Circadian Disruption Through Light–Dark Cycle Alteration Induced Alzheimer’s Disease-like Pathology in Mice

TL;DR

Disrupting the light-dark cycle in mice caused Alzheimer's-like brain changes, suggesting that circadian rhythm disruption may contribute to the disease.

Contribution

Demonstrates that circadian disruption induces Alzheimer’s-like pathology in mice through specific molecular mechanisms.

Findings

CD increased amyloid-beta and hyperphosphorylated tau in the hippocampus.

CD disrupted circadian clock genes and neurotransmitter rhythms in the hippocampus.

CD caused neuroinflammation and reduced blood-brain barrier proteins in mice.

Abstract

Circadian disruption (CD) has emerged as a critical factor compromising human health in contemporary society. Increasing evidence suggests that disturbances in circadian rhythms are involved in the pathogenesis of neurodegenerative diseases, such as Alzheimer’s disease (AD). The hyperphosphorylation of tau and the deposition of amyloid-β (Aβ) are recognized as major pathological hallmarks of AD. In this study, we aimed to explore the impact of long-term CD on AD-like pathological changes and to explore the underlying molecular mechanisms using a mouse model. To mimic the CD experienced by shift workers, mice were subjected to lighting conditions involving repeated reversals of the light–dark cycle. In this study, qPCR was to employed detect the expression profile of clock genes in the hippocampus. Subsequently, Western blotting and immunohistochemical analyses were used to evaluate AD-like pathological changes in the hippocampus following CD. For elucidating the underlying mechanisms, we assessed circadian expression patterns of major neurotransmitters, activation of microglia and astrocytes, and alterations of tight junction proteins within the hippocampus. Our findings demonstrated that light–dark cycle disruption triggered CD in mice, and then CD led to increased expression of Aβ protein and tau hyperphosphorylation. CD significantly disrupted the circadian expression profiles of hippocampal clock genes and major neurotransmitters, induced microglial and astrocytic activation, and decreased the expression of the tight junction proteins zonula occludens-1 and occludin in the hippocampus. These results suggest that changes in the light–dark cycles induced abnormal expression of hippocampal clock genes involved in circadian rhythm regulation, suggesting that the body is in a state of endogenous CD. CD induces AD-like pathological changes in mice, potentially mediated by dysregulated circadian oscillations of clock genes, neuroinflammation, loss of key blood–brain barrier proteins, and disturbed neurotransmitter expression in the hippocampus. Collectively, this study underscores the importance of circadian stability for brain health, and highlights the necessity for deeper exploration into the connection between AD and CD.

Figures

Full text

1. Introduction

Alzheimer’s disease (AD) is a permanent neurodegenerative disease marked clinically by cognitive impairments, such as memory impairment and aphasia. In 2019, around 50 million people worldwide were impacted by AD and other forms of dementia, with China accounting for 13 million cases, or approximately 25.5% of the global total. As the world faces an increasingly aging population, the number of affected individuals is projected to rise to 150 million by 2050 [1,2]. The pathogenesis of AD is widely believed to stem from the interaction between internal factors, such as genetic predisposition and aging, and external environmental factors, including physical and chemical hazards and lifestyle changes. The hallmark pathological features of AD include deposition of amyloid-β (Aβ) plaque, neurofibrillary tangles (NFTs) constituted by hyperphosphorylated tau protein, and neuronal loss in the brain. Pathophysiological processes underlying AD involve synaptic damage, mitochondrial dysfunction, neuroinflammation, hormonal alterations, and neurotransmitter imbalances [3,4]. Recent studies have shown that circadian and sleep cycle dysregulation play important roles in promoting Aβ deposition and tau hyperphosphorylation [5]. Therefore, environmental health hazards, such as circadian disruption (CD) and sleep dysregulation, could significantly elevate the risk of developing AD.

Circadian rhythms have evolved over time to enable living organisms to adapt to environmental cues, including light, temperature, and feeding patterns. These approximately 24 h cycles govern numerous behavioral and physiological processes in most living organisms [6,7]. Research in mammalian models has demonstrated that circadian rhythms are essential for memory formation and consolidation [8]. On a molecular scale, circadian rhythms are governed by transcription and translation feedback loops involving “core” clock genes. These include basic helix–loop–helix ARNT-like protein 1 (Bmal1) and circadian locomotor output cycles kaput (Clock), which encode activators, as well as period circadian regulator (Per)1, Per2, cryptochrome (Cry)1, and Cry2, which code for transcriptional repressors. This core clockwork operates not only in the central pacemaker of the mammalian hypothalamus—the suprachiasmatic nucleus—but also in almost all cell types, including astrocytes and neurons [9,10]. In physiological states, endogenous circadian rhythms are synchronized with external environmental cues, primarily light, to maintain alignment with the day–night cycle. However, exposure to atypical work schedules, such as shift work, night work, or frequent travel across time zones, can desynchronize endogenous rhythms from the external environment, leading to adverse effects on physiological and behavioral functions [11,12].

Many studies have identified CD and sleep deprivation as common features of AD. As single-cell oscillators within an organism, neurons of the central nervous system (CNS) coordinate with specific brain regions to regulate activities such as body temperature, memory formation and maintenance, and melatonin secretion [13]. Increasing evidence suggests that synaptic plasticity, particular neuronal firing frequency, may be regulated by the circadian clock. Disruption of circadian oscillations during cellular memory consolidation has been shown to impair the persistence of hippocampus-dependent memory [14]. Chronic CD, acting as a long-term stressor, may impair hippocampal memory through pathological mechanisms, including neuroinflammation, neuronal loss, neurotransmitter dysregulation, and synaptic degradation, potentially contributing to the neuropathological progression of AD [15,16]. Neuroinflammation, broadly defined as inflammation of the CNS involving activation of astrocytes and microglia, is increasingly recognized as a contributor to cognitive decline. Furthermore, as neurotransmitters are critical mediators of neuronal communication, impairments in their uptake and storage can lead to cognitive dysfunction [17]. Although CD and sleep disturbances are often regarded as late manifestations of AD, growing evidence implies that CD may precede the onset of clinical symptoms [18]. Previous studies revealed an association rather than a direct causal relationship regarding CD and AD, and further research is needed to elucidate this link.

This study intended to explore the causal relationship regarding CD and AD-like pathological changes. Following 90 consecutive days of circadian reversal, the expression levels of Aβ and tau proteins within the mouse hippocampus were measured to assess AD-like pathological alterations. To further examine the pathological events associated with neuroinflammation, we evaluated the activation states of astrocytes and microglia. Additionally, we explored the mechanisms underlying the development of AD pathological changes by assessing the effects of CD on tight junction proteins and the circadian oscillations of clock genes as well as major neurotransmitters.

2. Materials and Methods

2.1. Animals and Light–Dark Conditions

Seventy-two 5-week-old male C57BL/6J mice (19–21 g) were obtained from GemPharmatech Co. Ltd. (Nanjing, China). The mice were fed in the specific pathogen-free animal facility at the Institute of Environmental Medicine and Occupational Medicine, Academy of Military Medicine, Tianjin Academy of Military Sciences. The room conditions were maintained at a temperature of 20–23 °C and 40–60% humidity, with food and water available ad libitum. The experimental setup employed standard lighting conditions of 400–450 lux, supplemented by white LED lamps with a color temperature of 6000 K. After an interval of acclimatization, the mice were randomly assigned to either the control group or the circadian disruption (CD) group. Zeitgeber time zero (ZT0) was designated as the onset of the light phase, aligning with 8:00 h, while ZT12 was defined as the commencement of the dark phase, aligning with 20:00 h. Mice in the control group were maintained under a standard light–dark cycle (light: 8:00–20:00; dark: 20:00–8:00) throughout the experiment. In the CD group, mice were subjected to a reversed light–dark schedule: for the first 3 days, the light cycle was inverted (dark: 8:00–20:00; light: 20:00–8:00), followed by reversion to the normal schedule (light: 8:00–20:00; dark: 20:00–8:00) for the subsequent 3 days. This 6-day cycle was repeated 15 times for a total duration of 90 days. Upon completion of the modeling, six mice in each group were sacrificed by cervical dislocation, and the hippocampi were collected every four hours within one day, then stored at −80 °C for further analysis (Figure 1) [19]. The research was approved by the Animal Ethics Committee of the Tianjin Institute of Environmental Medicine and Occupational Medicine (IACUC of AMMS-04-2021-010).

2.2. Western Blotting

The hippocampal tissue (collected at ZT0) was lysed in radioimmunoprecipitation assay lysis buffer (Solarbio, Beijing, China), and the total protein concentration was determined using a bicinchoninic acid quantification kit (Solarbio, Beijing, China). Equal amounts of protein were separated by polyacrylamide gel electrophoresis and then transferred to polyvinylidene difluoride membranes. Primary antibodies (Santa Cruz Biotechnology, Shanghai, China) were used: mouse anti-β-amyloid (B-4) (1:500), mouse anti-TauC (1:500), rabbit anti-phospho-tau (Ser396) (PS396; 1:500), rabbit anti-PS404 (1:500), rabbit anti-glial fibrillary acidic protein (GFAP; 1:500), rabbit anti-ionized calcium-binding adaptor molecule 1 (IBA1; 1:1000), mouse anti-claudin 1 (1:1000), mouse anti-zonula occludens-1 (ZO-1; 1:500), and anti-glyceraldehyde 3-phosphate dehydrogenase (GAPDH; 1:10,000). ImmunoPure Goat Anti-Mouse IgG (H + L) or horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (H + L) (1:10,000) served as secondary antibodies. Protein band gray value was measured using Amersham Imager 680 software (GE Healthcare, Chicago, IL, USA). Relative protein expression was determined via normalizing the gray value of each target protein to that of GAPDH.

2.3. Quantitative Real-Time Polymerase Chain Reaction (qRT-PCR)

qRT-PCR was performed as previously described, and the detailed procedures can be found in the Supplementary data file [19]. The sequences of qRT-PCR primers are shown in Supplementary Table S1.

2.4. Immunohistochemistry Analyses

The hippocampal tissue (collected at ZT0) were paraffin-embedded and coronally sectioned at a thickness of 4 μm. Sections were deparaffinized and then rehydrated through a graded ethanol series. Antigenic retrieval was performed using bovine serum albumin (BSA) blocking solution. Endogenous peroxidase activity was blocked through incubating sections in 3% hydrogen peroxide for 25 min at 20–24 °C, followed by triple rinsing using phosphate-buffered saline (PBS). Sections were incubated overnight at 4 °C with monoclonal anti-Aβ (1:500) or anti-tau (1:1000) antibodies (Servicebio, Wuhan, China). After washing three times with PBS, sections were incubated with HRP-conjugated goat anti-rabbit secondary antibody (1:300; Servicebio, Wuhan, China) for 50 min at 20–24 °C. Following four PBS washes, 3,3′-diaminodbenzidine chromogenic substrate (Servicebio, Wuhan, China) was applied until a brownish-yellow coloration appeared under the microscope. The sections were dehydrated and then sealed with neutral gum after being counterstained with hematoxylin for 1 min. Stained sections were detected using a NIKON microscope (E100, Shanghai, China). For each group, five fields per section were randomly selected and imaged at 400 magnification. The ImageJ software (version 1.54p) was employed to analyze the absorbance of positive staining.

2.5. Immunofluorescence Analyses

The hippocampal tissue (collected at ZT0) were sectioned coronally with a thickness of 4 μm after being embedded in paraffin. Sections were dewaxed, rehydrated by a graded ethanol series, and blocked with 5% BSA to prevent nonspecific binding. Following triple rinsing with PBS, sections were incubated using monoclonal anti-IBA1 antibody (1:800, Servicebio, Wuhan, China) or monoclonal anti-GFAP antibody (1:500, Servicebio, Wuhan, China) overnight at 4 °C. After incubation, sections were rinsed thrice with PBS and then incubated with fluorescently labeled secondary antibodies (1:400, Servicebio, Wuhan, China) for 1 h at 20–24 °C. Following thrice additional PBS washes, 4′,6-diamidino-2-phenylindole (DAPI; Servicebio, Wuhan, China) was employed to counterstain the sections. Images were captured via a fluorescence microscope (NIKON ECLIPSE C1, Shanghai, China). For each group, four fields per section were randomly selected and captured at 200 magnification. The fluorescence intensity was analyzed and quantified by ImageJ software (version 1.54p).

2.6. Ultra-High Performance Liquid Chromatography–Mass Spectrometry Metabolomics (UHPLC-MS)

The expression levels of dopamine (DA), serotonin (5-HT), acetylcholine (Ach), glutamate (Glu), and γ-aminobutyric acid (GABA) were detected using UHPLC-MS. UHPLC-MS was carried out following a reported method, and the detailed procedures are also presented in the Supplementary Data file [20].

2.7. Statistical Analyses

All measurement data are showed as mean ± standard deviation (SD). Statistical analyses were carried out with SPSS software (version 25.0; IBM, Armonk, NY, USA). Two-group comparisons were performed using Student’s t-test. Differences in the expression levels of clock genes and neurotransmitters were evaluated through one- or two-way analysis of variance (ANOVA) followed by post hoc Least Significant Difference (LSD) tests. GraphPad Prism 8 software was used to generate figures and tables. A p value < 0.05 was considered statistically significant.

3. Results

3.1. CD Altered the Expression Profiles of Core Clock Genes in the Mouse Hippocampus

To explore CD effects on clock gene expression profiles in the mouse hippocampus, we examined 24 h expression levels of clock genes. Under normal light conditions, one-way ANOVA showed that time had a significant influence on the circadian profiles of Bmal1, Clock, Per1, Per2, Cry1, NR1D1 or nuclear receptor subfamily 1 group D member 1 (Rev-erbα), Rev-erbβ, D-site albumin promoter binding protein (Dbp), and Circadian associated repressor of transcription (Ciart) mRNA (p < 0.05). The R^2^ values of the fitted cosine curves for all clock genes studied exceeded the significance threshold (R^2^ > 0.25). Thus, based on both the p and R^2^ values, robust circadian rhythms of clock genes were observed in the hippocampus of control mice (Figure 2, Supplementary Table S1). Under reversed light conditions, one-way ANOVA revealed that the 24 h profiles of Bmal1, Per2, Cry1, Rev-erbα, Dbp, and Ciart were still significantly influenced by time (p < 0.05), whereas Clock, Per1 and Rev-erbβ showed no significant time-dependent variation (p > 0.05). The R^2^ values and amplitudes of the fitted cosine curves were generally low, indicating that the oscillations in mRNA levels were markedly weakened. Among these, the R^2^ values for Bmal1, Per2, Cry1, and Ciart exceeded the threshold (R^2^ > 0.25), whereas Rev-erbα and Dbp did not. Thus, based on the combination of p-values and R^2^ values, Bmal1, Per2, Cry1, and Ciart retained circadian rhythms, while the rhythmicity of Clock, Per1, Rev-erbα, Rev-erbβ, and Dbp was lost in the hippocampus of CD mice. In addition, compared with the LD group, the circadian acrophases of the clock genes bmal1, per2, cry1, and ciart in the CD group showed significant differences, with phase shifts of 6.1 h, 10.7 h, 11.5 h, and 9.0 h in sequence (Figure 2, Supplementary Table S1). These results suggest that CD exposure impairs the circadian oscillations of hippocampal clock genes. Alterations in the transcriptional profiles of these clock genes may disrupt brain homeostasis and contribute to the exacerbation of AD-like pathological changes.

3.2. CD Induced AD-like Pathological Changes

3.2.1. Increased Expression of Aβ Protein in the Hippocampus Caused by CD

To assess CD exposure effects on Aβ protein expression, we measured the relative levels of hippocampal amyloid precursor protein (APP) and Aβ using Western blotting (Supplementary Figure S1A) and immunohistochemistry (Supplementary Figure S2A). In the CD group, APP and Aβ protein levels were significantly higher than the control group (Figure 3A,C,D). To further evaluate the expression of Aβ in the hippocampus, we performed immunohistochemical analysis. In comparison with the control group, Aβ expression was notably higher in the hippocampus of CD-exposed mice (Figure 3B,E). Overall, the immunohistochemistry findings were consistent with the Western blotting results.

3.2.2. Tau Phosphorylation in Hippocampus Caused by CD

Similarly, to assess the impact of CD exposure on Tau phosphorylation, Western blotting (Supplementary Figure S1B) and immunohistochemistry (Supplementary Figure S2B) were used to assay total Tau5 protein (t-Tau) and phosphorylated Tau (p-Tau) at the Tau396 and Tau404 sites in the hippocampus. Both immunohistochemical and Western blotting assays showed that the expression level of hippocampal Tau5 the CD group was markedly increased in comparison to the control group (Figure 4A,B,D,F). Western blotting assay findings further revealed that the expression levels of Tau phosphorylated at the Tau396 and Tau404 sites were also significantly elevated in the CD group (Figure 4A,C,D). These results suggest that CD exposure induces AD-like pathological changes in wild-type mice.

3.3. CD-Induced Astrogliosis and Microglia Activation in the Mouse Hippocampus

Glial cell activation is a key pathological feature of neurodegenerative diseases, including AD [21]. To assess microglial and astrocyte activation, we measured expression levels of the astrocyte marker GFAP as well as the microglia activation marker IBA1. Western blotting results showed that GFAP and IBA1 protein levels were significantly increased in the CD mice compared with the control group (Figure 5A–C, Supplementary Figure S1C). Consistently, immunofluorescence staining revealed enhanced activation, and significantly increased expression of GFAP and IBA1 in astrocytes and microglia, respectively, compared with the control group (Figure 5D–G, Supplementary Figure S3). Overall, these results suggest that CD exposure induces astrocyte and microglia activation, which may exacerbate AD-like pathological changes.

3.4. CD Altered the Expression of Tight Junction Proteins in the Hippocampus

In order to investigate the impact of CD on the permeability of the hippocampal blood–brain barrier (BBB), we measured the expression of major tight junction—related genes via RT—PCR and analyzed the levels of corresponding proteins through Western blotting (Supplementary Figure S1D). In comparison to the control group, the mRNA expression levels of ZO-1, Occludin, Claudin-1, and Claudin-5 were significantly decreased in the hippocampus of CD mice (Figure 6A–D). Consistently, Western blotting results confirmed that CD exposure led to reduced expression of ZO-1 and Occludin proteins within the hippocampus (Figure 6E–G). These results suggest that CD disrupts tight junction integrity, thereby increasing BBB permeability and potentially aggravating AD-like pathological changes.

3.5. CD Induced Abnormal Expression of Hippocampal Neurotransmitters in Mice

The brain’s functional activity depends on the transduction of signals by neurotransmitters between different types of neurons and glial cells. Neurotransmitters are intricately involved in multiple cerebral functions, encompassing memory, learning, emotion, and movement [22]. We used targeted UHPLC-MS to measure major neurotransmitter concentrations in the hippocampus every 4 h throughout a 24 h period. The concentration curves of DA, 5-HT, Ach, Glu, and GABA over 24 h in hippocampal samples were depicted in Figure 7. In comparison to the control group, DA, 5-HT, and Ach levels were decreased, while Glu and GABA levels were increased in the CD group (Supplementary Figure S4). Differences in metabolite concentrations between the groups are further illustrated in the heat map (Supplementary Figure S5). We assessed the circadian rhythms of the five neurotransmitters over a 24 h period using cosine analysis. In the control group, DA, Ach, and Glu displayed circadian rhythmicity, while GABA and 5-HT did not (Figure 7, Supplementary Table S3). In the CD group, the expression of GABA and 5-HT also did not exhibit circadian rhythmicity. In contrast, the CD group exhibited loss of circadian rhythmicity in three neurotransmitters (DA, Ach and Glu) (Figure 7, Supplementary Table S3). Thus, CD disrupts circadian oscillations of major hippocampal neurotransmitters, which may impair hippocampal neural conduction, learning, and memory.

4. Discussion

With the rapid development of the global economy, CD has become a common phenomenon in modern society. In this study, we simulated CD, a condition frequently encountered in human work or daily life, by reversing the light and dark environments around the mice. The results showed that changes in the light–dark cycles induced abnormal expression of hippocampal clock genes involved in circadian rhythm regulation, suggesting that the body is in a state of endogenous CD. In addition, CD led to increased expression of Aβ protein and Tau hyperphosphorylation in mouse hippocampus, disrupted the expression of neurotransmitters related to signal transduction, triggered neuroinflammatory changes associated with cognitive impairment, and caused a decreased expression of key tight junction proteins in the BBB, indicating that CD may raise the risk of AD. Together, these findings highlight a novel pathway through which CD contributes to the onset or progression of AD, accompanied by dysregulated circadian oscillations of clock genes, increased expression of Aβ protein, Tau hyperphosphorylation, neuroinflammation, loss of key blood–brain barrier proteins, and disturbed neurotransmitter expression.

Light is the most important zeitgeber factor influencing circadian rhythms. An irregular light–dark cycle causes desynchronization between the external environment and the internal biological clock, thereby disrupting the expression of clock genes, including their acrophases and amplitude. When mice are exposed to a repeatedly altered light–dark environment, they continuously adjust the expression patterns of internal clock genes to adapt to changes in the external light–dark conditions. At the molecular level, our previous study has reported that an inverted light–dark cycle disrupts the rhythmic expression of two core circadian rhythm markers, hypothalamic clock genes and serum melatonin. Over time, the ability of the biological clock to drive circadian rhythm output is at least partially impaired, ultimately leaving the organism in a state of endogenous CD [19]. While many studies have emphasized that CD and sleep disturbances signify common clinical features among individuals with AD, these factors may also exacerbate cognitive impairment. Animal model studies have similarly suggested a potential pathological link between circadian rhythm disruption and AD [23]. Genetic disruption of clock function accelerates the accumulation of amyloid plaques and the occurrence of Tau hyperphosphorylation in mice [24]. However, the precise role of circadian clock disruption in mediating the pathological changes associated with AD following CD remains an open question in the research field. Clock genes are crucial for driving and maintaining circadian rhythms. Chronobiology research has demonstrated that endogenous circadian oscillators regulate learning and memory through neurotransmitter signaling and synaptic remodeling, ultimately affecting cognitive function [25]. Environmental factors such as dietary restrictions, jet lag, shift work, and irregular living habits can all lead to CD in the body [26]. In this study, we found that CD resulted in increased expression of Aβ protein and Tau hyperphosphorylation in the hippocampus, accompanied by altered clock gene expression levels, suggesting that circadian clock dysfunction may be one of the factors causing AD-like pathological changes. However, there is no data to address if there is Abeta aggregation caused by CD, only that Aβ staining increases (no plaques). Studies have found that the expression and distribution of Aβ and Tau protein in the brain have a circadian rhythm [27]. Loss of central circadian rhythm disrupts the daily oscillatory rhythm of Aβ protein in the hippocampal interstitial fluid, thereby accelerating the accumulation of amyloid plaques. Deletion or dysregulated expression of Bmal1 leads to impaired Aβ clearance function, significantly promoting the deposition of fibrillar Aβ plaques [28]. In addition, Per2 is negatively correlated with the phosphorylation level of tau protein, and decreased expression of Bmal1 causes downregulation of Per2, ultimately resulting in enhanced tau protein phosphorylation [29]. Therefore, CD may cause a sharp increase and aggregation of these proteins, accelerating the process of neurodegeneration in AD. In addition, it has been shown that Presenilin-2 normally cleaves APP, whose expression is activated by Clock and Bmal1 [30]. This indicates that CD and alterations in transcription/translation feedback loops may also impact amyloid clearance. Relevant studies have pointed out that there may be a connection between the abnormal expression of Bmal1 following CD and the abnormal phosphorylation of Tau protein, with which the results of this study are consistent [31]. Therefore, improving circadian rhythm disruptions could become a promising approach to impede the further exacerbation of AD-like pathological changes and may help slow the progression of neurodegenerative diseases.

Recent research has recognized neuroinflammation as the third significant pathological hallmark of AD, following Aβ deposition and NFTs, with the inflammatory response is pivotal in the onset and progression of the disease [32]. Neuroinflammation in the brain predominantly emanates from activated microglia and reactive astrocytes. In this study, we show that CD can induce the over-expression of markers of reactive microglia (Iba1) and astrocytes (GFAP). Clock genes can directly regulate neuroinflammation, and the disorder of clock genes will lead to the obvious activation of astrocytes. Importantly, CD caused by genetic mutations or environmental factors leads to neurodegeneration and cognitive impairment. For instance, genetic ablation of Bmal1 results in significant astrocyte activation and increased amyloid burden. Astrogliosis and microgliosis are responses to brain insults, including neurodegeneration, which may lead to neurotoxic consequences such as exacerbated neuroinflammation and impaired Aβ clearance [33]. Evidence suggests that neuroinflammation accelerates the progression of amyloid and tau lesions, which are further exacerbated by amyloid plaques and Tau tangles [34]. Clinical studies have also shown that microglia activation is positively correlated with Aβ and tau load in patients with AD. GFAP, a marker of astrocyte activation, is associated with amyloidosis, and its expression correlates with Aβ plaque density [35]. The BBB supplies nutrients to the CNS, maintains homeostasis, and regulates its communication with the periphery, thereby forming a protective barrier for the CNS. In addition, recent findings suggest that destruction of BBB integrity in AD occurs years before the onset of symptoms of cognitive impairment [36]. Early BBB impairment is independent of Aβ and tau pathologies, among which cerebral capillary damage and BBB disruption in the hippocampus are regarded as early biomarkers of cognitive dysfunction. The permeability of the BBB is dynamically regulated by circadian rhythms and sleep. BBB dysfunction induced by CD plays a crucial role in CNS neurodegenerative diseases [37]. Our findings indicated that CD may disrupt the BBB by reducing the expression of tight junction proteins (occludin and ZO-1). Disruption of BBB integrity triggers neurovascular uncoupling, reduces capillary density, decreases regional cerebral blood flow, and significantly promotes AD-related pathological depositions and neuronal loss [38]. The change in the permeability of the BBB in the hippocampus may lead to the entry of immune cells and pro-inflammatory factors from the periphery into the central nervous system, which can further accelerate the progression of cognitive dysfunction [39]. Under normal physiological conditions, an intact BBB facilitates the clearance of Aβ and prevents the transfer of peripheral Aβ into the brain, thus avoiding toxic accumulation. However, when BBB permeability is increased, Aβ clearance is impaired, leading to Aβ accumulation and NFTs [40]. Therefore, neuroinflammation and loss of BBB tight junction proteins play crucial roles in the pathophysiological mechanism of CD, contributing to AD-like pathological changes in mice.

Neurotransmitters are chemical substances released and recycled by neurons to transmit information, which are crucial for the function of complex nervous systems [41]. Brain function relies on the communication via signals between glial cells and different types of neurons, which is primarily mediated by neurotransmitters. These neurotransmitters can be excitatory, such as Glu, Ach, and DA, or inhibitory, such as GABA, glycine, and 5-HT. Neurotransmitters are actively involved in various brain functions, including movement, emotion, learning, and memory [42]. Studies have shown that the content of neurotransmitters and the expression of related genes and proteins show an approximately 24 h periodic fluctuation. Light is an important environmental factor in regulating the circadian rhythm of neurotransmitters. Light signals are transmitted to the SCN through the retinohypothalamic tract, and then the expression of clock genes is regulated, indirectly affecting the rhythm of neurotransmitters [43]. Numerous studies have demonstrated that dysregulation of neurotransmitters, including DA, 5-HT, Ach, Glu, and GABA, is related to cognitive impairment in AD [44]. The present study demonstrates that CD disrupts the circadian expression of major neurotransmitters. Ach and DA are key excitatory neurotransmitters involved in learning, memory, and other higher cognitive functions. Recent clinical data indicate that patients with AD exhibit severe deficiencies of both Ach and DA. Multiple neurotransmitter abnormalities exist in the brains of AD patients, with cholinergic system dysfunction being the most severe and most closely associated with the patients’ cognitive and behavioral impairments. DA is crucial for memory encoding and consolidation in hippocampal circuits, while abnormal DA expression reduces memory encoding and prevents memory enhancement [45]. Additionally, 5-HT is an important neurotransmitter widely distributed in the brain, which is involved in learning, memory, and cognitive processes. A decrease in 5-HT precursor 5-hydroxytryptophan (5-HTP) has shown a positive correlation with cognitive impairment in AD. In the brains of AD patients, the 5-HT content is significantly reduced, and the expression of related receptors is decreased, which accelerates cognitive impairment in AD patients [46,47]. These findings align with our observation that neurotransmitter levels are significantly reduced in the brains of mice exposed to CD. In addition, the imbalance of neurotransmitters such as Glu and GABA may promote the phosphorylation of Tau protein by activating related signaling pathways including the calcium signaling pathway and the glycogen synthase kinase-3β pathway, accelerate the formation of NFTs, and further damage the structure and function of neurons [48]. Therefore, CD may contribute to or exacerbate AD-like pathological changes by disrupting the normal regulation of neurotransmitter expression.

However, this study does have certain limitations. First, male mice were used to construct a CD model, and previous studies have confirmed significant sex differences in the regulatory mechanisms of the circadian system, which may limit the model’s generalizability to female subjects. Second, Aβ in conventional experimental mice rarely forms aggregates spontaneously. This means histological detection methods can only identify intracellular Aβ, presumed to be in monomeric form, and fail to capture AD-specific pathological features such as Aβ plaques and neurofibrillary tangles. To address this limitation, future studies could employ Western blotting analysis under non-denaturing conditions to detect high-molecular-weight Aβ aggregates in mice, thereby providing evidence for evaluating the pathological relevance of Aβ in this model. Third, only samples from a single time point (ZT0) were used to detect the expression levels of Aβ, APP, tau protein and their phosphorylated forms. The expression level at a single time point is susceptible to phase shift interference, making it difficult to fully capture the dynamic variation characteristics of amplitude, mesor and acrophase related to the rhythms of the aforementioned molecules. Additionally, although this study revealed that CD may induce AD-like pathological changes in mice through dysregulation of core clock genes’ circadian rhythms (such as Bmal1 and Per1), the specific roles of clock genes with region-specific expression in different brain areas (e.g., the suprachiasmatic nucleus vs. the hippocampus) in regulating AD pathological cascades remain unclear. This scientific question warrants systematic investigation in future research using tissue-specific gene knockout models and single-cell RNA sequencing.

5. Conclusions

In conclusion, repeated light–dark reversal induces abnormal expression of hippocampal clock genes involved in circadian rhythm regulation, suggesting that the body is in a state of endogenous CD. CD leads to increased expression of Aβ protein and Tau hyperphosphorylation in mouse hippocampus. Additionally, CD either induces or exacerbates AD-like pathological changes by activating microglia and astrocytes, reducing the expression levels of key tight junction proteins in the BBB, and causing dysregulation of neurotransmitter expression. With the increasing incidence of neurodegenerative diseases, deeper insights into AD pathogenesis and risk factors may facilitate the development of targeted interventions for vulnerable populations.