Oxidative stress, inflammation, neurogenesis, immune responses, dysbacteriosis and infection all about above 30 factors have been associated with AD [24]. Gut microbiota can release significant amounts of harmful metabolites as amyloids and lipopolysaccharides, which might modulate signaling pathways and induce the production of proinflammatory cytokines related to AD pathogenesis. The gut microbiome, leaky gut, and bacterial translocation could be involved in AD. In this study, we also found evidence of dysbacteriosis in both AD patients and APP/PS1 mice (Figs. 1–3), especially an increase in pathogenic and conditioned pathogens in AD patients (Fig. 1). The behavior, oxidative stress, inflammation, neurogenesis, and immune response of AD-cohousing mice were altered compared to normal control mice (Figs. 3–7). However, some of them are beneficial or exert different functions dependent on the numbers of other bacteria, as members of the Alistipes genera show high abundance in the most frail individuals and in middle-aged and older mice and are decreased in autism spectrum disorders, colitis and colorectal cancer, chronic hepatitis B patients, and appendicitis [1, 25]. Several studies reported that Bacteroides fragilis (B. fragilis), members of Bacteroides, and its immunomodulatory capsular polysaccharide A are equally effective in preventing colitis and experimental allergic encephalomyelitis in murine models [26]. These species also orchestrate robust protective anti-inflammatory responses during viral infections [22]. Rodents with high abundance of Alloprevotella genera in early life have high risk of behavioral phenotypes, with males but not females exhibiting deficits in social behavior [27]. Clinical investigation showed that the genera Lactobacillus, Clostridium IV, Paraprevotella, Clostridium sensu stricto, Desulfovibrio, and Alloprevotella were enriched in fecal samples from patients with chronic kidney disease [28].
Serum levels of many cytokines (TNF-α, EGF, IL-17α, prolactin, FGF-basic, MCP-1, IL-6, G-CSF, and MIP-1α) measured after cohousing (Fig. 5), and brain levels of inflammatory factors were also altered, including TREM2, IL-12α, IL-23, NF-κB p65, NLRP3 and TLP4 (Fig. 6 and Fig. 7). Extracts of AD-cohousing mice feces influenced levels of GSK3β, NF-κB, and LC3A/B in BV2 cells. Collectively, the results shows that gut microbiota change modulate signaling pathways and the production of proinflammatory cytokines related to AD pathogenesis. Although these limited microbiota animal models do not fully represent the situation in humans, there is considerable evidence of a role of gut microbiota in AD progression.
Recent human studies have investigated gut bacterial taxa and shown altered abundance in patients with AD [29]. A Chinese cohort had distinct microbial communities of Gammaproteobacteria, Enterobacteriales, and Enterobacteriaceae[4]. Li et al. studied AD patients and found that the fecal abundance of six genera increased (Dorea, Lactobacillus, Streptococcus, Bifidobacterium, Blautia, and Escherichia), while five decreased (Alistipes, Bacteroides, Parabacteroides, Sutterella, and Paraprevotella) [28]. They also found that blood samples had different abundances between control and AD groups; Propionibacterium, Pseudomonas, Glutamicibacter, Escherichia, and Acidovora increased, while Acinetobacter, Aliihoeflea, Halomonas, Pannonibacter, Leucobacter, and Ochrobactrum decreased [28]. We found that the genera of Megamonas, Faecalibacterium,Lachnospira, Fusicatenibacter༌ Prevotella 9 and Ruminococcus_2 were decreased, while Rhodococcus were increase compared to the control group (Fig. 1B). The commonly changed bacteria in AD-like mice and AD patients only belonged to the Lactobacillus genera, the more pathogenic bacteria in the AD patients were not found in specific pathogen-free (SPF) AD-like mice, and few special bacteria found were different from previous studies[4, 28, 29]. L. plantarum-derived lactic acid triggered the activation of the intestinal NADPH oxidase Nox and ROS generation. In turn, ROS production promoted intestinal damage, increased intestinal stem cell proliferation, and dysplasia. Nox-mediated ROS production required lactate oxidation by the host intestinal lactate dehydrogenase, revealing host-commensal metabolic crosstalk that is probably broadly conserved [3]. The increase in intestinal permeability coincided with higher plasma levels of LPS, serum IL-1, and TNF-α. Clinical studies suggested that individuals with microbial dysbiosis due to intestinal diseases have a higher risk of AD [28]. In this study, the fecal metabolites of AD-cohousing mice influenced PGRP-L and NOX1 expression in BV2 cells (Fig. 8A). Systemic inflammation was also observed. The study reveals marked sex differences in a multifactorial model of early-life adversity, both on emotional behaviors and gut microbiota and Lactobacillus genera was regulated by early adversity both in male and female[27]. Previous studies suggest that gut microbiota is associated with neuropsychiatric disorders, such as Parkinson’disease, amyotrophic lateral sclerosis, and depression. In the present study, clinical fecal samples were collected and analysed from AD patients for 16S ,which show gut microbiota is altered in AD patients and may be involved in the pathogenesis of AD[29].
Major group of microbes linked to AD include bacteria: Chlamydia pneumoniae, Helicobacter pylori, Porphyromonas gingivalis, Fusobacterium nucleatum, Prevotella intermedia, Actinomyces naeslundii, and spirochete group; fungi: Candida sp., Cryptococcus sp., Saccharomyces sp., Malassezia sp., Botrytis sp., and viruses: herpes simplex virus type 1 (HSV-1), human cytomegalovirus, and hepatitis C virus [10]. We also found that the pathogenic bacteria including Escherichia, Klebsiella, Pseudomonas, Rhodococcus, and Akkermansia were increased in AD patients, which indicated that these infections might an inducers and/or accelerators for oxidative stress, inflammation, autophagy, and neurodegeneration. Escherichia was increased at the genus level in both fecal and blood samples from subjects with AD and MCI [28]. Postmortem brain tissue from patients with AD showed that both LPS and gram-negative Escherichia coli fragments colocalize with amyloid plaques, and an increase in the abundance of a pro-inflammatory gut microbiota of Escherichia-Shigella and a reduction in anti-inflammatory Eubacterium rectale are possibly associated with a peripheral inflammatory state in patients with cognitive impairment and brain amyloidosis [10, 28, 30]. In this study, we found a higher abundance of Escherichia - Shigella in AD patients, and the expressions of GSK3β, NF-κB, LC3A/B, Tau, and p-Tau correlated with metabolites of feces of AD-cohousing mice in BV2 cells. IgG antibody levels to seven oral bacteria associated with periodontitis showed that abundance of Fusobacterium nucleatum and Prevotella intermedia were significantly increased at baseline serum draw in the AD patients compared to controls, and these remained significant when controlling for baseline age, Mini-Mental State Exam score, and APOE ε4 status, which suggested that chronic inflammation in periodontal disease could be a risk factor for AD [10, 28, 30]. F. nucleatum is an anaerobic oral commensal and a periodontal pathogen associated with a wide spectrum of human diseases. It is implicated in adverse pregnancy outcomes (chorioamnionitis, preterm birth, stillbirth, neonatal sepsis, preeclampsia), gastrointestinal disorders (colorectal cancer, inflammatory bowel disease, appendicitis), cardiovascular disease, rheumatoid arthritis, respiratory tract infections, Lemierre's syndrome, and AD [10, 28, 30]. Subtractive genomics analysis demonstrated that F. nucleatum infection could simultaneously regulate multiple signaling cascades that could upregulate proinflammatory responses, oncogenes, modulation of host immune defense mechanisms, and suppression of DNA repair system [28]. Microcin E492, a peptide naturally produced by Klebsiella pneumonia, assembles into amyloid-like fibrils in vitro, and these have the same structural, morphological, tinctorial, and biochemical properties as the aggregates observed in AD [10, 28, 30]. Low levels of the amino acid L-arginine in astrocytes surrounding amyloid plaques may be observed because arginine deiminase from Pseudomonas aeruginosa and peptidylarginine deiminase from bovine brain are inhibited by amyloid peptides that contain arginine (amyloid 1–42), and enhanced peptidylarginine deiminase activity is noted with free L-arginine [4, 10, 28]. Most bacteria in AD patients were not found in SPF model mice, strongly suggesting that studies of gut microbiota in aseptic mice do not tell the full story. Investigations in wild gut microbiota mice and long-term clinical studies are urgently needed to determine if these bacterial alterations are a cause or effect of AD. A recently report ensured that the important role of microglia and NLRP3 inflammasome activation in the pathogenesis of tauopathies and support the amyloid-cascade hypothesis in Alzheimer’s disease, and demonstrated that neurofibrillary tangles develop downstream of amyloid-beta- induced microglial activation [4, 10, 28], and NLRP3 is considered as an intracellular sensor that senses multiple microbial antigens and endogenous danger signals[30, 31]. In this study, serum levels of the cytokines MIP-1α was altered after cohousing(Fig. 5B). There were also changes in brain levels of inflammatory factors including, TREM2, IL-12α, IL-23, NF-κB p65, NLRP3and TLP4, (Fig. 6 and Fig. 7). The AD biomarkers Amyloid-β-A4, Tau, p-Tau, and APOE were upregulated. So, we sure that the gut microbiota appear to play an indispensable role in modulating the gut-brain axis and could be an important pathogenic factor of AD, and target on the microbiome-gut-brain axis will be an effective action. However, it is not clear if this is a useful diagnostic biomarkers as the AD gut microbiota could be distinguished from the healthy group but not the disease control groups, it perhaps due to the small sample size.
Tau hyperphosphorylation is associated with abnormal Aβ aggregation in AD, there are specific temporal patterns of phosphorylated Tau in different parts of the brain [4, 10]. Tau hyperphosphorylation plays a vital role in regulating synaptic function and maintaining cytoskeletal integrity [4, 10, 28, 30]. In this study, phosphorylation at sites Ser404 and Ser416 were upregulated after cohousing, indicating that the AD gut microbiota enhances Tau protein hyperphosphorylation at multiple sites. There are many factors regulating Tau phosphorylation level, including oxidative stress, inflammation, neurogenesis, immune response, dysbacteriosis, infection and autophagy dysfunction. Tau phosphorylation is also affected by oxidative stress, endoplasmic reticulum protein folding dysfunction, and protein clearance ability decreases mediated by the proteasome and autophagy [4, 10, 28, 30]. Normally, this abundant soluble protein can promote microtubule assembly and stability in axons, but this balance is upset when the Tau protein is hyperphosphorylated due to infection, metabolic disease, or chronic inflammation. Tau will be hyperphosphorylated and/or depolymerized depending on the activities of GSK-3β and the cell cycle protein-dependent kinase p25 [4, 10, 28]. As in Fig. 8A, the expression of GSK-3β was upregulated by AD feces extracts, and the autophagy was dysfunction.
Autophagy is a critical cellular process of internal degradation and recycling harmful or damaged components[31]. Autophagy dysfunction and tissue inflammation make people more susceptible to diseases, especially to intestinal diseases[32]. As a conservative serine/threonine protein kinase, mTOR is the junction of upstream pathways to regulate the cell growth, proliferation, movement, survival and autophagy [33], mTORC1 promotes the cell growth and metabolism, and inhibits autophagy by binding ULK1 complex [33]; and previous study have demonstrated that the leucine can activate the mTOR to regulate the autophagy and mitochondrial function [23]. In this study, we found that the levels of leucine in feces were reduced, while norleucine levels were increased in serum, expressions of mTOR and TORC1 were up-regulated in cohousing mice (Fig. 7A), and autophagy dysfunction (Fig. 7A), which indicate that AD-cohousing disorder the leucine metabolism, activate the mTOR to unregulated the autophagy. Dysfunction of autophagy aggravates the accumulation of pathological products and inflammation levels (Figs. 6–7). Moreover, systemic inflammatory reactions caused by compounds secreted by bacteria promote oxidative stress, neuroinflammation, autophagy and/or neurodegeneration as demonstrated in previous studies [28]. So, we conclude that harmful microbes and/or their metabolites from APP/PS1 mice implanted in the newborn mice, caused metabolic imbalance, activated chronic inflammatory responses and affected autophagy and tau protein hyperphosphorylation (cover). Therefore, targeting the gut microbiota could be an effective treatment to slow AD progression. The role of the complex gut microbiota in AD still requires further investigation both at the community and/or strain level.
Multivariable-adjusted analyses showed that sphingomyelins and ether-containing phosphatidylcholines were altered in preclinical biomarker-defined AD stages, whereas acylcarnitines and several amines, including the branched-chain amino acid valine and α-aminoadipic acid, changed in symptomatic stages [4, 10, 28, 30, 34]. Decreased neuronal glucose metabolism that occurs in AD brain could play a central role in disease progression [4, 10, 28, 30, 34]. And more evidences showed that amino acid oxidation can temporarily compensate for the decreased glucose metabolism, but eventually altered amino acid and amino acid catabolite levels likely lead to toxicities contributing to AD progression. Because amino acids are involved in so many cellular metabolic and signaling pathways, the effects of altered amino acid metabolism in AD brain are far-reaching [4, 10, 28, 30, 34]. In this study, we found that amino acid metabolism and lipid metabolism were imbalanced (Fig. 9, table S3), and we demonstrated that the leucine metabolism imbalance induced the autophagy dysfunction, which aggravates the accumulation of pathological products and inflammation levels, these also indicated that regulate the metabolism balance targeting on the gut-brain axis is important for AD prevention and treatment.
Dietary, microbial, and inflammatory factors modulate the gut-brain axis and influence physiological processes ranging from metabolism to cognition [35]. Nutrients affect gut microbiota composition and the formation and aggregation of cerebral Aβ [35]. In a transgenic AD mouse model, AD pathology shifted gut microbiota composition toward an inflammation-related bacterial profile, suggesting that these changes could contribute to disease progression and severity [7]. The gut microbiota has been shown to mediate the anti-epileptic effect of a ketogenic diet [7, 35]. Metabolites of dietary tryptophan produced by microflora control microglial activation, affect TGFα and vascular endothelial growth factor-β production, regulate transcription in astrocytes, and modulate CNS inflammation via the aryl hydrocarbon receptor, with implications for anxiety and depression [36]. The diet also impacts AD progression; germ-free mice demonstrated deficits in nonspatial and working memory tasks, as well as reduced hippocampal expression of brain-derived neurotrophic factor[37]. Uridine- and docosahexaenoic acid-containing diets could prevent rotenone-induced motor and gastrointestinal abnormalities associated with the pathogenesis of PD[37]. Altered gut microbiota composition has been associated with the onset of AD, which is characterized by the cerebral accumulation of amyloid-β fibrils[37]. It is therefore possible that modulation of the gut microbiome by specific nutritional intervention may prove to be an effective strategy to prevent or reduce the risk of neurodegenerative disorders, such as PD and AD.