Sleep, circadian rhythm and gut microbiota: alterations in Alzheimer’s disease and their potential links in the pathogenesis

The pro-inflammatory taxa Escherichia and Shigella of Enterobacteriaceae have long been proposed to contribute to series of gastrointestinal diseases.¹⁰ Increased level of E. coli LPS has also been detected in the postmortem brain samples of AD patients.⁴⁰ The exotoxin of Escherichia and Shigella could disrupt the integrity of epithelial cell further leading to leaky gut and facilitates the translocation of bacteria into the blood.⁴¹E. coli along with several gram-negative bacteria possess systems for producing bacterial Aβ which is able to penetrate intestinal barrier and BBB and initiate cross-seeding in the CNS.^42,43 In addition to Escherichia, bacterial Aβ producing systems have also been found in Staphylococcus, highlighting its potential role in contributing to AD pathogenesis.⁴⁴ Although Staphylococcus was not detected in human fecal sample, its higher abundance was found in the blood of AD patients.⁹ Studies have reported that strains of Ruminococcus gnavus which belong to the family Lachnospiraceae use terminal mucin glycans to degrade mucus layer of intestinal barrier.⁴⁵ Increased level of Ruminococcus gnavus has been associated with inflammatory bowel disease, suggesting the potential role of Ruminococcus gnavus in promoting inflammation.⁴⁶

The two families Ruminococcaceae and Clostridiaceae, major SCFA-producing taxa in mammalian GM, have been reported to be decreased in various metabolic and neurodegenerative diseases.⁴⁷ The relative abundance of Ruminococcaceae was found to be positively correlated with higher Mini-mental State Examination (MMSE) and Montreal Cognitive Assessment (MoCA) scores, which indicates better cognitive functions.¹⁰ Lower level of anti-inflammatory taxa Eubacterium rectale and Bacteroides fragilis along with increased pro-inflammatory cytokines such as IL-1β, NLRP3 and CXCL2 have been also detected in AD patients.²⁸Lactobacillus and Bifidobacterium are two common probiotic taxa capable of producing neurotransmitter gamma-amino butyrate (GABA) whose metabolism has been reported to be disrupted in AD patients.⁴⁸Lactobacillus and Bifidobacterium play an important role in protecting intestinal cells and inducing anti-inflammatory responses.^49,50 Studies have shown that probiotic treatment using strains of Lactobacillus and Bifidobacterium was able to ameliorate symptoms associated with AD.^51,52

GM alterations in human and animal models caused by sleep disturbance or related to sleep quality are presented in Table 3 (top)^71–74 and Table 3 (bottom),^75–80 respectively. To date, only a few studies explored the effects of sleep impacting on GM in humans, restricting their focus on the association between specific bacterial taxa and sleep quality based on Pittsburgh sleep quality index (PSQI) or sleep physiology. Two studies compared the GM of individuals after short-term sleep deprivation with baseline data collected before deprivation.^71,81 But their findings are largely inconsistent, likely owing to distinct experimental designs and several uncontrolled variables, including daily dietary and energy intake of the subjects. Therefore, few commonalities in GM changes can be concluded from human studies. In contrast, multiple animal-based experimental studies that focus on the impacts of long-term sleep deprivation and fragmentation on GM composition have been conducted, with largely identical results of GM alterations.

In addition to sleep loss, circadian rhythm disruption is also receiving increasing attention, given the increased prevalence of altered sleep-wake cycle and jet lag, which are largely due to working night shift and traveling across time zones. Aberrant light exposure, high fat diet, alcohol consumption and irregular eating behavior have been found to induce circadian misalignment.⁸⁶ Numerous studies have indicated a link between circadian rhythm disruption with higher risk of pathological conditions including obesity, cardiovascular diseases and neurodegenerative diseases. The diurnal oscillation of human GM is partially controlled by central clock,⁶⁸ indicating the regulatory roles of circadian in GM eubiosis. Thus, we summarized recent studies focusing on the effects of circadian rhythm disruption on GM components in Table 4.^68,87–91

What is the role of GM dysbiosis in AD? It remains debatable whether GM dysbiosis plays as causal or merely consequential role in AD. Recently, studies have started to support the idea that GM dysbiosis precedes the onset of AD and even contributes to AD pathogenesis. Li et al. found that AD and MCI groups had distinct GM compositions from healthy controls in both fecal and blood samples, largely consistent with a previous report by another group.^9,10 These findings provide a new perspective that GM dysbiosis starting at early MCI is a developing process with the cumulation and depletion of specific bacterial taxa. Studies of GM intervention in AD including probiotic supplement, antibiotic treatment, germ-free animals and FMT further reinforced the causal role of GM dysbiosis in AD pathogenesis.

What causes GM dysbiosis before the onset of AD? Human GM is determined by multiple factors including early life exposure, medical intervention, diet, stress, sleep and circadian rhythm.²¹ Many studies have associated these factors with GM eubiosis, and their potential impacts on AD pathogenesis. A recent paper proposed a perspective that diet-induced GM dysbiosis plays a role in the pathogenesis of AD.⁴⁴ Multiple reviews summarized GM alterations in AD and SCRD, respectively, but no reviews to date have systematically analyzed the patterns of GM changes in AD and SCRD simultaneously, or made a hypothesis linking SCRD, GM dysbiosis and AD.

First, we check the uniformity in GM alterations and their potential contributions to health and disease under AD and SCRD conditions. We compared the GM alterations and their potential roles (beneficial bacteria, pathobionts or controversial taxa) in a taxonomic view under distinct conditions: AD, sleep and circadian disruption (Table 5). We observe higher abundance of highly immunogenic Erysiopelotrichaceae at family level in both human and rodents in each condition, but most other changes in individual bacteria were inconsistent between human and rodent (Table 5), which may be caused by the differences in GM components between these two species.¹⁰⁰ Thus, when analyzing the overlapping of GM alterations in different conditions, we conduct separate evaluations in humans and rodents. In humans, SCFAs-producing Ruminococcaceae at family or genus level is shown to be significantly lower in either condition, whereas highly immunogenic bacteria including Erysiopelotrichaceae and Coriobacteriaceae at family level are shown to be significantly higher in each condition. Most other GM components are inconsistent between different conditions, sometimes due to no relevant data available at present (Table 5). In animal models, similar trends are observed in several bacteria individuals between different conditions. For example, beneficial bacteria including Lactobacillaceae, Bifidobacteriaceae, Turicibacteraceae and Lachnospiraceae at family and/or genus level are significantly decreased in AD, sleep disturbance and/or circadian disruption, and other parts of pathobionts are uniformly increased, with the exception of Ruminococcaceae. As stated above, the increase in Ruminococcaceae during sleep disturbance was probably due to aberrant food intake.

Next, we elucidate the potential role of GM dysbiosis in the development of AD by providing the evidence of how GM interventions, including probiotics, antibiotics, germ-free treatment and FMT, restore cognitive functions and alleviate AD pathology (Table 2) (Figure 3). Although various factors modulate GM composition, emerging evidence has indicated that SCRD could disturb GM and lead to GM dysbiosis. Most human studies merely investigated the correlation between SCRD and GM dysbiosis, while animal studies provided more insights into GM alterations under different SCRD conditions such as sleep deprivation, sleep fragmentation and circadian rhythm reversal. Studies have also revealed several possible mechanisms underlying how SCRD contributes to GM dysbiosis, including increased food intake, decreased physical activity, activation of HPA axis and compromised gut barrier integrity, and this topic has been reviewed elsewhere.^21,101

Studies have implicated that GM can be altered at lower taxonomic level without achieving alteration at high taxonomic level.³⁹ For example, Firmicutes and Bacteroidetes are the two largest bacterial phyla of the mammalian gastrointestinal tract, and their ratio (F/B) was commonly used in GM analysis.¹⁰² However, reviews have reported inconsistent changes in F/B ratio across a series of neurodegenerative diseases and metabolic disorders, making F/B ratio a debatable and controversial criterion.^6,99,103,104 In agreement with our findings, one review summarizing the GM alterations in patients with PD found that, at high taxonomic ranks like phylum and class level, the changes in bacterial taxa are neither disease-specific nor consistent among different studies, but a more concordant trend was observed at family and genus level.³⁹

Additionally, α-diversity was thought to be a good indicator of health and diseases, and has been frequently investigated in GM analysis.¹⁰⁵ However, we found that neither AD studies nor SCRD studies showed concordant variation of GM α-diversity. And α-diversity analysis was not included in several studies. This is supported by another review which examines the association between GM and PD. They found that the confounding results of α-diversity alteration reported by different studies did not substantiate the role of α-diversity analysis as reliable methods for identifying PD and its progression, suggesting that higher α-diversity was not necessarily a predictor of better health.⁶

Most studies examining GM alterations in AD or SCRD only evaluated compositional changes of GM, and few conducted function-related analyses such as Kyoto Encyclopedia of Genes and Genomes (KEGG) test or metabolite screening. However, reviews have indicated that two taxonomically distinct bacterial taxa could share similar functions, while two closely related taxa may act antagonistically.^92,106 This suggests that phylogenetic analysis which is based on the hypervariable regions of bacterial 16s RNA gene cannot alone represent GM alterations at both taxonomic and functional level. It is possible that an increase of one genus could be neutralized or even reversed by a decrease of predominant genus in the same family. Thus, it would be confusing and misleading to simply conduct compositional analysis in discussing GM alterations. Moreover, metabolic and functional analysis have provided some important molecular and signaling pathways including possible interaction mechanisms between SCRD and GM and how GM dysbiosis could contribute to AD development.^10,28,30,33

Lachnospiraceae and Akkermansia muciniphila, two taxa frequently investigated by the abovementioned studies, still remain controversial in their functions. As a core component of mammalian GM, Lachnospiraceae acts as a double-edged sword in health and disease.⁹² On the one hand, several members of Lachnospiraceae like Blautia, Coprococcus and Roseburia are crucial producers of butyrate and acetate, which induce anti-inflammatory responses, modulate insulin and lipid metabolism, and serve as the main nutrition source for colonic epithelial cells.^107–109 But on the other hand, other members, especially those capable of both producing propionate and degrading mucin, such as Dorea spp, Ruminococcus gnavus and Ruminococcus torques, have been associated with series of inflammation-related disorders and increased gut barrier permeability.^93,94 Unfortunately, the phylogenetic analyses in most studies were limited to the family level, possibly leading to the inconsistent data regarding the role of Lachnospiraceae in health and disease.

Akkermansia muciniphila (A. muciniphila) is another important SCFA-producer that utilizes mucin as carbon source.¹¹⁰ However, reduced abundance of A. muciniphila has been associated with inflammatory bowel diseases and elevated inflammation.⁸⁵ Several reviews have also suggested A. muciniphila as a promising probiotic in treating metabolic disorders and modulating immune responses.^111,112 Different from other mucin-degrading taxa, A. muciniphila was also found to promote mucin production, despite its ability to breakdown mucus layer.¹¹³ Nevertheless, increased level of A. muciniphila was found in PD patients and some opposite effects have been reported.^6,85

At compositional level, a weak connection of GM changes between human and animal studies can be established since human and murine harbor similar yet distinct microorganisms, although a shared trend of GM alterations was observed at functional level. However, compared to human, animal models exhibited more consistent GM alterations in both AD and SCRD studies. This discrepancy is mainly due to the limited studies available, heterogeneous samples and different methodologies applied in human studies.

In animal studies, mice and rats were born with identical genetic background, housed in constant environment and fed with unified food, and variables that could compromise the study have been carefully controlled as possible. Whereas in human studies, multiple factors including race, nationality, culture background and education may have substantial impacts on the lifestyle, daily diet and eating habit of participants, which directly affect GM composition.¹¹⁴ For example, participants of the five AD patients studies we have discussed above were from three continents with diverse culture background. It has been reported that diet plays a fundamental role in health and is a key determinant of GM.^115,116 Western-style diet, high in animal protein, sugar and fat and low in vegetables, favors the growth of Bacteroidetes, especially Prevotella, which has been associated with colon cancer and several bowel diseases.¹¹⁷ Mediterranean diet, featured by fruit, plant fiber and unsaturated fat, shifts GM toward more abundant Akkermansia, Bifidobacterium and Lactobacillus.¹¹⁷ Also, food rich in dietary fiber and carbohydrates promotes the growth of highly fermentative bacteria such as Lachnospiraceae, Lactobacillaceae and Ruminococcaceae in the phylum Firmicutes.⁹² Thus, the diverse dietary could contribute to the discrepant GM alterations in AD patients from different countries. Moreover, the varied experimental designs and heterogeneous methods, including fecal sample acquirement, DNA extraction and sequencing, as well as the criteria in determining cognitive function and sleep quality, make it difficult to conclude a consistent trend of GM alterations from different studies.

Therefore, it seems improper to compare GM alterations in human studies solely based on low-level phylogenetic analysis, which can be easily affected by the abovementioned factors. However, we observed a coherent trend by taking the perspective of metabolism and functions (Table 5, Figure 4).