Cross-Disease Analysis of Depression, Ataxia and Dystonia

University of Groningen

Cross-disease analysis of depression, ataxia and dystonia highlights a role for synaptic plasticity and the cerebellum in the pathophysiology of these comorbid diseases Huang, Miaozhen; de Koning, Tom J; Tijssen, Marina A J; Verbeek, Dineke S

Published in: Biochimica et biophysica acta-Molecular basis of disease

DOI: 10.1016/j.bbadis.2020.165976

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Citation for published version (APA): Huang, M., de Koning, T. J., Tijssen, M. A. J., & Verbeek, D. S. (2021). Cross-disease analysis of depression, ataxia and dystonia highlights a role for synaptic plasticity and the cerebellum in the pathophysiology of these comorbid diseases. Biochimica et biophysica acta-Molecular basis of disease, 1867(1), [165976]. https://doi.org/10.1016/j.bbadis.2020.165976

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BBA - Molecular Basis of Disease

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Cross-disease analysis of depression, ataxia and dystonia highlights a role for T synaptic plasticity and the cerebellum in the pathophysiology of these comorbid diseases ⁎ Miaozhen Huanga, Tom J. de Koninga,b,c, Marina A.J. Tijssenb, Dineke S. Verbeeka, a Department of Genetics, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands b Department of Neurology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands c Pediatrics, Department of Clinical Sciences, Lund University, Sweden

ARTICLE INFO ABSTRACT

Keywords: Background: There is growing evidence that the neuropsychiatric and neurological disorders depression, ataxia Cross-disease analysis and dystonia share common biological pathways. We therefore aimed to increase our understanding of their Depression shared pathophysiology by investigating their shared biological pathways and molecular networks. Ataxia Methods: We constructed gene sets for depression, ataxia, and dystonia using the Human Phenotype Ontology Dystonia database and genome-wide association studies, and identified shared genes between the three diseases. Wethen Synaptic plasticity assessed shared genes in terms of functional enrichment, pathway analysis, molecular connectivity, expression Cerebellum comorbidity profiles and brain-tissue-specific gene co-expression networks. Results: The 33 genes shared by depression, ataxia and dystonia are enriched in shared biological pathways and connected through molecular complexes in protein–protein interaction networks. Biological processes common/ shared to all three diseases were identified across different brain tissues, highlighting roles for synaptic transmission, synaptic plasticity and nervous system development. The average expression of shared genes was significantly higher in the cerebellum compared to other brain regions, suggesting these genes have distinctcer- ebellar functions. Several shared genes also showed high expression in the cerebellum during prenatal stages, pointing to a functional role during development. Conclusions: The shared pathophysiology of depression, ataxia and dystonia seems to converge onto the cerebellum that maybe particularly vulnerable to changes in synaptic transmission, regulation of synaptic plasticity and nervous system development. Consequently, in addition to regulating motor coordination and motor function, the cerebellum may likely play a role in mood processing.

1. Introduction genes associated with depression. Many of these genes were linked to synaptic structures and neurotransmission, and an integrative func- Depression is a disabling neuropsychiatric disease that affects more tional genomic analysis revealed that the origins of neuropsychiatric than 264 million people worldwide [1]. Current estimates are that disorders, including depression, are closely related to genes involved in ~20% of the general population will experience at least one episode of the development of the human brain [9]. None-the-less, the causal re- depression in their lifetime [2]. At present, fewer than half of patients lationships between genome-wide association study (GWAS) variants/ with depressive symptoms receive effective treatment, in part because genes and depression remain to be determined. the pathophysiology of depression is not fully understood [3]. Comorbid depression is also frequently seen in patients suffering Neuroanatomical and imaging studies in patients have shown that from ataxia and dystonia [10–15], neurological disorders that affect depression is a disease with a complex pathophysiology that seems to motor movement and motor function. For ataxia this is due to dys- involve multiple brain regions, including the hippocampus, prefrontal function of the cerebellum, while dystonia has been linked to dys- cortex and cerebellum [4–7]. A recently published genome-wide meta- function of the basal ganglia [16,17]. The anatomical structures in- analysis further illustrated the complexity of the genetic background of volved in both ataxia and dystonia are not strictly separated, because depression [8]. In this study, hundreds of independent variants and there is growing evidence that the cerebellum has a role in dystonia as

⁎ Corresponding author at: University Medical Center Groningen, Department of Genetics, Antonius Deusinglaan 1, 9700RB Groningen, the Netherlands. E-mail address: [email protected] (D.S. Verbeek). https://doi.org/10.1016/j.bbadis.2020.165976 Received 10 July 2020; Accepted 15 September 2020 Available online 02 October 2020 0925-4439/ © 2020 The Author(s). Published by Elsevier B.V. This is an open access article under the CC BY license (http://creativecommons.org/licenses/BY/4.0/). M. Huang, et al. BBA - Molecular Basis of Disease 1867 (2021) 165976 well [18]. Many disease genes have been identified for both disorders, (PPI) using the following databases: BioGrid, InWeb_IM and OmniPath. and ataxia and dystonia have a profoundly shared genetic background The enrichment network was visualized by Cytoscape (v3.7.2) [25]. converging into shared molecular pathways [19],which may also con- The related enrichment terms were manually clustered and marked tribute to comorbid depression. Common biochemical etiologies are with different colors. suggested to underlie the motor and psychiatric symptoms in cervical dystonia [20], but the severity of the motor symptoms severity not 2.4. Brain-tissue-specific gene co-expression network analysis necessarily correlates with the severity of non-motor symptoms suggesting that the depression is not the directly linked to the motor Gene co-expression network analysis of multiple brain tissues was symptoms or the cause of the motor symptoms. Patients with dystonia performed using NetworkAnalyst (assessed May 2019) [28]. We gen- can have mild motor symptoms but significant psychiatric/depression erated tissue-specific gene co-expression networks for cerebellum, symptoms. And this is not only the case in dystonia, a higher risk for cortex, nucleus accumbens/basal ganglia and hippocampus. Networ- depression was correlated with the severity of disease in dominantly kAnalyst uses data from the TCSBN, a database of tissue- and cancer- inherited cerebellar ataxias [21]. So more and more data becomes specific biological networks for 46 tissues and 17 cancers [29]. The available over depression in dystonia and ataxia, however the common intersecting pathways between the brain-tissue-specific gene co-ex- pathophysiological mechanisms underlying the comorbidity of these pression networks of the shared genes were plotted using the R-Package disorders remain unknown. UpSetR (v1.4.0) [30], and the analyses were performed in R (v3.5.2) The comorbidity between depression, ataxia and dystonia and the using RStudio (v1.1.463). profound shared genetic basis of ataxia and dystonia, motivated us to carry out a cross-disease analysis to discover their shared pathophy- 2.5. Protein-protein interaction network siological mechanisms. We identified the similarities and differences among the distinct disease gene sets associated with all three diseases, The STRING database (v11.0) [31] was used to obtain PPIs between and we then explored the function of gene shared between all three 163AD and 497Dep. The interactions are based on associated evidence, diseases within co-expression networks across different brain tissues including gene neighborhoods, gene fusions, gene co-occurrence, co- (Fig. 1). Our data advocate that there are molecular processes that are expression, text mining, biochemical and genetic data and protein shared between neuropsychiatric disorders such as depression and complex knowledge extracted from databases. The UniProtKB Retrieve/ neurological disorders like ataxia and dystonia and highlight a role for ID mapping tool (https://www.uniprot.org/uploadlists/) was used to the cerebellum and synaptic plasticity in its disease pathophysiology. convert gene symbols to UniProtKB entry name, and the PPI network was generated using the K-means clustering algorithm. The protein- 2. Materials and methods protein interactions were weighted by “edge weights” represented by confidence scores scaled between zero and one. 2.1. Ataxia, dystonia and depression gene collection 2.6. GTEx brain tissue transcriptome analysis We extracted genes associated with the terms ‘depression’, ‘ataxia’ and ‘dystonia’ from the Human Phenotype Ontology database (HPO) Publicly available multi-tissue transcriptome RNA sequencing (assessed February 2019) [22]. This identified 646 ataxia genes and (RNAseq) data from the Genotype-Tissue Expression (GTEx) project 298 dystonia genes (Tables S1 and S2). To complete the gene list as- (v7) was used for gene expression analysis [32]. GTEx provides the sociated with depression, we added genes identified by two recent RNAseq data as median TPM (transcripts per million) per tissue type, GWAS studies [8,23] to the HPO ‘depression’ gene list. In total, 497 and median TPM RNAseq data was available for 44 human tissues, genes linked to depression were identified (Table S3). Gene duplica- including 13 brain tissues from 216 donors (mixed racial and ethnic tions were removed, and gene names were manually adjusted when group and sex, age range 21–70). For 163AD and 497Dep, we analyzed necessary using GeneCards (https://www.genecards.org) and OMIM the gene expression levels for the cerebellum, cortex, nucleus ac- (https://omim.org/). To curate the data gene names not recognized by cumbens/basal ganglia and hippocampus separately. The differences in these webtools were excluded from the analyses. expression levels of all genes were determined using a one-way ANOVA and graphically illustrated by GraphPad Prism v.8.0 for Mac OS X 2.2. Gene ontology analysis and pathway enrichment analysis and (GraphPad Software, La Jolla California, USA). visualization 2.7. BrainSpan developmental transcriptome analysis Pathway enrichment analysis was performed using the web-based tool g:Profiler on two sets of genes: the 497 depression genes (497Dep; RNAseq data from BrainSpan (Atlas of the Developing Human Brain, TableS4) and the 163 genes shared between ataxia and dystonia www.brainspan.org) was used to perform temporal gene expression (163AD; Table S5) [24]. For this study, we used the Gene Ontology analysis of the depression genes present in the enriched pathways of the (GO), biological pathways (KEGG, Reactome and WikiPathways), pro- shared ataxia and dystonia gene cerebellar co-expression network. The tein databases (Human Protein Atlas and CORUM) and HPO that were RNAseq data is available as reads per kilobase per million (RPKM) of 16 significantly enriched (p < 10−5). Enrichment Map, a plugin in the human brain regions over 29 developmental stages. Additional details Cytoscape (v3.7.2) network analysis environment, was used to visualize on the samples can be found at the BrainSpan website. Hierarchical and interpret the results [25,26]. The WordCloud plugin was used as an clustering was carried out using the Pearson correlation method and a auxiliary drawing tool. heatmap of the clustering was generated using the Morpheus analysis software (https://software.broadinstitute.org/morpheus/). 2.3. Enrichment clustering 3. Results Enrichment clustering was performed on the shared gene set between ataxia and dystonia and the depression genes using Metascape 3.1. Common biological pathways underlie depression, ataxia, and dystonia [27]. The analysis was carried out with the following sources: KEGG Pathway, GO Biological Processes, Reactome Gene Sets, Canonical To assess common biological pathways underlying depression, Pathways and CORUM. The Molecular Complex Detection (MCODE) ataxia and dystonia, we generated list of Human Phenotype Ontology algorithm was applied to conduct protein–protein interaction analysis (HPO) genes for the terms ‘ataxia’, ‘dystonia’ and “depression” (Tables

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Fig. 1. Schematic representation of data analysis. Genes linked to Human Phenotype Ontology (HPO) terms ataxia, dystonia and depression were extracted from the HPO database, and the depression gene list was supplemented with genes reported in two recent GWAS on depression. Integrated enrichment analysis was performed on the two distinct sets of genes – those shared between ataxia and dystonia (163AD) and depression genes (497Dep) – and 33 genes shared between all three diseases were identified. Brain tissue transcriptomic analysis was performed using RNAseq data from the GTEx project, and protein–protein interaction (PPI) network analysis was performed using the STRING database. A gene co- expression network using RNAseq data of cerebellum (CB), cortex (CTX), basal ganglia (BG) and hippocampus (HIP) was built for the 33 genes shared between ataxia, dystonia and depression. The func- tionality of these networks was assessed using pathway enrichment analysis by testing the over- representation of specific Gene Ontology biological pathways and processes.

S1–S3). To complete the gene list associated with depression, we also 497Dep gene sets, we generated a single integrated enrichment map to included genes reported by two recently published depression GWAS reveal the enrichment analysis results (Fig. 2). This analysis identified [8,23], resulting in a final list of 497 genes associated with depression - biological pathways enriched in both gene sets, including modulation of the 497Dep gene set (Table S4). In the HPO gene lists for the term chemical synaptic transmission, cellular ion homeostasis, regulation of ‘ataxia’ and ‘dystonia’, 163 genes are linked to both ataxia and dystonia, neuron death, reactive oxygen species metabolic process, response to hereafter referred to as the 163AD gene set (Table S5). Given the pro- oxidative stress, response to inorganic substances, aerobic respiration found overlap between the ataxia HPO and dystonia HPO gene sets, we and behavior. This shared contribution to many pathways supports an continued our analysis with the 163AD gene set. overlap in the molecular pathology of ataxia, dystonia and depression. To explore the similarities and differences among the 163AD and However, we also saw differences between the 163AD and 497Dep

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Fig. 2. Enrichment map for shared ataxia and dystonia and depression gene sets. The map displays the enriched gene set for ataxia and dystonia (163AD, red) and depression (497Dep, blue). Nodes represent enriched gene sets. Node size corresponds to the number of genes in the set. The center of the node corresponds to the respective disease gene set, while the border of the node represents the Log10(P)value.

Table 1 Results of the pathway analysis of the enrichment map of the shared ataxia and dystonia and depression gene sets.

Gene sets GO Pathway description Log10(P)value

163AD|497Dep GO:0045333 Cellular respiration −42.76 163AD GO:0009060 Aerobic respiration −19.76 163AD|497Dep GO:0007610 Behavior −16.2 163AD CORUM:2904 Respiratory chain complex I (intermediate VII/650kD), mitochondrial −15.79 163AD|497Dep GO:0050804 Modulation of chemical synaptic transmission −15.36 163AD|497Dep GO:0006979 Response to oxidative stress −13.28 163AD|497Dep GO:1901214 Regulation of neuron death −12.33 497Dep GO:0050808 Synapse organization −12.01 497Dep hsa05322 Systemic lupus erythematosus −11.46 163AD|497Dep GO:0050905 Neuromuscular process −11.44 163AD|497Dep GO:0060322 Head development −10.35 163AD|497Dep GO:0006873 Cellular ion homeostasis −10.29 163AD|497Dep GO:0051640 Organelle localization −9.9 163AD CORUM:2919 Respiratory chain complex I (gamma subunit) mitochondrial −9.85 163AD|497Dep GO:0098662 Inorganic cation transmembrane transport −9.57 163AD|497Dep GO:0010035 Response to inorganic substance −9.09 163AD|497Dep GO:0042391 Regulation of membrane potential −9.07 163AD|497Dep GO:0072593 Reactive oxygen species metabolic process −8.82 497Dep GO:0048871 Multicellular organismal homeostasis −8.72 163AD|497Dep GO:0023061 Signal release −8.27 gene sets. The cellular respiration and inorganic cation transmembrane 33 genes that were present in both 497Dep and 163AD (Fig. 3A; transport pathways were selectively observed for ataxia and dystonia, Table 2). To examine to which distinct biological pathways these genes while the regulation of membrane potential and synapse organization, associate, we performed GO pathway enrichment analysis. These signal release and multicellular organismal homeostasis pathways were shared genes were significantly enriched for the mitochondrial orga- only observed for depression (Fig. 2; Table 1). nization (p = 2.0 × 10−5), polyol metabolic process (p = 0.0001), autophagy (p = 0.0002) and metal ion homeostasis (p = 0.002) pathways (Figs. 3B; S1). 3.2. Shared genes between depression, ataxia, and dystonia show high To expose the basis for a shared pathology in brain regions involved average relative expression in the cerebellum in depression, ataxia and dystonia, we used Genotype-Tissue Expression (GTEx) RNAseq data to investigate the relative expression levels of Given the high overlap in molecular pathways, we investigated the these shared genes in the cerebellum, cortex, basal ganglia and genetic overlap between depression, ataxia and dystonia, and identified

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Fig. 3. Shared genes between ataxia, dystonia and depression show high average expression in the cerebellum. Circos plot showing the differences and similarities between 163AD and 497Dep. Thirty-three shared genes between ataxia, dystonia and depression wereidentified (dark orange). B) Pathway analysis of the 33 shared genes. Significant biological pathways up toa p-value = 0.05 are shown. C) Boxplot showing the average gene expression levels of the shared genes in the cerebellum (CB), cortex (CTX), basal ganglia (BG), and hippocampus (HIP) (**** p < 0.0001;*** p = 0.001; ordinary one-way ANOVA followed by Tukey's multiple-comparisons test) (data extracted from GTEx database). D) Heatmap showing expression levels of the shared genes across 13 different brain tissues (data extracted from GTEx database) Note: ATXN8 is not present in the GTEx dataset. hippocampus. 3.3. Shared genes between depression, ataxia, and dystonia are The average relative expression for the shared genes was sig- interconnected at the protein level nificantly higher in the cerebellum compared to the other three brain regions (Fig. 3C). We also observed significantly higher cerebellar ex- We next investigated if the proteins coded by the shared genes expression of the 497Dep separately, suggesting that these genes them- hibit substantial molecular interactions. PPI data was collected from the selves may distinct functions in the cerebellum irrespective of the STRING database, and we combined this data into a network using K- overlap with the 163AD gene set (Fig. S2A and B). Notably, the average means clustering [31]. Of these 33 proteins, 28 were connected in six genome-wide expression levels did not differ between all four brain smaller subnetworks comprised of three to eight proteins (Fig. 4). The tissues (data not shown), which suggests that the high expression of this overall PPI network of these shared proteins showed significantly more specific set of genes in the cerebellum is a functional signal andnotan interaction than expected (p < 1 × 10−16). Four of these smaller artifact. Hierarchical clustering of the average expression levels of the subnetworks were connected into a larger PPI subnetwork. shared genes across 13 different brain tissues revealed two clusters that We then investigated if we could identify specific biological pro- contain genes with low or high relative expression levels in the cere- cesses underlying these subnetworks of proteins. The larger subnet- bellum and cerebellar hemisphere (Fig. 3D). work, comprised of four smaller subnetworks, was enriched for proteins Next, we used GO-term enrichment analysis to investigate which involved in autophagic mechanisms (p = 4.836 × 10−4, subnetwork biological pathways underlie these two clusters of genes. Genes with 2), aggregaphy (p = 7.78 × 10−3, subnetwork 1), metal ion home- high relative expression in the cerebellum were significantly enriched ostasis (p = 5.6 × 10−3, subnetwork 3) and tetrahydrobiopteric me- for mitochondrion organization (p = 0.0001) and autophagy process tabolic processes (p = 2.35 × 10−4, subnetwork 4). Two smaller (p = 0.002), while genes with low relative expression were sig- subnetworks (5 and 6) were not connected to the larger subnetwork and nificantly enriched for the polyol metabolic process (p = 4.9 × 10−6), were not significantly enriched for proteins involved in specific mole- polyol biosynthetic process (p = 7.5 × 10−5), diol biosynthetic process cular processes. This data thus suggests that the shared depression, (p = 0.0002), diol metabolic process (p = 0.0002) and alcohol meta- ataxia and dystonia genes function in common biological processes. bolic process (p = 0.001) (Fig. S2C and S2D).

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Table 2 p = 9.5 × 10−33, respectively) (Table S7). This suggest that the shared List of 33 shared genes between the 163AD and 497Dep gene sets. genes between ataxia, dystonia and depression function in biological Gene symbol Approved gene name HGNC ID processes that are tissue-specific. However, we did observe substantial overlap in subnetwork genes between the basal ganglia and hippo- AARS2 alanyl-tRNA synthetase 2, mitochondrial HGNC:21022 campus, which is reflected by the similarities in enrichment of biolo- AFG3L2 AFG3 like matrix AAA peptidase subunit 2 HGNC:315 gical processes (Fig. 5B). Overall, the shared biological processes across ARSA arylsulfatase A HGNC:713 ATP13A2 ATPase cation transporting 13A2 HGNC:30213 the four brain tissues include synaptic signaling, anterograde trans-sy- ATP1A3 ATPase Na+/K+ transporting subunit alpha 3 HGNC:801 naptic transmission, cell-cell signaling, synapse organization, nervous ATXN2 ataxin 2 HGNC:10555 system development and regulation of synaptic plasticity. ATXN8 ataxin 8 HGNC:32925 ATXN8OS ATXN8 opposite strand lncRNA HGNC:10561 3.5. Cerebellar gene co-expression networks expose key genes and biological BCS1L BCS1 homolog, ubiquinol-cytochrome c reductase HGNC:1020 complex chaperone pathways in disease-specific context C19orf12 chromosome 19 open reading frame 12 HGNC:25443 COQ2 coenzyme Q2, polyprenyltransferase HGNC:25223 Since many of the shared genes between depression, ataxia and CP ceruloplasmin HGNC:2295 dystonia showed high relative expression in the cerebellum, we in- CYP27A1 cytochrome P450 family 27 subfamily A member HGNC:2605 1 vestigated the molecular interaction between the proteins encoded by GBA glucosylceramidase beta HGNC:4177 these genes in the cerebellar co-expression networks of the 163AD and GCH1 GTP cyclohydrolase 1 HGNC:4193 497Dep gene sets. To do so, we performed MCODE analysis to detect HTT huntingtin HGNC:4851 densely connected proteins that correspond to known molecular com- MECP2 methyl-CpG binding protein 2 HGNC:6990 plexes in a large protein–protein interaction (PPI) network (Fig. S3A). PANK2 pantothenate kinase 2 HGNC:15894 PDGFB platelet derived growth factor subunit B HGNC:8800 The most prominent individual molecular complexes within this large PDGFRB platelet derived growth factor receptor beta HGNC:8804 PPI network were most enriched for processes such as trafficking and PLA2G6 phospholipase A2 group VI HGNC:9039 activation of AMPA receptors, synaptic plasticity, ephrin receptor sig- PPP2R2B protein phosphatase 2 regulatory subunit Bbeta HGNC:9305 naling pathway and the Rho GTPase cycle (Fig. S3B). The proteins of PRKCG protein kinase C gamma HGNC:9402 PSAP prosaposin HGNC:9498 these molecular complexes are equally represented in both the 497Dep PSEN1 presenilin 1 HGNC:9508 and 163AD gene sets (Fig. S3C). PTS 6-pyruvoyltetrahydropterin synthase HGNC:9689 We next investigated the presence of disease-specific genes in the SLC20A2 solute carrier family 20 member 2 HGNC:10947 cerebellar gene co-expression networks of the 163AD and 497Dep gene SLC30A9 solute carrier family 30 member 9 HGNC:1329 sets. Depression genes GRM5, SOX5, MEF2C and PPP2R2B were present SQSTM1 sequestosome 1 HGNC:11280 TBP TATA-box binding protein HGNC:11588 in the cerebellar gene co-expression network of 163AD (Fig. S4A; Table TMEM106B transmembrane protein 106B HGNC:22407 S8), and were present in several nodes enriched for genes involved in TTC19 tetratricopeptide repeat domain 19 HGNC:26006 modulation of chemical synaptic transmission, (trans-)synaptic sig- VCP valosin containing protein HGNC:12666 naling and nervous system development. The AD genes PRKCG, ATP1A2 and PPP2R2B were observed in the cerebellar gene co-expression network of 497Dep (Fig. S4B; Table S8) and were detected in 3.4. Gene co-expression network analysis shows common biological several nodes involved in (antero-grade-)synaptic transmission and - pathways between depression, ataxia, and dystonia across different brain signaling and nervous system development, but also in nodes enriched tissues for genes involved in learning, memory and cognition (Table S9). In the AD gene network, PPP2R2B was present in one module enriched for The tissue-specific expression patterns of genes may contribute genes involved in regulation of cell communication, whereas in the significantly to shared pathology in the brain. We investigated the depression gene network PPP2R2B was seen in two nodes enriched for brain-tissue-specific relationships between the 33 shared genes, and genes involved in the phosphorus metabolic process and the phosphate- examined the properties of these genes in gene co-expression networks containing compound metabolic process. Of note, the relative expres- using data from cerebellum, cortex, basal ganglia and hippocampus. We sion levels of GRM5, SOX5, MEF2C and PPP2R2B are higher in the detected 12 subnetworks in cerebellum, 14 in the cortex, two in the cortex compared to the cerebellum that was not the case for PRKCG and basal ganglia and three in the hippocampus (data not shown), and the ATP1A2 (Fig. S4C). main subnetwork per tissue is shown in Fig. 5A. A) Schematic representation of the main subnetwork of the cere- 3.6. Shared genes between depression, ataxia, and dystonia can be clustered bellum (CB), cortex (CTX), basal ganglia (BG) and hippocampus (HIP). by temporal gene expression pattern in the cerebellum Seeds are the 33 shared genes between ataxia, dystonia and depression. For example, the main subnetwork of the cerebellum has one seed, We next investigated the temporal expression of the shared genes PPP2R2B, whereas the cortex has nine seeds, of which seven are shown between depression, ataxia and dystonia in the cerebellum using the (ATP13A2, PRKCG, ATP1A3, C19orf12, HTT, AFG3L2 and SLC30A9). B) BrainSpan Transcriptional Atlas of the Developing Human Brain. We UpSetR plot indicating the number of intersections of GO biological hierarchically clustered the cerebellar temporal expression pattern of pathways across the four brain tissue subnetworks. Ten shared GO the shared genes over the different developmental stages. This analysis biological pathways were identified, including main processes in neu- revealed two clusters (Fig. 6A). Cluster 1 showed relatively low ex- rotransmission and neurodevelopment. pression that increased modestly over the course of brain development. The main subnetwork in the cerebellum (1 seed, 97 edges) is most Cluster 2 showed higher expression during prenatal stages compared significantly enriched for genes involved in synaptic signaling with the genes in cluster 1. Cluster 1 is enriched for genes involved in (p = 1.1 × 10−5) and chemical synaptic transmission organelle disassembly (p = 0.0001), mitochondrion organization (p = 3.8 × 10−5), while the main subnetwork in the cortex (9 seeds, (p = 0.001), cation transport (p = 0.003), autophagy (p = 0.001) and 548 edges) is enriched for vesicle-mediated transport in the synapse autophagy of mitochondrion (p = 0.003) (Fig. 6B). In contrast, the (p = 8.3 × 10−17) and trans-synaptic signaling (p = 1.2 × 10−15). genes expressed at higher levels during prenatal stages were enriched Additionally, the main subnetworks for the basal ganglia (28 seeds, for tetrahydropbiopterin biosynthetic and metabolic processes 3188 edges) and hippocampus (26 seeds, 4183 edges) are both enriched (p = 0.009) and mitochondrial respiratory chain complex III assembly for genes involved in cellular localization (p = 3.8 × 10−27 and (p = 0.02) (Fig. 6C).

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AARS2

TMEM106B

ATXN2 SQSTM1

AFG3L2 TBP VCP PPP2R2B SLC20A2

HTT ARSA SLC30A9 PRKCG PDGFB PDGFRB

PSEN1 CYP27A1 ATP1A3 GCH1

PSAP MECP2 PLA2G6 PTS

GBA COQ2 PANK2

ATP13A2 CP Node Color Subnetwork 1 Aggregaphy Subnetwork 2 Autophagy C19orf12 BCS1L Subnetwork 3 Metal ion homeostasis Subnetwork 4 Tetrahydrobiopteric metabolic process Subnetwork 5 Basal ganglia calcification Subnetwork 6 Mitochondrial Diseases TTC19 Edge Confidence low(0.15) high(0.70) medium(0.40) highest(0.90) Predicted interactions Known interactions Others gene neighborhood from curated databases textmining gene co-occurrence experiementally determined coexpression

Fig. 4. Genes shared between ataxia, dystonia and depression are interconnected and function in common pathways. Protein-protein interaction (PPI) network of the 33 genes shared between ataxia, dystonia and depression. Six subnetworks were identified using the K-means clustering algorithm. Colored lines show the interaction of proteins with Gene Ontology (GO) term pathways.

We next investigated the temporal expression of the depression, investigating the average expression levels of the 497Dep gene set, we ataxia and dystonia genes present in the disease-specific cerebellar gene also observed that these depression genes were on average higher ex- co-expression networks. The depression genes GRM5, SOX5, MEFC2 and pressed in the cerebellum compared for example the hippocampus. This PPPR2B showed significantly higher expression during the prenatal may suggest that in addition to the current knowledge on the role of stages of cerebellar development compared to postnatal stages, whereas neuroplasticity of the hippocampus in the etiology of depression [3], a the ataxia and dystonia genes ATP1A2 and PRKCG showed, on average, yet unexplored role may exist for the cerebellum in particular for co- higher expression levels during postnatal stages (Fig. 6D). morbid depression in ataxia and dystonia cases. We also identified substantial overlap in biological processes in our brain-tissue-specific gene co-expression networks of the shared genes, which suggests that 4. Discussion they may also act in common pathways across different brain tissues. The role of the cerebellum in the pathology of ataxia is well es- We have described the molecular pathways common between de- tablished [33], but there is also increasing evidence that the cerebellum pression, ataxia and dystonia, which seemingly converge onto the is also involved in dystonia [18]. Moreover, the cerebellum was re- susceptibility of the cerebellum to changes in synaptic transmission, cently shown to play a role in processing traumatic memories [34], and cellular ion transport, synapse organization, regulation of synaptic abnormal cerebellar structure and function have been reported in pa- plasticity and nervous system development. By investigating the genetic tients with depression [7]. These findings may suggest that the cere- overlap between these three disorders, we identified 33 shared genes bellum plays an important function in modulating physiological re- that were, on average, more highly expressed in the cerebellum com- sponses under stress and demonstrate the presence of cognitive pared to the cortex, basal ganglia and hippocampus. The high relative functions within the cerebellum. expression of these shared genes in the cerebellum suggests they have a We also observed that the cerebellar temporal expression pattern of distinct cerebellar function and that there is a shared pathology be- the 33 shared genes over different developmental stages was tween these three comorbid disorders in this brain region. Notably, by

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Fig. 5. Brain-tissue-specific co-expression networks of genes shared between ataxia, dystonia and depression expose both common and distinct brain-tissue-specific pathways. hierarchically clustered into two clusters with relatively low and high depend on intact autophagic machinery and proper mitochondrial expression during prenatal stages. This suggests that a subset of the content exchange, organelle distribution and thereby mitochondrial shared genes may play a role in cerebellar development. function [35,36]. For example, in mice the mitochondrial fusion gene Spatiotemporal or temporal specificity was also found for major de- Msf2 is required for postnatal development of the cerebellum as genetic pressive disorder (MDD) associated gene co-expression modules [9], depletion of Msf2 lead to severe defect in postnatal cerebellar growth further linking brain development with neuropsychiatric disease. and cerebellar Purkinje cell degeneration [36]. Moreover, light - and The observation that the biological processes autophagy and mi- electron microscopy studies showed morphological alterations of the tochondrial organization are enriched among these shared genes fur- mitochondria in the muscles of major depression disorder patients [37], ther supports a role for proper cerebellar development in the shared but if this also holds true for mitochondria in the cerebellum or other pathology of depression, ataxia and dystonia, as correct central nervous brain regions remains unknown. Recently, it was reported that the ul- system development, including of the cerebellum, has been shown to trastructure of mitochondria is correlated with the synaptic activity of

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Fig. 6. One-third of the genes shared between ataxia, dystonia and depression show high average expression in the prenatal cerebellum. Heatmap showing the expression of the genes shared between ataxia, dystonia and depression over 29 stages of cerebellar development (data extracted from the BrainSpan Transcriptional Atlas of the Developing Human Brain). The map is hierarchically clustered, and the developmental stages are chronologically ordered from prenatal to postnatal. Note: ATXN8 is not present in the BrainSpan dataset B) Significantly enriched Gene Ontology (GO) terms and pathways for shared genesnot highly expressed during the prenatal stages of cerebellum development (Cluster 1). C) Significantly enriched GO terms and pathways for shared genes highly expressed during prenatal stages of cerebellum development (Cluster 2). D) Heatmap showing the temporal expression of depression genes (GRM5, SOX5, MEF2C and PPP2R2B) and ataxia and dystonia genes (PPP2R2B, PRKCG and ATP2A1) in enriched pathways in cerebellar-derived co-expression networks using 163AD and 497Dep as seeds. neurons, and thus mitochondria contribute to neuroplasticity and sy- and reverse the loss of synapses caused by stress (reviewed in [54]). naptic performance [38]. Furthermore, damage to the mitochondrial Thus, evidence exists for a link between autophagy, synaptic plasticity electron transport chain and corresponding mitochondrial dysfunction and the pathogenesis of depression, but the role of the cerebellum re- has been recognized as the cause of a series of neuropsychiatric dis- mains to be investigated. eases, including depression [39–41]. Therefore, we may speculate that Our work further advocates that the co-morbidity between depres- alterations of the structure of mitochondria correlate with mitochon- sion, ataxia, and dystonia may be caused by mechanisms that alter drial dysfunction and oxidative stress and ultimately cause alterations synaptic plasticity since we observed regulation of synaptic plasticity in synaptic - and neuronal functioning. and subsequent modulation of synaptic transmission as the main mo- The activity of autophagy regulator Ulk1 in the growth cones of lecular process in common in our genetic enrichment comparison be- cerebellar granule cells was shown to be important for proper parallel tween these disorders. We also observed synaptic plasticity to be a fiber growth and thus in the progression of cerebellar development feature of one of the most prominent individual molecular complexes in [37]. Autophagy also has a protective role in neurodegeneration, as the cerebellar PPI networks of both 163AD and 497Dep and as an cerebellar Purkinje-cell-specific depletion of the autophagy machinery overlapping pathway across the four different brain-tissue-specific gene component Atg7 led to axonal degeneration in motor dysfunction and co-expression networks based on the 33 shared genes. Furthermore, two ataxia in the respective genetically modified Atg7 mice [42]. But the depression genes, metabotropic glutamate receptor 5 (GRM5) and the question still remains if there is a role for autophagy in depression. transcription factor Myocyte Enhancer Factor 2C (MEF2C), are present Alterations in the mRNA expression of autophagy machinery compo- in the cerebellar gene co-expression network of 163AD. Dendritic nents in blood and aberrant mTOR signaling in the prefrontal cortex mGluR5 activity has been shown to be required for MEF2-dependent have been reported in individuals suffering from major depression synapse elimination in hippocampal neurons, and these findings de- [43,44]. Additionally, several studies have reported an altered autop- monstrate that transcription factor activation regulates synaptic re- hagy process in depression-relevant mouse models [45,46], and similar finement via dendritic synapse activity [55]. Synaptic activity and evidence was obtained from treatment studies using anti-depressants to neuronal calcium influx via N-methyl-D-aspartate (NMDA) receptors alter autophagy [47–49]. Notably, suppression of autophagy was re- and voltage-gated calcium channels initiate diverse intracellular cas- ported to rescue cell death of neurons expressing the shared gene cades that include transcription factors. Notably, activation of NMDA Protein Phosphatase 2 Regulatory subunit beta (PPP2R2B) that was receptors in the synapse has already been linked with synapse elim- induced by oxidative stress and mitochondrial dysfunction [50,51]. ination [56], but whether NMDA signaling also plays a role in MEF2- Autophagy has also been reported to be involved in AMPA receptor induced synapse elimination and synaptic plasticity in the cerebellum degradation upon neuronal stimulation, affecting synaptic plasticity in remains to be investigated. hippocampal neurons, and suggest that autophagy contributes to sy- Overall our work suggests that the role of the shared genes in the co- naptic plasticity and brain functions [52]. Decreased expression of sy- morbidity of depression, ataxia and dystonia is mediated by the speci- napse-related genes and decreased synapse numbers were observed in ficity and timing of their expression. Based on this data, weproposea post-mortem brains of individuals with depression [53], and rapid- role for cerebellar development in the shared pathology of depression, acting anti-depressants were reported to increase synaptic connections ataxia and dystonia.

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