Comparative rhythmic transcriptome profiling of human and mouse striatal subregions
The human striatum can be subdivided into the caudate, putamen, and nucleus accumbens (NAc). In mice, this roughly corresponds to the dorsal medial striatum (DMS), dorsal lateral striatum (DLS), and ventral striatum (NAc). Each of these structures have some overlapping and 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 these three human striatal subregions. Here, we identify molecular rhythms across similar striatal subregions collected from C57BL/6J mice across 6 times of day and compare results to the human striatum. Pathway analysis indicates a large degree of overlap between species in rhythmic transcripts involved in processes like cellular stress, energy metabolism, and translation. Notably, a striking finding in humans is that small nucleolar RNAs (snoRNAs) and long non-coding RNAs (lncRNAs) are among the most highly rhythmic transcripts in the NAc and this is not conserved in mice, suggesting the rhythmicity of RNA processing in this region could be uniquely human. Furthermore, the peak timing of overlapping rhythmic genes is altered between species, but not consistently in one direction. Taken together, these studies reveal conserved as well as distinct transcriptome rhythms across the human and mouse striatum and are an important step in understanding the normal function of diurnal rhythms in humans and model organisms in these regions and how disruption could lead to pathology. Overall design: Adult male and female C57BL/6J mice (Jax stock no: 000664) (~10 wks old) were maintained on a 12:12 light/dark schedule (lights on at 7 AM (zeitgeber time 0 (ZT0)) and off at 7 PM) and were provided with food and water ad libitum. All animal use was conducted in accordance with the National Institute of Health guidelines and approved by the Institutional Animal Care and Use Committees of the University of Pittsburgh.Mice were sacrificed across 6 times of day, 4 h apart (ZT 2, 6, 10, 14, 18, and 22; n=5–6 mice/sex/timepoint). Bilateral 1-mm punches were taken centered over the NAc, DMS, and DLS. Total RNA was extracted using the RNeasy Plus Micro Kit (Qiagen). RNA quantity and quality were assessed using fluorometry (Qubit RNA Broad Range Assay Kit and Fluorometer; Invitrogen) and chromatography (Bioanalyzer and RNA 6000 Nano Kit; Agilent), respectively. Libraries were prepared using the Smartseq stranded total RNA ultra-low input sample preparation kits (Illumina). Paired-end dual-indexed sequencing (75bp) was performed using NextSeq 500 platform (Illumina) at the University of Pittsburgh Health Sciences Sequencing Core at UPMC Children's Hospital of Pittsburgh. A total of 40 million reads per sample was targeted. After quality control, HISAT2 (HISAT2v2.1.0) was used to align reads to the reference (Mus musculus Ensembl GRCm38) using default parameters. The resulting bam files were converted to expression count data using HTSeq (HTSeq v0.10.0) with default union mode. Filtering and normal- ization were performed separately for NAc, DMS, and DLS samples. After filtering, 11,867, 12,101, and 11,395 genes remained for NAc, DMS, and DLS samples, respectively. DESeq2 was then used to normalize for sequencing depth and RNA composition followed by a log2 transformation.