The organization of biological activities into daily cycles is universal in organisms as diverse as cyanobacteria, fungi, algae, plants, flies, birds and man. Comparisons of circadian clocks in unicellular and multicellular organisms using molecular genetics and genomics have provided new insights into the mechanisms and complexity of clock systems. Whereas unicellular organisms require stand-alone clocks that can generate 24-hour rhythms for diverse processes, organisms with differentiated tissues can partition clock function to generate and coordinate different rhythms. In both cases, the temporal coordination of a multi-oscillator system is essential for producing robust circadian rhythms of gene expression and biological activity.The temporal coordination of internal biological processes, both among these processes and with external environmental cycles, is crucial to the health and survival of diverse organisms, from bacteria to humans. Central to this coordination is an internal CLOCK that controls CIRCADIAN RHYTHMS of gene expression and the resulting biological activity (BOX 1). Despite disparate phylogenetic origins and vast differences in complexity among the species that show circadian rhythmicity, at the core of all circadian clocks is at least one internal autonomous circadian OSCILLATOR. These oscillators contain positive and negative elements that form autoregulatory feedback loops, and in many cases these loops are used to generate 24-hour timing circuits 1, 2 . Components of these loops can directly or indirectly receive environmental input to allow ENTRAINMENT of the clock to environmental time and transfer temporal information through output Competing interests statementThe authors declare no competing financial interests. NIH Public Access Author ManuscriptNat Rev Genet. Author manuscript; available in PMC 2009 September 1. Published in final edited form as:Nat Rev Genet. 2005 July ; 6(7): 544-556. doi:10.1038/nrg1633. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript pathways to regulate rhythmic clock-controlled gene (CCG) expression and rhythmic biological activity.Whereas a self-contained clock in single-celled organisms programmes 24-hour rhythms in diverse processes, multicellular organisms with differentiated tissues can partition clock function among different cell types to coordinate tissue-specific rhythms and maintain precision. Now that individual molecular circadian oscillators have been sufficiently described, it has become possible to go beyond single oscillators to try and understand how multiple oscillators are integrated into circadian systems. Evidence accumulated in recent years indicates that the intracellular oscillator systems of single-celled organisms might be more complex than those of higher eukaryotes, whereas the complexity of circadian outputs in multicellular organisms is an emergent property of intercellular interactions. In this review, we discuss the complexity of the circadian clocks on the basis of molecular genetic and geno...

The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) of the mammalian brain controls system-level rhythms in animal physiology. Specific SCN outputs synchronize circadian physiological rhythms in other brain regions. Within the SCN, communication among neural cells provides for the coordination of autonomous cellular oscillations into ensemble rhythms. Adenosine triphosphate (ATP) is a neural transmitter involved in local communication among astrocytes and between astrocytes and neurons. Using a luciferinluciferase chemiluminescent assay, we have demonstrated that ATP levels fluctuate rhythmically within both SCN2.2 cell cultures and the rat SCN in vivo. SCN2.2 cells generated circadian oscillations in both the production and extracellular accumulation of ATP. Circadian fluctuations in ATP accumulation persisted with an average period (τ) of 23.7hr in untreated as well as vehicleand forskolin-treated SCN2.2 cells, indicating that treatment with an inductive stimulus is not necessary to propagate these rhythms. ATP levels in the rat SCN in vivo were marked by rhythmic variation during exposure to LD12:12 or constant darkness, with peak accumulation occurring during the latter half of the dark phase or subjective night. Primary cultures of cortical astrocytes similarly expressed circadian oscillations in extracellular ATP accumulation that persisted for multiple cycles with periods of about 23hr. These results suggest that circadian oscillations in extracellular ATP levels represent a physiological output of the mammalian cellular clock, common to the SCN pacemaker and astrocytes from at least some brain regions, and thus may provide a mechanism for clock control of gliotransmission between astrocytes and to neurons.

In vertebrates, sialylated glycans participate in a wide range of biological processes and affect nervous system’s development and function. While the complexity of glycosylation and the functional redundancy among sialyltransferases provide obstacles for revealing biological roles of sialylation in mammals, Drosophila possesses a sole vertebrate-type sialyltransferase, DSiaT, with significant homology to its mammalian counterparts, suggesting that Drosophila could be a suitable model to investigate the function of sialylation. To explore this possibility and investigate the role of sialylation in Drosophila, we inactivated DSiaT in vivo by gene targeting and analyzed phenotypes of DSiaT mutants using a combination of behavioural, immunolabeling, electrophysiological and pharmacological approaches. Our experiments demonstrated that DSiaT expression is restricted to a subset of CNS neurons throughout development. We found that DSiaT mutations result in significantly decreased life span, locomotor abnormalities, temperature-sensitive paralysis and defects of neuromuscular junctions. Our results indicate that DSiaT regulates neuronal excitability and affects the function of a voltage-gated sodium channel. Finally, we showed that sialyltransferase activity is required for DSiaT function in vivo, which suggests that DSiaT mutant phenotypes result from a defect in sialylation of N-glycans. This work provided the first evidence that sialylation has an important biological function in protostomes, while also revealing a novel, nervous system-specific function of α2,6 sialylation. Thus, our data shed light on one of the most ancient functions of sialic acids in metazoan organisms and suggest a possibility that this function is evolutionarily conserved between flies and mammals.

The master circadian pacemaker located within the suprachiasmatic nuclei (SCN) controls neural and neuroendocrine rhythms in the mammalian brain. Astrocytes are abundant in the SCN and this cell type displays circadian rhythms in clock gene expression and extracellular accumulation of adenosine triphosphate (ATP). Still, the intracellular signaling pathways that link the SCN clockworks to circadian rhythms in extracellular ATP accumulation remain unclear. Since ATP release from astrocytes is a calcium-dependent process, we investigated the relationship between intracellular Ca2+ and ATP accumulation and have demonstrated that intracellular Ca2+ levels fluctuate in an antiphase relationship with rhythmic ATP accumulation in rat SCN2.2 cell cultures. Furthermore, mitochondrial Ca2+ levels were rhythmic and maximal in precise antiphase with the peak in cytosolic Ca2+. In contrast, our finding that peak mitochondrial Ca2+ occurred during maximal extracellular ATP accumulation suggests a link between these cellular rhythms. Inhibition of the mitochondrial Ca2+uniporter disrupted the rhythmic production and extracellular accumulation of ATP. ATP, calcium and the biological clock affect cell division and have been implicated in cell death processes. Nonetheless, rhythmic extracellular ATP accumulation was not disrupted by cell cycle arrest and was not correlated with caspase activity in SCN2.2 cell cultures. Taken together, these results demonstrate that mitochondrial Ca2+mediates SCN2.2 rhythms in extracellular ATP accumulation and suggest a role for circadian gliotransmission in SCN clock function.

While sialylation plays important functions in the nervous system, the complexity of glycosylation pathways and limitations of genetic approaches preclude the efficient analysis of these functions in mammalian organisms. Drosophila has recently emerged as a promising model for studying neural sialylation. Drosophila sialyltransferase, DSiaT, was shown to be involved in the regulation of neural transmission. However, the sialylation pathway was not investigated in Drosophila beyond the DSiaT-mediated step. Here we focused on the function of Drosophila cytidine monophosphate-sialic acid synthetase (CSAS), the enzyme providing a sugar donor for DSiaT. Our results revealed that the expression of CSAS is tightly regulated and restricted to the CNS throughout development and in adult flies. We generated CSAS mutants and analyzed their phenotypes using behavioral and physiological approaches. Our experiments demonstrated that mutant phenotypes of CSAS are similar to those of DSiaT, including decreased longevity, temperature-induced paralysis, locomotor abnormalities, and defects of neural transmission at neuromuscular junctions. Genetic interactions between CSAS, DSiaT, and voltagegated channel genes paralytic and seizure were consistent with the hypothesis that CSAS and DSiaT function within the same pathway regulating neural excitability. Intriguingly, these interactions also suggested that CSAS and DSiaT have some additional, independent functions. Moreover, unlike its mammalian counterparts that work in the nucleus, Drosophila CSAS was found to be a glycoproteinbearing N-glycans and predominantly localized in vivo to the Golgi compartment. Our work provides the first systematic analysis of in vivo functions of a eukaryotic CSAS gene and sheds light on evolutionary relationships among metazoan CSAS proteins.