Abstract
Hyperimmunity drives the development of Alzheimer disease (AD). The immune system is under the circadian control, and circadian abnormalities aggravate AD progress. Here, we investigate how an AD-linked mutation deregulates expression of circadian genes and induces cognitive decline using the knock-in (KI) mice heterozygous for presenilin 2 N141I mutation. This mutation causes selective overproduction of clock gene-controlled cytokines through the DNA hypermethylation-mediated repression of REV-ERBα in innate immune cells. The KI/+ mice are vulnerable to otherwise innocuous, mild immune challenges. The antipsychotic chlorpromazine restores the REV-ERBα level by normalizing DNA methylation through the inhibition of PI3K/AKT1 pathway, and prevents the overexcitation of innate immune cells and cognitive decline in KI/+ mice. These results highlight a pathogenic link between this AD mutation and immune cell overactivation through the epigenetic suppression of REV-ERBα.
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Introduction
Alzheimer disease (AD) is the most common neurological disorder characterized by progressive loss of cognitive functions. The key dual pathological hallmarks of AD are extracellular amyloid plaques and intracellular neurofibrillary tangles1. With the increase in lifespan, AD poses a severe burden on global public health. Most cases of AD are sporadic, but a small percentage is inherited and is caused by the mutated versions of the genes including APP, PSEN1, and PSEN2, which encode amyloid precursor protein, presenilin 1, and presenilin 2, respectively2. Identification of amyloid β (Aβ) peptide in the senile plaques and widespread occurrence of amyloid deposition have led to the prevailing amyloid cascade hypothesis3. Genetic studies also suggested that familial AD (FAD) mutations in the above genes elevate amyloidogenic Aβ42 production4. However, intensive efforts over the last several decades aimed at clearing or preventing amyloid deposits have failed to prove the central role of Aβ production and amyloid deposition in the pathogenesis of AD5.
There is increasing recognition that innate immune response contributes to AD pathogenesis6. Systemic inflammation markers are strongly associated with an increased likelihood of dementia7, and high levels of inflammatory mediators including interleukin-6 (IL-6) are found in the plasma of AD patients8. Cognitive status in patients with AD is inversely correlated with microglial activation but not Aβ load9,10. Furthermore, genome-wide association studies have clearly implicated innate immunity and inflammation in the pathogenesis of AD11,12. Therefore, it is now widely appreciated that inflammation is not only a central epiphenomenon in AD, but represents a major risk factor for the pathogenesis of AD7.
Innate immunity is under circadian control, and circadian proteins can directly control the expression of genes involved in the innate immune system13,14. Circadian rhythm is an endogenous biological rhythm with an approximately 24-h period and regulated by interlocked transcription–translation feedback loop system15,16. REV-ERBα (encoded by the NR1D1 gene) is a core inhibitory component of this circadian system and functions as a transcriptional repressor in the auxiliary loop by suppressing the expression of BMAL1 (encoded by the ARNTL gene)17.
Interestingly, recent studies have revealed an intimate link between circadian abnormalities and AD18,19,20,21,22,23,24,25. In the brain, molecular clock machineries are found not only in neurons, but also in glia, suggesting the importance of a functional molecular clockwork for proper glial function26,27. Therefore, it is imperative to understand the functional roles of the immune cell clock genes in the progress of AD from the aspect of hyperimmunity.
PSEN has been implicated in inflammation28,29. Especially, PSEN2 seems to be the main PSEN in microglia29. However, how the PSEN2 FAD mutations affect the innate immune system remains largely unknown. Therefore, mouse models mimicking human PSEN2 mutations would be desirable to delineate the molecular pathways underlying hyperimmunity-driven AD pathogenesis and cognitive impairment. The Asn residue at position 141 in PSEN2 is conserved between human and mouse, and its substitution by Ile (N141I) is tightly linked to a form of early-onset FAD with strong penetrance30.
Here, we generated knock-in (KI) mice expressing Psen2 with the N141I mutation and provide evidence that mice heterozygous for Psen2 N141I are vulnerable to otherwise innocuous, mild inflammatory challenges and exhibit a hyperactive immune response and memory deficits because of selective overproduction of clock gene-controlled cytokines. This hyperimmunity is due to the DNA methylation-mediated epigenetic suppression of the clock protein REV-ERBα. Therefore, we identify epigenetic repression of REV-ERBα as a missing link between the Psen2 N141I FAD mutation and hyperimmune activity.
Results
Psen2 N141I/+ mice overproduce clock gene-controlled cytokines in response to LPS
To investigate the pathogenic function of the Psen2 N141I mutation in vivo, we generated KI mice harboring the Psen2 N141I allele (Psen2N141I/+ and Psen2N141I/N141I) (Fig. 1a). The substitution of N to I (AAC to ATC) was confirmed by genomic sequencing (Fig. 1b). Human AD patients with FAD mutations are heterozygous30. Therefore, to more accurately recapitulate human AD and maintain the endogenous expression level, we used heterozygous Psen2N141I/+ (KI/+) mice for all experiments. The protein and mRNA levels of PSEN2 in KI/+ primary microglia and bone marrow-derived macrophages (BMDM) were comparable to those in WT cells (Supplementary Fig. 1a, b), indicating that the N141I mutation did not affect PSEN2 expression. Also, this mutation did not induce a compensatory change in PSEN1 expression (Supplementary Fig. 1a, b). We estimated γ-secretase activity on the basis of the cleavage of N-cadherin and mRNA expression level of Hes5 as a downstream target of the Notch pathway31, and found no difference between N141I KI/+ and WT genotypes in microglia and BMDM (Supplementary Fig. 1c, d).
To explore the functional consequences of the Psen2 N141I mutation in innate immunity, we examined whether this mutation affects inflammatory responses in Psen2N141I/+ mice following systemic injection of lipopolysaccharide (LPS) derived from Escherichia coli O111:B4. In mice, the immune response peaks in the hours around the beginning of the active phase27. Therefore, we injected WT and KI/+ mice intraperitoneally (i.p.) with various doses of LPS at 6:00 PM with light-on at 7:00 AM (Fig. 1c) and monitored the inflammatory response after 20 h. Compared with WT mice, KI/+ mice exhibited a higher circulating level of IL-6 at all LPS doses tested, and the relative difference between the genotypes was more pronounced at the lower doses (Fig. 1d). On the other hand, the blood levels of TNF-α were the same in both genotypes at all doses (Fig. 1e). The lowest dose (0.35 μg/kg body weight) we tested did not induce TNF-α secretion (Fig. 1f) in either WT or KI/+ mice, but still increased the blood level of IL-6 in KI/+ mice (Fig. 1f).
To explore the mechanisms underlying the overactive response of IL-6 by Psen2 N141I mutation, we compared the profiles of secreted cytokines using enzyme-linked immunosorbent assay (ELISA)-based cytokine protein array between WT and KI/+ microglia after LPS treatment. Among 40 targets, we detected 13 cytokines and observed increased secretion of C-X-C motif chemokine ligand 1 (CXCL1), C-C motif chemokine ligand 2 (CCL2), and CCL5 in addition to IL-6 in KI/+ microglia (Fig. 2a, b). Furthermore, their transcript levels were significantly upregulated in KI/+ microglia and BMDM relative to WT (Fig. 2c, d). To examine whether this set of cytokines is also more produced in KI/+ mice than WT, we measure their blood levels in mice injected with the dose of LPS that induced only IL-6 but not TNF-α secretion, and observed the same hyper response of this set of cytokines (Fig. 2e).
On the other hand, the secretion of CCL3, CCL4, and CXCL2 was lower in KI/+ microglia than WT by LPS treatment (Fig. 2b), but these cytokines showed no difference in blood serum levels between WT and KI/+ mice following LPS injection (Fig. 2f). Therefore, reduced secretion of these cytokines seems limited to in vitro microglia culture and subject to additional regulation in vivo.
Interestingly, all four increased cytokines (IL-6, CXCL1, CCL2, and CCL5) are known to be regulated by the circadian rhythm32. In contrast, secretion of TNF-α and its mRNA level were changed by LPS treatment to the same extent between WT and KI/+ genotypes in both microglia and BMDM (Fig. 2b–d). IL-1β is another major cytokine and is released through the inflammasome complex, such as the NLRP3 inflammasome in innate immune cells33. We activated the NLRP3 inflammasome by stimulating LPS-primed microglia with nigericin, as in our previous study33, and found a similar amount of released IL-1β and its transcript levels between WT and KI/+ microglia (Supplementary Fig. 2). Of note, the expression of both Tnf and Il-1b is circadian cycle–independent32. To sum up, the Psen2 N141I mutation induced hyperactive responses of clock gene-controlled cytokines in microglia and BMDM, innate immune cells representative of the brain and periphery, respectively.
Psen2 N141I/+ mice display proinflammatory microglial morphology and suffer memory deficit in response to the low dose of LPS ineffective in WT mice
Microglia morphology is closely related to their function and microglial activation is characterized by cell shape change34. Therefore, we wondered whether the increased production of four cytokines is associated with changes in the morphology in KI/+ microglia. We examined microglia shapes in the hippocampus of KI/+ and WT mice by immunohistochemical analyses with an antibody against a microglia-specific marker, IBA-1. In the WT mice, microglia had a small cell body with highly ramified processes; consistent with no induction of cytokine release, a low dose of LPS (0.35 μg/kg body weight) did not change their morphology (Fig. 3a). On the other hand, hippocampal microglia in KI/+ mice, even in the absence of LPS challenge, already had a round enlarged soma with shorter processes, and these morphological features were furthered by LPS injection at the dose ineffective in WT mice (Fig. 3a). We used the confocal images of microglia to reconstruct their 3D morphology and measured morphological parameters using IMARIS software (Fig. 3b). Total dendrite length and the number of dendrite terminal points of each microglial cell were lower in KI/+ than in WT mice and further reduced by LPS injection (Fig. 3c). Sholl analysis measures the average number of dendrite intersections on concentric rings spaced at 1-μm intervals from the center of the cell body. Sholl analysis revealed that the hippocampal microglia of Psen2N141I/+ mice had fewer dendrite intersections than those of WT mice and exhibited dwindled morphology, and these characteristics became more distinct in response to LPS injection (Fig. 3c), consistent with microglial activation. Therefore, on the basis of morphology, microglial activation was evident in KI/+ mice at baseline and was further induced by mild LPS challenge.
To determine whether the differential increase in the production of a subset of inflammatory cytokines in KI/+ is reflected by cognitive decline, we injected mice with the same low dose of LPS (0.35 μg/kg body weight), and conducted a Y-maze test 20 h after LPS injection. The arm alternation in the Y-maze was significantly decreased by LPS only in KI/+ mice (Fig. 3d), while the total number of arm entries was similar across all the groups, indicating normal locomotor function (Fig. 3e). To further examine learning and memory, we conducted a T-maze test with a food reward 20 h after LPS injection. Similar to the Y-maze test, the success rate for entering the correct arm was significantly lower in LPS-injected KI/+ mice than in the other groups (Fig. 3f, g). KI/+ mice developed no sickness behavior nor did they differ from WT mice in spontaneous home-cage behaviors at this low dose of LPS, when monitored by LABORAS (Supplementary Fig. 3a, b). Therefore, there was no difference with respect to basal activity patterns between the genotypes. In sum, a low dose of LPS induced a hyperactive immune response and caused memory deficit through the overproduction of clock gene-controlled cytokines including IL-6 in Psen2N141I/+ mice, while the same dose of LPS was innocuous to WT mice.
Psen2 N141I mutation induces secretion of clock gene-controlled cytokines and memory deficits in responses to fibrillar Aβ
To establish a link between Psen2 N141I mutation and AD immunopathology, we examined whether synthetic Aβ also induces hyperactive immune responses in Psen2N141I/+ mice. Fibrillar form of Aβ42 (fAβ42) was injected intracerebroventricularly (i.c.v.) in the right lateral ventricle (Fig. 4a), according to the previous studies35,36, and immune responses and memory deficits were analyzed 7 days later. The dosage of fAβ42 used in the current study did not elicit an immune response or memory impairment in WT mice (Fig. 4b, d). On the other hand, the arm alternation in the Y-maze was declined in fAβ42- injected KI/+ mice (Fig. 4b) with a similar total number of arm entries across all experimental groups (Fig. 4c). Also, increases in the circulating levels of clock gene-controlled cytokines including IL-6 were observed in fAβ42-injected KI/+ mice (Fig. 4d), while the TNF-α levels remained the same as a control in both genotypes (Fig. 4d).
In a similar manner to LPS treatment, fAβ42 treatment induced more production of clock gene-controlled cytokines (IL-6, CXCL1, CCL2, and CCL5) in KI/+ microglia than WT, while an increase in the TNF-α level was similar between two genotypes (Fig. 4e). Therefore, Psen2 N141I mutation with fAβ42 i.c.v. injection induces hyperactive immune responses and memory decline, thus explaining the link between hyperimmunity and AD pathology.
Psen2 N141I mutation decreases the expression of clock genes, but innate immune cells remain rhythmic
Because the Psen2 N141I mutation increased the production of clock gene-controlled cytokines, we examined whether there was an alteration in the expression levels of clock genes in WT and KI/+ microglia and BMDM without cell synchronization. The steady-state mRNA levels of Nr1d1, Clock, Cry1, Per1, and Per2 genes were significantly reduced and conversely the Arntl (encoding BMAL1) level was increased in KI/+ microglia and BMDM (Fig. 5a, b). Among the altered genes, the oscillation of Nr1d1 was severely impaired and its amplitude was greatly decreased in KI/+ microglia (Fig. 5c). These results suggest that the Psen2 N141I mutation impairs the expression of microglial clock genes. To validate the decrease of Nr1d1 expression in KI/+ immune cells under more physiological conditions, we performed acute isolation of microglia and peritoneal macrophages from Psen2N141I/+ mice and WT controls, and confirmed a significant decrease in the mRNA levels of Nr1d1 in KI/+ cells (Fig. 5d). Thus, compared with WT, Nr1d1 expression is suppressed in Psen2 N141I KI/+ immune cells.
To compare the robustness of daily oscillations in microglia and BMDM between the WT and KI/+ genotypes, we monitored circadian clock oscillations in primary microglia and BMDM derived from Per2::Luc mice crossed with KI/+ (Per2::Luc;Psen2N141I/+) or WT littermates (Per2::Luc;Psen2+/+). Per2::Luc reporter mice, in which the luciferase (Luc) gene is fused in-frame to the 3′ end of the endogenous mouse Per2 gene, show robust circadian rhythms in both SCN and peripheral tissues37. Since cell-autonomous circadian clock dampens gradually over time if it is not synchronized with the master SCN clock through neuronal signaling38, this reporter line is a good system to compare and analyze the dynamics and robustness of cell-autonomous circadian oscillations by bioluminescence recording. WT microglia cultures exhibited robust luminescence oscillations for 3 days, whereas KI/+ microglia cultures displayed two robust circadian peaks followed by a barely recognizable third peak (Fig. 5e). Statistical analysis revealed that the amplitude was reduced in KI/+ microglia compared to WT, while the circadian period was similar between the two groups (Fig. 5e). BMDM also exhibited similar alterations in circadian oscillation, comparable period but decreased amplitude in KI/+ cells relative to WT (Fig. 5f). To examine the effects of Psen2 mutation on non-immune cells, we monitored the robustness of daily oscillation in mouse embryonic fibroblasts (MEF). Interestingly, KI/+ MEF exhibited the same circadian oscillation period and amplitude as those of WT cells with an equal expression level of REV-ERBα (Supplementary Fig. 4). Therefore, Psen2 N141I mutation seems to affect Nr1d1 level only in immune cells; nevertheless, immune cells derived from Psen2N141I/+ mice remain rhythmic. Since REV-ERBα can control the immune response and is known as a transcriptional suppressor of IL-6 induction32,39,40, these data suggest that immunomodulatory but not circadian effects of REV-ERBα may contribute to the hyperimmunity phenotypes of KI/+ immune cells.
Downregulation of REV-ERBα underlies selective overproduction of IL-6 in KI/+ microglia
Consistent with the lower Nr1d1 mRNA level in KI/+ cells (Fig. 6a, b), protein levels of REV-ERBα were also much lower in KI/+ microglia and BMDM than in WT counterparts (Fig. 6a). Consistent with a previous report39, the chromatin immunoprecipitation (ChIP) assay showed that REV-ERBα bound directly to the proximal RORE (AGGTCA) promoter site of the Il-6 but not Tnf gene (Fig. 6b). Promoters of Arntl and the non-specific TATA box-binding protein (Tbp) gene were used as a positive and negative control of promoter binding by REV-ERBα, respectively41. The REV-ERBα binding level was significantly lower in KI/+ microglia than in WT (Fig. 6b), suggesting that downregulation of REV-ERBα may underlie the marked increase in IL-6 response to LPS. To confirm this notion, we knocked down the Nr1d1 gene in WT microglia by transduction with lentivirus expressing Nr1d1-targeting shRNA (Fig. 6c). Similar to the Psen2 N141I mutation, suppression of Nr1d1 expression in WT microglia potentiated the inflammatory response, as shown by an increase in the secretion (Fig. 6d) and mRNA expression (Fig. 6e) of IL-6 in response to LPS. In addition, the mRNA (Supplementary Fig. 5a) and secretion levels (Supplementary Fig. 5b) of other clock gene-controlled cytokines (CXCL1, CCL2, and CCL5) were increased with the suppression of Nr1d1. Thus, the knockdown of REV-ERBα was sufficient to induce a selective increase in the production of clock gene-controlled cytokines. We next overexpressed REV-ERBα in KI/+ microglia using lentivirus (Fig. 6f). As expected, the expression of human NR1D1 in KI/+ microglia was sufficient to revert the response of IL-6 to LPS to the WT levels (Fig. 6g, h). In addition, the mRNA (Supplementary Fig. 5c) and secretion levels (Supplementary Fig. 5d) of other clock-controlled cytokines were reduced with overexpression of Nr1d1. In contrast, the secretion and transcript level of TNF-α were not impacted by modulating REV-ERBα expression in microglia (Fig. 6d, e, g, h). These data indicate that the Psen2 N141I mutation selectively elevates clock-controlled cytokines production in response to LPS through downregulation of REV-ERBα in microglia.
Nr1d1 promoter is hypermethylated in KI/+ innate immune cells
Recently, the role of epigenetic mechanisms in the pathogenesis of AD has been gaining attention42. Notably, the methylation of DNA, which occurs mostly on CpG dinucleotides (CpG islands) and results in transcriptional repression, is altered in AD brains43. To determine whether Psen2 N141I mutation represses Nr1d1 expression via DNA hypermethylation, we performed whole-genome DNA methylation sequencing of WT and KI/+ microglia. Next, we searched CpG islands in the Nr1d1 promoter using the prediction algorithm at www.urogene.org/methprimer and estimated methylation levels in the predicted CpG islands through differential cytosine methylation analysis. As a result, the overall methylation ratio of Nr1d1 ~2 kbp upstream of the transcription start site was significantly higher in KI/+ microglia than in WT (Fig. 7a). On the contrary, the same differential cytosine methylation analysis of the promoters of other clock genes revealed similar levels of DNA methylation (Fig. 7b).
To examine whether promoter methylation is associated with REV-ERBα expression, we demethylated DNA in KI/+ microglia using 5-azacytidine (5-Aza) and determined Nr1d1 transcript level by qRT-PCR. Treatment of KI/+ microglia with 5-Aza for 24 h rescued Nr1d1 mRNA (Fig. 7c) and REV-ERBα protein levels (Fig. 7d), and subsequently decreased IL-6 expression (Fig. 7e) and secretion (Fig. 7f) to the levels similar to those in WT, without affecting TNF-α expression (Fig. 7e) or secretion (Fig. 7f). The same treatment with 5-Aza also restored the Nr1d1 transcript (Fig. 7c) and REV-ERBα protein levels (Supplementary Fig. 6a), and reverted elevated expression and secretion profiles of IL-6 without affecting TNF-α in KI/+ BMDM (Supplementary Fig. 6b, c). 5-Aza treatment had no effects in WT cells (Fig. 7c–f and Supplementary Fig. 6a–c). Taken together, these data suggest that DNA hypermethylation of the promoter of the Nr1d1 gene is critical for reducing its expression in KI/+ innate immune cells.
The antipsychotic chlorpromazine restores REV-ERBα expression, and prevents hyperactive immune response and cognitive decline in Psen2 N141I/+ mice
Aiming at restoring the REV-ERBα level and thereby preventing the hyperactive immune response, we screened a chemical library containing 2150 compounds, which consists of clinical phase I-III compounds and FDA-approved, commercially available drugs. We chose compounds that preferentially reduced IL-6 secretion over TNF-α, and selected compounds for further testing after eliminating compounds showing cytotoxicity and already known to have anti-inflammatory or anti-AD effects. Among selected compounds, we narrowed the list to the compounds that are predicted to cross the blood–brain barrier (Supplementary Fig. 7a).
Among the drugs filtered, the antipsychotic chlorpromazine (CPZ, 0.5 μM for microglia and 1 μM for BMDM) efficiently reduced IL-6 secretion and mRNA level (Fig. 8a and Supplementary Fig. 7b) without affecting those of TNF-α (Supplementary Fig. 7c, d) in KI/+ microglia and BMDM. Also, CPZ reversed LPS-induced increases in the mRNA levels of other clock gene-controlled cytokines in KI/+ microglia and BMDM (Supplementary Fig. 7e–g). At these concentrations, CPZ was not toxic to the cells (cell death of 2.76% ± 1.78% in WT and 2.39% ± 0.42% in KI/+ microglia; 2.4% ± 0.69% in WT and 2.26% ± 0.19% in KI/+ BMDM). Furthermore, CPZ treatment of KI/+ microglia rescued Nr1d1 mRNA (Fig. 8c) and protein levels (Fig. 8d). CPZ treatment of KI/+ BMDM also restored Nr1d1 mRNA (Supplementary Fig. 7h) and protein levels (Supplementary Fig. 7i). Moreover, CPZ treatment in KI/+ microglia rescued fAβ42-induced hyperimmunity without affecting TNF-α levels (Fig. 8e). Of note, CPZ treatment did not alter mRNA levels of other clock genes in KI/+ microglia and BMDM as well as WT cells (Supplementary Fig. 8a, b).
To examine whether CPZ can attenuate the overproduction of clock gene-controlled cytokines in vivo, we injected CPZ into WT and KI/+ mice 4 h before LPS injection (Fig. 8f). Administration of CPZ decreased the blood levels of clock gene-controlled cytokines in KI/+ mice without affecting TNF-α (Fig. 8g). To examine whether CPZ can rescue LPS-induced memory deficits in KI/+ mice, we conducted Y-maze test (Fig. 8h, i). CPZ recovered the arm alternation in LPS-injected KI/+ mice (Fig. 8h). The total number of arm entries was the same among all groups, indicating that CPZ-injected mice had normal locomotor activity (Fig. 8i).
In addition, CPZ injection in KI/+ mice recovered microglial morphology (Fig. 9a) and rescued the total dendrite length, the number of dendrite terminal points and dendrite intersections at the basal state and prevented LPS-induced morphological changes (Fig. 9b, c). CPZ at the current low dose did not alter microglial morphology in WT mice (Supplementary Fig. 8c, d). Taken together, these data show that CPZ prevents LPS-induced hyperactive inflammation and memory deficits in Psen2N141I/+ mice, revealing a previously unexplored action of this antipsychotic drug CPZ.
CPZ rescues REV-ERBα expression by rectifying DNA hypermethylation through the inhibition of PI3K/AKT1 pathway in KI/+ microglia
To examine whether CPZ restores Nr1d1 expression via demethylation of its promoter, we performed methylation-specific PCR on the Nr1d1 promoter. We designed methylation primers based on the regions showing higher methylation ratios in KI/+ microglia (Fig. 7a). Hypermethylation was observed in genomic DNA extracted from KI/+ microglia (Fig. 10a) and CPZ treatment effectively demethylated Nr1d1 promoter in KI/+ microglia (Fig. 10a). 5-Aza was used as a positive control for demethylation (Fig. 10a). DNA methylation is regulated by the enzymes called DNA methyltransferases (DNMT) and ten-eleven translocation (TET). DNMTs convert unmodified cytosine to 5-methylcytosine (5mC), while TET enzymes convert back the oxidized 5mC into 5-hydroxymethylcytosine and thereby catalyze demethylation44. Thus, we examined the mRNA expression levels of DNMTs and TETs and observed higher levels of Dnmt3a and Dnmt3b while Tet1 was lower in KI/+ microglia than WT cells (Fig. 10b). Similarly, mRNA expression levels of Dnmt3a and Dnmt3b were upregulated but Tet1 was downregulated in acutely isolated microglia (Supplementary Fig. 9a) and peritoneal macrophages (Supplementary Fig. 9b). Importantly, CPZ reverted a significantly high expression level of Dnmt3a to that of WT (Fig. 10c) and downregulated its protein amount (Fig. 10d).
To identify the signaling pathway responsible for the DNA hypermethylation, we focused on MAPK1/3, GSK3β, and PI3K/ATK1 since they are known to regulate DNMT3A expression45,46,47,48. All these kinases were highly phosphorylated in KI/+ microglia (Fig. 10e), but only phosphorylation of AKT1 was reduced by CPZ (Fig. 10f).
Consistently, pharmacological inhibition of PI3K downregulated DNMT3A and rescued Nr1d1 level (Fig. 10g, h). However, inhibition of MAPK1/3 by PD98059, as shown by the decreased phosphorylation of MAPK1/3 at T202/Y204 (Supplementary Fig.10a), did not affect AKT1 phosphorylation and failed to rescue DNMT3A and Nr1d1 levels in KI/+ microglia (Supplementary Fig. 10a, b). An increase in the inhibitory phosphorylation of S9 indicates decreased GK3β activity in KI/+ microglia (Fig. 10e). There are only a few GSK3β activators reported. Differentiation-inducing factor-3 (DIF-3) was shown to activate GSK3β independently of AKT1 or MAPK cascade49. So, we used DIF-3 to examine whether GSK3β activation can rescue Nr1d1 suppression in KI/+ microglia. Low dose (5 μM) of DIF-3 did not activate GSK3β (Supplementary Fig. 10d). DIF-3 at the concentration of 7.5 μM was able to activate GSK3β, as shown by the dephosphorylation of S9, but did not affect AKT1 phosphorylation and failed to rescue DNMT3A and Nr1d1 levels in KI/+ microglia (Supplementary Fig. 10d, e). We treated primary microglia with DIF-3 at this concentration for 3 h due to the cytotoxicity after longer treatment, and DIF-3 at higher doses than 7.5 μM was highly toxic to microglia. PD and DIF-3 decreased mRNA levels of Il-6 and Tnf in both WT and KI/+ microglia in a dose-dependent manner (Supplementary Fig. 10c, f). Therefore MAPK1/3 or GSK3β was not closely associated with PI3K/AKT1 overactivation under our experimental condition. These data indicate that the overactivation of PI3K/AKT1 pathway is the proximal event involved in Nr1d1 suppression in KI/+ microglia. To sum up, CPZ restored REV-ERBα expression levels by correcting DNA hypermethylation through the inhibition of PI3K/AKT1 pathway in Psen2 N141I KI/+ microglia.
Discussion
Here, we show that Psen2 N141I mutation downregulates REV-ERBα in innate immune cells and mutant mice fail to optimize the innate immune response to an otherwise innocuous immune and fAβ42 challenges, leading to selective overproduction of clock gene-controlled cytokines and cognitive decline (Fig. 10i). We revealed hypermethylation of the Nr1d1 promoter through the PI3K/AKT1 pathway and upregulation of DNMTs as the underlying epigenetic repressive mechanism. Therefore, our findings highlight a mechanism linking the Psen2 N141I FAD mutation to altered expression of REV-ERBα, which renders mutant mice vulnerable to AD immunopathology.
In addition to Nr1d1, other clock genes including Per1, Per2, Cry, and Clock were also markedly downregulated in KI/+ microglia and BMDM. However, overactive response of IL-6 and other clock gene-controlled cytokines is not readily explained by their downregulation. CLOCK forms a complex with the NF-κB subunit p65 and increases the transcriptional activity of NF-κB50. Therefore, Clock-deficient BMDM is less responsive to LPS51. PER2 can also promote inflammation, and Per2-/- mice are protected from LPS-induced endotoxic shock and show decreased serum levels of several cytokines, but the IL-6 level remains normal52. Cry1-/-Cry2-/- BMDM show increased inflammatory features, but without selectivity for IL-6 or clock gene-controlled cytokines, and the levels of both IL-6 and TNF-α are increased by the absence of CRY53. Therefore, the selectively enhanced production of clock gene-controlled cytokines in Psen2N141I/+ mice is most likely due to the repression of REV-ERBα. In addition, DNA methylation sequencing and differential cytosine methylation analysis in the promoter regions of clock genes revealed hypermethylation only in the promoter of Nr1d1 gene in KI/+ microglia. As such, CPZ recovered expression of only Nr1d1 among the clock genes, further lending support to our conclusion.
The mediation of circadian regulation by REV-ERBα in innate immune cells has been associated with inflammation in the periphery32. However, the role of REV-ERBα in central immunity has not been well known. Recently, the link between REV-ERBα and microglia function has been studied using gene knockout approaches40. Deletion of REV-ERBα activates NF-κB signaling, and multiple proinflammatory genes, including TNF-α and circadian-regulated cytokine genes, are upregulated, which is different from the selective elevation of expression of only circadian gene-controlled cytokines observed in this study. Also, Psen2N141I/+ mice showed intact memory function and normal baseline behavior patterns (Figs. 3, 4, and 8 and Supplementary Fig. 3), which were different from the mildly disturbed circadian locomotor behaviors, neurodegeneration, or mood dysregulation of REV-ERBα KO mice17,54. Therefore, the repression of REV-ERBα due to the Psen2 N141I mutation did not phenocopy REV-ERBα KO mice, suggesting that the residual level of REV-ERBα is enough to maintain normal central clock and neural function. There has been great interest in understanding how circadian proteins go awry during the progress of neurodegeneration and the cause of their dysfunction. Our study uncovers the effects of the Psen2 N141I FAD mutation on the expression of REV-ERBα through unbalancing of DNA methylation/demethylation toward hypermethylation, and reveals a functionally important pathogenic pathway leading to hyperreactive innate immunity in AD.
CPZ was introduced into psychiatry for the treatment of schizophrenia and other psychoses in the 1950s55. Its psychotropic effects were mainly attributed to the blockade of dopamine receptors in neurons56, and partially to the inhibition of neuroinflammation57. However, the action of CPZ on innate immune cells is largely unknown and the rescue of REV-ERBα expression reveals a previously unrecognized effect of CPZ.
It is known that antipsychotic drugs such as CPZ, when taken for a long time at high doses, increase mortality in elderly patients with dementia-related psychotic symptoms58. CPZ has been used in the concentration ranges of 1~25 mg/kg in previous animal studies59,60,61. However, in our study, we used CPZ at the dose of 0.35 μg/kg body weight, much lower than the reported doses. Therefore, further studies would be desirable to optimize the CPZ regimen for the prevention of immunopathology with reduced risk of toxicity.
Psen2 N141I mutation can alter various pathways, including autophagy62, intracellular Ca2+ level63, and oxidative stress64, all of which may lead to overactivation of PI3K/AKT1 pathway. However, the effects of Psen2 N141I mutation on these pathways in immunocompetent cells remain poorly understood. Interestingly, CPZ was identified as an autophagy inducer through a cell-based high-throughput image screening65. CPZ can negatively regulate store-operated Ca2+ entry and inhibit proton channel66,67. These effects of CPZ may contribute to its immunomodulatory action. However, these studies adopted higher concentrations of CPZ than our current report, and sometime in non-immune cells. Therefore, further study will be highly warranted to identify which process(es) is impaired by Psen2 N141I mutation and how CPZ subdues overactivated PI3K/AKT1 pathway in microglia and other immune cells.
An interesting question is regarding the relative contributions of REV-ERBα’s circadian or immunomodulatory effects to the phenotypes. Importantly, PER2::Luc bioluminescence recordings from KI/+ microglia and BMDM confirmed that these cells remain rhythmic, although their amplitudes were decreased (Fig. 5e, f). Therefore, we propose that the hyperimmune response of a subset of cytokines was not due to a general disruption of the cellular clockwork, but REV-ERBα’s diminished immunomodulatory activity.
Overall, our findings show that the Psen2 N141I mutation leads to dysregulated innate immunity through the epigenetic suppression of REV-ERBα and overproduction of a selective subset of clock gene-regulated cytokines and, mutant mice are highly vulnerable to immune challenge and predisposed to cognitive decline and AD immunopathology.
Methods
Animals
All protocols and ethical regulations for the care and use of laboratory animals were approved by and in accordance with the guidelines established by the Institutional Animal Care and Use Committee of DGIST (DGIST-IACUC-21051804-0001). Animals were maintained in a specific pathogen-free environment under a standard 12-h light/12-h dark cycle at the DGIST animal facility. Psen2N141I/+ mice were generated using homologous recombination from C57BL/6J. Targeting vector included the I141 mutation in exon 4 and the Neor-loxp sequence, and mutated sequence in the targeting vector was inserted into Psen2 of the WT allele. Psen2N141I/N141I;loxp-Neor-loxp mice were crossed with Cre mice to generate KI mice carrying the Psen2 N141I mutation using the Cre-loxp system. Per2::Luc KI mice were a generous gift from Joseph Takahashi37. Psen2N141I/+ mice were crossed with Per2::Luc mice to monitor real-time circadian dynamics using bioluminescence recording. In all experiments, 8-week-old male mice were used and the number of mice in each experiment is provided in the figure legend.
Reagents and antibodies
The following antibodies were used: horseradish peroxidase–conjugated β-Actin (sc47778; 1:5000) from Santa Cruz Biotechnology; PSEN1 (5643; 1:1000), REV-ERBα (13418; 1:1000), DNMT3A (3598; 1:1000), AKT1 (2938; 1:2000), phosphorylated AKT1 (S473, 9271; 1:1000), MAPK1/3 (4969; 1:1000), phosphorylated MAPK1/3 (T203/Y205, 4370; 1:2000), GSK3β (9315; 1:1000) and phosphorylated GSK3β (9336; 1:1000) from Cell Signaling Technology; IBA-1 (019-19741; 1:250) from Wako Chemicals; N-cadherin (610920; 1:1000) from BD Bioscience; PSEN2 (ab51249; 1:2000) from Abcam; REV-ERBα (PA5-29865; 1:1000) from Thermo Fisher Scientific. The following reagents were used: LPS from E. coli 0111:B4 (L4391), DEX (50-02-2), LY294002 (L9908), DIF-3 (D0567) and chlorpromazine hydrochloride (C8138) were purchased from Sigma-Aldrich; 5-azacytidine (ab142744) from Abcam; and D-luciferin (E1601) from Promega; PD98059 (9900) from Cell Signaling Technology.
Cell culture
Primary microglia were obtained from 1–3-day-old neonatal mice brain with trypsinization and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Corning) supplemented with 10% heat-inactivated fetal bovine serum (HI-FBS, Hyclone) and 1% penicillin–streptomycin (Hyclone). Microglia were isolated at days in vitro 12 by tapping. Purity of primary microglia and no contamination by astrocytes were confirmed by immunostaining with antibodies against IBA-1 and GFAP, specific microglia and astrocyte markers, respectively68. BMDM were obtained from femurs and tibias of 6–7-week-old mice with flushing to extrude bone marrow and were differentiated and grown in RPMI 1640 (GIBCO) medium supplemented with 10% HI-FBS and 1% penicillin–streptomycin69.
MEF cells were obtained from pregnant female mice at gestational day 14.5–15.5. Embryos were harvested from the uterus and the head and visible internal organs were removed. Next, the tissue was rinsed with PBS, minced using a razor blade in 0.25% Trypsin-EDTA (T/E) and incubated for 25 min with pipetting intermittently for dissociation. Suspended cells were pelleted at 1000 × g for 3 min and collected cells were cultured in the MEF medium containing DMEM supplemented with 10% HI-FBS, 1% penicillin–streptomycin (Hyclone), and 2 mM L-glutamine for more than 3 days.
Acute isolation of primary microglia and peritoneal macrophages
The protocol was modified from the previous study69. PBS-perfused brains were extracted from 8-week-old mice and washed with 1× HBSS, chopped by surgical scissors and dissociated with tissue dissociation buffer (0.05% collagenase D, 5 mM CaCl2, 20 U/mL DNase I, 15 mM HEPES and 0.5% glucose in HBSS) for 15 min at 37 °C. The dissociated tissues were passed through 70-μm pore mesh and pelleted at 500 × g for 3 min. Then, microglia cells were isolated using Percoll (P4937, Sigma-Aldrich) gradient (70%, 37%, and 30% Percoll in HBSS). The interphase between 70% and 37% Percoll was transferred to a new tube and centrifuged for 5 min at 500 × g. Each pellet was resuspended in MACS buffer, transferred to a new microcentrifuge tube, and microglia were isolated using CD11b+ microbeads (130-093-634, Miltenyi Biotec). Peritoneal macrophages were obtained from peritoneum of 8-week-old C57BL/6 mice. Four days after i.p. injection of 3% Brewer thioglycolate medium70, the needle filled with cold PBS was inserted through peritoneal wall into each anesthetized mice and peritoneal fluid was withdrawn slowly. Cell pellets were harvested after centrifugation.
Quantitative RT-PCR
RNA was isolated using the ImProm-II Reverse Transcriptase kit (Promega) and cDNA was synthesized using oligo dT. PCR primers were commercially synthesized (Cosmo Genetech). qRT-PCR was performed on the reverse transcribed product using Taq Polymerase (Invitrogen) and primers (Supplementary Table 1) specific for mouse cDNAs. TOPrealTM qPCR 2 × PreMIX (SYBR Green with low ROX) (Enzynomics) was used. Amplification (50 cycles) was performed in a CFX96 Real-Time System (Bio-Rad). Actb or 18S rRNA was used as the reference genes for normalization. CFX-manager (Bio-Rad) was used for analysis. The primers used are listed in Supplementary Table 1.
Enzyme-linked immunosorbent assay
ELISA kits for mouse IL-6, TNFα, IL-1β, CCL2, CXCL1, CCL5, CCL3, CCL4, and CXCL2, and a Proteome Profiler Mouse Cytokine Array Panel A kit were purchased from R&D Systems and used to measure cytokines in culture media and blood serum according to the manufacturer’s instructions. Synergy HTX (Agilent Technologies) reader was used for the measurement of absorbance with Gen5 software (version 2.04) for data collection and analyses.
Western blot analysis
Cells were lysed in lysis buffer (1% Triton X-100, 250 mM sucrose, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride, and 50 mM NaCl in 20 mM Tris-HCl, pH 7.4) with 1× protease and phosphatase inhibitors (Thermo Fisher Scientific) and 0.1 M dithiothreitol (Sigma-Aldrich). For REV-ERBα detection, cells were lysed in radio-immunoprecipitation assay buffer with 1× protease and phosphatase inhibitors and without 1 mM phenylmethylsulfonyl fluoride and 0.1 M dithiothreitol. Cell lysates were separated by SDS–polyacrylamide gel electrophoresis and transferred to polyvinylidene fluoride membranes. Membranes were incubated with the appropriate primary antibodies, and bound antibodies were detected by species-specific, horseradish peroxidase–conjugated secondary antibodies (1:5000). Chemiluminescence detection was performed to analyze the protein bands of interest. The blots were quantified using Image Studio lite 4.0 (LI-COR Biosciences).
ChIP-qPCR analysis
Cells were fixed and proteins were cross-linked to DNA using 0.75% paraformaldehyde (PFA) in PBS for 30 min. Glycine (125 mM) was added to quench cross-linking reaction, and cells were collected and lysed in radio-immunoprecipitation assay buffer (Sigma-Aldrich) containing 1× protease and phosphatase inhibitor cocktail, and DNA was fragmented by sonication to 500–1000-bp size. Protein A-agarose/salmon sperm DNA beads (Millipore) were incubated with DNA and antibody against the protein of interest or IgG, and the samples were immunoprecipitated overnight with rotation at 4 °C. DNA was eluted by adding elution buffer (10 mM EDTA, 1% SDS, 50 mM Tris-HCl, pH 8.0). Input samples were incubated with RNase A (20 μg/mL) and Proteinase K (0.5 mg/mL) at 65 °C overnight to reverse cross-linking. DNA was precipitated with isopropanol, and qRT-PCR was performed to assess the binding of the protein to the anticipated binding sites in DNA using the primers used listed in Supplementary Table 1.
Y-maze assay
Y-maze was used to evaluate spatial working memory. The assay was conducted in white plastic arms of a Y-shaped maze. A mouse was placed in the center and was allowed to freely explore the arms for 5 min. The experiment was recorded with EthoVision software 11.5 (Noldus). The number of arm entries and the number of triads were analyzed to calculate the percentage of alternation by dividing the number of three consecutive arm entries (triads) by the number of possible triads × 100 (total arm entries – 2).
T-maze assay
T-maze was used to evaluate spatial learning and memory with reward alternation. The assay was conducted in white plastic arms of a T-shaped maze. Mice were familiarized with the maze and food reward for 5 min before the test. Then, in the test run, rewards were placed in both arms, and one arm was blocked. Mice were placed at the base and ran to open arms to eat the reward. At the next trial, the previously closed arm was opened. Mice were placed back again at the base and chose one arm. If mice chose the newly opened arm, they were able to eat the reward. If mice incorrectly chose the previously visited arm, they did not get any rewards. The number of trials in which the correct arm was visited was expressed as a percentage of total arm entries. The experiment was recorded with EthoVision software 11.5 (Noldus).
LABORAS
Mice were placed in every single cage and recorded for 24 h starting from the night cycle at 7:00 PM. Activities of mice including locomotion, climbing, rearing, grooming, and eating were recorded and automatically analyzed using the Laboratory Animal Behavior Observation Registration and Analysis System (LABORAS, Metris).
Microglial transduction
shNr1d1 (22747) was purchased from Addgene. Human NR1D1 cDNA was cloned into the PLJM1-EGFP vector to generate lentiviruses. Lentiviruses were produced by transfection of Lenti-X 293T cells (632180, Clontech) with the transfer vector (PLKO.1-shNr1d1 or PLJM1-NR1D1-EGFP), packaging vector (psPAX2, Addgene), and VSV-G envelope-expressing vector (PMD2.G, Addgene). After 3 days, the supernatant was harvested and ultracentrifuged at 25,000 × g for 2 h (Optima XPN-100, Beckman Coulter) for virus concentration. Primary microglia were infected with the lentiviruses in a medium supplemented with hexadimethrine bromide (8 μg/mL). After 24 h, the medium was replaced with a fresh culture medium. eGFP expression was monitored using fluorescence microscopy after 72 h to estimate the lentiviral transduction efficiency.
Immunohistochemistry
Mice were anesthetized by injection of a mixture of Zoletil (Virbac, 50 mg/kg) and Rompun (Bayer, 10 mg/kg). Then, the mice were perfused with PBS, followed by 4% PFA. Brains were collected, post-fixed in 4% PFA for 16 h, transferred to 30% sucrose until they sank to the bottom of the tube, and frozen with optimal cutting temperature compound. Embedded brains were cut into 50-μm-thick coronal sections and slices were floated in PBS. Samples were incubated in sodium citrate buffer (10 mM tri-sodium citrate dihydrate, 1 N HCl, 0.05% Tween-20 in water) in a 1.5-mL tube at 95 °C for 5 min for antigen retrieval. Samples were washed with PBS three times and blocked with PBS containing 5% normal donkey serum and 0.01% Triton X-100. Slices were incubated with IBA-1 antibody in PBS containing 3% bovine serum albumin for 24 h at 4 °C and then with an appropriate secondary antibody for 2 h at room temperature. Images were acquired with LSM 7 and LSM 700 confocal laser scanning microscope and analyzed with ZEN 2010 software (Carl Zeiss).
Stereotaxic injection
FITC-conjugated Aβ1−42 (4033502.05, BACHEM) was fibrilized in DMEM supplemented with 5% HI-FBS by overnight incubation at 37 °C71. Mice were anesthetized with Zoletil and Rumpun, and stereotaxic surgery was performed for i.c.v. injection of 2.5 μL of fAβ42 (1.25 μM) in the right lateral ventricle at the speed of 0.25 μL/min using 10 μL Hamilton syringe and 27G needle. Coordinates were: AP –0.82 mm, ML +0.34 mm, DV –2.0 mm from the bregma.
Microglia morphology analysis
Confocal images were obtained along the entire Z-axis of a randomly selected field. Then, 3D images were reconstructed from confocal images using IMARIS software (version 9.2.1, Bitplane AG). Dendrite length, dendrite branch points, and Sholl radius of microglia were quantified using FilamentTracer function following IBA-1 signals.
Bioluminescence recording
To monitor real-time circadian dynamics in cells derived from Per2::Luc reporter mice, luminescence was continuously measured at 36 °C with a cell culture incubator–incorporated AB-2550 Kronos Dio luminometer (Atto) equipped with analysis software (version 2.10.233). Light emission was integrated for 1 min with 10-min intervals between measurements. Cells were synchronized with DEX (150 nM) in culture media. At 15 min after DEX addition, cells were washed and the medium was changed to a fresh recording medium (culture medium supplemented with 0.3 mM D-luciferin). Bioluminescence was analyzed in real time by the cosinor procedure72,73.
Methylation sequencing
Genomic DNA was isolated from 10 × 106 microglial cells from WT and KI/+ mice using the MasterPure DNA purification kit (Epicentre) according to the manufacturer’s instructions. The extracted genomic DNA was resuspended in TE buffer and quantified by fluorometry. Bisulphite conversion of genomic DNA was conducted using the EZ DNA Methylation Lightning kit (Zymo Research). In this method, non-methylated cytosine residues are converted into uracil by bisulphite treatment and read as thymine when sequenced. Methylated cytosines, protected from conversion, are still read as cytosines. Bisulphite-converted DNA was purified and used to prepare a sequencing library using the EpiGnome Methyl-Seq kit (Epicentre). In this procedure, to synthesize DNA containing a specific sequence tag, DNA is randomly primed using a polymerase able to read uracil nucleotides. Only the complement to the original bisulphite-treated DNA is used as the sequencing template; thus, the resulting Read 1 will always be the same sequence as the original bisulphite-treated strand.
Methylation-specific PCR
Genomic DNA was extracted from cells using genomic DNA extraction kit purchased from QIAGEN according to the manufacturer’s instructions. Incubation of the genomic DNA with sodium bisulfite for conversion of all unmodified cytosine to uracil using bisulfite conversion kit purchased from New England Biolabs. PCR was performed on bisulfite-conversed DNA using primers designed as in Supplementary Table 1. PCR conditions were as follows: 95 °C for 3 min; 34 cycles of 95 °C for 30 s the specific annealing temperature (unmethyl, 49 °C; methyl, 54 °C) for 30 s, and 72 °C for 1 min; and a final extension of 5 min at 72 °C.
Statistical analysis
Data were acquired in at least three independent experiments and are presented as mean ± standard error of the mean values. Statistical significance and n values are described in each figure legend. Statistical analysis was performed by using Student’s unpaired t-test, or one-way ANOVA and Tukey’s post-test, or two-way ANOVA and Bonferroni post-test. Statistical significance was analyzed using GraphPad Prism 8.0.1 (GraphPad Software).
Reporting summary
Further information on research design is available in the Nature Research Reporting Summary linked to this article.
Data availability
All the data supporting this study are available in the article and Supplementary information. The methylation sequencing data generated in this study have been deposited in NCBI Sequence Read Archive (SRA) PRJNA808254 (WT microglia: SRR18071758, SRR18071757, and SRR18071756; KI/+ microglia: SRR18071755, SRR18071754, and SRR18071753)74. Source Data are provided with this paper.
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Acknowledgements
We thank Dr Joseph Takahashi for providing Per2::Luc knock-in mice. This work was supported by the National Research Foundation of Korea (NRF) of the Ministry of Science and ICT of Korea through grants 2018M3C7A1056275 (to S.-W.Y., and E.-K.K.), 2020R1A2C4002156 (to E.-K.K.), and 2017R1C1B2008775 (to H.K.C.), and OATC Inc (S.-W.Y.). The funder had no role in the design, data collection and analyses, and publication of the study.
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S.-W.Y. designed the experiments, analyzed and interpreted the data, and wrote the manuscript. H.N. designed and performed most of the experiments, collected and analyzed the data, wrote the manuscript, and illustrated the schematic diagrams. Y.L., J.-W.L., Y.P., S.H., and B.K. performed additional experiments. B.K. and H.K.C. provided mice, and analyzed and interpreted the data. H.-K.A., K.M.C., and J.H. generated and provided Psen2N141I/+ mice. K.K., E.-K.K., and H.K.C. provided critical experimental materials and revised the manuscript. All authors read and approved the final manuscript.
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S.-W.Y. and E.-K.K. are the scientific advisors to OATC Inc. S.-W.Y., H.N., and Y.L. have pending patent applications: international patent PCT/KR2021/015855 and Korean patent 10-2021-0150049. No potential conflict of interest was reported by all other authors.
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Nam, H., Lee, Y., Kim, B. et al. Presenilin 2 N141I mutation induces hyperactive immune response through the epigenetic repression of REV-ERBα. Nat Commun 13, 1972 (2022). https://doi.org/10.1038/s41467-022-29653-2
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DOI: https://doi.org/10.1038/s41467-022-29653-2
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