Exp Neurobiol 2023; 32(4): 259-270
Published online August 31, 2023
© The Korean Society for Brain and Neural Sciences
Hyeri Nam†, Boil Kim†, Younghwan Lee, Han Kyoung Choe* and Seong-Woon Yu*
Department of Brain Sciences, Daegu Gyeongbuk Institute of Science and Technology (DGIST), Daegu 42988, Korea
Correspondence to: *To whom correspondence should be addressed.
Han Kyoung Choe, TEL: 82-53-785-6150, FAX: 82-53-785-6109
Seong-Woon Yu, TEL: 82-53-785-6113, FAX: 82-53-785-6109
†These authors contributed equally to this article.
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/4.0) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.
Circadian rhythm is a 24-hour cycle of behavioral and physiological changes. Disrupted sleep-wake patterns and circadian dysfunction are common in patients of Alzheimer Disease (AD) and are closely related with neuroinflammation. However, it is not well known how circadian rhythm of immune cells is altered during the progress of AD. Previously, we found presenilin 2 (Psen2) N141I mutation, one of familial AD (FAD) risk genes, induces hyperimmunity through the epigenetic repression of REV-ERBα expression in microglia and bone marrow-derived macrophage (BMDM) cells. Here, we investigated whether repression of REV-ERBα is associated with dysfunction of immune cell-endogenous or central circadian rhythm by analyses of clock genes expression and cytokine secretion, bioluminescence recording of rhythmic PER2::LUC expression, and monitoring of animal behavioral rhythm. Psen2 N141I mutation down-regulated REV-ERBα and induced selective over-production of IL-6 (a well-known clock-dependent cytokine) following the treatment of toll-like receptor (TLR) ligands in microglia, astrocytes, and BMDM. Psen2 N141I mutation also lowered amplitude of intrinsic daily oscillation in these immune cells representatives of brain and periphery. Of interest, however, the period of daily rhythm remained intact in immune cells. Furthermore, analyses of the central clock and animal behavioral rhythms revealed that central clock remained normal without down-regulation of REV-ERBα. These results suggest that Psen2 N141I mutation induces hyperimmunity mainly through the suppression of REV-ERBα in immune cells, which have lowered amplitude but normal period of rhythmic oscillation. Furthermore, our data reveal that central circadian clock is not affected by Psen2 N141I mutation.
Circadian rhythm is an endogenous biological oscillator with an approximately 24-h period that allows the organisms to optimize their behavior and physiology to accommodate daily environmental changes [1, 2]. In mammals, the hypothalamic suprachiasmatic nucleus (SCN) is the circadian pacemaker and serves as the master clock by synchronizing peripheral clocks present in the various peripheral tissues and cell types throughout the body [2, 3]. Recent studies have revealed an intimate link between circadian abnormalities and Alzheimer disease (AD). Circadian disruptions, including sleep-wake cycle disturbance, sundowning, and altered daily rhythms of body activity and core temperature, are highly prevalent among AD patients . Circadian rhythms also are altered in various AD animal models [5-7]. Distinct AD animal models exhibited diverse circadian and sleep/wake phenotypes, such as alteration in circadian period, changes in sleep phase length, changes in activity levels, and misregulated clock gene expression. Although these studies provide valuable insight on the correlation between AD pathophysiology and circadian rhythm, it still remains unclear whether the crosstalk between AD and circadian rhythm occurs at either the systemic temporal mismatch between environmental and physiological events or misalignment among internal peripheral oscillators.
Presenilins (PSENs) are multi-pass membrane proteins and play important roles in AD by generating amyloid beta (Aβ) peptides as the catalytic core component of γ-secretase complex [8-10]. Various mutations of distinct domains of
REV-ERBα acts as a transcriptional repressor on RevDR2 and retinoic acid-related orphan receptor-binding elements (RORE) in competition with transcriptional activator, RORs. In addition to cytokines as its targets, REV-ERBα suppresses the expression of
This study is aimed to demonstrate the circadian properties of central pacemaker and immune cell-specific peripheral oscillators in
All procedures for the care and use of laboratory animals were approved by the Institutional Animal Care and Use Committee of DGIST. Mice were housed under standard 12-h light/dark cycle with a specific pathogen-free environment at DGIST animal facility.
Microglia and astrocytes were obtained from neonatal mice (ages 1~3 days) brains and prepared by trypsinization. Cells were cultured with Dulbecco’s modified eagle medium (Corning, NY, USA), supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS; Hyclone, Logan, UT, USA) and 1% penicillin-streptomycin (Hyclone). Cells were isolated and used as described previously [13, 19]. Bone marrow-derived macrophages (BMDM) were obtained by femurs and tibias from 6~7 weeks old mice, as previously described .
Dexamethasone (DEX) was purchased from Sigma-Aldrich (St. Louis, MO, USA). D-Luciferin was purchased from Promega (Madison, WI, USA). N-palmitoyl-S-dipalmitoylglyceryl Cys-Ser-(Lys)4 (Pam3CSK4; 100 ng/ml), heat-killed Listeria monocytogenes (HKLM; 107 cells/ml), polyinosinic-polycytidylic acid high molecular weight (poly (I:C) HMW; 10 μg/ml), and low molecular weight (poly (I:C) LMW; 1 μg/ml), lipopolysaccharide purified from
RNA was isolated from cells using Qiazol (Qiagen; Hilden, Germany). cDNA was synthesized with oligo dT and ImProm-II Reverse Transcriptase kit (Promega; Madison, WI, USA). qRT-PCR was performed with TOPrealTM qPCR 2xPreMIX (SYBR Green with lox ROX; Enzynomics; Republic of Korea). Primers targeted specifically interested mouse cDNAs and were used as designed previously .
Cells were lysed in radio-immunoprecipitation assay buffer with 1× protease and phosphatase inhibitors and 1 mM phenylmethylsulfonylfluoride and 0.1 M dithiothreitol. Cell lysates were separated by SDS-page gel and transferred to polyvinylidene fluoride membranes. Membranes were incubated with REV-ERBα antibody (Thermo Fisher Scientific; Waltham, MA, USA) and detected by species-speciﬁc, horseradish peroxidase–conjugated secondary antibodies. The blots were quantiﬁed using Image Studio lite 4.0 (LI-COR Biosciences; Lincoln, NE, USA).
ELISA kits for mouse IL-6 and TNF-α were purchased from R&D Systems (Minneapolis, MN, USA). The supernatants of the cultured cells were used to measure the cytokines according to manufacturer’s instructions.
SCN explant cultures were prepared and monitored in a similar manner to that described previously, with minor modifications . One-week-old WT or
To monitor real-time circadian rhythms with the cells and SCN slices from
The locomotor activity and body temperature of the mice were measured using E-mitter, a radio transmitter-based telemetry system (Starr Life Science; Oakmont, PA, USA). E-mitter was implanted beneath the skin on the backs of the mice using aseptic techniques under general anesthesia induced by intraperitoneal ketamine (100 mg/kg) and xylazine (10 mg/kg) injection . After implantation, the mice recovered for at least one week and acclimatized in a regular 12 h light/dark cycle. Activity and temperature data detected by the implanted sensor were transmitted to a receiver (ER-4000 Energizer/Receiver). Data acquisition and digital transformation was performed using VitalView software every 6 min (Starr Life Science). Chi-square periodogram analysis was performed using the xsp package  in R .
Data acquisition and analysis were performed in GraphPad Prism (GraphPad Software, San Diego, USA) with at least 3 independent experiments. Statistical analysis was determined by Student’s unpaired t-test, and presented as mean±standard error of the mean values (SEM). Real-time bioluminescence was analyzed by the cosinor procedure [22, 23]. Chi-square periodogram analysis in circadian behavior recording was performed using the xsp package  in R .
Microglia have an intrinsic molecular clock [17, 27, 28]. Other immune cells, astrocytes and BMDM, also have an intrinsic molecular clock and are associated with circadian regulation [16, 29]. Because
Disruption of rhythmic expression of clock genes in microglia by
Previously, we examined that
Our results indicate that REV-ERBα expression is reduced in immune cells by
It has long been conceived that circadian disturbance and AD progression are reciprocally related. Genetic or environmental degenerative cues can lead to circadian rhythm disturbance . Conversely, circadian rhythm affects Aβ dynamics , and circadian alterations precede the onset of AD symptoms . Circadian alterations can contribute to the pathogenesis from the early stages and exacerbate cognitive impairment, as demonstrated in clock gene knockout animal models [37-39]. Clock gene deletion abolishes molecular rhythmicity and induces a wide range of pathological phenotypes, some of which may be irrelevant to AD. Therefore, model system carrying clock gene deletion have limitations in representing the pathological changes occurring to clock components or rhythmicity during neurodegeneration, and may not be well suited to studying the AD-related circadian deficits . On the other hand, various AD models exhibited circadian and sleep disruption, but the defect of circadian rhythm has not been dissected in terms of the hierarchy of circadian system [5-7]. In this study, we utilized
REV-ERBα plays critical roles in maintaining fine tuning of circadian molecular machinery oscillation and in the proper expression of various physiological functions of clock-controlled genes. In
We thank Dr. Joseph Takahashi for providing