Transcriptome Analysis of the Human Striatum in Tourette Syndrome

Abstract

Background: Genome-wide association studies have not revealed any risk-conferring common genetic variants in Tourette syndrome (TS), requiring the adoption of alternative approaches to investigate the pathophysiology of this disorder.

Methods: We obtained the basal ganglia transcriptome by RNA sequencing in the caudate and putamen of nine TS and nine matched normal control subjects.

Results: We found 309 downregulated and 822 upregulated genes in the caudate and putamen (striatum) of TS individuals. Using data-driven gene network analysis, we identified 17 gene coexpression modules associated with TS. The top-scoring downregulated module in TS was enriched in striatal interneuron transcripts, which was confirmed by decreased numbers of cholinergic and gamma-aminobutyric acidergic interneurons by immunohistochemistry in the same regions. The top-scoring upregulated module was enriched in immune-related genes, consistent with activation of microglia in patients' striatum. Genes implicated by copy number variants in TS were enriched in the interneuron module, as well as in a protocadherin module. Module clustering revealed that the interneuron module was correlated with a neuronal metabolism module.

Conclusions: Convergence of differential expression, network analyses, and module clustering, together with copy number variants implicated in TS, strongly implicates disrupted interneuron signaling in the pathophysiology of severe TS and suggests that metabolic alterations may be linked to their death or dysfunction.

Keywords: Basal ganglia; Immune system; Interneuron; NOS; Network; Nitric oxide synthase; RNA-Seq.

Figure 1. General scheme of our analyses

Analysis flow diagram for RNA-seq data analysis. Boxes: pre-analysis and post-analysis data; arrows: methods applied to pre-analysis data to get post-analysis data/results. Red text: results. RNA-seq reads are converted into read counts and the Reads Per Kilobase of transcript per Million mapped reads, or RPKM, for each transcript in the Gencode annotation. edgeR is applied to counts to infer differentially gene expression (DGE). Weighted Gene Co-expression Network Analysis, or WGCNA, in combination with Gene Set Enrichment Analysis, or GSEA, is applied to RPKM to infer modules of co-expressed genes enriched in DEGs.

Figure 2. Differential NOS+ cell density, elevated CD45/PTPRC+ cell density, and microglial activation in Tourette syndrome patients

(A-C) Immunostaining for NOS⁺ cells in the caudate of representative normal control (A) and TS case (B). Scale bars 100μm. (C) Stereological estimates of NOS⁺ cell density (cells per mm³) in the caudate and putamen of normal control (n=7) and TS (n=7) cases. (D-F) Immunostaining for CD45/PTPRC⁺ cells in the caudate of representative normal control (D) and Tourette syndrome case (E). Scale bars 100 μm. (F) Stereological estimates of PTPRC ⁺ cell density (cells per mm³) in the caudate of control (n=10) and TS (n=8) cases. (G-I) Immunostaining for IBA⁺ cells in caudate of representative normal control (G) and Tourette syndrome case (H). Scale bars 100 μm. (I) IBA⁺ cell body size (μm²) and dendrite length (μm) in the caudate in control (n=7) and TS (n=5) cases. * p<0.05, one-tailed student's t-test.

Figure 3. Differentially co-expressed gene networks in TS

The center of the figure shows the global network where the nodes (green circles) are WGCNA modules and the edges are significant correlations between the modules eigen-genes. The four inserts show the detailed network within four relevant modules; Top left: blue (neuronal metabolism) module. Top right: magenta (immune/microglia) module. Bottom right: lightcyan (cell adhesion/protocadherin) module. Bottom left: turquoise module (synaptic transmission/interneuron). Circles: network genes. Large Circles: top five genes with highest intra-modular connectivity (hubs). Squares: network genes overlapping with CNVs genes. Red: up-regulated gene. Blue: down-regulated gene. Concentric circle arrangement in the case of the turquoise (bottom left) and magenta (top right) modules reflects intra-modular connectivity: genes closer to the center have higher intra-modular connectivity than genes further away from the center.