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Circadian Rhythms, Neurons, and Sleep

Research Summary

Michael Rosbash is interested in circadian rhythms, which reflect a circa 24-hour pacemaker that is nearly ubiquitous among higher organisms. The same mechanism and proteins function in mammals and flies, indicating deep conservation and making Drosophila an important model system. A myriad of tissues and systems—including much of animal biochemistry, endocrinology, and physiology—are under clock control. Circadian clocks also function within the brain to influence sleep.

Our current work has several major goals: (1) to understand fly brain circadian circuitry and its relationship to behavior, phase shifting, and the molecular clock; (2) to understand the mechanism and importance of circadian neuronal plasticity; (3) to understand the why and how of sleep and its relationship to circadian rhythms; (4) to understand the contribution of posttranscriptional regulation to circadian biology, sleep, and neuronal function; and (5) to understand better circadian timing, circadian transcriptional regulation, and the enigmatic process of temperature compensation.

Fly Brain Circadian Circuitry and Its Relationship to Activity Rhythms, Phase Shifting, and the Molecular Clock
With respect to activity rhythms, many insect species, including Drosophila, manifest a crepuscular pattern: two locomotor activity peaks are centered roughly on dawn and dusk. This activity pattern is governed by approximately 75 pairs of circadian neurons. Some years ago we established a relationship between two groups of these neurons, morning and evening cells, and the morning and evening peaks of activity. We subsequently profiled gene expression at different times of day on Affymetrix arrays, concentrating on the two subgroups of morning cells. Our current work is focused on extending these observations in three directions. First, we have substantially refined the key evening cell population. Four key evening cells in combination with four morning cells are sufficient to recapitulate all known features of circadian locomotor behavior. Second, we are profiling gene expression from the four evening neurons by around-the-clock RNA-Seq and comparing this with the same profiling from morning cells. Third, we are advancing our understanding of the relationship between morning and evening cells. Current experiments suggest not surprisingly that these cells fire at opposite times of day, the morning cells in the morning and the evening cells in the evening, and hint at how firing is maintained out of phase.

The study of Drosophila phase shifting by light has been dominated for 15 years by a cell-autonomous model: light causes the photoreceptor Cryptochrome (CRY) to undergo a conformational change, which causes timeless protein (TIM) degradation. Our recent work indicates, however, that the firing of specific circadian neurons mimics light and causes proper phase shifting, including TIM degradation. These results and others indicate that Drosophila phase shifting, like that in mammals, relies on a neuronal circuit. We are also interested in how firing more generally intersects with the clock transcription-translation program.

Genetic manipulations of Mef2 levels disrupt circadian and activity-dependent changes in axonal fasciculation of the s-LNv dorsal projections.

The Mechanism and Importance of Circadian Neuronal Plasticity
The dorsal processes of fly circadian neurons undergo a prominent daily cycle of neuronal process bundling and unbundling (fasciculation and defasciculation). Although neuronal firing is important for this morphological cycle, we have recently shown that the core transcriptional clock regulates it quite directly. We would like to know more about this circadian plasticity cycle, its functions, and the role played by postsynaptic partners.

The Why and How of Sleep and Its Relationship to Circadian Rhythms
Although sleep is still an enigma, fly sleep appears to mimic mammalian sleep in most of its key features, which include regulation by the circadian clock as well as regulation by a poorly understood homeostatic mechanism. The latter keeps track of sleep so that we, or flies, can make up lost sleep (sleep rebound or recovery sleep). Although the two regulatory systems are believed to be distinct, we have evidence that some circadian neurons are important for regulating total sleep levels. We are also engaged in genetic as well as molecular studies to identify molecules involved in sleep regulation, especially in sleep deprivation and in recovery sleep. Lastly, we are studying sleep regulation in the context of aging. Old flies, like old humans, sleep more poorly than young flies. We would like to understand why, which might provide insight into an important aspect of aging, into sleep regulation, or both.

The Contribution of Posttranscriptional Regulation to Circadian Biology, Sleep, and Neuronal Function
Our recent studies analyzing nascent RNA (Nascent-Seq) from fly heads as well as mouse liver indicate considerable posttranscriptional regulation, i.e., mRNA circadian oscillations that are not strictly due to transcriptional regulation. We had also approached this topic from another direction, by finding microRNAs (miRNAs) that undergo circadian oscillations in level. To pursue these topics, the posttranscriptional regulation of mRNAs and cycling miRNAs in the context of circadian neurons, we recently sequenced miRNAs from isolated circadian neurons around-the-clock and validated several cycling miRNAs. Their mechanism of cycling, molecular targets, and functions are being pursued.

Our recent Nascent-Seq studies addressed mRNA editing. We became interested in this topic not only because of its relationship to neuroscience (many important neuronal mRNAs are edited) but also because it is not well studied in the context of specific neurons. We are therefore analyzing the editing of mRNA isolated from discrete neuronal populations, including circadian neurons.

Circadian Transcriptional Regulation, Circadian Timing, and Temperature Compensation
There are two enigmatic features of the circadian transcription factor CLOCK:CYCLE binding to DNA (CLOCK:BMAL1 binding in mammals): the enormous number of target sites and genes as well as a disconnect between the uniform phase of DNA binding and the heterogeneous phases of cycling transcription. Our recent work in mouse liver suggests that the disconnect is due in part to a CLOCK:BMAL1 "pioneer-like" activity; i.e., the transcription factor opens chromatin to allow the binding of additional transcription factors. In fly heads, the large number of binding sites reflects in part tissue-specific binding. We are keen to compare binding sites and target genes between different categories of circadian neurons. We are also keen to track detailed clock gene transcription within different circadian neurons in real time.

There are still many unexplained aspects of circadian timing: for example, why is there so little period-length variation between individuals, and why is there so little change with temperature (temperature compensation)? We are interested in both of these issues and beginning genetic screens, focusing on a few circadian neurons of interest.

Grants from the National Institutes of Health and the Ellison Medical Foundation provided support for ongoing projects.

As of October 7, 2013

Scientist Profile

Investigator
Brandeis University
Molecular Biology, Neuroscience