Timed Exercise and Clocks: Does a Minimally Functional Suprachiasmatic Clock Support 24h Behavioral Rhythms?

The brain’s suprachiasmatic nucleus (SCN) is the master clock driving circadian rhythms in mammals. Vasoactive intestinal polypeptide (VIP) and its cognate receptor, VPAC2, are expressed in SCN neurons and mice with genetically targeted deletion of VPAC2 (Vipr2 -/-animals) show aberrant resetting to light, abnormal behavioral rhythms, and diminished SCN clock gene expression. Timed daily access to a running-wheel (scheduled voluntary exercise; SVE) promotes Vipr2 -/- SCN clock cell synchrony and 24h behavioral rhythms. We hypothesized that timed exercise alters the SCN transcriptome. Here, in control (Vipr2+/+) and Vipr2-/- mice under freely exercising and SVE conditions, RNAseq and qRT-PCR were used to measured gene expression of laser-dissected SCN. Compared to Vipr2+/+ mice, hundreds of genes were differentially expressed in the SCN from Vipr2-/- mice rhythmic in the freely exercising condition. Unexpectedly, SVE did not promote a Vipr2+/+-like SCN transcriptome in Vipr2-/- mice and many transcripts involved in SCN function including Avp, C1ql3, Gpr176, Prok2, Sst, Per2, and Nr1d1 remained dysregulated in the SVE condition. By contrast, circadian oscillators in the liver and lung were mostly intact in Vipr2-/- mice. This study indicates that marked molecular deficits in the SCN are sustained in behaviorally rhythmic Vipr2-/- mice, raising the possibility that a minimal functional SCN circadian clock can underpin whole animal rhythms. Samples of laser-microdissected SCN from four experimental groups were used to assess gene expression by bulk RNAseq analysis and included: Vipr2+/+ nSVE CT14 (n=4), Vipr2+/+ SVE ZT14/CT24 (n=4), Vipr2-/- nSVE CT14 (n=3), and Vipr2-/- SVE ZT14/CT14 (n=3). RNA extraction was carried out with ReliaPrep Kit (Promega, UK), checked with RNA 6000 Pico Kit (Agilent Technologies, USA), and amplified with with Sense Total RNA-Seq Library Prep Kit (Lexogen, USA). RNA sequencing was done by paired-end sequencing on an Illumina HiSeq 4000 NGS platform (Genomic Technologies Core Facility, University of Manchester, UK). Raw reads were uploaded to the Galaxy server at the University of Manchester (https://centaurus.itservices.manchester.ac.uk/). Reads were trimmed with Trimmomatic, aligned to the mouse genome (Version GRCm38) using HISAT2 aligner, and counts calculated using Stringtie and annotated using GENCODE. Differential gene expression was analysed using DeSeq2 was used. After obtaining the differentially expressed genes, the p-value was corrected with the Benjamini Hochberg method to obtain the False Discovery Rate.