Date of Award
Doctor of Philosophy (PhD)
Circadian rhythms are cycles of physiological activity that are conserved across all of life’s taxa – ranging from cyanobacteria to humans - due to their importance. They are conserved to allow organisms to maximize their capacity to obtain resources in their environment. In mammals, light and dark input into the retina is the strongest synchronizer of circadian rhythms. On the molecular level, this tightly regulated transcriptional-translational feedback loop is orchestrated by proteins with cyclical expression. The loss of these proteins has functional consequences on human health and diseases.
Recently, associations have been made between circadian proteins and a host of pathological conditions including cancer, diabetes, obesity, inflammation, and neurodegeneration. In 2013, Dr. Erik Musiek established a link between the core circadian clock protein BMAL1 and neurodegeneration as well as redox homeostasis (Musiek et al., 2013). Since then, other work has also shown circadian dysfunction to be a prominent symptom of Alzheimer’s disease. In establishing the role of BMAL1 in neurodegeneration, Dr. Musiek also noted a stark age-dependent increase in astrocyte and microglial activation. These glial cells are essential for brain homeostasis and play roles in central nervous disease pathogenesis.
To further understand the role of the circadian clock in regulating glial activation, we investigated the circadian protein REV-ERBa. Canonically, BMAL1 drives the transcription of a number of genes. One of those genes is Nr1d1, which codes for REV-ERBa - a dedicated transcriptional repressor. Although REV-ERBa was not initially described in the core clock machinery, this protein can also inhibit production of BMAL1 by binding to consensus sequences. Since REV-ERBa is downstream of BMAL1, we asked the question, does REV-ERBa play a role in regulating glial activation and if so, what are the consequences to neuronal health?
In the periphery, REV-ERBa had already been shown to play a role in regulating macrophage activation (Gibbs et al., 2012). Therefore, we investigated microglia first for a functional role of REV-ERBa. Deletion of REV-ERBa induced spontaneous microglial activation in areas of the mouse brain such as the hippocampus. This spontaneous activation abrogated time-of-day oscillation in microglial activation. In culture, isolated microglial lacking REV-ERBa also exhibited pro-inflammatory phenotypes and increased basal NFkB signaling. We also found through Chromatin immunoprecipitation (ChIP) that REV-ERBa physically interacted with several NFkB genes in microglia. Primary microglia lacking REV-ERBa also exhibited an exacerbated response to the inflammogen lipopolysaccharide (LPS).
Within the brain parenchyma, microglia and astrocytes interact closely. Activated microglia can in turn induce astrocyte activation (Liddelow et al., 2017). In the brain, knocking out REV-ERBa also resulted in astrocyte activation. However, in contrast to the microglia, primary astrocytes isolated from REV-ERBa knockout mice did not show a basal activated phenotype in culture but did respond to an LPS challenge. Since REV-ERBa is a ligand sensitive nuclear receptor, it can be targeted by small molecules. One such molecule is SR9009, which was synthesized by our collaborators in the Burris lab (Solt et al., 2012). Application of this drug reduced LPS induced neuroinflammation in mice and IL-1b release from primary microglia, but was ineffective in cells lacking REV-ERBa.
Activated glia can influence neuronal health in the brain (Andreasson et al., 2016). Accordingly, primary neurons grown in conditioned media from REV-ERBa deficient primary glial cultures were more susceptible to oxidative damage. In the CA3 region of the hippocampus, we also noted a striking reduction in synaptic volume in the REV-ERBa knockout mice. Notably, presynaptic terminals showed a decrease in immunoreactivity by multiple methods. Synapses can be tagged for microglia-mediated removal by complement molecules (Stevens et al., 2007). In both BMAL1 knockout and REV-ERBa knockout mice, we noted increases in complement gene expression as well as complement protein expression in astrocytes and microglia. We then observed striking increases in microglia-mediated synaptic pruning in the REV-ERBa knockout mice. Interestingly, REV-ERBa deletion also abrogated oscillations in microglia-mediated synaptic pruning. Since synaptic connections are used for communication between brain regions, we also assessed the overall functional brain connectivity. In REV-ERBa knockout mice, we observed decreases in resting state functional connectivity in brain areas associated with neurodegeneration. Overall, the work here highlights a novel role for a circadian protein in various facets of glial and neuronal health. It also establishes a pharmacologically accessible link between the circadian clock and neuroinflammation, which can potentially be leveraged to treat neurodegenerative disease
Chair and Committee
Erik S. Musiek
Joseph Dougherty, Abhinav Diwan, Marco Colonna, Timothy Miller,
Griffin, Percy, "The Role of the Circadian Clock Protein Rev-erba in Neuroinflammation & Synaptic Health" (2020). Arts & Sciences Electronic Theses and Dissertations. 2318.