Circadian dysfunction is a common attribute of many neurodegenerative diseases, most of which are associated with neuroinflammation. Circadian rhythm dysfunction has been associated with inflammation in the periphery, but the role of the core clock in neuroinflammation remains poorly understood. Here we demonstrate that Rev-erbα, a nuclear receptor and circadian clock component, is a mediator of microglial activation and neuroinflammation. We observed time-of-day oscillation in microglial immunoreactivity in the hippocampus, which was disrupted in Rev-erbα −/− mice. Rev-erbα deletion caused spontaneous microglial activation in the hippocampus and increased expression of proinflammatory transcripts, as well as secondary astrogliosis. Transcriptomic analysis of hippocampus from Rev-erbα −/− mice revealed a predominant inflammatory phenotype and suggested dysregulated NF-κB signaling. Primary Rev-erbα −/− microglia exhibited proinflammatory phenotypes and increased basal NF-κB activation. Chromatin immunoprecipitation revealed that Reverbα physically interacts with the promoter regions of several NF-κB-related genes in primary microglia. Loss of Rev-erbα in primary astrocytes had no effect on basal activation but did potentiate the inflammatory response to lipopolysaccharide (LPS). In vivo, Reverbα −/− mice exhibited enhanced hippocampal neuroinflammatory responses to peripheral LPS injection, while pharmacologic activation of Rev-erbs with the small molecule agonist SR9009 suppressed LPSinduced hippocampal neuroinflammation. Rev-erbα deletion influenced neuronal health, as conditioned media from Rev-erbα-deficient primary glial cultures exacerbated oxidative damage in cultured neurons. Reverbα −/− mice also exhibited significantly altered cortical resting-state functional connectivity, similar to that observed in neurodegenerative models. Our results reveal Rev-erbα as a pharmacologically accessible link between the circadian clock and neuroinflammation.Rev-erbα | circadian | microglia | neuroinflammation C ircadian clocks allow organisms to precisely synchronize internal physiological processes with their external environment. A conserved transcriptional-translational feedback loop known as the core circadian clock controls cycles of protein expression that produce transcriptional and physiologic rhythms. This core circadian clock consists of the transcriptional activators BMAL1 and CLOCK, which drive transcription of their own transcriptional repressors, including CRYPTOCHROME (CRY), PE-RIOD (PER), and REV-ERB proteins (1). The circadian system regulates a variety of critical cellular processes, including aspects of metabolism, inflammation, and redox homeostasis (2). Disruptions of the clock or its associated proteins have been implicated in pathological conditions ranging from cancer to neurodegenerative diseases (2-4). However, the roles of cellular circadian clocks in brain health and neuroinflammation are still poorly understood.Aberrant glial cell activation and neuroinflammation are hallmarks of many neuro...

Background Alzheimer’s disease is characterized by two main neuropathological hallmarks: extracellular plaques of amyloid-β (Aβ) protein and intracellular aggregates of tau protein. Although tau is normally a soluble monomer that bind microtubules, in disease it forms insoluble, hyperphosphorylated aggregates in the cell body. Aside from its role in AD, tau is also involved in several other neurodegenerative disorders collectively called tauopathies, such as progressive supranuclear palsy (PSP), corticobasal degeneration (CBD), some forms of frontotemporal dementia, and argyrophilic grain disease (AGD). The prion hypothesis suggests that after an initial trigger event, misfolded forms of tau are released into the extracellular space, where they spread through different brain regions, enter cells, and seeding previously normal forms. Thus understanding mechanisms regulating the clearance of extracellular tau from the CNS is important. The discovery of a true lymphatic system in the dura and its potential role in mediating Aβ pathology prompted us to investigate its role in regulating extracellular tau clearance. Methods To study clearance of extracellular tau from the brain, we conjugated monomeric human tau with a near-infrared dye cypate, and injected this labeled tau in the parenchyma of both wild-type and K14-VEGFR3-Ig transgenic mice, which lack a functional CNS lymphatic system. Following injection we performed longitudinal imaging using fluorescence molecular tomography (FMT) and quantified fluorescence to calculate clearance of tau from the brain. To complement this, we also measured tau clearance to the periphery by measuring plasma tau in both groups of mice. Results Our results show that a significantly higher amount of tau is retained in the brains of K14-VEGFR3-Ig vs. wild type mice at 48 and 72 h post-injection and its subsequent clearance to the periphery is delayed. We found that clearance of reference tracer human serum albumin (HSA) was also significantly delayed in the K14-VEGFR3-Ig mice. Conclusions The dural lymphatic system appears to play an important role in clearance of extracellular tau, since tau clearance is impaired in the absence of functional lymphatics. Based on our baseline characterization of extracellular tau clearance, future studies are warranted to look at the interaction between tau pathology and efficiency of lymphatic function.

SUMMARY Circadian clock dysfunction is a common symptom of aging and neurodegenerative diseases, though its impact on brain health is poorly understood. Astrocyte activation occurs in response to diverse insults and plays a critical role in brain health and disease. We report that the core circadian clock protein BMAL1 regulates astrogliosis in a synergistic manner via a cell-autonomous mechanism and a lesser non-cell-autonomous signal from neurons. Astrocyte-specific Bmal1 deletion induces astrocyte activation and inflammatory gene expression in vitro and in vivo, mediated in part by suppression of glutathione-S-transferase signaling. Functionally, loss of Bmal1 in astrocytes promotes neuronal death in vitro. Our results demonstrate that the core clock protein BMAL1 regulates astrocyte activation and function in vivo, elucidating a mechanism by which the circadian clock could influence many aspects of brain function and neurological disease.

A promising new therapeutic target for the treatment of Alzheimer's disease (AD) is the circadian system. Although patients with AD are known to have abnormal circadian rhythms and suffer sleep disturbances, the role of the molecular clock in regulating amyloid‐beta (Aβ) pathology is still poorly understood. Here, we explored how the circadian repressors REV‐ERBα and β affected Aβ clearance in mouse microglia. We discovered that, at Circadian time 4 (CT4), microglia expressed higher levels of the master clock protein BMAL1 and more rapidly phagocytosed fibrillary Aβ1‐42 (fAβ1‐42) than at CT12. BMAL1 directly drives transcription of REV‐ERB proteins, which are implicated in microglial activation. Interestingly, pharmacological inhibition of REV‐ERBs with the small molecule antagonist SR8278 or genetic knockdown of REV‐ERBs‐accelerated microglial uptake of fAβ1‐42 and increased transcription of BMAL1. SR8278 also promoted microglia polarization toward a phagocytic M2‐like phenotype with increased P2Y12 receptor expression. Finally, constitutive deletion of Rev‐erbα in the 5XFAD model of AD decreased amyloid plaque number and size and prevented plaque‐associated increases in disease‐associated microglia markers including TREM2, CD45, and Clec7a. Altogether, our work suggests a novel strategy for controlling Aβ clearance and neuroinflammation by targeting REV‐ERBs and provides new insights into the role of REV‐ERBs in AD.

The circadian clock regulates various aspects of brain health including microglial and astrocyte activation. Here we report that deletion of the master clock protein BMAL1 in mice robustly increases expression of complement genes, including C4b and C3, in the hippocampus. BMAL1 regulates expression of the transcriptional repressor REV-ERBa, and deletion of REV-ERBa causes increased expression of C4b transcript in neurons and astrocytes as well as C3 protein primarily in astrocytes. REV-ERBa deletion increased microglial phagocytosis of synapses and synapse loss in the CA3 region of the hippocampus. Finally, we observed diurnal variation in the degree of microglial synaptic phagocytosis which was antiphase to REV-ERBα expression. This daily variation in microglial synaptic phagocytosis was abrogated by global REV-ERBα deletion, which caused persistently elevated synaptic phagocytosis. This work uncovers the BMAL1-REV-ERBa axis as a regulator of complement expression and synaptic phagocytosis in the brain, linking circadian proteins to synaptic regulation.

Circadian clock dysfunction is a common symptom of aging and neurodegenerative diseases, though its impact on brain health is poorly understood. Astrocyte activation occurs in response to diverse insults, and plays a critical role in brain health and disease. We report that the core clock protein BMAL1 regulates astrogliosis in a synergistic manner via a cell-autonomous mechanism, and via a lesser non-cell-autonomous signal from neurons. Astrocyte-specific Bmal1 deletion induces astrocyte activation in vitro and in vivo, mediated in part by suppression of glutathione-s-transferase signaling. Functionally, loss of Bmal1 in astrocytes promotes neuronal death in vitro. Our results demonstrate that the core clock protein BMAL1 regulates astrocyte activation and function in vivo, elucidating a novel mechanism by which the circadian clock could influence many aspects of brain function and neurologic disease. Highlights:• Circadian disruption promotes astrocyte activation.• Astrocyte-specific deletion of the circadian clock gene BMAL1 induces astrocyte activation. • BMAL1 regulates astrocyte activation by altering glutathione-s-transferase signaling.• Loss of astrocyte BMAL1 enhances neuronal cell death in a co-culture system. eTOC blurb:Lananna et al. show that the circadian clock protein BMAL1 regulates astrocyte activation via a cell autonomous-mechanism involving diminished glutathione-s-transferase signaling. This finding elucidates a novel function of the core circadian clock in astrocytes, and reveals a BMAL1 as a modulator of astrogliosis.

Circadian dysfunction has been described in patients with symptomatic Alzheimer's disease (AD), as well as in presymptomatic phases of the disease. Modeling this circadian dysfunction in mouse models would provide an optimal platform for understanding mechanisms and developing therapies. While numerous studies have examined behavioral circadian function, and in some cases clock gene oscillation, in mouse models of AD, the results are variable and inconsistent across models, ages, and conditions. Ultimately, circadian changes observed in APP/PS1 models are inconsistent across studies and do not always replicate circadian phenotypes observed in human AD. Other models, including the 3xTG mouse, tau transgenic lines, and the accelerated aging SAMP8 line, show circadian phenotypes more consistent with human AD, although the literature is either inconsistent or minimal. We summarize these data and provide some recommendations to improve and standardize future studies of circadian function in AD mouse models.

The development of vaccines for infectious diseases for which we currently have none, including HIV, will likely require the use of adjuvants that strongly promote germinal center responses and somatic hypermutation to produce broadly neutralizing antibodies. Here we compared the outcome of immunization with the T-cell dependent antigen, NP-conjugated to chicken gamma globulin (NP-CGG) adjuvanted with the toll-like receptor 9 (TLR9) ligands, CpG-A or CpG-B, alone or conjugated with the cationic lipid carrier, DOTAP. We provide evidence that only NP-CGG adjuvanted with DOTAP-CpG-B was an effective vaccine in mice resulting in robust germinal center responses, isotype switching and high affinity NP-specific antibodies. The effectiveness of DOTAP-CpG-B as an adjuvant was dependent on the expression of the TLR9 signaling adaptor MyD88 in immunized mice. These results indicate DOTAP-CpG-B but not DOTAP-CpG-A is an effective adjuvant for T cell-dependent protein antigen-based vaccines.