The E3 ubiquitin ligase adaptor Tango10 links the core circadian clock to neuropeptide and behavioral rhythms

Circadian transcriptional timekeepers in pacemaker neurons drive profound daily rhythms in sleep and wake. Here we reveal a molecular pathway that links core transcriptional oscillators to neuronal and behavioral rhythms. Using two independent genetic screens, we identified mutants of Transport and Golgi organization 10 (Tango10) with poor behavioral rhythmicity. Tango10 expression in pacemaker neurons expressing the neuropeptide PIGMENT-DISPERSING FACTOR (PDF) is required for robust rhythms. Loss of Tango10 results in elevated PDF accumulation in nerve terminals even in mutants lacking a functional core clock. TANGO10 protein itself is rhythmically expressed in PDF terminals. Mass spectrometry of TANGO10 complexes reveals interactions with the E3 ubiquitin ligase CULLIN 3 (CUL3). CUL3 depletion phenocopies Tango10 mutant effects on PDF even in the absence of the core clock gene timeless. Patch clamp electrophysiology in Tango10 mutant neurons demonstrates elevated spontaneous firing potentially due to reduced voltage-gated Shaker-like potassium currents. We propose that Tango10/Cul3 transduces molecular oscillations from the core clock to neuropeptide release important for behavioral rhythms.

High-Salt Diet Impairs the Neurons Plasticity and the Neurotransmitters-Related Biological Processes

Salt, commonly known as sodium chloride, is an important ingredient that the body requires in relatively minute quantities. However, consuming too much salt can lead to high blood pressure, heart disease and even disruption of circadian rhythms. The biological process of the circadian rhythm was first studied in Drosophila melanogaster and is well understood. Their locomotor activity gradually increases before the light is switched on and off, a phenomenon called anticipation. In a previous study, we showed that a high-salt diet (HSD) impairs morning anticipation behavior in Drosophila. Here, we found that HSD did not significantly disrupt clock gene oscillation in the heads of flies, nor did it disrupt PERIOD protein oscillation in clock neurons or peripheral tissues. Remarkably, we found that HSD impairs neuronal plasticity in the axonal projections of circadian pacemaker neurons. Interestingly, we showed that increased excitability in PDF neurons mimics HSD, which causes morning anticipation impairment. Moreover, we found that HSD significantly disrupts neurotransmitter-related biological processes in the brain. Taken together, our data show that an HSD affects the multiple functions of neurons and impairs physiological behaviors.

Chloride oscillation in pacemaker neurons regulates circadian rhythms through a chloride-sensing WNK kinase signaling cascade

Central pacemaker neurons regulate circadian rhythms and undergo diurnal variation in electrical activity in mammals and flies. In mammals, circadian variation in the intracellular chloride concentration of pacemaker neurons has been proposed to influence the response to GABAergic neurotransmission through GABAA receptor chloride channels. However, results have been contradictory, and a recent study demonstrated circadian variation in pacemaker neuron chloride without an effect on GABA response. Therefore, whether and how intracellular chloride regulates circadian rhythms remains controversial. Here, we demonstrate a signaling role for intracellular chloride in the Drosophila ventral lateral (LNv) pacemaker neurons. In control flies, intracellular chloride increases in LNv neurons over the course of the morning. Chloride transport through the sodium-potassium-2-chloride (NKCC) and potassium-chloride (KCC) cotransporters is a major determinant of intracellular chloride concentrations. Drosophila melanogaster with loss-of-function mutations in the NKCC encoded by Ncc69 have abnormally low intracellular chloride six hours after lights on, and a lengthened circadian period. Loss of kcc, which is expected to increase intracellular chloride, suppresses the long-period phenotype of Ncc69 mutant flies. Activation of a chloride-inhibited kinase cascade, consisting of the WNK (With No Lysine (K)) kinase and its downstream substrate, Fray, is necessary and sufficient to prolong period length. Fray activation of an inwardly rectifying potassium channel, Irk1, is also required for the long-period phenotype. These results indicate that the NKCC-dependent rise in intracellular chloride in Drosophila LNv pacemaker neurons restrains WNK-Fray signaling and overactivation of an inwardly rectifying potassium channel to maintain normal circadian period length.

A screen for sleep and starvation resistance identifies a wake-promoting role for the auxiliary channel unc79

The regulation of sleep and metabolism are highly interconnected, and dysregulation of sleep is linked to metabolic diseases that include obesity, diabetes, and heart disease. Further, both acute and long-term changes in diet potently impact sleep duration and quality. To identify novel factors that modulate interactions between sleep and metabolic state, we performed a genetic screen for their roles in regulating sleep duration, starvation resistance, and starvation-dependent modulation of sleep. This screen identified a number of genes with potential roles in regulating sleep, metabolism or both processes. One such gene encodes the auxiliary ion channel UNC79, which was implicated in both the regulation of sleep and starvation resistance. Genetic knockdown or mutation of unc79 results in flies with increased sleep duration, as well as increased starvation resistance. Previous findings have shown that unc79 is required in pacemaker for 24-hour circadian rhythms. Here, we find that unc79 functions in the mushroom body, but not pacemaker neurons, to regulate sleep duration and starvation resistance. Together, these findings reveal spatially localized separable functions of unc79 in the regulation of circadian behavior, sleep, and metabolic function.

Uncovering the Roles of Clocks and Neural Transmission in the Resilience of Drosophila Circadian Network

Studies of circadian locomotor rhythms in Drosophila melanogaster gave evidence to the preceding theoretical predictions on circadian rhythms. The molecular oscillator in flies, as in virtually all organisms, operates using transcriptional-translational feedback loops together with intricate post-transcriptional processes. Approximately150 pacemaker neurons, each equipped with a molecular oscillator, form a circuit that functions as the central pacemaker for locomotor rhythms. Input and output pathways to and from the pacemaker circuit are dissected to the level of individual neurons. Pacemaker neurons consist of functionally diverse subclasses, including those designated as the Morning/Master (M)-oscillator essential for driving free-running locomotor rhythms in constant darkness and the Evening (E)-oscillator that drives evening activity. However, accumulating evidence challenges this dual-oscillator model for the circadian circuit organization and propose the view that multiple oscillators are coordinated through network interactions. Here we attempt to provide further evidence to the revised model of the circadian network. We demonstrate that the disruption of molecular clocks or neural output of the M-oscillator during adulthood dampens free-running behavior surprisingly slowly, whereas the disruption of both functions results in an immediate arrhythmia. Therefore, clocks and neural communication of the M-oscillator act additively to sustain rhythmic locomotor output. This phenomenon also suggests that M-oscillator can be a pacemaker or a downstream path that passively receives rhythmic inputs from another pacemaker and convey output signals. Our results support the distributed network model and highlight the remarkable resilience of the Drosophila circadian pacemaker circuit, which can alter its topology to maintain locomotor rhythms.

Emergent population activity in metric-free and metric networks of neurons with stochastic spontaneous spikes and dynamic synapses

We show that networks of excitatory neurons with stochastic spontaneous spiking activity and short-term synaptic plasticity can exhibit spontaneous repetitive synchronization in so-called population spikes. The major reason for this is that synaptic plasticity nonlinearly modulates the interaction between neurons. For large-scale two-dimensional networks, where the connection probability decreases exponentially with increasing distance between the neurons resulting in a small-world network connectome, a population spike occurs in the form of circular traveling waves diverging from seemingly non-stationary nucleation sites. The latter is in drastic contrast to the case of networks with a fixed fraction of steady pacemaker neurons, where the set of a few spontaneously formed nucleation sites is stationary. Despite the spatial non-stationarity of their nucleation, population spikes may occur surprisingly regularly. From a theoretical viewpoint, these findings show that the regime of nearly-periodic population spikes, which mimics respiratory rhythm, can occur strictly without stochastic resonance. In addition, the observed spatiotemporal effects serve as an example of transient chimera patterns.