Molecular Biology&Biochemistry Neurology
Circadian clocks control essential aspects of animal physiology that oscillate with a roughly 24h period, and aberrant clock function is linked to various human pathologies including metabolic, proliferative, psychiatric and neurodegenerative disorders. In humans and flies, the circadian clock is driven by daily cycles of gene expression produced by oscillations of the PERIOD (PER) transcription factor, which binds to a positively acting transcription factor to antagonize per expression as well as the expression of many other circadian genes. The protein kinase DOUBLETIME (DBT) plays a central role in the Drosophila clock by controlling the level of PER via PER phosphorylation state and is the ortholog of the human clock components casein kinase I (CKI) d and e. My lab’s analysis of various dbt mutants has raised the possibility that the activity of DBT may be modulated by proteins with which it interacts. We have recently identified novel components of the Drosophila circadian clock called BRIDE OF DOUBLETIME (BDBT) and SPAGHETTI (SPAG) using proteomics and genetic screens for DBT-binding proteins. Knocking down the expression of BDBT leads to reduced phosphorylation and over-accumulation of PER, and BDBT is necessary for circadian behavioral and molecular rhythms. Complementary structural analysis has shown that BDBT resembles FK506-binding proteins, a class of enzymes that catalyze the cis-trans isomerization of proline residues. However, the residues responsible for catalytic activity are not conserved in BDBT, demonstrating that BDBT enhances DBT’s circadian function in a manner that does not involve the prolyl isomerase activity of canonical members of this protein family. Instead, the DBT/BDBT complex may control regulated nuclear localization of clock protein complexes. SPAG, an HSP90 cochaperone, is also a DBT-interacting protein, and RNAi knock-down of its mRNA produces long circadian periods, increased DBT autophosphorylation and reduced DBT levels in the middle of the day .
It is clear that a relationship exists between circadian dysfunction and neurodegenerative conditions like Alzheimer’s Disease (AD), but the reasons for and nature of the relationship are not clear. AD patients often exhibit disrupted sleep-wake behavior, but it is not known whether the disruptions are caused by AD or contribute to AD. Aggregates of the microtubule-binding protein tau are found in AD patients and are thought to contribute to its pathology. Our recently published work has established a relationship between apoptosis pathways and circadian components that is likely to be relevant to tauopathies, including AD. We have shown that reductions in activity of the circadian DBT kinase leads to light-dependent activation of the caspase DRONC by cleavage. The reductions in DBT activity are produced either by expression of a catalytically inactive form of DBT or by RNAi knock-down of spag. When DBT activity is down-regulated in the eye together with human tau (htau) expression, the htau protein is cleaved, DRONC is activated by cleavage and eye-specific degeneration is enhanced. Reduced DBT activity most likely works cell autonomously in Drosophila S2 cells and eye photoreceptors to activate DRONC but also stimulates DRONC activation non-autonomously in adjacent cells via signaling from the circadian neuropeptide PDF. In summary, my lab has proposed that DBT may inhibit DRONC activation by phosphorylating it to prevent its cleavage, and that DRONC-mediated cleavage of htau may connect light-induced processes involving circadian components to enhance tauopathy. These processes are stimulated by the circadian neuropeptide PDF and by aging, which leads to DBT phosphorylation and DRONC activation even in wild type flies and potentially in humans.