Circadian Biology and the Neurovascular Unit.

Mammalian physiology and cellular function are subject to significant oscillations over the course of every 24-hour day. It is likely that these daily rhythms will affect function as well as mechanisms of disease in the central nervous system. In this review, we attempt to survey and synthesize emerging studies that investigate how circadian biology may influence the neurovascular unit. We examine how circadian clocks may operate in neural, glial, and vascular compartments, review how circadian mechanisms regulate cell-cell signaling, assess interactions with aging and vascular comorbidities, and finally ask whether and how circadian effects and disruptions in rhythms may influence the risk and progression of pathophysiology in cerebrovascular disease. Overcoming identified challenges and leveraging opportunities for future research might support the development of novel circadian-based treatments for stroke.

Beyond the symptom: the biology of fatigue.

A workshop titled "Beyond the Symptom: The Biology of Fatigue" was held virtually September 27-28, 2021. It was jointly organized by the Sleep Research Society and the Neurobiology of Fatigue Working Group of the NIH Blueprint Neuroscience Research Program. For access to the presentations and video recordings, see: https://neuroscienceblueprint.nih.gov/about/event/beyond-symptom-biology-fatigue. The goals of this workshop were to bring together clinicians and scientists who use a variety of research approaches to understand fatigue in multiple conditions and to identify key gaps in our understanding of the biology of fatigue. This workshop summary distills key issues discussed in this workshop and provides a list of promising directions for future research on this topic. We do not attempt to provide a comprehensive review of the state of our understanding of fatigue, nor to provide a comprehensive reprise of the many excellent presentations. Rather, our goal is to highlight key advances and to focus on questions and future approaches to answering them.

Rhythms in barriers and fluids: Circadian clock regulation in the aging neurovascular unit.

The neurovascular unit is where two very distinct physiological systems meet: The central nervous system (CNS) and the blood. The permeability of the barriers separating these systems is regulated by time, including both the 24 h circadian clock and the longer processes of aging. An endogenous circadian rhythm regulates the transport of molecules across the blood-brain barrier and the circulation of the cerebrospinal fluid and the glymphatic system. These fluid dynamics change with time of day, and with age, and especially in the context of neurodegeneration. Factors may differ depending on brain region, as can be highlighted by consideration of circadian regulation of the neurovascular niche in white matter. As an example of a potential target for clinical applications, we highlight chaperone-mediated autophagy as one mechanism at the intersection of circadian dysregulation, aging and neurodegenerative disease. In this review we emphasize key areas for future research.

Keeping an eye on circadian time in clinical research and medicine.

BACKGROUND: Daily rhythms are observed in humans and almost all other organisms. Most of these observed rhythms reflect both underlying endogenous circadian rhythms and evoked responses from behaviours such as sleep/wake, eating/fasting, rest/activity, posture changes and exercise. For many research and clinical purposes, it is important to understand the contribution of the endogenous circadian component to these observed rhythms. CONTENT: The goal of this manuscript is to provide guidance on best practices in measuring metrics of endogenous circadian rhythms in humans and promote the inclusion of circadian rhythms assessments in studies of health and disease. Circadian rhythms affect all aspects of physiology. By specifying minimal experimental conditions for studies, we aim to improve the quality, reliability and interpretability of research into circadian and daily (i.e., time-of-day) rhythms and facilitate the interpretation of clinical and translational findings within the context of human circadian rhythms. We describe protocols, variables and analyses commonly used for studying human daily rhythms, including how to assess the relative contributions of the endogenous circadian system and other daily patterns in behaviours or the environment. We conclude with recommendations for protocols, variables, analyses, definitions and examples of circadian terminology. CONCLUSION: Although circadian rhythms and daily effects on health outcomes can be challenging to distinguish in practice, this distinction may be important in many clinical settings. Identifying and targeting the appropriate underlying (patho)physiology is a medical goal. This review provides methods for identifying circadian effects to aid in the interpretation of published work and the inclusion of circadian factors in clinical research and practice.

Cell-Type-Specific Circadian Bioluminescence Rhythms in Dbp Reporter Mice.

Circadian rhythms are endogenously generated physiological and molecular rhythms with a cycle length of about 24 h. Bioluminescent reporters have been exceptionally useful for studying circadian rhythms in numerous species. Here, we report development of a reporter mouse generated by modification of a widely expressed and highly rhythmic gene encoding D-site albumin promoter binding protein (Dbp). In this line of mice, firefly luciferase is expressed from the Dbp locus in a Cre recombinase-dependent manner, allowing assessment of bioluminescence rhythms in specific cellular populations. A mouse line in which luciferase expression was Cre-independent was also generated. The Dbp reporter alleles do not alter Dbp gene expression rhythms in liver or circadian locomotor activity rhythms. In vivo and ex vivo studies show the utility of the reporter alleles for monitoring rhythmicity. Our studies reveal cell-type-specific characteristics of rhythms among neuronal populations within the suprachiasmatic nuclei ex vivo. In vivo studies show Dbp-driven bioluminescence rhythms in the liver of Albumin-Cre;DbpKI/+ "liver reporter" mice. After a shift of the lighting schedule, locomotor activity achieved the proper phase relationship with the new lighting cycle more rapidly than hepatic bioluminescence did. As previously shown, restricting food access to the daytime altered the phase of hepatic rhythmicity. Our model allowed assessment of the rate of recovery from misalignment once animals were provided with food ad libitum. These studies confirm the previously demonstrated circadian misalignment following environmental perturbations and reveal the utility of this model for minimally invasive, longitudinal monitoring of rhythmicity from specific mouse tissues.

Integrating Neuropsychology into Interprofessional Geriatrics Clinics.

Interprofessional healthcare teams are increasingly viewed as a clinical approach to meet the complex medical, psychological, and psychosocial needs of older adult patients. Despite the fact that older adults are at risk for cognitive difficulties, neuropsychologists are not routinely included on Geriatrics consult teams. The primary aim of this paper is to highlight the utility of neuropsychology within an interprofessional Geriatrics consult clinic. To address this aim, we describe specific benefits to patient care that may be associated with the inclusion of neuropsychologists on Geriatrics consult teams, including differential diagnosis, enhanced patient care, and reduced barriers to care. We provide a description of the integration of neuropsychology within a Veterans Health Administration (VA) interprofessional Geriatrics consult clinic team in order to illustrate the implementation of this model.

Methods for Detecting PER2:LUCIFERASE Bioluminescence Rhythms in Freely Moving Mice.

Circadian rhythms are driven by daily oscillations of gene expression. An important tool for studying cellular and tissue circadian rhythms is the use of a gene reporter, such as bioluminescence from the reporter gene luciferase controlled by a rhythmically expressed gene of interest. Here we describe methods that allow measurement of circadian bioluminescence from a freely moving mouse housed in a standard cage. Using a LumiCycle In Vivo (Actimetrics), we determined conditions that allow detection of circadian rhythms of bioluminescence from the PER2 reporter, PER2::LUC, in freely behaving mice. The LumiCycle In Vivo applies a background subtraction that corrects for effects of room temperature on photomultiplier tube (PMT) output. We tested delivery of d-luciferin via a subcutaneous minipump and in the drinking water. We demonstrate spikes in bioluminescence associated with drinking bouts. Further, we demonstrate that a synthetic luciferase substrate, CycLuc1, can support circadian rhythms of bioluminescence, even when delivered at a lower concentration than d-luciferin, and can support longer-term studies. A small difference in phase of the PER2::LUC bioluminescence rhythms, with females phase leading males, can be detected with this technique. We share our analysis scripts and suggestions for further improvements in this method. This approach will be straightforward to apply to mice with tissue-specific reporters, allowing insights into responses of specific peripheral clocks to perturbations such as environmental or pharmacological manipulations.

Dim light in the evening causes coordinated realignment of circadian rhythms, sleep, and short-term memory.

Light provides the primary signal for entraining circadian rhythms to the day/night cycle. In addition to rods and cones, the retina contains a small population of photosensitive retinal ganglion cells (pRGCs) expressing the photopigment melanopsin (OPN4). Concerns have been raised that exposure to dim artificial lighting in the evening (DLE) may perturb circadian rhythms and sleep patterns, and OPN4 is presumed to mediate these effects. Here, we examine the effects of 4-h, 20-lux DLE on circadian physiology and behavior in mice and the role of OPN4 in these responses. We show that 2 wk of DLE induces a phase delay of ∼2 to 3 h in mice, comparable to that reported in humans. DLE-induced phase shifts are unaffected in Opn4-/- mice, indicating that rods and cones are capable of driving these responses in the absence of melanopsin. DLE delays molecular clock rhythms in the heart, liver, adrenal gland, and dorsal hippocampus. It also reverses short-term recognition memory performance, which is associated with changes in preceding sleep history. In addition, DLE modifies patterns of hypothalamic and cortical cFos signals, a molecular correlate of recent neuronal activity. Together, our data show that DLE causes coordinated realignment of circadian rhythms, sleep patterns, and short-term memory process in mice. These effects are particularly relevant as DLE conditions-due to artificial light exposure-are experienced by the majority of the populace on a daily basis.

A functional context for heterogeneity of the circadian clock in cells.

Characterization of circadian systems at the organism level-a top-down approach-has led to definition of unifying properties, a hallmark of the science of chronobiology. The next challenge is to use a bottom-up approach to show how the molecular workings of the cellular circadian clock work as building blocks of those properties. We review new studies, including a recently published PLOS Biology paper by Nikhil and colleagues, that show how programmed but also stochastic generation of variation in cellular circadian period explain important adaptive features of entrained circadian phase.

Nobiletin affects circadian rhythms and oncogenic characteristics in a cell-dependent manner.

The natural product nobiletin is a small molecule, widely studied with regard to its therapeutic effects, including in cancer cell lines and tumors. Recently, nobiletin has also been shown to affect circadian rhythms via their enhancement, resulting in protection against metabolic syndrome. We hypothesized that nobiletin's anti-oncogenic effects, such as prevention of cell migration and formation of anchorage independent colonies, are correspondingly accompanied by modulation of circadian rhythms. Concurrently, we wished to determine whether the circadian and anti-oncogenic effects of nobiletin differed across cancer cell lines. In this study, we assessed nobiletin's circadian and therapeutic characteristics to ascertain whether these effects depend on cell line, which here also varied in terms of baseline circadian rhythmicity. Three cell culture models where nobiletin's effects on cell proliferation and migration have been studied previously were evaluated: U2OS (bone osteosarcoma), which possesses robust circadian rhythms; MCF7 (breast adenocarcinoma), which has weak circadian rhythms; and MDA-MB-231 (breast adenocarcinoma), which is arrhythmic. We found that circadian, migration, and proliferative effects following nobiletin treatment were subtle in the U2OS and MCF7 cells. On the other hand, changes were clear in MDA-MB-231s, where nobiletin rescued rhythmicity and substantially reduced oncogenic features, specifically two-dimensional cell motility and anchorage-independent growth. Based on these results and those previously described, we posit that the effects of nobiletin are indeed cell-type dependent, and that a positive correlation may exist between nobiletin's circadian and therapeutic effects.

Deconstructing circadian disruption: Assessing the contribution of reduced peripheral oscillator amplitude on obesity and glucose intolerance in mice.

Disturbing the circadian regulation of physiology by disruption of the rhythmic environment is associated with adverse health outcomes but the underlying mechanisms are unknown. Here, the response of central and peripheral circadian clocks to an advance or delay of the light-dark cycle was determined in mice. This identified transient damping of peripheral clocks as a consequence of an advanced light-dark cycle. Similar depression of peripheral rhythm amplitude was observed in mice exposed to repeated phase shifts. To assess the metabolic consequences of such peripheral amplitude depression in isolation, temporally chimeric mice lacking a functional central clock (Vgat-Cre+ Bmal1fl/fl ) were housed in the absence of environmental rhythmicity. In vivo PER2::LUC bioluminescence imaging of anesthetized and freely moving mice revealed that this resulted in a state of peripheral amplitude depression, similar in severity to that observed transiently following an advance of the light-dark cycle. Surprisingly, our mice did not show alterations in body mass or glucose tolerance in males or females on regular or high-fat diets. Overall, our results identify transient damping of peripheral rhythm amplitude as a consequence of exposure to an advanced light-dark cycle but chronic damping of peripheral clocks in isolation is insufficient to induce adverse metabolic outcomes in mice.

CIRCADA: Shiny Apps for Exploration of Experimental and Synthetic Circadian Time Series with an Educational Emphasis.

Circadian rhythms are daily oscillations in physiology and behavior that can be assessed by recording body temperature, locomotor activity, or bioluminescent reporters, among other measures. These different types of data can vary greatly in waveform, noise characteristics, typical sampling rate, and length of recording. We developed 2 Shiny apps for exploration of these data, enabling visualization and analysis of circadian parameters such as period and phase. Methods include the discrete wavelet transform, sine fitting, the Lomb-Scargle periodogram, autocorrelation, and maximum entropy spectral analysis, giving a sense of how well each method works on each type of data. The apps also provide educational overviews and guidance for these methods, supporting the training of those new to this type of analysis. CIRCADA-E (Circadian App for Data Analysis-Experimental Time Series) allows users to explore a large curated experimental data set with mouse body temperature, locomotor activity, and PER2::LUC rhythms recorded from multiple tissues. CIRCADA-S (Circadian App for Data Analysis-Synthetic Time Series) generates and analyzes time series with user-specified parameters, thereby demonstrating how the accuracy of period and phase estimation depends on the type and level of noise, sampling rate, length of recording, and method. We demonstrate the potential uses of the apps through 2 in silico case studies.

Recurring circadian disruption alters circadian clock sensitivity to resetting.

A single phase advance of the light:dark (LD) cycle can temporarily disrupt synchrony of neural circadian rhythms within the suprachiasmatic nucleus (SCN) and between the SCN and peripheral tissues. Compounding this, modern life can involve repeated disruptive light conditions. To model chronic disruption to the circadian system, we exposed male mice to more than a month of a 20-hr light cycle (LD10:10), which mice typically cannot entrain to. Control animals were housed under LD12:12. We measured locomotor activity and body temperature rhythms in vivo, and rhythms of PER2::LUC bioluminescence in SCN and peripheral tissues ex vivo. Unexpectedly, we discovered strong effects of the time of dissection on circadian phase of PER2::LUC bioluminescent rhythms, which varied across tissues. White adipose tissue was strongly reset by dissection, while thymus phase appeared independent of dissection timing. Prior light exposure impacted the SCN, resulting in strong resetting of SCN phase by dissection for mice housed under LD10:10, and weak phase shifts by time of dissection in SCN from control LD12:12 mice. These findings suggest that exposure to circadian disruption may desynchronize SCN neurons, increasing network sensitivity to perturbations. We propose that tissues with a weakened circadian network, such as the SCN under disruptive light conditions, or with little to no coupling, for example, some peripheral tissues, will show increased resetting effects. In particular, exposure to light at inconsistent circadian times on a recurring weekly basis disrupts circadian rhythms and alters sensitivity of the SCN neural pacemaker to dissection time.

Evidence for Internal Desynchrony Caused by Circadian Clock Resetting.

Circadian disruption has been linked to markers for poor health outcomes in humans and animal models. What is it about circadian disruption that is problematic? One hypothesis is that phase resetting of the circadian system, which occurs in response to changes in environmental timing cues, leads to internal desynchrony within the organism. Internal desynchrony is understood as acute changes in phase relationships between biological rhythms from different cell groups, tissues, or organs within the body. Do we have strong evidence for internal desynchrony associated with or caused by circadian clock resetting? Here we review the literature, highlighting several key studies from measures of gene expression in laboratory rodents. We conclude that current evidence offers strong support for the premise that some protocols for light-induced resetting are associated with internal desynchrony. It is important to continue research to test whether internal desynchrony is necessary and/or sufficient for negative health impact of circadian disruption.

Biomarkers of oxidative stress, inflammation, and vascular dysfunction in inherited cystathionine ß-synthase deficient homocystinuria and the impact of taurine treatment in a phase 1/2 human clinical trial.

STUDY OBJECTIVE: A phase 1/2 clinical trial was performed in individuals with cystathionine ß synthase (CBS) deficient homocystinuria with aims to: (a) assess pharmacokinetics and safety of taurine therapy, (b) evaluate oxidative stress, inflammation, and vascular function in CBS deficiency, and (c) evaluate the impact of short-term taurine treatment. METHODS: Individuals with pyridoxine-nonresponsive CBS deficiency with homocysteine >50 µM, without inflammatory disorder or on antioxidant therapy were enrolled. Biomarkers of oxidative stress and inflammation, endothelial function (brachial artery flow-mediated dilation [FMD]), and disease-related metabolites obtained at baseline were compared to normal values. While maintaining current treatment, patients were treated with 75 mg/kg taurine twice daily, and treatment response assessed after 4 hours and 4 days. RESULTS: Fourteen patients (8-35 years; 8 males, 6 females) were enrolled with baseline homocysteine levels 161 ± 67 µM. The study found high-dose taurine to be safe when excluding preexisting hypertriglyceridemia. Taurine pharmacokinetics showed a rapid peak level returning to near normal levels at 12 hours, but had slow accumulation and elevated predosing levels after 4 days of treatment. Only a single parameter of oxidative stress, 2,3-dinor-8-isoprostaglandin-F2α, was elevated at baseline, with no elevated inflammatory parameters, and no change in FMD values overall. Taurine had no effect on any of these parameters. However, the effect of taurine was strongly related to pretreatment FMD values; and taurine significantly improved FMD in the subset of individuals with pretreatment FMD values <10% and in individuals with homocysteine levels >125 µM, pertinent to endothelial function. CONCLUSION: Taurine improves endothelial function in CBS-deficient homocystinuria in patients with preexisting reduced function.