2.2.1 Clock-BMAL1

The suprachiasmatic nucleus (SCN) within the hypothalamus serves as the primary coordinator of most circadian rhythms. Light stimuli prompt the SCN to synchronize with the ambient light–dark cycle. Additionally, the expression of circadian clock genes has been detected in nearly all cells outside the SCN, and these rhythmic expressions persist even in vitro. Circadian rhythms refer to the rhythmic changes in a mammal's 24-h cycle, including sleep patterns, dietary habits, hormone secretion, and body temperature fluctuations [11-13]. Disruptions in circadian rhythm cause significant risks for aberrant sleep–wake cycles, diabetes, obesity, cancer, and other diseases [14]. Biological clocks are regulated by core clock genes, such as Bmal1, circadian locomotor output cycles kaput (CLOCK), cyclic circadian rhythm proteins (Pers; Per1, Per2 and Per3), and cryptochrome (Crys; Cry1 and Cry2). These fundamental clock genes are widely conserved across plants, animals, and microorganisms [15]. The Clock protein interacts with Bmal1 to form a heterodimer known as the Clock-Bmal1 complex. This complex enhances the expression of Per 1/2/3 and Cry 1/2 in the cytoplasm. Subsequently, this complex translocates into the nucleus where it functions as a transcriptional inhibitor to suppress Clock-Bmal1-mediated gene transcription, thereby establishing a negative feedback loop (Figure 1). Additionally, the Clock-Bmal1 heterodimer positively regulates nuclear hormone receptors (Rev-erbs; Rev-erα/β), which act as transcriptional repressors. These receptors enter the nucleus and competitively bind to the Bmal1 promoter element to inhibit Bmal1 expression, thus forming another negative feedback loop.

2.2.2 Circadian Rhythms Affect Lipid Metabolism

It has been reported that disruption of circadian regulation leads to increased levels of various phospholipids in plasma, consequently disturbing membrane lipid homeostasis [16, 17]. Rev-erbα controls the rhythmic expression of genes involved in fatty acid biosynthesis and phosphatidyl biosynthesis, such as choline kinaseα(Chkα) (Figure 1) [18]. Circadian misalignment is commonly observed in individuals with obesity who tend to have nocturnal overeating [19]. Disruptions in circadian eating behavior are induced by mutations in the Bmal1 and Clock genes, ultimately leading to overall hyperactivity in diet-induced obese mice [20, 21]. Reduced Clock expression diminishes leptin sensitivity in obese mice; alterations are observed in Clock-regulated gene expression such as glucose transporter 2 (GLUT2) and glucose isomer genes, contributing to fasting hypoglycemia (Figure 1) [22]. Knockout of the Bmal1 gene leads to a reduction in lipid gene expression, such as stearoyl-CoA desaturase, and decreases synthesis of long-chain polyunsaturated fatty acids (PUFA) in adipose tissue [23]. PUFA display a circadian rhythm, with peak levels observed 9 h after exposure to light; diurnal oscillations are evident in nuclear lipids (34%) and mitochondrial lipids (31%), primarily composed of phospholipids [24].

2.2.3 Circadian Rhythms Affect Intestinal Mucosal Barrier and Cognitive Function

AD is characterized by tau aggregation and is associated with disrupted circadian rhythms and suppression of clock gene expression. It has been reported that the circadian clock protein Rev-erbα is a key regulator between the biological clock, glial activation, and neuroinflammation, and loss of rev-erbα triggers activation of microglia, increases nuclear factor κB(NF-κB) signaling in microglia, leading to neuronal damage [25]. Lee found in male mice that Rev-erbα is involved in lipid metabolism and macrophage function, and that Rev-erbα deletion causes abnormal lipid metabolism in microglia, induces lipid droplet accumulation, and leads to lipid droplet accumulation specifically in male microglia, which damages tau phagocytosis in microglia and exacerbates neuroinflammation [26].

Furthermore, in intestinal immune cells, the biological clock collaborates with the microbiome to regulate the circadian rhythm of dietary lipid absorption, which reaches its maximum during nighttime [27]. The transcription suppressor nuclear factor interleukin 3 (NFiL3), a leucine zipper transcription factor, is expressed in various immune cells and regulates lipid absorption and export in small intestinal epithelial cells. The expression of Nfil3 is regulated by the Clock transcription suppressor REV-ERBα, resulting in a rhythmic diurnal pattern of Nfil3 expression [28].

The intestinal barrier plays a critical role in maintaining intestinal homeostasis. Changes in tight junctions or reduced mucin protection can impact the permeability of the intestinal barrier, which is influenced by circadian rhythm. CLOCK-BMAL1 binding to the E-box on the promoter region positively regulates nuclear hormone receptors Rev-erbα, which induce occludin and claudin 1 expression in mouse and human intestinal epithelial cells [10, 29]. The expressions of claudin 1 and occludin were regulated by the circadian clock, with both mRNA and protein levels reaching their lowest point at night and peaking during the day [29]. Tan et al. investigated the mechanism of acrylamide-induced neurotoxicity related to the circadian clock in mice brains. Their study revealed that an acrylamide-containing diet significantly decreased occludin protein levels and exacerbated cognitive dysfunction and spatial memory impairment during the night phase. This effect suggested that circadian rhythm disruptor compromised intestinal barrier integrity and increased circulating levels of LPS, Interleukin1β(IL-1β), and tumor necrosis factorα(TNF-α) [30]. In another study, disruption of the circadian rhythm was shown to modulate the composition of gut microbiota, impair intestinal barrier integrity, and induce both peripheral and central inflammation as well as cognitive impairment in mice. These alterations were accompanied by AD-like tau hyperphosphorylation, defects in neurogenesis, and a loss of synaptic proteins in the hippocampus in mice with disrupted circadian rhythms [31]. Additionally, Song et al. reported that oolong tea polyphenols alleviated intestinal barrier damage and reduced LPS transport to the serum by regulating gut microbiota composition, thereby mitigating circadian rhythm disruption-induced cognitive decline [32]. Therefore, maintaining a regular circadian rhythm enhances intestinal barrier integrity and effectively suppresses pathological changes and dysfunctions.

2.3.1 Circadian Rhythms Mediate the Effects of ERS on Cognitive Function

In certain physiological or pathological conditions, the demand for protein biosynthesis can increase dramatically, surpassing the folding capacity of the endoplasmic reticulum (ER), leading to the accumulation of partially folded, misfolded, or unfolded proteins, a condition known as ERS. The buildup of these aberrant proteins within the ER lumen triggers the activation of the unfolded protein response (UPR) signaling pathway. Mild ER stress elicits an “adaptive or protective UPR” that prevents excessive accumulation of misfolded/unfolded proteins in the ER. However, excessive or prolonged ERS can lead to sustained activation of the UPR, referred to as “maladaptive or terminal UPR”, which exacerbates neuroinflammation and triggers cell death [33]. In recent years, a large amount of evidence has shown that ERS is important in the development of AD [34], and that protein degradation regulated by UPR affects the excessive phosphoric acid of neurofibrillary tangles. Circadian rhythm disruptions can occur at an early stage in specific neurodegenerative conditions, the misalignment of circadian rhythms cause ERS [35]. Melatonin is known for its role in regulating circadian rhythms, sleep, and aging. The deficiency of melatonin in cerebrospinal fluid promotes the progression of cognitive impairment, and melatonin supplementation can effectively slow down the process of degeneration of hippocampal neurons and improve cognitive ability. Melatonin protects neurons by alleviating Ca²⁺ overload and ERS, regulating intracellular Ca²⁺ levels and cell homeostasis [36]. Kainic Acid induced ERS, affecting CDK5/GSK-3β and tau phosphorylation. Melatonin plays a role in Ka-induced neuronal degeneration and tau hyperphosphorylation by relieving ERS, further emphasizing the protective role of melatonin in Ka-induced neurons. Furthermore, in rats exposed to long-term light, circadian rhythms were disrupted, ERS-related proteins were increased, and melatonin supplementation prevented molecular damage [37]. Therefore, the interaction between circadian rhythm and ERS is a target for melatonin in therapy in neurodegenerative diseases.

2.3.2 Circadian Rhythms Mediate Effects of Oxidative Stress on Cognitive Function

Meanwhile, disruption of the circadian rhythm is associated with increased neuronal oxidative stress, resulting in lipid peroxidation as well as protein and nucleic acid oxidation, which are implicated in the early pathogenesis of AD [34, 38]. Bmal1 coordinates the transcription of genes related to REDOX processes in the brain, including quinone oxidoreductase antibody and acetaldehyde dehydrogenase. Moreover, reduced Bmal1 expression exacerbates neuronal cell death induced by oxidative stress both in vivo and in vitro [39]. Neuroinflammation spreads through the activation of astrocytes and microglia, contributing to neurodegeneration [39]. The inflammatory response exhibited by microglia indicates that inflammation in the hippocampus or cortex can directly hinder Bmal1 expression in peripheral neurons and glial cells, thus impairing the expression of Bmal1-mediated oxidative stress genes and leading to neurodegeneration in these cells. Rev-erbα-Bmal1 regulates pro-inflammatory cytokine production in macrophages [40]. Additionally, inflammation disrupts the circadian clock as LPS inhibits Bmal1 and REV-ERBα levels in macrophages.

3.1.1 Phospholipids

The ER is vital in lipid biosynthesis, particularly crucial membrane lipids such as phosphatidylcholine (PC) and phosphatidylethanolamine (PE). Maintaining lipid homeostasis within the ER is closely associated with the regulation of the unfolded protein response (UPR) [49, 50]. Phospholipids predominantly constitute the membranes of mitochondria and the ER, where PC and PE regulate membrane integrity. Perturbations in PC and PE levels ultimately induce stress on the lipid bilayer, leading to ER stress (Figure 2) [51]. Low ratios of PC/PE have been linked to non-alcoholic fatty liver disease, type 2 diabetes, cardiac disorders, and muscular dystrophy [52, 53]. Mammalian cells possess two nascent biosynthetic pathways for PC production. The cytidine diphosphate-choline pathway, discovered in the 1950s, is primarily responsible for tissue-specific synthesis of PC; meanwhile, PEN-methyltransferase (PEMT) catalyzes the continuous methylation of the amino head group of PE to synthesize PC [54, 55]. Exogenous intake of PC has been shown to inhibit proinflammatory transcription in TNF-α treated Caco2 cells. In addition, the low concentration of the major membrane lipid PC in gastrointestinal mucous membranes observed in patients with ulcerative colitis suggests that defects in colon PC metabolism may be involved in its pathogenesis and progression [56]. Aberrant lipid metabolism is implicated in the pathophysiology of AD. Whiley et al. investigated plasma lipids in AD using a multiplatform screening approach and identified three PC molecules—PC 16:0/20:5, PC 16:0/22:6, and PC 18:0/22:6—that were significantly diminished in AD cases [57]. Phosphatidylserine (PS), which constitutes 13%–15% of phospholipids in the human cerebral cortex, is a major acidic phospholipid class. As an essential component of protein docking sites in signaling pathways, PS interacts with Akt and protein kinase C to facilitate the growth and differentiation of nerve cells [58]. Studies involving PS supplementation in rodents have demonstrated improvements in memory, learning capacity, and other cognitive parameters [59].

3.1.2 Fatty Acids

Differences in lipid composition and fatty acid structure can impact ER homeostasis, potentially disrupting the Toll-like receptor 4 (TLR4) pathway when saturated fatty acids are replaced by Ω-3 PUFA. This substitution may lead to a reduction in pro-inflammatory markers in the aorta, including Interleukin-1β (IL-1β), TNF-α, phosphorylated nuclear factor kappa B (pIκBα), phosphorylated κB inhibits protein kinase β (pIKKβ), as well as unfolded protein response markers ATF6 and GRP78 [60]. As essential components of brain nerve cell lipids and potent anti-inflammatory bioactivators, Docosahexaenoic acid (DHA) and Eicosapentaenoic acid (EPA) are crucial for maintaining cognitive function and protecting against biological damage. PC composed of various fatty acids can mitigate cognitive decline and provide neuroprotective effects, with EPA and DHA contributing significantly to these benefits [61]. DHA influences neuronal development and neuroprotection in the formation of PC, PE, and PS [62]. Numerous studies have demonstrated that Ω-3 PUFA, including DHA, possess anti-inflammatory and immunomodulatory properties and show promise in the prevention or treatment of chronic inflammatory conditions such as inflammatory bowel disease, rheumatoid arthritis, asthma, and neurodegenerative diseases [63, 64]. Tan et al. reported that both exogenous and endogenous DHA can protect neurons from Aβoligomer induced damage in the pathogenesis of AD by reducing ERS and preventing cell apoptosis [65]. Therefore, it can be concluded that DHA exhibits neuroprotective effects by promoting cerebral cell growth and enhancing memory function.

3.1.3 Cholesterol

Cholesterol, the most abundant sterol, regulates membrane fluidity and permeability, as well as neuronal plasticity and synaptic remodeling [66]. Among all tissues in the body, the central nervous system contains the highest concentration of cholesterol, with its synthesis and circulation being crucial for maintaining normal neurophysiology. Conversely, aberrant cholesterol metabolism in the brain has been implicated in various neurodegenerative disorders [67]. Cholesterol, predominantly present in neuronal membranes and lipid rafts, is highly susceptible to oxidation [68]. The resultant oxidized derivatives of cholesterol significantly influence cellular function, with their synthesis primarily occurring within the ER. Disturbances in cholesterol homeostasis disrupt proper ER functioning and trigger abnormal activation of the UPR (Figure 2) [49]. Lipid rafts, characterized by their high content of cholesterol, are particularly susceptible to oxidative stress and modulate ER protein activity as well as protein folding or transport through localized alterations in membrane structure.

It is widely acknowledged that ERS disrupt the regulation of lipid metabolism and lipogenesis genes, while there is a bidirectional relationship between lipid metabolism and ERS [69]. However, the precise underlying mechanism remains elusive. Mechanistically, the accumulation of free cholesterol in the lumen of the endoplasmic reticulum induces ERS [70]. Excessive retention of free cholesterol in macrophages leads to cholesterol accumulation, thereby triggering the UPR, which occurs at various stages of atherosclerotic lesions [71]. Furthermore, animal studies have demonstrated that decreasing cholesterol levels is associated with a reduction in amyloid deposition in the central nervous system, while increasing dietary cholesterol intake promotes the accumulation of amyloid. In a sample of patients with AD, elevated cholesterol levels were found to be correlated with lower Mini-mental state examination scores compared to normal individuals [72].

As cholesterol-lowering medications, statins have been proposed as potential therapeutic agents for neurodegenerative disorders. Salari evaluated the neuroprotective effects of simvastatin against trimethyltin (TMT) induced memory decline and hippocampal neurodegeneration. In TMT-treated animals, simvastatin administration resulted in reduced learning and memory deficits in behavioral tasks, decreased acetylcholinesterase activity, and diminished Alzheimer's pathology markers such as presenilin-1 and hyperphosphorylated Tau. Additionally, simvastatin treatment reduced astrocytic and microglial reactivity in the TMT group [73]. The misfolding of Aβ is a key feature of AD. Gao demonstrated that Aβ ion channels facilitated Ca²⁺ influx in the presence of 15% cholesterol but inhibited this influx at higher cholesterol levels. Consequently, excessive cholesterol may disrupt the regular distribution of the lipid bilayer, preventing ion flux through the membrane [74].

3.2.1 The Lipid Chain Structure and the Thickness Affect Membrane Fluidity

The lipid chain structure and the thickness of the lipid bilayer are important in determining membrane fluidity (Figure 3) [93]. Molecular dynamics simulations revealed that the oligomerization of SERCA is influenced by the thickness of the lipid bilayer. Cholesterol or 14:0–18:0 PC in ER enhances the lipid order within the membrane. SERCA2b, as a conformationally active protein, loses its activity due to reduced conformational freedom in the cholesterol-ordered membrane [94]. Membrane thickness, head group composition, and lipid saturation directly impact the maximum flow rate and Ca²⁺ affinity of the SERCA pump. The proper functioning of SERCA relies on the fluidity of liquid crystalline bilayers, with higher activity observed in 18:1 PC compared to 18:0 PC [95]. The maximum activity (Vmax) of SERCA depends on this bilayer thickness and can adapt through subtle conformational changes. The presence of anionic lipids PS and phosphatidylinositol phosphoric acid in lower concentrations in the endoplasmic membrane increased Vmax of SERCA [96]. Analysis of the SERCA-Phospholamban (PLB) system reveals that phosphorylation of PLB, an endogenous regulator, induces lipid disordering, attenuates SERCA inhibition, and modulates the charge of lipid head groups to control the balance between order and disorder in SR membrane [97].

3.2.2 Nannular or Nonannular Lipid Interacts With SERCA2b Protein, and Affects the Activity of SERCA2b

Proteins are adapted to a high membrane fluidity environment of lipid bilayer with the presence of an annular lipid shell, consisting of lipid molecules bound tightly to the surface of integral membrane proteins. Marsh utilized the electron spin resonance technique to determine the quantity of lipid molecules binding to the SERCA2b protein, and their number was found to be consistent with the perimeter of the protein's transmembrane region. This observation provides evidence for the existence of a distinct lipid molecular shell resembling a ring surrounding the SERCA2b protein [98].

The regulation of ion channel activity by specific lipid molecules, such as the modulation of K⁺ channels by PIP2, is widely recognized as an essential component of cellular electrical signaling [99]. Arginine residues utilize phospholipids as anchoring points for conformational changes, thereby revealing the presence of a non-cyclic lipid binding site located within the luminal regions of transmembrane helices M2 and M4 in ER/mitochondrial membrane-cytoplasmic lobules [78]. The research provides evidence that the SERCA2b protein is surrounded by both annular and non-annular lipids, which interact with the SERCA2b protein (Figure 3). Additionally, it demonstrates that the unsaturation and acyl chain length of lipids have an impact on the activity of SERCA2b.

3.3.1 ERS Mediates Intestinal Inflammation

Excessive ERS is a characteristic feature of enteritis pathogenesis, which can lead to the apoptosis of intestinal epithelial cells and compromise epithelial integrity. Previous studies have demonstrated the effectiveness of probiotics in ameliorating intestinal inflammation. Notably, prevotella histicola, an emerging probiotic strain, exhibits significant anti-inflammatory activity by inhibiting the IRE1α-JNK and NF-κB signaling pathways to alleviate chronic colitis induced by sodium glucan sulfate [104]. Furthermore, mannose facilitates the normalization of protein N–glycosylation and enhances the binding of glyceraldehyde phosphate dehydrogenase to TNF-α mRNA, while directly reducing glyceraldehyde 3-phosphate levels to inhibit TNF-α production in macrophages and reduce such stress [105]. PC, primarily synthesized through the cytidine diphosphate-choline pathway in mammalian tissues, prevents ERS across diverse cell types. Studies utilizing mouse models with impaired intestinal PC synthesis have demonstrated that reduced colon PC concentration leads to adult-onset colitis [106, 107].

3.3.2 ERS Mediates Brain Inflammation

Neuronal ERS has been demonstrated to exhibit a positive correlation with microglial activation, thereby promoting the inflammatory microenvironment in neurodegenerative diseases and leading to neurotoxicity (Figure 4). Recombinant NLR Family, Pyrin Domain Containing Protein 3 (NLRP3), an inflammasome present in the central nervous system, can be activated by Aβ protein. The initial initiation signal induced by the toll-like receptor/nuclear factor NF-κB pathway enhances the expression of IL-1β and NLRP3, thus promoting inflammasome formation [108, 109]. Aβ oligomer is considered a ligand for TNF-α receptor and TLR4 [110].

Numerous studies have demonstrated that Aβ oligomers can increase apoptosis by activating the TNF-α pathway and inducing ERS [111]. Chronic dysfunction of the ER is strongly associated with impairment in memory and cognition, while downregulation of PERK expression prevents aberrant phosphorylation of eIF2α (Figure 4) [112]. Perturbations in cellular Ca²⁺ homeostasis are a prominent characteristic of AD, characterized by elevated levels of Ca²⁺ observed in neurons and astrocytes within mice. Particularly, basal Ca²⁺ levels surrounding astrocyte Aβ plaques are significantly heightened [113]. Under conditions of ERS, disruptions of brain tissue's Ca²⁺ homeostasis promote abnormal accumulation of Aβ protein. Additionally, IRE1-induced NLRP3 facilitates the aberrant aggregation of Aβ protein and the formation of neurofibrils composed of hyperphosphorylated Tau protein [114].