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Original Article

Exp Neurobiol 2023; 32(4): 259-270

Published online August 31, 2023

https://doi.org/10.5607/en23012

© The Korean Society for Brain and Neural Sciences

Presenilin 2 N141I Mutation Induces Hyperimmunity by Immune Cell-specific Suppression of REV-ERBα without Altering Central Circadian Rhythm

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
e-mail: choehank@dgist.ac.kr
Seong-Woon Yu, TEL: 82-53-785-6113, FAX: 82-53-785-6109
e-mail: yusw@dgist.ac.kr
†These authors contributed equally to this article.

Received: April 12, 2023; Revised: June 21, 2023; Accepted: July 7, 2023

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.

Abstract

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.

Graphical Abstract


Keywords: Circadian rhythm, Presenilin 2, Alzheimer disease, Hyperimmunity, REV-ERBα

INTRODUCTION

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 [4]. 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 PSEN1 (e.g., N135D, L166P, M233T and C410Y) and PSEN2 (e.g., T122P, N141I, M239V, and M239I) can significantly increase Aβ42 production or Aβ42/Aβ40 ratio [11, 12]. We previously revealed that Psen2 N141I mutation induced hyperimmunity with increased production of clock gene-controlled cytokines including interleukin-6 (IL-6). Mechanistically, Psen2 N141I mutation suppressed expression of Nr1d1 (a gene encoding REV-ERBα) , but not the expression of Nr1d2 (a gene encoding REV-ERBβ), through the hypermethylation of the Nr1d1 promoter [13].

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 Bmal1 through binding of RORE in Bmal1 promoter in core molecular clock machinery. REV-ERBα serves as a transcriptional repressor of the cytokines controlled by circadian rhythm [14]. The systemic and circadian clock functionally oscillates with alteration of period in the knockout of Nr1d1 [15], raising a possibility that Psen2 N141I mutant may exhibit a dissociation of circadian phenotype between central and peripheral oscillator. Our previous finding reported the decreased amplitude of molecular circadian clock in microglia, one of major types of glia. It is noteworthy that the glial cells harbor functional peripheral clock that regulates inflammation and cytokine production and that another type of glial cells, astrocytes, plays a crucial role in time-keeping of the SCN [16, 17]. The evidence raises the question to what extent the circadian system is affected in Psen2 N141I mutant, an AD model.

This study is aimed to demonstrate the circadian properties of central pacemaker and immune cell-specific peripheral oscillators in Psen2N141I/+ mice with suppressed level of REV-ERBα. We identify that Psen2 N141I mutation modulates amplitude, but not period of local clocks in immune cells. Psen2 N141I mutation also does not alter either central molecular clock or behavioral rhythms.

MATERIALS AND METHODS

Animals

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. Psen2 N141I knock-in mice were generated as described previously [13]. Per2::Luc knock-in mice were a generous gift from Joseph Takahashi [18]. Psen2N141I/N141I and Psen2+/+ mice were crossed with Per2::Luc mice for bioluminescence recording.

Cell culture

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 [20].

Reagents

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 Escherichia coli O111:B4 (LPS; 1 μg/ml), flagellin from Salmonella typhimurium (FAL-ST; 1 μg/ml), Pam2CGDPKHPKSF (FSL-1; 100 ng/ml), 20-mer single-strand RNA derived from HIV-1 long terminal repeat (ssRNA) (1 μg/ml), and oligonucleotides containing unmethylated CpG dinucleotides (ODN 1826; 0.5 μm) were purchased from Invivogen (San Diego, CA, USA).

Quantitative RT-PCR

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 [13]. Actb was used as the reference gene for normalization.

Western blotting analysis

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).

Enzyme-linked immunosorbent assay (ELISA)

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 slice culture

SCN explant cultures were prepared and monitored in a similar manner to that described previously, with minor modifications [21]. One-week-old WT or Psen2N141I/+ mice with Per2::Luc knock-in allele [18] were sacrificed and their brains were quickly removed before being chilled and moistened in Gey’s balanced salt solution (GBSS), supplemented with 0.01 M HEPES and 36 mM D-glucose, and aerated with 5% CO2 and 95% O2. The brains were then coronally cut into 400-µm thick slices using a Leica VT1000 S vibratome (Leica; Germany). The slices were maintained on a culture insert membrane (Millicell-CM, Millipore; Burlington, MA, USA) and dipped into culture medium (50% minimum essential medium, 25% GBSS, 25% horse serum, 36 mM glucose, and 1× antibiotic-antimycotic) at 37°C. The SCN slices were cultivated for more than two weeks before being used in experiments.

Bioluminescence recording

To monitor real-time circadian rhythms with the cells and SCN slices from Per2::Luc knock-in mice, luminescence was continuously measured with cell culture incubator-incorporated luminometer (Kronos Dio, Atto; Amherst, MA, USA). Light emission was measured and integrated for 1 min at 10-min intervals at 36°C. Astrocytes were synchronized with dexamethasone (DEX, 150 nM) during 2 h. Astrocytes and SCN slices were changed with 1 ml fresh recording media supplemented with 0.3 mM D-luciferin in a 35-mm petri dish. Real-time bioluminescence was analyzed by the cosinor procedure [22, 23].

Behavior recording

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 [24]. 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 [25] in R [26].

Statistical analysis

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 [25] in R [26].

RESULTS

Psen2 N141I mutation alters rhythmic expression pattern of clock genes in immune cells

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 Psen2 N141I mutation induces the over-production of clock-controlled cytokines upon immune challenges [13], we measured rhythmic expression pattern of representative clock genes in WT and KI/+ immune cells to understand the circadian alteration associated with the hyperactivation of KI/+ immune cells. We listed up the representative clock molecules [30] and main regulator of clock genes, Csnk1a1 [31] and examined the intrinsic oscillation of clock genes after cell synchronization. Immune cells were exposed to DEX (100 nM) for 2 h to synchronize cellular clock rhythm by mimicking serum shock [32] and were further cultured for 48 h in the absence of DEX with sampling every 4 h to measure clock gene mRNA levels. DEX treatment was not toxic to all immune cells (cell death of microglia without DEX treatment: 6.97%±0.85% in WT and 7.99%±1.17% in KI/+, 24 h after DEX treatment: 9.07%±2.18% in WT and 9.01%±2.72%; astrocytes without DEX treatment: 7%±0.05% in WT and 6.3%±1.16% in KI/+, 24 h after DEX treatment: 3.22%±0.63% in WT and 2.63%±0.43% in KI/+; BMDM without DEX treatment: 3.33%±1% in WT and 4.61%±0.87% in KI/+, 24 h after DEX treatment: 5.56%±1.35% in WT and 4.15%±0.66% in KI/+). Psen2 N141I mutation downregulated the steady-state mRNA levels of Nr1d1, Clock, Cry1, Per1, and Per2 genes and conversely upregulated mRNA levels of Arntl [13]. Likewise, the oscillation of Nr1d1 was diminished in KI/+ microglia (Fig. 1A), which is consistent with our previous report [13] . Rhythmic expression of Arntl showed tendency to slightly increase in KI/+ microglia (Fig. 1C). Oscillation of other genes was also diminished in KI/+ microglia (Fig. 1B, D, E, G, H) while oscillation of Clock and Cry was shifted (Fig. 1D, F). The daily rhythm of Dbp and Csnk1a1 was least affected (Fig. 1I, J). The oscillation of Nr1d1 in KI/+ astrocytes and BMDM also disappeared (Fig. 2A, G). Psen2 N141I mutation induced change of rhythmic expression of Arntl (Fig. 2B, H) and Clock (Fig. 2C, I) in both KI/+ astrocytes and BMDM compared with WT cells. Oscillation of Per1 and Per2, but not Cry were slightly shifted in both KI/+ astrocytes (Fig. 2D~F) and BMDM (Fig. 2J~L).

Psen2 N141I mutation dampens intrinsic robustness, but not period of daily oscillation rhythm in immune cells

Disruption of rhythmic expression of clock genes in microglia by Psen2 N141I mutation may suggest that Psen2 N141I mutation impairs intrinsic clock rhythm of immune cells. However, in our previous study, we observed normal circadian periods with significant reduction in the amplitude of PER2::LUC oscillations in KI/+ microglia and BMDM [13]. We extended these analyses to primary astrocytes derived from Psen2N141I/+ mice. In a similar manner to microglia and BMDM, REV-ERBα level was down-regulated in KI/+ astrocytes (Fig. 3A). Likewise, intrinsic amplitude of cell-autonomous circadian oscillation of primary astrocytes was also significantly reduced by Psen2 N141I mutation (Fig. 3B), although period was not different between genotypes (Fig. 3C). In sum, these results show that circadian rhythm remained intact but with lowered amplitude in immune cells derived from Psen2N141I/+ mice. This is in accordance with a widely accepted view that REV-ERBα renders robustness by fine tuning the entire molecular circadian clock network [33, 34]. Therefore, Psen2 N141I-mediated suppression of REV-ERBα seems to be the primary cause of hyperimmunity and downregulation of circadian amplitude and shifted or diminished rhythmic oscillation of some clock genes may be due to the indirect effects of REV-ERBα suppression.

IL-6 but not TNF-α is over-produced in immune cells derived from Psen2N141I/+ mice in comparison with WT following toll-like receptors (TLRs) activation

Previously, we examined that Psen2 N141I mutation induced hyper immune responses, specifically IL-6 not TNF-α of microglia and BMDM after LPS administration, an agonist of TLR4 [13]. To further explore the functional consequences of down-regulation of REV-ERBα [13] and declined circadian robustness in innate immunity, we examined whether Psen2 N141I mutation affects inflammatory functions of immune cells following activation of various TLRs [19]. Microglia, BMDM, and astrocytes derived from WT or Psen2N141I/+ mice (Fig. 4A) were treated with Pam3CSK4 for TLR1/2, HKLM for TLR2, Poly (I:C) HMW or Poly (I:C) LMW for TLR3, LPS for TLR4, ST-FLA for TLR5, FSL-1 for TLR6, ssRNA for TLR7, and ODN1826 for TLR9, respectively. All TLR ligands induced significantly higher increases in the IL-6 secretion in KI/+ microglia than WT (Fig. 4B). However, this mutation-dependent effect on the magnitude of the TLR ligands response was not observed in TNF-α release (Fig. 4B). More robust release of IL-6, but not TNF-α by the TLR ligands was also observed in BMDM (Fig. 4C) and primary astrocytes (Fig. 4D). Therefore, markedly increased secretion of IL-6, but not TNF-α, is a common response among typical innate immune cells harboring Psen2 N141I mutation.

The molecular oscillation of central clock remains intact in Psen2N141I/+ mice

Our results indicate that REV-ERBα expression is reduced in immune cells by Psen2 N141I mutation, which is associated with reduced robustness of immune cell-intrinsic circadian oscillation. To examine whether Psen2 N141I mutation also affects the central expression of REV-ERBα, we measured mRNA level of Nr1d1 in the SCN tissue. Of interest, Nr1d1 transcript level was the same between WT and KI/+ SCNs (Fig. 5A). To examine the molecular oscillation of central clock, we obtained SCN tissue slices from Per2::Luc;Psen2N141I/+ and Per2::Luc;Psen2+/+ mice for explant study. In contrast to faster dampening in Psen2N141I/+ immune cells along with lower circadian amplitude than those in WT cells, we found that Psen2 mutation altered neither period nor amplitude of PER2::LUC oscillation of SCN cultures (Fig. 5B, C). On the basis of this result of SCN PER2::LUC rhythm, we expected the normal circadian behavior at the animal locomotor activity. As such, actogram of mouse rhythmic locomotor activities under both 12-hour/12-hour light/dark (LD) and constant darkness (DD) for another 14 days revealed no apparent differences in the locomotor activity rhythm between WT and Psen2N141I/+ mouse (Fig. 6A~C). Likewise, body temperature fluctuations were the same between genotypes (Fig. 6D, E). The periodogram analyses showed no differences for amplitude in locomotor activities (Fig. 6A, B) and body temperature (Fig. 6C, D). These data indicate that Psen2 N141I mutation does not affect systemic circadian rhythm.

DISCUSSION

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 [4]. Conversely, circadian rhythm affects Aβ dynamics [35], and circadian alterations precede the onset of AD symptoms [36]. 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 [40]. 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 Psen2N141I/+ mice, knock-in mice heterozygous for Psen2 N141I mutation to address the disruption of central and peripheral clock in AD model. We previously generated this mouse line to mimic heterozygosity of human FAD patients and maintain endogenous expression level of mutant PSEN2 protein, overcoming the shortcoming of excessively high level of transgenes in other AD models [13]. We observed that Psen2 N141I mutation reduces the amplitude of peripheral clock in immune cells, but did not affect the central clock. To our knowledge, this is the first example that AD model animals exhibit specific clock phenotype in peripheral oscillator without central and systemic circadian phenotypes.

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 Nr1d1 knockout homozygote mice, the locomotor activity and molecular clock functionally oscillate with a slightly short period, while the malfunctions of various peripheral organs were reported including immune, metabolism, and brain functions [15, 41, 42]. In locomotor activity and SCN PER2::LUC oscillation, the circadian phenotypes of Psen2 N141I is comparable to wildtype and restricted to peripheral oscillators in immune cells. These phenotypes are in well accordance with our previous results of the suppressed expression of Nr1d1 through its promoter hypermethylation in Psen2 N141I mutant as primary mechanism of hyperimmune pathophysiology. These results indicate that the circadian effect of Nr1d1 suppression is tissue specific. For example, the central oscillator in the SCN is less susceptible and maintains period length comparable to wildtype, while the peripheral oscillator in the immune cells are more susceptible and exhibit reduced amplitude. This points to the more prevalent circadian effect in AD progression than previously considered. In addition to systemic evaluation of circadian function by the determination of sleep-wake cycle or daily activity pattern, more detailed examination of internal alignment of oscillator is required [43, 44], as tissue-specific dosage effect may predispose misalignment of internal organs. To sum, Psen2 N141I mutant mice provides a unique opportunity to address whether the link between circadian system and familial AD risk factor lies in the systemic timing discordant to environmental time or in the misalignment among central and peripheral oscillators. Further study will be warranted to elucidate the sophisticated relationship between reduced robustness of circadian oscillation and hyperimmunity in immune cells harboring Psen2 N141I.

ACKNOWLEDGEMENTS

We thank Dr. Joseph Takahashi for providing Per2::Luc knock-in mice. This research was supported by Basic Science Research Program through the National Research Foundation of Korea (NRF) funded by the Ministry of Education (2021R1A6A3A13043826 to H.N.) and the Ministry of Science and ICT of Korea (2018M3C7A1056275 to S.-W.Y., and 2019M3C1B8090845 to H.K.C.).

Figures

Fig. 1. Psen2 N141I mutation alters rhythmic expression patterns of clock genes in microglia. (A~J) Comparison of transcript abundance of representative circadian clock genes at different times after synchronization during 24 h (A, Nr1d1; B, Nr1d2; C, Arntl; D, Clock; E, Cry; F, Rora; G, Per1; H, Per2; I, Dbp; J, Csnk1a1). For RNA extraction, microglia were collected every 4 h after 2 h synchronization by dexamethasone (DEX, 100 nM) treatment (n=4). mRNA levels were normalized to Actb. Data are mean±SEM.
Fig. 2. Psen2 N141I mutation alters rhythmic expression patterns of clock genes in astrocytes and BMDM. (A~F) Comparison of transcript abundance of representative circadian clock genes at different times after synchronization during 24 h in primary astrocytes (A, Nr1d1; B, Arntl; C, Clock; D, Per1; E, Per2; F, Cry) and (G~L) BMDM (G, Nr1d1; H, Arntl; I, Clock; J, Per1; K, Per2; L, Cry). For RNA extraction, cells were collected every 4 h after 2 h synchronization by dexamethasone (DEX, 100 nM) treatment (n=3). mRNA levels were normalized to Actb. Data are mean±SEM.
Fig. 3. Psen2 N141I mutation decreases robustness of circadian oscillation in primary astrocytes. (A) Analyses of REV-ERBα protein levels by western blotting in primary astrocytes (n=3). Signals on western blots were normalized to those of ACTB. (B) Representative bioluminescence recording of rhythmic Per2::Luc expressed astrocytes. Primary astrocytes were isolated from Per2::Luc;Psen2+/+ (WT) or Per2::Luc;Psen2N141I/+ mice (KI/+) and exposed to DEX (100 nM) for 2 h, followed by measurement of luciferase bioluminescence every 10 min (measurement for 60 sec and interval for 600 sec) over 4 days. (C) Circadian period and amplitude were analyzed (n=7). Data are mean±SEM. ##p<0.01 for indicated comparisons.
Fig. 4. Psen2 N141I mutation causes selective increase in the production of IL-6 in innate immune cells. (A) Schematic diagram suggesting culture system of primary mouse microglia, astrocytes and bone marrow-derived macrophages. (B~D) ELISA of IL-6 and TNF-α in culture supernatants from WT and KI/+ microglia (B), bone marrow-derived macrophages (BMDM; C), and astrocytes (D) after treatment with the TLR ligands for 12 h (n=4). Cells were stimulated with 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) or low molecular weight (poly (I:C) LMW; 1 μg/ml), lipopolysaccharide (LPS; 1 μg/ml), flagellin from Salmonella typhimurium (FAL-ST; 1 μg/ml), Pam2CGDPKHPKSF (FSL-1; 100 ng/ml), single-strand (ss) RNA (1 μg/ml), or oligonucleotides containing unmethylated CpG dinucleotides (ODN 1826; 0.5 μM). Data are mean±SEM. #p<0.05, ##p<0.01, and ###p<0.001 for the indicated comparisons.
Fig. 5. Psen2 N141I mutation does not down-regulate REV-ERBα or alter the central clock. (A) Relative mRNA expression levels of Nr1d1 in the suprachiasmatic nucleus (SCN) region from WT or Psen2N14I/+ mice (n=3). mRNA levels were normalized to Actb. (B) Representative results of bioluminescence recordings in the SCN slices from Per2::Luc;Psen2+/+ (WT) or Per2::Luc;Psen2N141I/+ mice (KI/+). (C) All recordings of SCN slices (n=8) were used to analyze the circadian period and amplitude. Data are mean±SEM.
Fig. 6. Psen2 N141I mutation does not affect circadian behavior and physiology. (A) Representative actograms of locomotor activity of WT and Psen2N141I/+ (KI/+) mice. The periods of constant darkness were revealed by a gray background. Mice were exposed to 12 h:12 h light/dark cycles (LD, 14 days), as indicated by the empty and filled bars shown above, followed by constant darkness (DD, 14 days; gray background). (B) Periodograms for locomotor activity under constant darkness. The number in the plot shows the average value (h) of peaks of each genotype. (23.9±2.275 h in WT and 23.9±0.212 h in KI/+; n=8). Gray line: periodogram for individual mouse; black line, average of each animal; red line, significance at p<0.001 using chi-square (Qp) statistics. (C) Total locomotor activity of WT and Psen2N141I/+ (KI/+) mice. Average activity was counted under 12 h:12 h light/dark cycles (LD) and constant darkness (DD). (D) Representative actograms of body temperature in WT and KI/+ mice. (E) Periodograms for body temperature under constant darkness (23.9±0.0025 h in WT and 23.8±0.160 h in KI/+; n=8). Gray line: periodogram for individual mouse; black line, average of each animal; red line, significance at p<0.001 using chi-square (Qp) statistics.

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© 2019. Experimental Neurobiology: the official journal of the Korean Society for Brain and Neural Sciences & the Korean Society for Neurodegenerative Disease.