An impaired intrinsic microglial clock system induces neuroinflammatory alterations in the early stage of amyloid precursor protein knock-in mouse brain

McGeer and McGeer [1] suggested the pathological importance of neuroinflammation mediated by microglia rather than amyloid β (Aβ) in the pathogenesis of Alzheimer’s disease (AD), as anti-inflammatory agents have a substantial sparing effect on AD [2]. Although the neuroinflammation regularly observed in AD has long been incorporated into the amyloid cascade hypothesis of AD, two recent reports have suggested an inflammatory hypothesis for AD. Treatment with the colony-stimulating factor 1 receptor (CSF1R) inhibitors, that block microglial proliferation and rescue inflammatory alterations, improved the performance in memory and behavioral tasks without affecting the accumulation of Aβ in mouse models of AD [3, 4]. Furthermore, previous studies have provided evidence that infection of the brain with microbes is linked to AD [5, 6]. Some microbes can remain latent in the nervous system with the potential for reactivation, and neuronal damage caused by direct microbial action and microbe-induced inflammation might occur years after the initial infection. In line with this infection hypothesis of AD, we previously reported that virulence factors of Porphylomonas gingivalis, a major pathogen in periodontal disease, including lipopolysaccharide (LPS) and gingipains, were able to activate microglia to induce neuroinflammation through the activation of Toll-like receptor 2 and protease-activated receptor-2, respectively, [7, 8]. These observations suggest that excessive neuroinflammation mediated by microglia is a key driver of AD rather than a result of the disease.

Circadian rhythm disturbances have long been considered a consequence of neurodegeneration associated with AD. AD patients exhibit profound disruptions in their circadian rhythm concerning sleep-wakefulness and other processes. In the post-mortem brain tissue of AD patients, altered synchronization in the rhythms of clock gene expression, including period 1 (PER1), period 2 (PER2), and brain and muscle Arnt-like protein-1 (BMAL1), was noted in different regions of AD patients compared with control subjects [9]. The clock gene system is known to be capable of influencing the inflammatory process in a number of ways. BMAL1 is the central mediator of the circadian control of the immune system and promotes an anti-inflammatory state [10]. A recent clinical study showed that the significant deregulation of BMAL1 in the brain was associated with early AD [11]. An intrinsic molecular clock also exists in microglia that control diurnal morphological changes in their processes, and these cells regulate the sleep-wake cycle-dependent changes in synaptic strengths [12‐14]. Furthermore, infection of P. gingivalis induced process extension of cortical microglia, and this response was significantly greater during the day than night [15], suggesting that the intrinsic microglial clock limits the over-reaction and inflammatory responses of microglia during the active phase. However, little is known about the possible contribution of microglial intrinsic molecular clock to the neuroinflammatory response in AD pathology.

Although a number of AD mouse models have been developed based on amyloid precursor protein (APP) overexpression, the overexpression paradigm may cause additional phenotypes unrelated to AD, including the overproduction of soluble N-terminal fragments, C-terminal fragment-α, C-terminal fragment-β, and APP intracellular fragments [16]. To overcome these drawbacks, two novel AD mouse models (single humanized APP knock-in (KI) mice carrying Swedish (NL), Beyreuther/Iberian (F), or Arctic (G) mutations in different combinations) were generated by knock-in (KI) of a humanized Aβ sequence bearing AD-associated mutations into the mouse APP locus [17]. These models exhibit unique pathophysiologic properties in the brain. For example, APP-KI^{NL-G-F/NL-G-F} mice, which bear all three mutations, show aggressive Aβ pathology starting at 2 months of age [18].

We showed for the first time in the present study that a reduced expression level of BMAL1 was responsible for the increase in the inflammatory phenotype of microglia in APP-KI mice through a reduction in RORα, which in turn reduced IκBα and enhanced NF-κB activation. To determine the pathological roles of REV-ERB in the inflammatory response and learning ability of APP-KI mice, we evaluated the effects of SR9009, a synthetic agonist for REV-ERB, because REV-ERBα nuclear receptor plays a pivotal role in the negative feedback loop regulating the BMAL1 and CLOCK expression. These results suggest that an impaired intrinsic microglial clock system contributes to neuroinflammatory responses and the resultant cognitive impairment in the early stage of AD.

There is increasing evidence that immune parameters change with the time of day and that disruption of the circadian rhythms is linked to inflammatory pathologies. Previous studies have noted the importance of the circadian clock in age-related neuroinflammatory sensitization [27, 28]. BMAL1 is the central mediator of the circadian control of the immune system and anti-inflammatory system, as it drives the expression of RORα, which can increase the levels of IκBα [10, 29], a major negative regulator of NF-κB [30]. Furthermore, REV-ERBα is also a transcriptional repressor that inhibits the BMAL1 expression [26]. LPS-induced sepsis is less severe when injection at ZT10 than at ZT12, coinciding with a decrease in pro-inflammatory cytokines TNF-α, IL-6, and CXCL1 at ZT12, and increase in the anti-inflammatory cytokine IL-10. This effect is blunted a myeloid-specific BMAL1^−/− mouse, indicating the anti-inflammatory role of BMAL1 in myeloid-lineage cells [31]. A recent report has demonstrated that activation of the NF-κB pathway in both primary microglia experiments and REV-ERBα^−/− brain, demonstrating that REV-ERBα negatively controls microglial activation and neuroinflammation [32]. These observations are consistent with the previous observations that the NF-κB-mediated transcriptional repression of the clock feedback limb could cause circadian disruption in response to inflammation [33, 34]. These results suggest that REV-ERBα/BMAL1 is a regulator of microglial activation and neuroinflammation. However, little is known about the impairment of the intrinsic microglial clock in the early phase of AD. Furthermore, the involvement of an altered circadian clock gene expression in the increased presence of microglia with an inflammatory phenotype in AD remains poorly understood. To address these questions, we explored mRNA expression profiles of circadian clock genes and pro-inflammatory genes in cortical microglia isolated from 2-month-old APP-KI and WT mice using a combination of CAGE sequencing and q-PCR analyses. We first found that the mean mRNA expression of BMAL1 and REV-ERBα was significantly lower in APP-KI microglia than in WT microglia. In contrast, the mean mRNA expression of pro-inflammatory genes, including TNF-α, IL-1β, and IL-6, at ZT14 was significantly higher in APP-KI microglia than in WT microglia. It was also noted that the diurnal mRNA expression of these pro-inflammatory genes was negatively associated with that of BMAL1. In contrast, the diurnal mRNA expression of IκBα was positively associated with that of BMAL1. Previous study reported that RORα inhibits NF-κB signaling by inducing IκBα gene expression [29]. We reported that chronic activation of NF-κB promoted increased expression of pro-inflammatory mediators including IL-1β, TNFα, and iNOS [21]. On the basis of these previous observations, it is likely to speculate that reduced expression of BMAL1 may promote the NF-κB activation and the subsequent increased expression of pro-inflammatory genes in APP-KI microglia.

A cross-sectional study had been conducted to examine the association between circadian function, aging, and preclinical AD pathology which included 189 cognitively normal participants. After correction for age and sex, the presence of preclinical amyloid plaque pathology, assessed by positive PiB imaging or increasing cerebrospinal fluid was associated with increased intradaily variability, suggesting preclinical AD is associated with rest-activity rhythm fragmentation and circadian dysfunction could contribute to the early pathogenesis of neurodegenerative disease [35]. In the present study, we used single humanized 2-month-old APP-KI mice. Compared to 6-month- old APP-KI mice, the 2-month-old mice did not exhibit Aβ deposition and activated microglia at the site of Aβ deposition in the hippocampus and cerebral cortex. Therefore, 2-month-old APP-KI mice were reasonable to be used to study the pathogenesis in the early stage of AD.

SR9009, which was developed as a synthetic REV-ERBα agonist, altered the circadian behavior and the circadian pattern of the core clock gene expression in the hypothalamus of mice [36]. Furthermore, LPS-induced IL-6 expression was attenuated in microglia from BMAL1-deficient mice. This phenotype was recapitulated by pharmacological disruption of oscillatory rhythmicity using SR9009 [37]. In a previous study, the REV-ERBα agonists, GSK4112, and SR9001 appeared to prevent neuroinflammation and cell death when infused into the brain [38]. These observations prompted us to examine whether or not the pharmacological activation of REV-ERBα could ameliorate neuroinflammatory response culminating in the disruption of learning and memory. The WT mice showed a response to the novel object and were able to discern a change in the object even after treatment with SR9009. Rather surprisingly, APP-KI mice did not show a response and could not discern a change in the object after treatment with SR9009, suggesting that pharmacological activation of REV-ERBα induced memory disruption in APP-KI mice but not in WT mice. On the other hand, SR9009 has been reported to have REV-ERB-independent proliferation and metabolism [39]. Therefore, additional experiments using REV-ERB deficient microglia are necessary to examine whether a memory impairment effect of SR9009 is due to an activation of REV-ERB.

It is interesting to speculate that OAβ enhances the effects of SR9009, as OAβ is generated even in the brain of 2-month old APP-KI mice. Furthermore, the APP expression is required for microglial activation, particularly in response to OAβ [40]. Our observations in the present study showed that treatment with SR9009 alone was able to increase the expression of IL-1β and IL-6 but not that of TNF-α in microglial cells. In contrast, OAβ failed to increase the expression of these pro-inflammatory genes. The combined treatment with SR9009 and OAβ significantly increased the TNF-α expression and further enhanced the expression of IL-1β and IL-6 in microglial cells. The present finding that OAβ significantly suppressed BMAL1 expression in microglia might suggest the possible involvement of decreased the BMAL1 expression and the subsequent activation of NF-κB in OAβ-induced neuroinflammation. Additional experiments will be needed to confirm this possibility. In the present study, systemic treatment with SR9009 induced activation of hippocampal microglia, increased expression of inflammatory mediators in the hippocampus, and memory disruption in APP-KI mice. SR9009 alone also increased the expression of IL-1β and IL-6 in MG6 microglia. The combined treatment with OAβ and SR9009 further increased expression of these cytokines. In contrast, Griffin et al. have reported that SR9009 exhibited dose-dependent suppression of LPS + ATP-induced IL-1β protein secretion in BV-2 cells and primary cultured microglia [32]. Moreover, an application of REV-ERBα agonist GSK4112 suppressed LPS-induced microglia activation through NF-κB pathway resulting in suppressing the expression and secretion of the pro-inflammatory cytokines [38]. In the present study, however, we could not see anti-inflammatory effects of SR9009. On the contrary, SR9009 induced pro-inflammatory mediators in both cultured microglia and the cerebral cortex of APP-KI mice. This discrepancy may stem from differences in experimental procedures. In our in vitro studies, MG6 microglia were synchronized by treatment with dexamethasone before treatment with SR9009. Dexamethasone was used in the concentration that does not induce anti-inflammatory effects. The inflammatory effect of SR9009 on cultured microglia corresponded well with our in vivo findings.

It has been reported that RORα can exert bi-directional regulation of IL-6 expression dependent on the activation state of astrocytes [41]. RORα may negatively regulate IL-6 expression through the NF-κB pathway in reactive astrocytes. On the other hand, RORα may trans-activate IL-6 expression by interacting with a RORE in the promoter of non-reactive astrocytes. For these two pathways, RORα competes with REV-ERBα that binds the same response elements with a repressor activity. These observations prompted further examination of an anti-inflammatory effect of SR9009 on the strongly activated microglia, because Griffin et al. used a high concentration of LPS (i.e., 50 ng/mL) to activate microglia [32]. As shown in (Additional file 2: Figure S1), SR9009 exerted an anti-inflammatory effect on non-synchronizing and strongly activated MG6 microglia treated with LPS (50 ng/mL). Therefore, it is considered that SR9009 activates NF-kB pathway in synchronizing and mildly activated microglia through activation of REV-ERBα, while SR9009 may repress NF-κB in non-synchronizing and strongly activated microglia by interacting with RORE through activation of REV-ERBα. However, the exact action mechanism of SR9009 on microglia is to be elucidated in future studies.

Several recent reports suggest that circadian dysfunction has a causal role in AD-related neurodegeneration [42‐44]. However, neuronal circadian dysfunction alone cannot fully explain the mechanistic relationship between circadian dysfunction and neurodegeneration, because neuroinflammation may play a crucial role in AD-related neurodegeneration [3, 4]. In the present study, we have found that microglia isolated from APP-KI mice exhibited circadian alterations that may be associated with excessive neuroinflammation. Therefore, it may be concluded that circadian disturbances of BMAL1/RORα/NF-κB crosslinking in microglia may contribute to the early stage of AD pathologies through induction of excessive neuroinflammation.

In summary, we found that microglial clock gene expression was disturbed in the early phase of APP-KI mice. The reduction expression of BMAL1 in the microglia from APP-KI mice at ZT14 or after SR9009 exposure was shown to be responsible for the increased presence of microglia with an inflammatory phenotype through a reduction in RORα, which in turn reduced IκBα and enhanced NF-κB activation. Therefore, microglial clock gene disturbance and resultant chronic neuroinflammation may contribute to the onset of AD.