each participant: the anterior cingulate cortex (ACC) and the left dorsal striatum. LC-model was used to process spectra and generate absolute glutamate concentrations. To determine if there was a significant effect of group on concentrations, a one-way analysis of variance was performed in R. Neither TD or ADHD or their combination was associated with differences in glutamate levels in either the ACC (F(3,132)=0.97, p=0.41) or striatum (F(3,121)=0.59, p=0.62). Variation in glutamate concentration was unrelated to age, sex, medication use, IQ, tic or ADHD severity. Obsessive-compulsive (OC) traits were positively correlated with ACC glutamate concentration within the participants with TD (r=0.45, pcorrected=0.02). We found no evidence that TD or ADHD are directly associated with alterations in glutamatergic transmission within the fronto-striatal circuits. However, OC-traits correlating with ACC glutamate concentration supports a role for involvement of glutamatergic transmission in OC-symptoms within TD. As previous literature is inconclusive, further investigation is warranted to determine if our finding  extends to OC-symptoms outside of TD.

Basal ganglia structure in Tourette’s Disorder and Attention-Deficit/Hyperactivity Disorder: Influence of comorbidity and medication use (ECNP 2016)

Structural variation of the basal ganglia has been associated with both Tourette’s disorder (TD)[1] and Attention-Deficit/Hyperactivity disorder (ADHD)[2]. However, previous studies have been inconsistent in their findings and often failed to account for comorbidity, medication use and other possible confounders. This is of particular importance considering the high rate of comorbidity of ADHD in TD (~40%) [3] and the common use of antipsychotic medication in the treatment of tics. Antipsychotics have previously been associated with grey matter volume reductions in other patient groups (i.e. schizophrenia) and animal models [4]. Herein, we aimed to determine the relationship of TD, ADHD and the combination thereof with basal ganglia volumes, taking medication into account. A total of 141 participants (age 8-12 years) were included in this analysis from whom good quality T1-weighted magnetic resonance (MR) datasets and full phenotypic information were available; Conners’ Parent Rating Scale, Yale Global Tic Severity Scale, Children’s Yale-Brown Obsessive Compulsive Scale, Children’s Social Behaviour Questionnaire, medication history and estimated full-scale IQ. These included participants with TD (n=47, age=10.5[1.4], 47% with comorbid ADHD), ADHD (n=39, age=10.7[1.3]) and controls (n=55, age=11.0[1.0]). T1-weighted datasets were processed within the FMRIB Software Library (FSL). This included registration to standard space and segmentation to isolate subcortical structures with the FMRIB integrated registration and segmentation tool (FIRST)[5]. Volumes for the caudate nucleus and putamen were then extracted for both the left and right hemisphere. Across all participants, basal ganglia volumes were estimated as a function of TD (categorical), ADHD severity (continuous measure across all participants) and TD-by-ADHD severity interaction. Total brain volume, sex, age and IQ were included as covariates. To assess left-right asymmetry, hemisphere was added as a repeated measures variable interacting with TD. Furthermore, a left-right asymmetry index (AI=(R-L)/(R+L)) was calculated to investigate associations between asymmetry and phenotype/medication within the TD group. Analysis revealed no difference in absolute caudate nucleus or putamen volumes between those with and without tics (F(1,134)=0.29, p=0.59; F(1,134)=0.27, p=0.60), no association with ADHD severity (F(1,134)=0.001, p=0.98; F(1,134)=0.04, p=0.85) or interaction between ADHD severity and TD (F(1,133)=2.55, p=0.11; F(1,133)=0.14, p=0.71). A trend towards a hemisphere-by-TD interaction in the caudate nucleus (F(1,139)=3.13, p=0.08) was seen. There was no association between caudate nucleus AI and tic severity, obsessive-compulsive symptoms, age of tics onset or duration since tic onset. The degree of AI did not differ with medication status regarding current antipsychotic use versus no-antipsychotic use (F(1,42)=0.53, p=0.47) or current stimulant versus no-stimulant use (F(1,42)=-0.22, p=0.64). In conclusion, we found no evidence that TD, ADHD or the combination thereof are associated with volume differences in the basal ganglia. A trend finding tentatively supports previous literature indicating alterations in left-right asymmetry associated with TD. However, the degree of asymmetry was unrelated to phenotypic measures or medication status. Further research is required to confirm this result regarding medication as the current study may have been underpowered to detect their effects.  

Cortical connectivity in adolescents and young adults with attention-deficit/hyperactivity disorder (ECNP 2015)

Attention-deficit/hyperactivity disorder (ADHD) is a common neurodevelopmental disorder affecting approximately 5% of the school age population and characterised by a pattern of pervasive inattention and/or hyperactivity and impulsivity that is associated with impairments of functioning. Neuroimaging studies have highlighted structural and functional abnormalities associated with ADHD; including cortical and connectivity differences based both on structural (diffusion magnetic resonance imaging [MRI]) and functional MRI data. The innovative approach of applying intrinsic curvature analysis to the cortical surface applied herein allowed us determine whether the structural connectivity abnormalities thus far identified at a centimeter scale also occur at a millimeter scale within the cortical surface. Differential expansion of the surface during development underlies both intrinsic curvature and also results in a higher proportion of shorter connections, from which we infer more efficient connectivity [1, 2]. Thus, we can use intrinsic curvature as a quantifiable measure of connectivity within the cortex. Furthermore, intrinsic curvature is related to gyrification [2]; however, it holds greater power to detect subtle shape differences in the cortex indicative of abnormal neurodevelopment [2]. We hypothesised that previous gyrification findings may have been obfuscated by the scale of morphological parameters employed; we therefore proposed that the intrinsic curvature may have increased power to detect subtle differences in cortical connectivity of ADHD, but that these would not be detectable with the related but larger-scale gyrification index. Structural MR images from NeuroIMAGE (www.neuroimage.nl) participants, whose scans were acquired at one of two test sites (Amsterdam and Nijmegen), were utilised in this study. 618 full, good quality datasets from 374 families were available. Of these, 164 were healthy controls [mean(SD) age 16.8 (3.2)], 306 had ADHD [17.2 (3.4)] and 148 were healthy siblings of a person with ADHD [17.7 (3.8)]. FreeSurfer was used to reconstruct the cortical surfaces and compute the local gyrification index (lGI) for each point on the surface (vertex) [3]. Intrinsic curvature was calculated from the FreeSurfer reconstructions per vertex using Caret software. The skew of the intrinsic curvature distribution was calculated per hemisphere. Because we were interested in the degree of intrinsic curvature independent of brain size, we used the skew of the distribution rather than the mean for analysis [2, 4]. A vertex-wise general linear model analysis of lGI was performed using glmfit in FreeSurfer with age, gender, scanner site and total brain volume as covariates and a monte carlo simulation for multiple comparison correction. An univariate analysis of co-variance (ANCOVA) was used to assess group differences in intrinsic curvature skew per hemisphere with age, gender, surface area of hemisphere and scanner site as covariates. No group differences were found in lGI or intrinsic curvature skew. Despite the increased sensitivity of intrinsic curvature to subtle morphological abnormalities of the cortical surface we found no differences between groups. This suggests that there are no milimeter scale connectivity abnormalities associated with ADHD. This highlights the diffference between ADHD and other neurodevelopmental disorders like schizophrenia and autism spectrum disorder that have shown differences in intrinsic curvature or related measures [2, 5].

Neural Structure and Function in Tourette syndrome: distinguishing neural structure and function in Tourette syndrome from healthy controls and ADHD (ESSTS 2014)

Rationale: Attention-Deficit/Hyperactivity Disorder (ADHD) is often found comorbid with Tourette syndrome (TS), similarly tic disorders often comprise secondary diagnoses in ADHD, making it difficult to eliminate the potential confounds of comorbidity on research findings in TS. Although the frontostriatal circuits and its main regions, the prefrontal cortex and the basal ganglia have been implicated in TS, there are multiple inconsistent findings in imaging measures and executive functioning in TS. Furthermore glutamate is a major neurotransmitter modulating the activity of the frontostriatal circuits, but its role in TS in unclear. Objectives: The aim of the study is to identify brain (1) structural, (2) functional and (3) connectivity abnormality differences, and (4) neuropsychological differences between TS and ADHD, to elucidate which neural correlates correspond to each condition, which are common and which unique. Similarly (5) glutamate concentrations from the frontostriatal region, acquired with magnetic resonance spectroscopy (MRS), will be compared between groups. Methods: 180 children (n=60 per group, 8-12 years old); (1) TS, (2) ADHD and (3) healthy controls will be recruited and undergo imaging and neuropsychological assessment. Structural, diffusion and functional (resting state and task specific) magnetic resonance images will be obtained as well as MRS glutamate and neuropsychological data. Advanced neuroimaging analysis techniques will be applied, including the use of FreeSurfer. Results from the three groups will be analysed in a multiple regression model. Outcome: This will be the first study to explicitly investigate the neural correlates of TS and ADHD in children.

Exploring neurodevelopmental aspects of tics in a juvenile rat model of repetitive behavior. Society for Neuroscience conference, 17-21 October 2015, Chicago, USA

Tourette Syndrome (TS) is a neurodevelopmental disorder which affects 0.4-1% of the population. It is defined by the presence of multiple motor and at least one vocal tic, typically starting during childood. TS is not only about tics. Indeed, up to 90% of all TS patients experience psychiatric comorbidities, mainly ADHD and OCD, but also behavioral disturbances. The exact cause of TS remains elusive, but tics are supposed to be caused by an abnormal regulation of the motor loop activity, which consequentially leads to the hyper-excitability of motor regions of the brain cortex . Dopamine (DA) appears to have a central role, more likely through its fine regulation of the direct and indirect striatal pathways, which in turn regulate voluntary and involuntary movements. Unilateral 6-hydroxidopamine (6-OHDA) lesion in adult rats is a well-established model used in Parkinson´s Disease research. In this model, selective degeneration of nigrostriatal dopaminergic neurons is chemically induced through the administration of 6-OHDA. Subsequent chronic application of dopaminergic agonists challenge leads to the development of repetitive involuntary movements. This is a consequence of striatal increased sensitivity to DA, which is also a putative pathological mechanism of TS, and is induced in this model via previous DA deprivation. The repetitive involuntary movements observed are sudden, rapid and brief, and occur repeatedly at irregular intervals, mainly involving the contralateral forepaw, face and mouth. During the period of levodopa efficacy, the movements change in intensity and frequency, but not in location. Animals can be distracted, and the involuntary movements reduce when they focus on something. The phenotype was challenged using different dopaminergic and non-dopaminergic agents, with a special emphasis on modulators of metabotropic glutamate receptors.  Several pharmacological approaches have been shown to interfere with the repetitive movements, either worsening or improving the phenotype, indicating possible lines of therapeutic actions, or co-pathogenic mechanisms. This model could allow the evaluation of the consequences of an altered striatal regulation occurring during development, and could also be employed in the investigation of new therapeutic options for tic disorders.

Finding developmental aspects tics in a neurodevelopmental rat model of repetitive behaviors. 1st world congress on Tourette syndrome and tic disorders. June 24-26 2015. London, UK.

Tourette Syndrome (TS) is a neurodevelopmental disorder affecting 0.4-1% of the population and is defined by the presence of multiple motor and at least one vocal tic, started before 18 years of age and lasting for at least 1 year. The exact cause of TS remains still elusive, but a central role seems to be played by dopamine (DA), a key regulator of the direct and indirect striatal pathway  of movement. The unilaterally lesioned 6-hydroxidopamine (6-OHDA) adult rat is a well-established model used in Parkinson´s Disease research. In this model a selective degeneration of nigrostriatal dopaminergic neurons is chemically induced by the administration of 6-OHDA. The following chronic application of levodopa leads to the development of repetitive involuntary movements, mainly involving the forepaw, the neck and the mouth. This appears as a consequence of the striatal super sensitivity to DA, which is also a putative pathological mechanism of TS and is induced in this model via previous DA deprivation. We propose to translate this adult model to juvenile rats, inducing the lesion in postnatal days and monitoring its neurodevelopmental consequences, in order to remedy the complete lack of juvenile animal models featuring a tic-like phenotype. This could allow evaluating the consequences of DA supersensitivity during development, and could represent a new tool to test therapeutic options for tic disorders. METHODS: 21days old male wistar rats received a double stereotaxic injection of 4.4µg of 6-OHDA hydrochloride (n=16) or vehicle (n=8) in the mFB. The injection coordinates had previously been scaled from an adult rat brain (3months old, 600g). Repetitive behavior was induced through the administration of a mixture of levodopa/benserazide (P.O. 6/15 mg/kg) 3 times a week for 3 weeks. The Abnormal Involuntary Movements observed involving the limb, neck and trunk, and the mouth were scored according to previously published protocols. Post-mortem analysis of the lesioned brain included staining with tyrosine hydroxylase (TH), and the quantification of the remaining DA in the denervated striatum via HPLC. RESULTS: Repetitive behavior was induced through the administration of a mixture of levodopa/benserazide (P.O. 6/15 mg/kg) 3 times a week for 3 weeks. The Abnormal Involuntary Movements observed were scored according to a previously published protocol.

Finding developmental aspects and possible drug targets of TS and OCD: metabotropic glutamatergic mechanisms in a neurodevelopmental rat model of repetitive behaviors. European Society for the study of Tourette Syndrome (ESSTS) annual meeting, April 25-26 2014, Paris, France.

Tourette syndrome (TS) is a neurodevelopmental disorder affecting 0.4-1% of the general population. Symptoms become evident during childhood and involve several motor tics and at least one vocal tic. Unfortunately, the etiology of the disease is still unclear, but a broad range of findings support the idea that tics in TS occur as a consequence of a deregulated activity of the corticostriatal-thalamocortical circuit, which leads to an increased sensitivity to dopamine in the striatum (Buse et al. 2013). Up to now, numerous animal models of TS have been published, but all in adult animals. The unilaterally lesioned 6-hydroxidopamine (6-OHDA) rat is a well-established model used in Parkinson´s Disease research. In this model a degeneration of nigrostriatal neurons is chemically induced by the intrastriatal or intranigral administration of 6-OHDA, which selectively targets monoaminergic neurons. Chronic application of L-dopa to 6-OHDA lesioned rats leads to the development of repetitive involuntary movements, mainly involving the forepaw, the neck and the mouth. This appears as a consequence of the striatal super sensitivity to DA, caused by higher surface expression of dopamine receptors, which is a putative pathological mechanism of TS and is induced in this model via previous DA deprivation. We propose to translate this model to juvenile rats, inducing the lesion in postnatal days and monitoring its neurodevelopmental consequences. This could provide a new insight about TS pathological mechanism, and a new tool to test therapeutic option for this disease.

Investigation of the in vivo effect of classical and new psychotherapeutic approaches on the behavioural and neurochemical profile of rat model for Tourette´s syndrome and control animals- an MR spectroscopy study. Spring meeting international graduate school in molecular medicine. April 7-8 2016. Ulm, Germany.

Neurodevelopmental disorders reflect a disequilibrium between excitatory and inhibitory neurotransmitters during brain development. Evidences support glutamate’s (Glu) involvement in Tourette Syndrome (TS) as well as in Attention Deficit and Hyperactivity Disorder (ADHD), where low GABA levels induce hyperglutamatergic state. Glutamate major role in cortico-striatal-thalamo-cortical circuits (CSTC) and the extensive interconnection with dopamine suggest overlapping pathways in tic-generation process and hyperactivity. Almost 90% of TS patients also have ADHD and vice versa 20-30% of ADHD patients also experience tics. Potential treatment options using neuroleptics (aripiprazole) and glutamatergic modulators (riluzole) are here investigated. Glu-GABA metabolism in Wistar Kyoto (WKY) and SHR (spontaneous hypertensive rats), a preclinical model for ADHD, is investigated using in vivo proton (1H) magnetic resonance spectroscopy (MRS). Analysis of the drug treatment effect (aripiprazole 1.5mg/kg; riluzole 6 mg/kg and vehicle) are conducted on a TS rat model from PND 35 to 50, covering their evolving period from childhood to early adulthood. Control animals are sham-operated. We want to compare the effect of neuroleptics (aripiprazole) and glutamatergic drugs (riluzole) on the behaviour and neurometabolites metabolism during adolescence on control rats and a TS rat model. The recently published model clearly satisfies the face validity criterion in modeling TS. This study will also provide information regarding its predictive validity. Results and conclusions: Surgery protocol established and tic session in the juvenile male rat achieved. Drug treatment is not toxic over adolescence period in control rats. Treatment with both aripiprazole and riluzole are effective on reducing grooming and climbing repetitive behaviours in the SHR but not in their normotensive controls WKY. Both drug treatments exert a reduction in GABA, Glutamate and Glutamine metabolites that is higher in the PFC more than in STR of SHR, while the treatment does not change these levels in the WKY strain in any of the region of interest.

Neurochemical effect of aripiprazole and riluzole in a juvenile rat model for Tourette Syndrome and comorbid ADHD, Eunethydis meeting 2015, 7-10 October 2015 Stockholm

Neurodevelopmental disorders reflect a disequilibrium between excitatory and inhibitory neurotransmitters during brain development. Evidences support glutamate’s (Glu) involvement in Tourette Syndrome (TS) as well as in Attention Deficit and Hyperactivity Disorder (ADHD), where low GABA levels induce hyper-glutamatergic state. Glu major role in cortico-striatal-thalamo-cortical circuits (CSTC) and the extensive interconnection with dopamine suggest overlapping pathways in tic-generation process and hyperactivity. Almost 90% of TS patients also have ADHD and viceversa 20-30% of ADHD patients also experience tics. Potential treatment options using neuroleptics (aripiprazole) and Glu-modulators (riluzole) are here investigated. Methods: Glu-GABA metabolism detection in Wistar Kyoto (WKY) and SHR, a preclinical model for ADHD, using magnetic resonance spectroscopy (MRS); establishment of a TS juvenile animal model and analysis of the drug treatment effect on neurometabolites from PND 35 to 50. Results: TS model is established in WKY and SHR juvenile rats; Glu and GABA concentration are longitudinally acquired in treated and untreated groups. Discussion: If validated, modulation of the glutamate system could provide a valuable new pharmacological approach in the treatment TS and comorbid ADHD.

Investigation of the effect of aripiprazole and riluzole on the behavioral and neurochemical profile of Tourette Syndrome juvenile rat model and control animals - an MR spectroscopy study. 1st TSA (Tourette Syndrome Association) world meeting, June 24-26, 2015 London, UK

Introduction and objectives: Pharmacologic intervention in Tourette syndrome (TS) is suggested when tics are distressing and/or interfere with function. According to the different guidelines available (Roessner et al., 2011; Pringsheim et al., 2012) neuroleptics and alpha-2 agonists are mainly used as first-choice drugs. Beside their efficacy on tics, such interventions show severe side effects. New treatments are required. Aripiprazole has been found to be effective on tic-management and to have a well tolerated side effect profile (Davies et al., 2006; Kawohl et al., 2009). Dopamine (DA) metabolism dysfunctions are well documented in TS, but imaging research data and genetic studies give definite hints that other neurotransmitters take part to tic generation: histamine, serotonin, norepinephrine, glutamate and GABA (Buse et al., 2013; Udvardi et 2013). The glutamate and DA metabolism are closely connected. The glutamatergic modulator Riluzole exerts neuroprotection from glutamate excito-toxicity both in vitro and in vivo and clinical trials are ongoing (Risterucci et al., 2006). We want to compare the effect of neuroleptics (Aripiprazole) and glutamatergic drugs (Riluzole) on the behaviour and cerebral glutamate metabolism during development using MR spectroscopy (MRS) on control rats and a TS rat model (Bronfeld et al., 2013). The recently published model clearly satisfies the face validity criterion in modelling TS. This study will also provide information regarding its predictive validity. Methods: WKY rats and spontaneous hypertensive rats (SHR), an ADHD animal model, undergo instrastriatal bicuculline (GABA antagonist) microinjection that induce an acute tic-session. Control animals are sham-operated. MRS scan are acquired in each group (ARI 1.5 mg/kg; RILU 6 mg/kg and vehicle) from PND 35 to 50, covering the evolving period from childhood to sexual maturation in rats. Results and discussion: Preliminary spectra acquisition and glutamate, GABA, glutamine and lactate absolute quantification in the left dorsal striatum and prefrontal cortex are on-going. Preliminary behavioural data treated groups compared to vehicle and sham operated are presented. No differences in the body weight in any of the treated groups compared to the vehicle are observed after 15 days of sub-chronic treatment with both drugs. Conclusion: Differences in the spectra acquired on the treated groups give hints on the mechanism in which drug classical treatments, such as neuroleptics (Aripiprazole) compared to new treatments, such as glutamatergic drugs (Riluzole) induce changes in the brain metabolism during its developmental stages. Acknowledgement This work was supported by the Marie Curie ITN TS-EUROTRAIN (FP7-PEOPLE-2012-ITN, under the REA grant agreement no. 316978).

Magnetic resonance spectroscopy (MRS) in Tourette Syndrome patients 1st TSA (Tourette Syndrome Association) world meeting, June 24-26, 2015 London, UK

Introduction and objectives: Tourette syndrome (TS) is a childhood-onset neuropsychiatric disorder characterized by the presence of multiple motor and vocal tics, commonly associated with ADHD (Attention Deficit and Hyperactivity Disease) and OCD (Obsessive Compulsive Disorders) (XXXX, 0000). TS pathophysiology involves disrupted neuronal circuits and neurotransmitter dysfunction involving the frontostriatal circuit but also limbic and associative circuits that are likely to arise during the brain connection reshape during neurodevelopment. Neuroimaging techniques are applied in TS to assess anatomical, functional and neurochemical aspect of the disorder. Non-invasive techniques like MRS can detect differences in metabolic patterns between grey and white matter. In 2005 the first study using proton magnetic resonance spectroscopy was conducted on TS patients and matched control and since then only a few publications followed (DeVito et al., 2005). They point their attention on GABA and glutamate metabolism, but also choline, creatine, N-acetylaspartate and myoinositol (Draper et al., 2014; Tinaz et al., 2014). The investigated brain regions involve frontal cortex, caudate nucleus, putamen, and thalamus. Results are insuffi cient and contraversive. The absolute quantification of metabolite at low concentration provides information about cell membrane turnover during myelination and cell proliferation, brain energy level, antioxidative defense and osmoregulation. Longitudinal studies are needed in order to correlate neurochemical profile and clinical aspects of the disease such as tic severity and drug resistance. Methods: We performed a narrative review since meta-analysis and systematic reviews are not possible due to the insufficient published data. Key words searched in the MEDLINE and EMBASE database are: “Tourette + spectroscopy,” “Tourette + MRS,” “tic + disorder + spectroscopy.” Results and discussion: Over eight neuroimaging clinical trials now on going in TS, only two performed MRS to define differences in metabolites level in TS children and adult patients compared to control. The most recent was concluded in February 2015, but results are not yet available (www.clinicaltrials.gov). Conclusion: The brain undergoes several structural and metabolic changes during the transition from childhood to adolescence and adulthood. MR spectroscopy provides an in vivo snapshot of those neurochemical alterations and represents a non-invasive tool able to track neurologic and/or neuropsychiatric disorders progression over time. Acknowledgement This work was supported by the Marie Curie ITN TS-EUROTRAIN (FP7-PEOPLE-2012-ITN, under the REA grant agreement no. 316978).

Investigation of the in vivo effect of classical and new psychotherapeutic approaches on the behavioural and neurochemical profile of a rat model of Tourette syndrome and control animals ­ an MRS study  Society for Neuroscience, Annual meeting, 17-21 October 2015, Chicago, USA

Tourette syndrome (TS) is a neurodevelopmental disorder characterized by motor and vocal tics and a high comorbidity­ rate with ADHD (attentional deficit/hyperactivity disorder). One third of children with TS experience an improvement of tics during adolescence until young adulthood but a substantial part of patients remains symptomatic after the second decade of life. In both, children and adults, pharmacologic intervention is suggested if tics are distressing and/or interfere with psychosocial function. Different guidelines suggest neuroleptics and alpha­2a agonists as drugs of first­choice. Beside their efficacy on tics, such interventions show severe side effects. New treatments are required. Aripiprazole, a partial D2 agonist, has been found to be effective in tic ­management. Dopamine (DA) metabolism is dysfunctional in TS, but imaging data give definite hints that other neurotransmitters take part in tic generation: histamine, serotonin, norepinephrine, glutamate and GABA, Glutamate and DA metabolism is closely interconnected. The glutamatergic modulator riluzole exerts neuroprotection against glutamate excito­toxicity both in vitro and in vivo. Clinical trials in TS are ongoing. We compare the effect of neuroleptics (aripiprazole) and glutamatergic drugs (riluzole) on the behavior and cerebral glutamate metabolism during development using MR spectroscopy (MRS) in a TS rat model versus control rats. The recently published model clearly satisfies the face validity criterion in modelling TS. This study will also provide information regarding its predictive validity. WKY rats and spontaneous hypertensive rats (SHR), an ADHD animal model, undergo bicuculline (GABA antagonist) microinjections that induce an acute tic­ session. Control animals are sham­ operated. MRS spectra are acquired in each group (ARI 1.5mg/kg; RILU 6 mg/kg and vehicle) from PND 35 to 50, covering the evolving period from childhood to young adulthood in rats. Preliminary data on glutamate, GABA, glutamine and lactate absolute quantification in the left dorsal striatum and prefrontal cortex are presented. Preliminary behavioral data of the treated groups ­ compared to vehicle and sham operated animals ­ are presented. No differences in the body weight or brain weight in any of the treated groups compared to the vehicle are observed after 15 days of sub­chronic treatment with both drugs. Differences in the MR spectra acquired in rats treated with aripiprazole (classical neuroleptic approach) and riluzole (possible new option) give hints on which cerebral metabolites are influenced by the two different compounds during brain development.

Investigation of the molecular effects of Aripiprazole and Riluzole in a TS rat model – An MR Spectroscopy Study. ESSTS annual meeting, April 25-26 2014, Paris, France.

Introduction: Dysfunctions in the dopamine metabolism in Tourette Syndrome (TS) are well documented, especially since the efficacy of dopamine antagonists in tic suppression and comorbid-related symptoms management has got a reasonable effect size. Aripiprazole, a partial dopamine agonist, and tiapried, a dopamine antagonist, are the drugs most often used in Germany for TS treatment. Research data from imaging and genetic studies give definite hints that other neurotransmitter systems are involved: histamine, serotonin, norepinephrine (also alpha 2A agonists are used as treatment in TS) and glutamate. The glutamate and dopamine metabolism are closely connected. More than 60% of the neuronal synapses are glutamatergic. Clinical trials with glutamatergic compounds are ongoing. In this study we want to investigate aripiprazole´s and riluzole´s effect on the cerebral glutamatergic metabolism in rats by MR spectroscopy. To reach this aim, we will use a new-established animal model recently published and that clearly satisfies the face validity criterion in modelling TS. Methods: Wistar rats and spontaneous hypertensive rats (SHR), an ADHD animal model, will stereotactically undergo intrastriatal microinjections of bicuculline, a GABA antagonist (Bronfeld et al., 2013). Control animals will be sham-operated. Animals will be treated with either aripiprazole or riluzole, a glutamatergic modulator. Behavioural test and MR spectroscopy will be conducted. Results: This study is ongoing. Data about set up, surgery, anaesthesia etc will be presented.

Mild White Matter Changes in Un-medicated Obsessive-Compulsive Disorder Patients and Their Unaffected Siblings.

Obsessive-compulsive disorder (OCD) is a common neuropsychiatric disorder with moderate genetic influences andwhite matter abnormalities in frontal-striatal and limbic regions. Inconsistencies in reported white matter results from diffusion tensor imaging (DTI) studies can be explained, at least partly, by medication use and between-group differences in disease profile and stage. We used a family design aiming to establish whether white matter abnormalities, if present in un-medicated OCD patients, also exist in their unaffected siblings. Forty-four OCD patients, un-medicated for at least the past 4 weeks, 15 of their unaffected siblings, and 37 healthy controls (HC) underwent DTI using a 3-Tesla MRI-scanner. Data analysis was done using tract-based spatial statistics (TBSS). Fractional anisotropy (FA), axial diffusivity (AD), radial diffusivity (RD), and mean diffusivity (MD) values were compared within seven skeletonised regions of interest (ROIs), i.e., corpus callosum, bilateral cingulum bundle, bilateral inferior longitudinal fasciculus/frontal-occipital fasciculus (ILF/FOF) and bilateral superior longitudinal fasciculus (SLF). Un-medicated OCD patients, compared with HC, had significantly lower FA in the left cingulum bundle. FA was trend-significantly lower in all other ROIs, except for the corpus callosum. Significant three-group differences in FA (and in RD at trend-significant level) were observed in the left cingulum bundle, with the unaffected siblings representing an intermediate group betweenOCD patients and HC. OCD patients showed lower FA in the left cingulum bundle, partly driven by trend-significantly higher values in RD. Since the unaffected siblings were found to be an intermediate group between OCD patients and HC, this white matter alteration may be considered an endophenotype for OCD.

Changes in Metabolite Concentrations in Tourette’s Disorder and Obsessive-Compulsive Disorder - A Proton Magnetic Resonance Spectroscopy Study.

Abnormal glutamatergic transmission in cortico-striato-thalamo-cortical (CSTC) circuits is thought to be involved in the pathophysiology of Tourette’s disorder (TD) and obsessive-compulsive disorder (OCD). Using proton magnetic resonance spectroscopy, the current study aimed to investigate regional concentrations of glutamatergic compounds in TD and OCD patients in comparison to healthy control subjects (HC). Twenty-three TD patients, 20 OCD patients and 22 HC were included. Short echo-time single-voxel 3T MRS was obtained from dorsal anterior cingulate cortex (dACC) and midline bilateral thalamus. The 3-group comparison showed a significant difference in choline concentration in the thalamus. Thalamic choline was highest in OCD patients, showing a significant difference with TD, and a trend compared to HC (post-hoc analyses). Glutamine in dACC correlated negatively with tic severity scores in TD patients, while glutamate in thalamus correlated positively with anxiety severity scores in OCD patients. These findings suggest subtle differences in metabolites in CSTC areas between TD and OCD. Alterations of choline concentrations seem to be both regional (only in thalamus, not in dACC) and disease specific in OCD pathology. The findings need replication in larger groups, but encourage further research into glutamatergic metabolites in TD and OCD.

Trans-diagnostic Comparison of Response Inhibition in Tourette’s Disorder and Obsessive-Compulsive Disorder

Impaired response inhibition is related to neurodevelopmental disorders, such as Tourette’s Disorder (TD) and obsessive-compulsive disorder (OCD). Unlike OCD, in which neural correlates of response inhibition have been extensively studied, TD literature is limited. By using a stop-signal task, we investigated the neural mechanisms underlying response inhibition deficits in TD compared to OCD and healthy controls (HC). Method: Twenty-three TD patients, 20 OCD patients and 22 HC were scanned (3T MRI). Region-of-interest analyses were performed between TD, OCD and HC. Results: Performance was similar across all subject groups. During inhibition TD compared with HC showed higher right inferior parietal cortex (IPC) activation. During error processing TD compared with HC showed hyperactivity in left cerebellum, right mesencephalon, and right insula. Three-group comparison showed an effect of group for error-related activation in supplementary motor area (SMA). Post-hoc analyses showed higher error-related SMA activity in TD compared with OCD and HC. Error-related left cerebellar activity correlated positively with tic severity. Hyperactivation of IPC during inhibition and a widespread hyperactivated network during error processing in TD suggest compensatory inhibition- and error-related circuit recruitment to boost task performance. The lack of overlap with activation pattern in OCD suggests such compensatory mechanism is TD-specific.

Altered Functional Connectivity in Resting State Networks  in Tourette’s Disorder

 

The brain is a complex network where brain regions are anatomically and functionally interconnected in order to facilitate important functions like cognition and movement. It remains incompletely understood how brain connectivity contributes to the pathophysiology of TD. By using resting-state functional MRI, we aimed to identify alterations in the default mode network (DMN), frontal-parietal network (FPN), sensori-motor network (SMN) and salience network (SN) in adult TD compared with healthy control (HC) subjects. In 23 adult TD patients and 22 HC resting state scans were obtained using a 3 Tesla MRI scanner. Independent component analysis was performed comparing TD and HC using dual regression, to investigate connectivity patterns within and between DMN, FPN, SMN and SN. TD patients showed significantly higher functional connectivity within the default mode network (DMN) and lower functional connectivity in the left frontal parietal network (FPN) when compared to HC. Moreover, TD patients showed a stronger coupling between the DMN and left FPN than HC. Finally, in TD patients, functional connectivity within the DMN correlated negatively with tic severity. We tentatively interpret the increased functional connectivity within the DMN in TD patients as compensatory to the lower functional connectivity within the left FPN. The stronger coupling between DMN and left FPN, together with the negative correlation between DMN and tic severity could potentially indicate that such a compensation mechanism is used to optimally sustain the DMN functioning during motor inhibition .

Putative neurochemical abnormalities associated with subcortical iron deficiency in Tourette syndrome. 23rd Annual Meeting of the Organization of Human Brain Mapping, Vancouver, Canada, June 25-29, 2017.

Iron is an essential trace element that is involved in varied biochemical processes. The atypical homeostasis of iron during specific developmental epochs has been shown to lead to diverse motor/affective/cognitive deficits as exhibited by various neuropsychiatric and movement disorders (1). Gilles de la Tourette syndrome (GTS) presents an example of a neuropsychiatric/movement disorder with a diverse symptomatology driven by putative abnormalities in the neurotransmission of dopamine, GABA and glutamate (2, 3). Given the significant role exhibited by iron in excitatory/inhibitory/modulatory signaling, we postulated that GTS patients exhibit abnormalities in iron metabolism, which may influence mechanisms of subcortical neurotransmission. Using a combination of Quantitative Susceptibility Mapping (QSM) and Magnetic Resonance Spectroscopy (1H-MRS), we investigated (a) whether GTS patients exhibit abnormalities in subcortical iron content, and (b) whether iron levels exhibit an influence on Gln:Glu; a relationship that has not been previously investigated in-vivo.QSM, 1H-MRS and MP2RAGE(4) data were acquired from 28 GTS patients and 22 age/gender matched healthy controls on a 3T Siemens Verio. A 10ml blood sample was also collected from each subject for the quantitation of serum Ferritin. Susceptibility-weighted data were acquired using FLASH with TR=30ms; TE=17ms; 256x256 matrix; flip-angle=13°; 0.8mm isotropic nominal resolution. High-quality phase maps were reconstructed using a data-driven coil-combination method (5). QSM was computed using the SDI approach via SHARP bias removal and TKD susceptibility calculation (6) with referencing to CSF (7). An MP2RAGE-QSM hybrid contrast image (8) was created to yield more robust segmentations of basal ganglia nuclei via FSL-FIRST. Brainstem and cerebellar nuclei were mapped onto native QSM space by non-linear transformation of masks that were carefully delineated on a QSM group-average. Group differences in bilateral median susceptibility were assessed via Wilcoxon rank-sum tests. Motion-affected data were removed using a multivariate robust Mahalanobis distance framework based on two structural image quality indices (9, 10). 1H-MRS data were acquired from a left striatal voxel as described previously (2). A multiple linear regression model accounting for age, gender and image quality was used to examine the relationship of susceptibility with Gln:Glu.We report significant reductions in the susceptibility of brainstem (p=0.001), basal ganglia (p=0.04) and all subcortical (p=0.0007) nuclei of GTS patients in comparison to controls. These effects were mainly driven by significant reductions of susceptibility in the subthalamic nucleus, substantia nigra, striatum, and red nucleus (Figure 1). Subcortical susceptibility reductions were mirrored by significant reductions in serum Ferritin (p=0.011). Correlation analyses revealed significant associations between striatal Gln:Glu and susceptibility in the striatum (R=0.65, p=0.002) and all subcortical nuclei (R=0.59, p=0.033). All correlations exhibited an approximate VIF of 1.5 indicating little to no multi-collinearity between predictor variables.Our results indicate that GTS patients exhibit abnormalities in subcrotical iron homeostasis. The observed association between striatal susceptibility and Gln:Glu indicates that iron exhibits and an influence on GABA-Glu-Gln cycling. As iron is involved in multiple biochemical pathways that regulate neurochemical signalling (1), alterations of iron hemostasis in GTS provide new clues on the biological basis of the demonstrated disruptions in dopaminergic/GABAergic/glutamateric signalling. Alterations in the mechanisms sustaining the typical spatio-temporal synergy of subcortical neurotransmission during specific development epochs may have a profound influence on reinforcement-learning and habit-formation systems, leading to the acquisition of abnormalities in motor and behavioural functions.

 

QSM meets MRS: The influence of subcortical iron on glutamatergic neurotransmission in a movement disorder population. 25th Annual Meeting of the International Society for Magnetic Resonance in Medicine (ISMRM), Honolulu, Hawaii, USA, April 22-27 2017.


 

Iron is a trace element that is essential to the vitality of an organism, as it is ideally suited for the catalysis of many biochemical reactions due to its ability to transition between two thermodynamically stable oxidation states. Within the brain, non-heme iron is abundant in subcortical nuclei, overlapping with regions that contain large proportions of dopamine, GABA and glutamate (Glu). The atypical homeostasis of iron during development may influence the biochemical mechanisms that sustain typical subcortical neurochemical signalling, providing a possible biological basis for deficits exhibited by varied neuropsychiatric and movement disorders. Gilles de la Tourette Syndrome (GTS) is an example of a neurodevelopmental movement disorder with reported reductions in serum Ferritin levels and abnormalities in the neurotransmission of dopamine and GABA. We have recently shown that GTS patients also exhibit reductions in striatal Glu and glutamine (Gln), indicating possible abnormalities in GABA- Glu-Gln cycling, which may lead to alterations in the spatio-temporal dynamics of excitatory, inhibitory and modulatory subcortical neurochemical signalling. Along this line, a comprehensive body of literature has indicated that iron deficiency alters the neurotransmission of dopamine and GABA by influencing the mechanisms involved in receptor function and neurotransmitter synthesis/transport. Here, we used a combination of Quantitative Susceptibility Mapping (QSM) and Magnetic Resonance Spectroscopy (1H-MRS) to investigate (a) whether GTS patients exhibit alterations in iron metabolism, and (b) whether iron levels exhibit a general influence on the Gln:Glu ratio.QSM, 1H-MRS and MP2RAGE data were acquired from 28 GTS patients and 22 age/gender matched healthy controls on a 3T Siemens MAGNETOM Verio using a 32-channel head coil. A 10ml blood sample was also collected from each subject for the quantitation of serum Ferritin. Susceptibility-weighted data were acquired using FLASH with !=30ms; !=17ms; 256x256 matrix; flip-angle=13°; 0.8mm isotropic nominal resolution. High-quality phase maps were reconstructed using an automated, data- driven coil combination method. QSM images were computed using the superfast dipole inversion approach via SHARP bias removal and TKD susceptibility calculation, with referencing to lateral ventricle CSF (Figure 1). An MP2RAGE-QSM hybrid contrast image was created to yield more robust/accurate segmentations of basal ganglia nuclei via FSL-FIRST. Brainstem and cerebellar nuclei were mapped onto native QSM space by non-linear transformation of masks that were carefully delineated on a QSM group-average template. Median susceptibility values were extracted from each ROI and Wilcoxon rank-sum tests were used to assess group differences. Motion-affected data were removed using a multivariate robust Mahalanobis distance framework based on two structural image quality indices. 1H-MRS data were acquired from a left striatal voxel (TE= 30ms, TR=3000ms, nav=128) employing automated voxel localization; frequency and phase-drift correction; and LCModel absolute metabolite quantitation with consideration of within-voxel compartmentation. A multiple linear regression model accounting for age, gender and two indices of image quality was used to examine the relationship of susceptibility with Ferritin and the Gln:Glu ratio. The variance inflation factor (VIF) was used to assess multi-collinearity between predictor variables.We report significant reductions in the susceptibility of brainstem (p=0.001), basal ganglia (p=0.04) and all subcortical (p=0.0007) nuclei of GTS patients in comparison to controls. These effects were mainly driven by significant reductions of susceptibility in the subthalamic nucleus, substantia nigra, striatum, and red nucleus (Figure 2). Subcortical susceptibility reductions were mirrored by significant reductions in serum Ferritin (Welch’s test, p=0.011; Figure 3). Correlation analyses in the whole sample revealed significant associations between Ferritin and susceptibility in all subcortical (R=0.65, p=0.009) and basal-ganglia (R=0.44,p=0.02) nuclei. Significant associations were also observed between striatal Gln:Glu and susceptibility in the striatum (R=0.65, p=0.002) and all subcortical nuclei (R=0.59, p=0.033). All correlations exhibited an approximate VIF of 1.5 indicating little to no multi-collinearity between predictor variables.Our results indicate that GTS patients exhibit abnormalities in cerebral iron homeostasis; and provide in-vivo evidence for a relationship between iron metabolism and glutamatergic neurotransmission. This result is supported by previous work indicating that gestational and lactational iron deficiency suppresses striatal and hippocampal glutamatergic neurotransmission, in addition to influencing enzymes involved in GABA-Glu-Gln cycling. As iron is involved in multiple biochemical pathways that regulate neurochemical signalling, alterations of its hemostasis in GTS provide new clues on the biological basis of the observed disruptions in excitatory, inhibitory and modulatory signalling. Although GTS is considered a polygenic hereditary disorder with multiple affected systems, it is plausible to suggest that iron holds an important role in the pathophysiology of GTS, as its deficiency has been related to a neurochemically driven influence on the acquisition motor and behavioral functions during development.

 

Effects of different coil reconstruction algorithms on Quantitative Susceptibility Mapping results. 4th International Workshop on MRI Phase Contrast & Quantitative Susceptibility Mapping. Graz, Austria. September 26-28, 2016.

 

The standard Quantitative Susceptibility Mapping (QSM) methods rely on the phase evolution of the signal from a FLASH sequence to obtain information on the susceptibility of the imaging source. Such images are often obtained through the combination of the signal detected separately by multiple receive coil elements (channels). Since each channel is subject to specific modulations of the complex signal, a simple linear combination of the signals can result in constructive or destructive interference due to the inconsistency of the phase offset across the different channels. To overcome this issue, several different coil reconstruction algorithms have been proposed. Some of them, including those implemented by a major MRI vendor, while performing well for obtaining magnitude images, are known to degrade the phase images. Here, we investigate the potential impact of degraded phase images on QSM results.FLASH complex images (TR: 30 ms, TE: 17 ms, α: 13°), of the full brain (0.8 mm iso.) were acquired in 22 healthy volunteers (6 females, aged 19 to 57 yr), with a MAGNETOM 3T Verio MRI system (Siemens, Erlangen, Germany) and a body Tx / 32-channels Rx coil. For each acquisition, both the vendor’s default reconstruction algorithm and a recently proposed extension of the ESPIRiT approach [4, 5] (using data driven virtual conjugate coils [6]) were applied to obtain the combined phase information. The two different phase images were used as input of the same QSM pipeline implementing the Superfast Dipole Inversion (SDI) approach (SHARP bias removal and TKD susceptibility calculation) [7], using the CSF of the lateral ventricles as reference. The two different susceptibility maps were compared both on a voxel-by-voxel basis and on a region-of-interest (ROI) basis. For the voxel-by-voxel analysis both single subject and grouped results are considered. The ROI analysis was performed on: basal ganglia, segmented with FSL-FIRST subcortical segmentation algorithm on optimized MP2RAGE-QSM hybrid contrast images [8, 9]; brain stem, segmented with nonlinear transformation of ATAG atlas (Red Nucleus, RN; Substantia Nigra, SN; Subthalamic Nucleus, STN) to native space; brain cortex, segmented with FREESURFER for extraction of insula and cingulum masks. The results indicate that the artifacts originating from the standard coil reconstruction algorithm influence the QSM results beyond the region where the phase information is obviously corrupted. This is finding is consistent across all subjects and does not average out completely. Additionally, the ROI analysis indicate that the susceptibility values are more consistent for the corrected phase images. In conclusion, this study indicates that degraded phase images suffer from serious local and non- local biases, that are consistent across subjects and that may lead to poor results in group studies, even when considering ROIs that do not seem degraded by visual inspection of either the input phase or the QSM results.

Quantitative Susceptibility Mapping in Gilles de la Tourette Syndrome. European Society for the study of Tourette Syndrome (ESSTS) 2016 Annual Meeting. Warsaw, Poland. June 8-11 2016. 


The bulk of current literature on Gilles de la Tourette Syndrome (GTS) pathophysiology supports the hypothesis of a dysregulated dopaminergic system, although more recent work has implicated extra- dopaminergic systems (e.g. GABAergic, glutamatergic, seretonergic and histaminergic). This data suggests that the complex symptomatology of GTS is a result of spatially focalized alterations in the interaction of neurochemical systems. Consequently, it is currently unclear whether dopaminergic abnormalities are primary or secondary in nature. Considerable evidence has linked alterations in the concentration of brain iron to several developmental and movements disorders. Various groups have shown that abnormalities in iron concentrations affect (i) dopaminergic neurotransmission during development; (ii) dopamine metabolism in the striatum; (iii) dopamine transporters; and (iv) the sensitivity and number of dopamine D1 and D2 receptors (Stankiewicz et al., 2007). In this work, we hypothesized that iron levels will exhibit differences between patients and controls in gray matter regions. We utilized Quantiative Suscpeibility Mapping (QSM) as an indirect measure of brain iron, as it allows determining the bulk magnetic susceptibility distribution of tissue in vivo, for which many studies have reported a reasonable correlation with brain iron concentrations (Langkammer et al., 2012).Forty-three adult GTS patients and 40 healthy controls were included. A 10 ml blood sample for measurement of serum ferritin levels and a comprehensive clinical battery were collected from each subject. MP2RAGE and FLASH images were acquired from each subject at using a 3T MAGNETOM Verio system. QSM maps were computed from the FLASH data by employing sophisticated harmonic artefact reduction for phase data (SHARP) and homogeneity enabled incremental dipole inversion (HEIDI). An MP2RAGE-QSM hybrid contrast image was created to yield more robust and accurate segmentations of subcortical nuclei via the FSL-FIRST algorithm. Masks for brain stem nuclei (substantia-nigra, subthalamic nucleus, red nucleus) were created for each subject by transforming the ATAG atlas (Keuken et al., 2014) from MNI space to native space. Cortical masks from insular and cingular regions of interest were extracted from FREESURFER’s whole brain segmentation. Mean susceptibility values were extracted from each ROI and Wilcoxon rank-sum tests were used to assess group differences.Significant differences in mean susceptibility values between patients and controls were observed for the amygdala in the right (RH) and left hemisphere (LH); RH hippocampus; RH and LH insular cortex; LH cingular cortex; LH red nucleus; and RH sub-thalamic nucleus. A significant negative correlation was observed between the Yale Global Tic Severity Scale (YGTSS-TTS) and susceptibility values of RH insular-cortex. Additionally, significant negative correlations were observed between YGTSS and serum ferritin values with RH red-nucleus susceptibility values.We expand on previous work that reported abnormalities in the T2 relaxation times of amygdalar and insular regions (Peterson et al., 1994) by (a) utilizing QSM as a more accurate measure for the assessment of iron concentrations in the brain (Deistung, 2013) and (b) investigating a larger number of ROIs. Our results indicate that GTS patients exhibit abnormalities in iron levels in brain regions involved in motor control and salience.

 

Pathological glutamatergic neurotransmission in Gilles de la Tourette Syndrome. 24th Annual Meeting of the International Society for Magnetic Resonance in Medicine (ISMRM), Abstract # 2420, Singapore, Singapore, May 7 - 13, 2016 


 

Gilles de le Tourette Syndrome (GTS) is a hereditary, neuropsychiatric movement disorder with fundamental alterations in the functional dynamics of cortico-striatal circuitry1. Motor and phonic tics are the hallmark features of GTS, with a majority of the patients presenting with associated conditions that include obsessive-compulsive and attention-deficit hyperactive behaviours. Pathophysiologically, alterations in (a) phasic dopamine transmission and (b) the density/binding potential of D2 receptors and dopamine transporters in striatal and cortical regions have suggested an abnormality in tonic and phasic dopamine release2. Transient phasic release of dopamine depends on a glutamatergic excitatory drive via the activation of NMDA receptors3, and has been implicated with motor control, reward prediction errors and reinforcement seeking behaviour4. The dynamic regulation of dopaminergic firing is driven by (a) extra-synaptic dopaminergic concentrations, which regulate dopamine release by acting on D2 autoreceptors5-7, and (b) GABAergic and glutamatergic input from cortical, striatal and mesencephalic regions8. Therefore, we hypothesized that glutamatergic signalling is related to pathophysiology of GTS, and aimed at investigating whether patients exhibit alterations in glutamate metabolism within regions implicated in GTS pathophysiology.3T MRS data were acquired from 37 GTS patients and 36 healthy controls on a Siemens MAGNETOM Verio using a 32-channel coil. Repeated acquisitions were obtained from 23 controls for test-retest reliability assessment and 15 patients following four weeks of pharmacotherapy with the D2 partial agonist aripiprazole. A landmark based GRE pre-scan (Auto-Align Head; AAH) was applied for automatic registration of all following protocols to the same geometry. T1-weighted images were acquired with MP2RAGE (TR=5s, TE=3.93ms, 1mm3 voxel dimensions). 1H-MRS spectra were obtained from 3 ROIs with PRESS (TE= 30ms, TR=3000ms, 80 or 128 averages). To minimize errors from bulk drift in the time lag between the anatomical and spectral acquisitions, single-shot ‘dummy’ spectra were localized on the MP2RAGE immediately after acquisition. The AAH sequence was applied again before each MRS acquisition to co-register the ‘dummy’ scan geometry to the newly defined space. Spectra were acquired from the anterior Mid-Cingulate Cortex (aMCC, 6.4 mL), the bi-lateral thalamus (7.2 mL) and the left striatum (3.4 mL) following FASTESTMAP shimming. To account for incoherent averaging due to head motion and temporal drifts in the B0 field, we implemented a non-linear least squares minimization operation to fit each signal average to a reference scan by adjusting the frequency and phase of the signal9. Absolute metabolite concentrations were calculated using the water signal as internal reference while considering compartmentation within the voxel10 (Fig. 1). Optimized masks including subcortical nuclei were generated from SP12 and FSL-FIRST. Relaxation effects of metabolites were ignored. Spectra were fit with LCModel11 in a 0.3-3.67ppm range to avoid spurious residual signals above 3.7ppm. Inclusion criteria for good quality were: (a) correct voxel prescription; (b) SNR>10; (c) FWHM<11Hz; and (d) CRLB<50%12. Group differences were assessed using a repeated measure analysis of variance (2 times × 3 regions × 2 groups) followed by post-hoc independent- and paired-sample t- tests.We observed a significant time x group interaction (F = 22.39, p = 0.000127) for glutamate+glutamaine (Glx) concentrations. Post-hoc independent sample t-tests revealed significant reductions of Glx concentrations in the left striatum (t61 =2.594, p = 0.0119) and the bilateral thalamus t58 =2.189, p = 0.0325) of GTS patients in comparison to normal controls (Fig. 3). Following treatment with aripiprazole, patients exhibited significant increases in striatal Glx concentrations (t10 =-3.241, p = 0.009) and a trend for increases in the thalamic voxel (t10 =2.189, p = 0.072) when compared to baseline (Fig. 2). Multiple regression analysis revealed a significant negative correlation between left striatal Glx levels (r = -0.451, p = 0.011) and tic severity (Fig 4). Thalamic Glx levels were negatively correlated with the premonitory urges preceding tics (r = -0.434, p =0.023) and depression (r = -0.589, p = 0.002).Our results implicate glutamatergic metabolism in the pathophysiology of GTS and indicate a possibly dysfunctional astrocytic-neuronal coupling system, a notion that has been observed by genetic13-15 and functional-based14 studies. Since the metabolic paths of glutamate, glutamine and GABA are intertwined, GTS patients may exhibit a perturbed balance between excitatory and inhibitory neurotransmission, which may ultimately affect the modulation of tonic and phasic dopamine release from the substania nigra pars compacta and the ventral tegmental area. This might have a profound effect on motor control, reward-prediction errors and goal-directed behaviour. Focal asymmetries in excitatory, inhibitory and modulatory neurotransmitter ratios in functionally distinct striatal regions may lead to the diverse symptomatology associated with GTS.

Test-Retest Quantitation of Absolute Metabolite Concentrations with Partial-Volume correction using different segmentation methods. International Society for Magnetic Resonance Imaging in Medicine (ISMRM), Abstract # 1974, Toronto, Ontario, Canada, May 30 - June 5, 2015.

 

The quantitation of absolute metabolite concentrations via Magnetic Resonance Spectroscopy using water as an internal concentration standard, requires the accurate determination of the compartmentation within the localized region of interest. Previous work has shown that different segmentation approaches yield different estimates of metabolite levels in grey matter [1]. In this study, we sought to investigate the test-retest reliability of absolute metabolite quantitation using two commonly used segmentation algorithms. Anatomical and MR spectroscopic data from 12 healthy controls (1 female, age range: 23-50) was acquired on a 3T Siemens MAGNETOM Verio Scanner using a 32 channel phased-array coil. T1-weighted images were acquired using an MP2RAGE sequence with the parameters: TR=5s, TE=3.93ms, FOV=192mm, 256x256 acquisition matrix, 1.0x1.0x1.0mm voxel dimensions. 1H MRS spectra from two regions of interest were obtained with a point-resolved-spectroscopy (PRESS) sequence with the following parameters: TE= 30ms, TR=3000ms, 80 water suppressed and 16 water-unsuppressed averages. A 25x16x16 mm3 cortical voxel was prescribed on the Anterior Mid-Cingulate Cortex (ACC) with the center of the voxel projecting to the genu of the corpu collosum, and an orientation that is parallel to the hippocampal axis. A 28x16x16 mm3 subcortical voxel was localized on the thalamus while maximizing the amount of grey matter within the voxel. Regions of interest were shimmed automatically with the FASTESTMAP Sequence [2,3] . The inbuilt AutoAlignHead Siemens sequence was used to align the geometry of the voxel to a standard. On the retest scan, the voxels were automatically localized using the saved voxel geometry information from the first scan. Averaged spectra were exported from the scanner as Raw Data Format (.rda) files. To register the MRS voxel onto the antomical image, the transformation matrix of the MRS voxel was first calculated from the rda file header. A binary mask representing the limits of the MRS voxel was then constructed to map the voxel onto the anatomical image [4]. SPM12 New Segment and Freesurfer (version 5.3.0) were used to segment the brain into different tissue compartments. Freesurfer grey matter, white matter and CSF tissue classes were extracted from the segmentation-generated tissue labels. GM, WM and CSF tissue fractions were then calculated within the registered SVS voxel from the segmentation outputs.Scanner-averaged frequency domain spectra were analyzed with LCModel [5], which implements a fully automated quantitation algorithm which does not account for partial volume effects. Inclusion criteria for good quality spectra were SNR higher than 15, an FWHM lower than 12Hz, and Cramer Rao lower bounds lower than 20%. Compartmentation within the MRS voxel was considered for the quantitation of absolute metabolite concentrations by applying equation (1) while ignoring relaxation effects of metabolites since they have similar T1/T2 times in GM and WM and areapproximately accounted for by LCModel [1]. Absolute metabolite concentrations  were calculated using tissue fraction percentages generated using SPM and Freesurfer. The reliability of the automatic MRS voxel re-localization method was assessed using the Sørensen–Dice metric which yielded mean ± standard deviation values of 0.78 ± 0.10 for the ACC voxel and 0.71 ± 0.28 for the Thalamic voxel. Anatomical image segmentation using SPM and Freesurfer yielded results that were significantly different between the two algorithms. Mean values of grey matter, (GM), white matter (WM) and corticospinal fluid (CSF) fractions within the two SVS voxel are illustrated in table 1. Within the same package, the segmentation algorithms exhibited high reliability across scanning time-points as the estimates for the tissue classes within the MRS voxel did not reached significance using a two sided paired t-test. Comparing the values between segmentation algorithms, results exhibited discrepancies of more than 20%. SPM's segmentation of the ACC voxel yielded CSF fractions that were 20% higher than Freesurfer's, while in the thalamic voxel, the opposite effect was observed, as Freesurfer's CSF tissue fractions were more than 30% higher than SPM's. Given that a criterion for localizing the thalamic voxel was to maximize the amount of grey matter, Freesurfer's results demonstrate better estimates based on visual inspection, as the amount of white matter with SPM segmentation was significantly high (Figure 1). As a result of peak resonance overlaps at 3T, five metabolite concentrations were considered as reliable: tNAA (NAA + NAAG), tCre (Cr + pCr), tCho (Cho + pCho), mI, Glx(Glu + Gln). Means and SDs of uncorrected and partial volume corrected metabolite spectra are presented in table 2. Overall, Freesurfer yielded higher estimates of absolute metabolite concentrations when compared to SPM. Pairwise absolute percentage differences were then calculated for uncorrected, Freesurfer- corrected and SPM-corrected metabolite concentrations. The averaged standard deviations of absolute percentage difference were consistently lower for Freesurfer-corrected concentrations for the ACC voxel. No general trend was observed for the thalamic voxel.In summary, our observations suggest that partial volume
correction may help improve absolute metabolite concentration estimates,
though there are substantial differences between the segmentation
algorithms of SPM and Freesurfer in cortical and subcortical regions, which
are reflected in the correction of metabolite concentrations. Careful consideration of which package to use for estimating compartmentation within different MRS regions of interest is recommended.

 

Retrospective control for motion-artefacts in functional neuroimaging datasets using Wavelet and ICA based methods, 1st World Congress on Tourette Syndrome & Tic Disorders, Abstract # 202, 1st Tourette World Congress. London, UK. June 24-26 2015.

 

It has recently been well established that head motion during fMRI imaging data acquisition introduces undesirable and artifactual effects that can significantly bias group comparisons of functional connectivity (Power, 2012). As a result, several groups have put forth different retrospective denoising strategies, such as nuisance regression, aCompCor, Scrubbing, and Global Signal Regression. Nonetheless, these strategies have their drawbacks as their implementation might lead to the alteration of the temporal structure of the data, the loss of temporal degrees of freedom, the inadvertent removal of the signal of interest or the increase of the likelihood of anti-correlations. (Power, 2015). In this work, we investigate the utility of two recently published methods – (i) the discrete wavelet transform (Patel, 2014) and (ii) automated independent-component analysis (ICA) feature classification (Pruim, 2015)– for the identification and removal of non-stationary events in resting-state fMRI time-series, while potentially circumventing common drawbacks.Anatomical and resting-state fMRI data from 10 healthy controls and 10 Gilles de la Tourette patients (GTS) was pre-processed with a custom-built processing pipeline as outlined in Figure 1. Three denoising strategies were utilized in the processing stream. Strategy Type-I included the regression of nuisance variables from linear/quadratic trends, 24 motion parameters, and WM/CSF signals via aCompCor. Type-II and Type-III strategies included the regression of nuisance variables of Type-I, with the addition of ICA-AROMA (ICA Automated Removal Of Motion Artifacts) and Wavelet Despike, respectively. The Derivative of the VAriance of the Root mean Square of the BOLD signal (DVARS) was used as a metric of motion as it indexes the variance in the fMRI signal between successive frames.Our results indicate that Type-I and Type-III denoising strategies do not completely remove group differences of BOLD signal variance as illustrated in Figure 2. On the other hand, data processed with ICA-AROMA (Type-2 stream) resulted in a significant reduction in the mean and standard deviation of the high-motion GTS group, and more importantly, a matching of DVARS group means. To interrogate the effects of the denoising strategy on removing distance-dependent connectivity artifacts (which increase locally and decrease globally), we used the delta-R plot (Power, 2012) on the GTS sample to inspect the change of correlation between Type-I/Type-II and Type-I/Type III with respect to Euclidian distance. A trend for a decrease in short-distance correlations was observed for Type-II ICA-AROMA denoising.Our results indicate that the ICA-AROMA denoising strategy performs well at decreasing group differences of the DVARS motion metric between a high motion clinical population and control population. Additionally, ICA-AROMA results in a decrease in short-distance correlations, which might affect group comparisons. On other hand, Wavelet Despike is a promising strategy for the removal of non-stationary events in fMRI time-series, however, its implementation on our data did not result in the matching of DVARS means between the groups. It is possible that the performance of the Discrete Wavelet Transform parameters is dependent the acquisition strategy. In this case, it is difficult to comment on the performance of the algorithm since the discrete wavelet filter parameters may need to be optimized for the specific sequence parameters. In sum, with sequence specific optimization, ICA and wavelet-based denoising methods perform well at removing non-stationary events in the fMRI time-series, while circumventing the drawbacks of the alteration of the temporal structure of the data or the loss of temporal degrees of freedom, which are associated with scrubbing.

 

Significant tic reduction in a treatment resistant Gilles de la Tourette Syndrome patient following treatment with nabiximols (Sativex). 1st World Congress on Tourette Syndrome & Tic Dis- orders, Abstract # 156. 1st Tourette World Congress. London, UK. June 24-26 2015.

 

Despite the fact that the range of treatments available for the management of the symptomatology of Gilles de la Tourette syndrome (GTS) have been expanding over the last decade, treatments are either ineffective or cause significant adverse effects in some patients. As a result, there is an urgent need for uncovering novel treatment strategies that are more effective in treatment resistant patients, cause less adverse effects and could potentially ameliorate both tic and behavioural symptoms. Early anecdotal reports provided evidence that patients with GTS choose cannabinoids as a form of self-medication either legally or illegally. Along this line, preliminary studies suggested that cannabinoid based medicine’s (CBMs) such as delta-9-tetrahydrocannabinol (THC) are effective in the treatment of GTS. Here we report the first case of an effective treatment of a patient with treatment resistant GTS using nabiximols (Sativex®) - a plant extract from Cannabis sativa L. containing THC and cannabidiol (CBD) at a 1:1 ratio. 
The 22 years old male patient suffered from severe treatment resistant GTS starting at age 17. The patient exhibited extreme simple and complex motor and vocal tics in addition to the presence of comorbid attention deficit hyperactivity disorder (ADHD), obsessive-compulsive disorder (OCD), pathological gambling, and sleep problems. Most pronounced of his symptoms were extreme vocal tics including coprolalia which pushed him to seek different pharmacological treatment strategies such as haloperidol, risperidone, aripirazole, and clonidine, but also methylphenidate, atomoxetine, , fluphenazine, and pramipexole (alone or in combination). All treatments were either ineffective or caused severe adverse effects. Therefore, treatment with nabiximols (Sativex®) was initiated. Several clinical measures were acquired before and after the treatment was commenced. These included the Yale Global Severity TS scale (YGTSS); Global Clinical Impairment (GCI); GCI for premonitory urges; Premonitory Urge for Tics Scale (PUTS); GTS-Quality of Life (GTS-QOL); GCI for GTS QOL and the Tourette Syndrome Symptom List (TSSL). Treatment with nabiximols commenced at a dose of 1 puff/day (=100μl containing 2.7 mg THC and 2.5 mg CBD) and slowly increased up to a dosage of 3x3 puffs/day (24.3 mg THC and 22.5 mg CBD). Clinical assessments were performed twice, once before treatment with nabiximols and once after 2 weeks of a stable dose of 3x3 puffs/days. As shown in table 1, treatment resulted in major improvement of both tics, premonitory urges, global impairment, and health related quality of life without causing relevant adverse effects.This is the first case study suggesting that treatment with nabiximols may be effective in the treatment of patients with severe and treatment resistant GTS. Anecdotal reports as well as preclinical data have provided some evidence that nabiximols may be more effective and better tolerated than pure THC. Nabiximols is formulated as an oromucosal spray and is partly absorbed sublingually, resulting in reduced first pass effects, faster absorption, accelerated onset, and stronger effect compared to orally administered pharmaceutical products such as pure THC. Given the positive response exhibited by the patient highlighted in this report, further investigation of the effects of nabiximols is proposed on a larger group of patients in clinical trial setting.

 

TS-EUROTRAIN: European wide investigation of the etiology and pathophysiology of Gilles de la Tourette syndrome and related disorders. 1st World Congress on Tourette Syndrome & Tic Disorders, London, UK. June 24-26 2015.

 

The majority of psychiatric disorders fall into the category of complex genetic disorders, which are likely caused by genetic susceptibility factors that interact with the environment to confer the total risk of acquiring complex phenotypes. Such complex traits do not usually exhibit clear-cut boundaries between affected and non-affected states, but exhibit a phenotype that extends over a range of severity and comorbidity. Over the past decade, it has become increasingly evident that a wide range of clinically distinct, developmental and neuropsychiatric disorders exhibit a significant degree of etiological overlap. Gilles de la Tourette Syndrome (GTS) provides an excellent model for the exploration of shared etiology across such disorders, as it is a complex developmental disorder that wavers along the fine margin between psychiatry and neurology. GTS is characterized by the presence of multiple motor tics and one or more vocal tics, and presents with comorbid conditions that frequently include Attention Deficit Hyperactivity Disorder (ADHD) and Obsessive Compulsive Disorder (OCD). TS-EUROTRAIN is a Marie Curie Initial Training Network (http://ts-eurotrain.eu) that aims to build on current knowledge in the field.Our consortium tackles several major challenges that include: (i) assembling a large genetic database for the evaluation of the complex etiology of GTS and related disorders with high statistical power; (ii) exploring the role of gene-environment interaction as the role of epigenetic phenomena in GTS have not even begun to be evaluated; (iii) employing endophenotypic based approaches to dissect the basis of shared etiology between GTS, OCD and ADHD; (iv) establishing new developmental animal models for GTS as such models are important and severely lacking; and (v) gaining new insights into the neurobiological mechanisms of GTS via transverse and longitudinal imaging based studies to pave the way for new treatment strategies.The project is pursued by ten partners, from academia and industry and 12 PhD students. Our ultimate aims are to elucidate the complex etiology of the onset and clinical course of GTS, investigate the neurobiological underpinnings of GTS and related disorders, translate research findings into clinical applications, and establish a Pan-European infrastructure for the study of GTS and associated disorders.

 

Elemental, neurochemical and network based anal- ysis of the pathophysiological mechanisms of Gilles de la Tourette Syn- drome. European Society for the study of Tourette Syndrome (ESSTS) 2014 Annual Meeting. Paris, France. April 25-26 2014.

 

Gilles de la Tourette syndrome (GTS) is a neurodevelopmental movement disorder whose cardinal features are the presence of fluctuating motor and phonic tics. Approximately 90% of GTS patients exhibit comorbidities that most commonly include Attention Deficit Hyperactivity Disorder (60%) and Obsessive Compulsive Disorder (20-60%). Currently, there is an ongoing effort to further characterize the pathophysiology of GTS and to identify new tic-suppressing therapies. Extensive evidence supports a role for the dopaminergic system within cortico-striatal-thalamo-cortical (CSTC) circuitry as a primary abnormality in GTS. However, given the heterogeneity of clinical symptoms associated with GTS, it has been postulated that other neurotransmitter systems and networks might be involved. In this study, we will focus on the role of the glutamatergic system, iron content and cognitive control networks, and use a procedure that is commonly used in the neuropsychiatric literature in which clinical and MRI data is acquired both off- and on-treatment. We aim to acquire (i) MR spectroscopy data to investigate alterations in glutamate metabolite levels; (ii) quantitative susceptibility maps (MP2RAGE-Multiecho) to quantify iron deficiency; (iii) resting state fMRI data to interrogate the functional decoupling between cognitive control and basal ganglia motor networks; and (iv) diffusion weighted imaging data for exploratory analysis of structural networks. This work will have implications on future glutamatergic modulatory therapies for tic-suppression and could potentially extend the current pathophysiological model of GTS beyond CSTC circuitry. 


 

Oral presentations

 

Rizzo F , Nespoli E, Subgang A, Abei A, Hengerer B, Bar-gad I, Rasche V , Ludolph AG, Boeckers T. Behavioural and 1 h MR spectroscopic preclinical investigation of psychopharmacologic approaches for Tourette´s Syndrome. European Society for the study of Tourette Syndrome (ESSTS) 2016 Annual Meeting, June 8-11 2016, Warsaw, Poland. (selected as invited talk)

Introduction: Dopamine metabolism dysfunctions are well documented in TS, but imaging research data and genetic studies give definite hints that other neurotransmitters take part to tic generation: histamine, serotonin, norepinephrine, glutamate and GABA. The glutamate and DA metabolism are closely connected. Haloperidol, a D1 and D2 complete antagonist, has been widely used for tic management, but its efficacy is shown together with severe side effects. New treatments are required. Aripiprazole, a partial dopamine antagonist, has been found to be effective on tics and to have a well-tolerated side effect profile. Riluzole, glutamatergic modulator, exerts neuroprotection from glutamate excito-toxicity both in vitro and ameliorates dyskinesia symptoms in vivo. Aim of the study: We want to compare the effect of neuroleptics (aripiprazole) and glutamatergic drugs (riluzole) on the behavior and cerebral glutamate metabolism during development using MR spectroscopy (MRS) on juvenile control rats and a TS rat model.7 Even though a unique preclinical model for TS is not yet available, the recently published model clearly satisfies the face validity criterion in modelling TS. This study will also provide information regarding its predictive validity. Methods: WKY rats and spontaneous hypertensive rats (SHR), an ADHD animal model, undergo instrastriatal bicuculline (GABA antagonist) microinjection that induce an acute tic-session. Control animals are sham-operated. Behavioral recording and MRS scans are acquired in each group (ari 1.5mg/kg; rilu 6 mg/kg and vehicle) from PND 35 to 50, covering the evolving period from childhood to sexual maturation in rats. Results: Spectra quantification obtained in the left dorsal striatum and prefrontal cortex of WKY and SHR rats treated with vehicle/aripiprazole/riluzole are presented together with behavioral data. Preliminary spectra acquired from sham/bicuculline injected rats are also shown. In WKY strain, both treatments results not toxic after 15 days of sub-chronic treatment. Both drugs reduce rearing frequency without affecting the spontaneous locomotor activity. Aripiprazole, but not riluzole, exert an effect on the neurochemical level of striatal GABA and prefrontal Glutamine absolute concentrations. SHR strain behavioral and neurochemical data acquisition on/off treatment are still ongoing. Conclusions: This study shows how I. 1H NMR spectroscopy is a valid tool to non-invasively detect even small changes in the neurochemical profile due to different conditions, as strain specificity, drug treatments and physiological/ticcing phenotype; II. Differences in the spectra acquired on the treated groups give hints on the way in which drug treatments induce changes in the brain metabolism during its developmental stages. Acknowledgements: This work was supported by the Marie Curie ITN TS-EUROTRAIN (FP7-PEOPLE-2012-ITN, under the REA grant agreement n°316978).

Kanaan AS, Gerasch S, García-García I, Lampe L, Pampel A, Anwander A, et al. Pathological glutamatergic neurotransmission in Gilles de la Tourette syndrome. BRAIN 2017; 140: 218–234.

Gilles de la Tourette syndrome is a hereditary, neuropsychiatric movement disorder with reported abnormalities in the neuro- transmission of dopamine and -aminobutyric acid (GABA). Spatially focalized alterations in excitatory, inhibitory and modula- tory neurochemical ratios within specific functional subdivisions of the basal ganglia, may lead to the expression of diverse motor and non-motor features as manifested in Gilles de la Tourette syndrome. Current treatment strategies are often unsatisfactory thus provoking the need for further elucidation of the underlying pathophysiology. In view of (i) the close spatio-temporal synergy exhibited between excitatory, inhibitory and modulatory neurotransmitter systems; (ii) the crucial role played by glutamate (Glu) in tonic/phasic dopaminergic signalling; and (iii) the interdependent metabolic relationship exhibited between Glu and GABA via glutamine (Gln); we postulated that glutamatergic signalling is related to the pathophysiology of Gilles de la Tourette syndrome. As such, we examined the neurochemical profile of three cortico-striato-thalamo-cortical regions in 37 well-characterized, drug-free adult patients and 36 age/gender-matched healthy control subjects via magnetic resonance spectroscopy at 3 T. To interrogate the influence of treatment on metabolite concentrations, spectral data were acquired from 15 patients undergoing a 4-week treatment with aripiprazole. Test-retest reliability measurements in 23 controls indicated high repeatability of voxel localization and metab- olite quantitation. We report significant reductions in striatal concentrations of Gln, Glu + Gln (Glx) and the Gln:Glu ratio, and thalamic concentrations of Glx in Gilles de la Tourette syndrome in comparison to controls. ON-treatment patients exhibited no significant metabolite differences when compared to controls but significant increases in striatal Glu and Glx, and trends for increases in striatal Gln and thalamic Glx compared to baseline measurements. Multiple regression analysis revealed a significant negative correlation between (i) striatal Gln and actual tic severity; and (ii) thalamic Glu and premonitory urges. Our results indicate that patients with Gilles de la Tourette syndrome exhibit an abnormality in the flux of metabolites in the GABA-Glu-Gln cycle, thus implying perturbations in astrocytic-neuronal coupling systems that maintain the subtle balance between excitatory and inhibitory neurotransmission within subcortical nuclei.

 

 

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