Investigating roles of the Top3b-Tdrd3 complex in regulation of sleep and circadian rhythms in mental health disorders

National Institute On Aging

Investigators

Abstract

Circadian rhythms are ~24-hour cycles in behavior and physiological processes that allow organisms to anticipate and adapt to environmental changes. These rhythms are generated by internal timing systems called circadian clocks that are comprised of three major components: the molecular clock, input pathways which synchronize the clock to the environment, and output pathways which transmit information to temporally organize molecular and physiological processes. The molecular clock functions as a transcriptional-translation feedback loop to drive rhythmic transcription of thousands of genes. In mammals, the transcription factors CLOCK and BMAL1 activate the transcription of clock-controlled genes (CCGs), including their own repressors, PERIOD (PER) and CRYPTOCHROME (CRY). PER and CRY accumulate in the cytoplasm, are post-translationally modified, and then translocate into the nucleus where they inhibit CLOCK and BMAL1 to prevent further transcription. Over time, PER and CRY degrade, allowing for the ~24-hour cyclic mechanism to repeat14. While most research on circadian control have focused on transcriptional and posttranslational mechanisms, less than 30% of circadian mRNAs are directly regulated by transcription, suggesting that other post-transcriptional, RNA-based mechanisms play critical roles. For instance, processes such as alternative splicing, mRNA stability, polyadenylation, and circadian miRNAs, have all been demonstrated to contribute to circadian gene expression. Notably, other RNA-binding proteins that regulate neurodevelopment, such as Ataxin-2 and FMRP, are key regulators of circadian activity patterns in both Drosophila and mice, highlighting the potential connection between neurodevelopment and circadian defects. Given the role of Top3b-Tdrd3 in RNA metabolisms, its links to neurodevelopment, and its interaction with FMRP, we hypothesize that the Top3b-Tdrd3 complex may resemble FMRP in playing a critical role in regulation of circadian rest-activity pathways in both Drosophila and mice. The findings from this project will provide a deeper understanding of RNAbased control of the circadian clock and its implications for human diseases such as autism and schizophrenia. Ultimately, elucidating these molecular mechanisms may present new therapeutic concepts and establish sleep and circadian dysfunctions as important foci for clinical intervention. We have made important progress for different aims of this project: we employed the Drosophila Activity Monitoring (DAM) system to monitor circadian behaviors of WT and Top3b-TDRD3 KO Drosophila. We found that Top3b-KO and Top3b-RGG deletion mutant (which lacks RNA-binding domain) mutant flies exhibit weakened rest-activity patterns, whereas Top3b-Y332F catalytic mutant display normal patterns, indicating that Top3b depends on its RNA-binding but not topoisomerase activity to regulate circadian rhythms. Moreover, Tdrd3-KO flies exhibit arrhythmic rest-activity patterns, which can be rescued by re-expression of Tdrd3. Thus, Tdrd3 plays a major, whereas Top3b plays minor, role for normal circadian rhythms. Our subsequent analysis revealed that Tdrd3-KO, but not Top3b-KO flies show aberrant branching and tract splitting of pdf-neurons, which are central pacemakers required to drive circadian behaviors. Moreover, our RNA-seq at two timepoints, one during the day and another during the night, identified 42% of transcripts in Top3b-KO and 36% of transcripts in Tdrd3-KO lost their normal rhythmic fluctuation patterns compared to WT, suggesting that Top3b-Tdrd3 may regulate circadian gene expression. Our data suggest that Top3b-Tdrd3 is a key regulator of rest-activity patterns, and infer that altered sleep and circadian dysfunctions could contribute to mental health disorders observed in individuals carrying their mutations. We are currently examining circadian behaviors of Top3b-mutant mice in collaboration with Ross McDevitt and Victoria Acosta-Rodriguez's groups.