Diurnal rhythms across the human dorsal and ventral striatum

Abstract

The human striatum can be subdivided into the caudate, putamen, and nucleus accumbens (NAc). Each of these structures have some overlapping and some distinct functions related to motor control, cognitive processing, motivation, and reward. Previously, we used a "time-of-death" approach to identify diurnal rhythms in RNA transcripts in human cortical regions. Here, we identify molecular rhythms across the three striatal subregions collected from postmortem human brain tissue in subjects without psychiatric or neurological disorders. Core circadian clock genes are rhythmic across all three regions and show strong phase concordance across regions. However, the putamen contains a much larger number of significantly rhythmic transcripts than the other two regions. Moreover, there are many differences in pathways that are rhythmic across regions. Strikingly, the top rhythmic transcripts in NAc (but not the other regions) are predominantly small nucleolar RNAs and long noncoding RNAs, suggesting that a completely different mechanism might be used for the regulation of diurnal rhythms in translation and/or RNA processing in the NAc versus the other regions. Further, although the NAc and putamen are generally in phase with regard to timing of expression rhythms, the NAc and caudate, and caudate and putamen, have several clusters of discordant rhythmic transcripts, suggesting a temporal wave of specific cellular processes across the striatum. Taken together, these studies reveal distinct transcriptome rhythms across the human striatum and are an important step in helping to understand the normal function of diurnal rhythms in these regions and how disruption could lead to pathology.

Keywords: circadian rhythms; gene expression; human postmortem; striatum.

Fig. 1.

Circadian gene expression patterns of four canonical circadian clock genes in the NAc (Top), caudate (Middle), and putamen (Bottom). In the scatterplots, each dot represents a subject, with the x axis indicating TOD on a ZT scale (−6 to 18 h) and the y axis indicating level of transcript expression. The red line is the fitted sinusoidal curve. Confidence intervals (90%) of peak estimates are depicted by the red bar in each plot. All four clock genes show rhythms in each striatal region, with consistent peak times across regions. Peak time and P values are located above each scatterplot.

Fig. 2.

Characterization of the top rhythmic transcripts in the NAc, caudate, and putamen. (A) Lists of the top 20 rhythmic transcripts in the NAc, caudate, and putamen. Many of the top rhythmic transcripts in the caudate are core circadian clock genes. In the NAc, most of the top rhythmic transcripts are noncoding RNAs, including snoRNAs, snRNAs, and lncRNAs. Ensembl IDs are listed for the lncRNAs, as all these transcripts were not associated with Human Genome Organisation Gene Nomenclature Committee symbols. (B) Biotype charts of the top 100 rhythmic transcripts revealed that the majority of the top rhythmic transcripts in the caudate and putamen are protein-coding, with a small percentage of noncoding RNAs (i.e., lncRNAs). In the NAc, a larger percentage of the top rhythmic transcripts were noncoding RNAs, particularly snoRNAs (14%).

Fig. 3.

Pathway and biological process enrichment for rhythmic transcripts in the NAc, caudate, and putamen. (A) Top five pathways and upstream regulators enriched for rhythmic transcripts in each striatal region (Dataset S4 provides a complete list of pathways and upstream regulators). A significance threshold of P < 0.01 (NAc, 1,344 transcripts; caudate, 1,053 transcripts; putamen, 3,097 transcripts) was used to determine rhythmicity for the transcript input list. The significance threshold for pathway and upstream regulator enrichment is P < 0.05 [−log₁₀(P value) > 1.3 in plots]. (B) Pathway enrichment for all three regions shown on the same plot, revealing both distinct and overlapping pathway enrichment between regions. (C) GO biological process enrichment via Metascape for the top 1,000 rhythmic transcripts in each region. Meta-analysis was used to compare process enrichment across striatal regions, depicted in the Cytoscape network plots. Terms with P < 0.01, a minimum count of 3, and enrichment factor >1.5 were grouped into clusters based on their membership similarities. The most statistically significant term within a cluster was chosen to represent the cluster. The top 10 significant terms were chosen for visualization (Dataset S5 provides a complete list of all enriched processes within each cluster). The nodes are represented as pie charts, where the size of the pie is proportional to the total number of gene hits for that specific term. The pie charts are color-coded based on the identity of the gene list, where the size of the slice represents the percentage of transcripts enriched for each corresponding term. Similar terms (kappa score >0.3) are connected by edges.

Fig. 4.

Overlap in rhythmic transcripts between striatal regions. (A) RRHO plot indicating a high degree of overlap in rhythmic transcripts between the NAc and putamen and the caudate and putamen. In contrast, there was a very small degree of overlap in rhythmic transcripts between the NAc and caudate. (B) Venn diagrams showing overlap of rhythmic transcripts at a significance threshold of P < 0.05. Consistent with the RRHO plots, transcripts in common between only the NAc and putamen (1,157 transcripts) and caudate and putamen (1,163 transcripts) show a higher degree of overlap than the NAc and caudate (347 transcripts). There were 424 transcripts in common between all three striatal regions. (C) Top five pathways and upstream regulators enriched for rhythmic transcripts in common between NAc and caudate, NAc and putamen, caudate and putamen, and all three regions (Dataset S7 provides a complete list of pathways and upstream regulators). Overlapping rhythmic transcripts were from the Venn diagram in B. (D) GO biological process enrichment via Metascape for the rhythmic transcripts in common between the NAc and caudate, NAc and putamen, caudate and putamen, and all three regions (Dataset S8 provides a complete list of all enriched processes within each cluster). Network plots were generated using the same parameters as in Fig. 3.

Fig. 5.

Phase relationships between striatal regions. (A–C, Top) Phase concordance plots showing the phase relationship in rhythmic transcripts between the NAc and caudate (A), NAc and putamen (B), and caudate and putamen (C). For a given transcript, phases were plotted between the two regions, and transcripts were considered concordant if their phase differences fell within a window of ±4 h. (A, Top) There was low phase concordance between the NAc and caudate (46%; 215 concordant transcripts and 250 discordant transcripts). The discordant transcripts appeared in three distinct clusters. (B, Top) There was very high phase concordance between the NAc and putamen (89%; 1,715 concordant transcripts and 211 discordant transcripts). (C, Top) There was also high phase concordance between the caudate and putamen (78%; 1,482 concordant transcripts and 411 discordant transcripts). The discordant transcripts appeared in two distinct clusters. (A–C, Middle) Top five pathways and upstream regulators enriched for concordant and discordant transcripts, with discordant clusters analyzed separately (Datasets S9, S12, and S15 provide complete lists of pathways and upstream regulators for concordant and discordant transcripts in each region comparison). (A–C, Bottom) GO biological process enrichment via Metascape for concordant and discordant transcripts between regions (Datasets S10, S13, and S16 provide complete lists of enriched processes within clusters for each region comparison). Network plots were generated using the same parameters as in Figs. 3 and 4.