Studying circadian (~24hr) rhythms offers a tremendous opportunity to understand a fundamental brain function at molecular, cellular and circuit levels as well as opportunities for novel therapies. Studies in Drosophila identified the first circadian clock gene, which is conserved in humans and is mutated in a familial sleep disorder. Clock genes function within pacemaker neurons in the brain to drive behavioral rhythms. Although we have a relatively good understanding of these molecular clocks, it is unknown how molecular clocks regulate rhythmic neuronal activity and how rhythmic outputs feed back to the molecular clock. To identify potential outputs of pacemaker neurons, we conducted gene expression profiling studies from a group of homogeneous master pacemaker neurons, the Drosophila LNvs. One gene rhythmically expressed in LNvs encodes an Inward rectifier K+ channel (Ir).
In Aim 1, we test the idea that Ir expression is directly regulated by the core clock transcription factors to regulate LNv excitability. Altered Ir expression in LNvs changes the period length of circadian rhythms. Thus the electrical activity of LNvs feeds back to regulate their molecular clocks. To test if the mechanism for this is transcriptional, we measured gene expression profiles in LNvs that were either constitutively hyperpolarized or hyperexcited. We found dramatic re-organization of circadian gene expression such that hyperpolarizing LNvs at dawn when they are normally most excitable makes their circadian gene expression resemble dusk, when they are normally inactive. Conversely, hyperexciting LNvs at dusk makes their circadian gene expression resemble dawn. Thus LNv neuronal activity can impose time-of-day to circadian gene expression and this suggests a major revision to our understanding of circadian molecular clocks.
In Aim 2, we propose to identify the transcription factor(s) and signaling pathway(s) that connect neuronal activity to circadian rhythms in gene expression. One group of genes mis-regulated by altering LNv excitability encode translation initiation regulators. This was striking because three protein translation regulators are highly expressed in LNvs and mutants in these factors alter circadian rhythms and/or clock resetting by light. Thus translation initiation is a previously unappreciated regulatory step in the molecular clock.
In Aim 3, we propose to test if these translation regulators affect translation of specific clock mRNAs and/or global translation in LNvs. We will also test if these regulators respond to signals such as light and neuronal activity to acutely regulate translation in LNvs. We believe that the endogenous neural and molecular rhythms of LNvs, combined with techniques we developed for whole-genome profiling of LNvs from an intact nervous system, make LNvs a unique model to study the dynamic bi-directional relationships between gene expression and neuronal excitability and plasticity.

Public Health Relevance

Disrupted circadian (~24hr) rhythms are associated with a wide range of human symptoms from sleep and mood disorders to increased incidences of cancer and obesity. These rhythms are controlled by intracellular clocks whose understanding at the molecular level may open up novel therapies. Here we seek to understand the molecules that regulate molecular clock speed via electrical activity and protein translation.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM063911-11
Application #
8303305
Study Section
Neurodifferentiation, Plasticity, and Regeneration Study Section (NDPR)
Program Officer
Sesma, Michael A
Project Start
2001-07-01
Project End
2015-05-31
Budget Start
2012-06-01
Budget End
2013-05-31
Support Year
11
Fiscal Year
2012
Total Cost
$335,934
Indirect Cost
$115,934
Name
New York University
Department
Biology
Type
Schools of Arts and Sciences
DUNS #
041968306
City
New York
State
NY
Country
United States
Zip Code
10012
Collins, Ben; Kaplan, Harris S; Cavey, Matthieu et al. (2014) Differentially timed extracellular signals synchronize pacemaker neuron clocks. PLoS Biol 12:e1001959
Collins, Ben; Blau, Justin (2013) A plastic clock. Neuron 78:580-2
Keene, Alex C; Mazzoni, Esteban O; Zhen, Jamie et al. (2011) Distinct visual pathways mediate Drosophila larval light avoidance and circadian clock entrainment. J Neurosci 31:6527-34
Dahdal, David; Reeves, David C; Ruben, Marc et al. (2010) Drosophila pacemaker neurons require g protein signaling and GABAergic inputs to generate twenty-four hour behavioral rhythms. Neuron 68:964-77
Keene, Alex C; Duboue, Erik R; McDonald, Daniel M et al. (2010) Clock and cycle limit starvation-induced sleep loss in Drosophila. Curr Biol 20:1209-15
Blanchard, Florence J; Collins, Ben; Cyran, Shawn A et al. (2010) The transcription factor Mef2 is required for normal circadian behavior in Drosophila. J Neurosci 30:5855-65
Knowles, Alyson; Koh, Kyunghee; Wu, June-Tai et al. (2009) The COP9 signalosome is required for light-dependent timeless degradation and Drosophila clock resetting. J Neurosci 29:1152-62
Blau, Justin (2008) PERspective on PER phosphorylation. Genes Dev 22:1737-40
Blau, J; Blanchard, F; Collins, B et al. (2007) What is there left to learn about the Drosophila clock? Cold Spring Harb Symp Quant Biol 72:243-50
Collins, Ben; Mazzoni, Esteban O; Stanewsky, Ralf et al. (2006) Drosophila CRYPTOCHROME is a circadian transcriptional repressor. Curr Biol 16:441-9

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