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Astrocyte regulation of circadian behavior

Final Report Summary - ASTROCLOCK (Astrocyte regulation of circadian behavior)

Biological rhythms govern the ebb and flow of life on the Earth. Animals have an internal timekeeping mechanism to anticipate daily changes associated with the transition of day to night, which is deeply involved in the regulation and maintenance of behavioral and physiological processes. In mammals, the circadian system is organized in a hierarchy of multiple oscillators at organism, cellular and molecular level. At the organism level, the suprachiasmatic nucleus (SCN) integrates light information to ultimately regulate over rhythms in gene expression, physiology and behavior (Fig.1). At cellular level, the SCN is composed of multiple oscillating neurons that are coupled to act as a single circadian unit leading to coordinated circadian signaling outputs. At molecular level, the circadian clock consists in the transcription- and translation-based interconnected feedback loops, in which the transcription factors Bmal1 and Clock drive the expression of Per and Cry genes, whose products lead to the inhibition of their own transcription.
The brain, including the SCN, is composed of a heterogeneous population of cells, including astrocytes, which have a well-documented role in regulating neuronal communication and thus, behavioral processes. Recent evidence suggests an involvement of astrocyte physiology in the regulation of circadian rhythms in Drosophila and in mammals. However, the contribution of astrocytes circadian clock (here referred to as “astroclock”) to the timekeeping system is largely unexplored. Identifying such a role for "astroclock" will not only reveal a more complex cellular signaling in brain than the considered so far, but would also have significant implications for therapeutic research on transient perturbations in circadian rhythms (such as jet lag), adverse effects of shift workers and in disorders associated with circadian rhythms dysfunctions. Our long-term goal is to open new avenues to treat disorders associated with circadian rhythms dysfunction based on the development of astrocyte-targeted drugs. Our short-term goal is to validate our hypothesis that "astroclock" modulate neuronal activity and behavior. To test our hypothesis we performed a systematic study in astrocytes at the molecular, cellular and system levels: from the transcriptional and post-transcriptional mechanisms that control circadian rhythms in astrocytes, to astrocyte-targeted manipulation of circadian gene expression and to the investigation of the effects of this manipulation on neurons and animal circadian behavior.
The work performed during the two years of ASTROCLOCK can be summarized in five major research lines:
1.- To define the role of “astroclock” in neuronal rhythmicity in vitro, the fellow performed co-cultures of synchronous (transfected with scramble control siRNAs) or arrhythmic (transfected with Bmal1 siRNAs) astrocytes, with asynchronous neurons grown onto physically separated layers, but sharing the same culture media. With the support of Berdondini’s team, the fellow is developing microfluidic devices that will allow the systematic co-culture of astrocytes and neurons expressing a Per2-luciferase (per-luc) or a Dbp-luciferase (dbp-luc) reporter respectively, for simultaneous monitoring of neuronal or astrocytic populations for drug screening or for recording of cells isolated from different brain areas (SCN, cortex and hippocampus).
2.- To investigate a possible direct role of astrocytes in modulating neuronal circadian rhythmicity in vivo, the fellow generated an inducible conditional knockout mouse in which Bmal1 gene was specifically deleted in astrocytes (Bmal1cKO). Phenotypic studies in Bmal1cKO, including molecular, cellular and behavioral analysis, were performed.
3.- To define the role of “astroclock” in neuronal activity, the fellow collaborated with Berdondini’s team to perform recordings on large-scale high-density electrode arrays (i.e. that records simultaneously from 4096 electrodes) in acute retinas of control and Bmal1cKO animals The computational analysis are still running with the support of Berdondini’s team.
4.- To dissect the transcriptional and post-transcriptional mechanisms of astrocyte to neuron circadian communication the fellow used Next-Generation Sequencing (NGS) technology. The global oscillating transcriptome and miRNome from cortex (peripheral pacemaker), at different time points of the day of Bmal1cKO and control mice were obtained and analyzed with the support of Francesco Nicassio’s team (IIT, SEEM, Milano).
5.- With the data sets obtained in the research line 4, the fellow identified the pathways required for astrocyte to neuron circadian communication by performing the functional validation in vitro and in vivo.
The results achieved in the five research lines (see above) of ASTROCLOCK, revealed an unexpected and important role of astrocytes in the control of circadian rhythms at cellular, tissue and organism level in mammals, leading to a deeper understanding on circadian rhythms in the brain. Specifically:
1.- From the first research line, the results provide general but fundamental insights about the role of astrocyte circadian rhythms in neuronal rhythmicity.
2.- From the second research line, the results reveal that disruption of circadian clock in astrocytes (Bmal1cKO) leads to altered behavioral phenotype.
3.- From the third research line, with the support of Alessandro Maccione and Stefano di Marco (researchers at Berdondini’s team), the fellow is evaluating the impact of “astroclock” in neuronal activity in vivo by performing electrophysiological recordings of acute retinas.
4.- By uncovering the global oscillating transcripts/miRNA that are deregulated in Bmal1cKO as compared to control mice, those ASTROCLOCK results are the first comprehensive study of the circadian oscillating transcripts in astrocytes, including miRNAs and mRNAs.
5.- From the five research line, the fellow identified the pathways involved on astrocyte to neuron communication by performing the functional validation in vitro and in vivo.
Currently, it is unclear how cellular signaling modulates the cycle-to-cycle precision of circadian rhythms. This lack of knowledge contributes to researchers inability to precisely define the role of circadian abnormalities in circadian-associated disorders. ASTROCLOCK results uncover the role of “astroclock” in the critical input signals of the central pacemaker (from the retina studies), in the impact on the oscillators of the central pacemaker and in the neurobehavioral outputs under circadian control. The involvement of “astroclock” in the modulation of neuronal behavior suggests that astrocytes might be a novel cell target for the development of novel drugs and treatment strategies for those disorders. Therefore, by developing new knowledge on molecular and cellular mechanisms of glial-neuron communication in circadian rhythms, ASTROCLOCK results will certainly pave the way to novel cellular and signaling targets (i.e. astrocytes, astrocyte-neuron signaling) for the pharmacological treatment of brain disorders involving alterations of circadian rhythmicity.
Alterations of circadian rhythmicity in humans, regardless of whether it results from voluntary (e.g. shift work or jetlag) or involuntary (e.g. sleep disorders) circumstances, has been associated with a variety of mental and physical disorders and also have a negative impact on safety (i.e. human errors), human performance and productivity. Economically, while the European costs resulting from these negative impacts of disrupted brain circadian rhythmicity are difficult to assess (i.e. some studies suggest that the resulting economic burden of insomnia is very high, with the largest proportion of all expenses (76%) attributable to insomnia-related work absences and reduced productivity), the overall direct costs of brain disorders in Europe was assessed in 2010 at €798 billion/year, with a trend of constantly increasing. Sleep disorders have a cost of €35.4 billion/year, but the costs induced by circadian rhythms alterations go far behind this specific brain disorder.
Thus, ASTROCLOCK results, as well as, the knowledge generated will certainly exert a positive impact on R&D of pharma-industry, a sector that depends on the better understanding of the molecular signaling in the brain, potentially allowing the development of novel pharmaceutical strategies to treat diseases induced by alteration of circadian rhythms. Hence, the results generated under this program will exert a positive impact on both economy and society.
FELLOW: Olga Barca Mayo (Senior Postdoc).
COORDINATOR: Davide De Pietri Tonelli (Team leader). Neurobiology of microRNAs lab.
SUPERVISOR: Luca Berdondini (Team leader). NetS3 Lab - NeuroEngineering & bio-arTificial Synergic SystemS Laboratory.