The link between gut microbiome and Alzheimer's disease: From the perspective of new revised criteria for diagnosis and staging of Alzheimer's disease

Abstract Over the past decades, accumulating evidence suggests that the gut microbiome exerts a key role in Alzheimer's disease (AD). The Alzheimer's Association Workgroup is updating the diagnostic criteria for AD, which changed the profiles and categorization of biomarkers from “AT(N)” to “ATNIVS.” Previously, most of studies focus on the correlation between the gut microbiome and amyloid beta deposition (“A”), the initial AD pathological feature triggering the “downstream” tauopathy and neurodegeneration. However, limited research investigated the interactions between the gut microbiome and other AD pathogenesis (“TNIVS”). In this review, we summarize current findings of the gut microbial characteristics in the whole spectrum of AD. Then, we describe the association of the gut microbiome with updated biomarker categories of AD pathogenesis. In addition, we outline the gut microbiome‐related therapeutic strategies for AD. Finally, we discuss current key issues of the gut microbiome research in the AD field and future research directions. Highlights The new revised criteria for Alzheimer's disease (AD) proposed by the Alzheimer's Association Workgroup have updated the profiles and categorization of biomarkers from “AT(N)” to “ATNIVS.” The associations of the gut microbiome with updated biomarker categories of AD pathogenesis are described. Current findings of the gut microbial characteristics in the whole spectrum of AD are summarized. Therapeutic strategies for AD based on the gut microbiome are proposed.


Highlights
• The new revised criteria for Alzheimer's disease (AD) proposed by the Alzheimer's Association Workgroup have updated the profiles and categorization of biomarkers from "AT(N)" to "ATNIVS." • The associations of the gut microbiome with updated biomarker categories of AD pathogenesis are described.
• Current findings of the gut microbial characteristics in the whole spectrum of AD are summarized.
• Therapeutic strategies for AD based on the gut microbiome are proposed.

INTRODUCTION
Alzheimer's disease (AD), mainly characterized by episodic memory loss, executive dysfunction, language impairment, and declined daily living ability, is the most common form of neurodegenerative disorder causing dementia. 1Currently, ≈ 6.7 million Americans aged ≥ 65 suffer from AD.By 2060, the number of individuals with AD dementia is projected to reach 13.8 million. 2The core pathological features of AD are extracellular amyloid beta (Aβ) deposition and intracellular neurofibrillary tangles derived from tau protein hyperphosphorylation. 3 Currently, most of the interventions for AD focus on targeting Aβ aggregation and clearance. 4,5Although some findings of phase II/III clinical trials are promising in terms of anti-Aβ therapy, [6][7][8][9][10] these investigations are still in the early stage, and need further validation for their long-term therapeutic effect and safety.In addition, numerous pathological mechanisms and risk factors (e.g., inflammation, mitochondrial dysfunction, obesity, loss of hearing) may be closely associated with the onset of AD, suggesting the potential of multi-target and comprehensive therapeutic strategies for AD. 1,11Therefore, exploring novel mechanisms of AD is increasingly attracting researchers' interest worldwide.[14] Intestinal microbiome refers to the microbial community colonizing the human and animal gut, including bacteria, fungi, viruses, and so on.Among them, bacteria account for the highest proportion, with four top phyla, including Proteobacteria, Firmicutes, Actinobacteria, and Bacteroidetes. 15Alterations of gut microbial compositions and diversity are regarded as "gut microbial dysbiosis."7][18][19] The impact of dysbiosis in gut microbiota on AD pathogenesis may be through the "microbiota-gut-brain" axis, which is a bidirectional communication pathway possibly mediated by the auto-nomic nervous system (ANS), neuroimmunity, enteroendocrine, shortchain fatty acids (SCFA), neurotransmitters, and so on. 20Remarkedly, gut microbiome abnormalities have been observed in the whole clinical spectrum of AD, and have shown the potential for early identification of AD, [21][22][23][24][25][26] suggesting that dysbiosis in gut microbiota may serve as a valuable biomarker of AD.However, the regulatory mechanism of gut microbiota on AD pathogenesis remains largely unclear.
Notably, most of the previous studies focused on investigating the correlation between gut microbial alterations and amyloidosis (one of the core AD biomarkers), while few studies summarized the association of gut microbiota with other AD-related pathological biomarkers.Accumulating evidence suggests that gut microbiome may also affect other AD-related pathophysiological changes, such as tauopathy, inflammation/immune response, brain atrophy, regional glucose metabolism, and so on.Thus, understanding the impact of gut microbiota on different AD biomarker categories may help to comprehensively elucidate the "gut-brain" communication pathways in AD, which may further provide multiple potentially targeted interventions for AD.
In this review, we first summarize the evolution of diagnostic criteria for AD.Then, we review current findings of gut microbiome characteristics in different clinical stages of AD, including preclinical AD, mild cognitive impairment (MCI), and AD dementia (Table 1).Additionally, the correlations between the gut microbiota and the updated three broad biomarker categories of AD pathogenesis are discussed.Furthermore, we discuss the microbiome-mediated therapeutic strategies for AD.Finally, we conclude with current issues of the gut microbiome research in AD and highlight future perspectives of this field.The aim of this review is: (1) to clarify the pathophysiological mechanisms of AD from the perspective of the "microbiota-gut-brain" axis, and (2) to explore novel therapeutic strategies for AD.

THE EVOLUTION OF DIAGNOSTIC CRITERIA FOR AD
The diagnostic criteria for AD have been evolving during the past  38 The definition of AD is mainly based on clinical symptoms and histopathologic evidence obtained from brain biopsy or autopsy.With the advances of available AD biomarkers in vivo, such as structural magnetic resonance imaging (MRI), molecular neuroimaging with positron emission tomography (PET), and cerebrospinal fluid (CSF) analysis of Aβ or tau proteins, the International Working Group (IWG) proposed a new conceptual framework for the diagnosis of AD in 2007, which highlighted the combination of clinical phenomenology and biological markers. 39In 2014, the IWG further divided biomarkers into two categories: diagnostic markers (Aβ 1-42 , total tau [t-tau], phosphorylated tau [p-tau] in CSF, amyloid PET), and progression markers (structural MRI and fluorodeoxyglucose [FDG]   PET]. 40 2011, the National Institute on Aging and the Alzheimer's Association (NIA-AA) published separate recommendations for the diagnosis of AD in its preclinical, MCI, and dementia states.[41][42][43] These criteria also proposed a model of the pathophysiological sequence of AD, of which Aβ accumulation is an "upstream" event in the cascade leading to the "downstream" synaptic dysfunction, neurodegeneration, and eventual neuronal loss.In 2018, the NIA-AA further categorized biomarkers into Aβ deposition, pathologic tau, and neurodegeneration [AT(N)].44 If an individual has an abnormal Aβ deposition evidence, but a biomarker for tau is not available, then the individual is placed into the "Alzheimer's continuum."The diagnostic framework is a purely biological definition of AD that relies on biomarkers.
Due to the occurrence of major developments in AD, the 2018 NIA-AA research framework has been further updated (see https://aaic.alz.org/nia-aa.asp).In addition to traditional CSF and imaging biomarkers, the present criteria have incorporated plasma biomarkers into updated biomarker categorization, disease diagnosis, and staging.This full multimodal biomarker profile is further categorized as ATNX."X" is added to the new biomarker categorization and refers to inflammation (I), vascular brain injury (V), and α-synuclein (S).The revised guidelines highlight that biological diagnosis and staging of AD is now transitioning from the purpose of research to the application in clinical practice.
The progression of diagnostic criteria for AD is briefly summarized in Table 2.

ALTERATIONS OF THE GUT MICROBIOTA IN AD
Currently, specific alterations in the composition and diversity of the gut microbiota have been found in different clinical stages of AD. 21,[23][24][25][26]27,28,30,34,35 Vogt et al. reported significant differences in the gut microbial composition of AD patients compared to healthy controls, with significantly reduced Firmicutes and Bifidobacterium, and increased Bacteroidetes in AD. 25 Liu et al. also confirmed the decreased fecal microbial diversity and proportion of Firmicutes in AD patients compared to healthy subjects. 23 Additionall, they first found a progressive enriched prevalence of Gammaproteobacteria, Enterobacteriales, and Enterobacteriaceae from controls to amnestic MCI (aMCI) and AD patients.23 Subjective cognitive decline (SCD), characterized by a self-reported decline in the memory and/or other cognitive domains without objective cognitive impairment, is considered the earliest clinical symptom of preclinical AD. 45 Current research has shown that individuals with SCD exhibit specific gut microbial profiles similar to patients with AD. 26 The abundance of phylum Firmicutes and its corresponding Clostridia, Clostridiales, Ruminococcaceae, and Faecalibacterium also showed a trend toward a progressive decline from controls to SCD individuals and cognitive impairment patients.Specifically, the abundance of the anti-inflammatory genus Faecalibacterium was significantly decreased in SCD compared to controls, suggesting that alterations of gut microbial composition may be present in preclinical AD.Interestingly, a few studies have also shown that gut microbiome composition may serve as an indicator of preclinical AD.For instance, one study reported that the relative abundance of phylum Bacteroidetes was significantly enriched, whereas phylum Firmicutes and class Deltaproteobacteria were significantly decreased in Aβ-positive cognitively normal individuals compared to that in Aβ-negative cognitively normal individuals.22 Using metagenomic sequencing analysis, Ferreiro et al.21 further identified some species most associated with preclinical AD status, including Dorea formicigenerans, Oscillibacter sp. 5720, Faecalibacterium prausnitzii, Coprococcus catus, and Anaerostipes hadrus.
Currently, a systematic review summarizes the gut microbiome characteristics in SCD, MCI, and AD patients.They found that the relative abundance of phylum Firmicutes was significantly lower in AD and MCI than controls, while the relative abundance of phylum Bacteroidetes was significantly higher in MCI than controls.An increasing trend for Enterobacteriaceae and a decreasing trend for Ruminococcaceae, Lachnospiraceae, and Lactobacillus were present during AD. 46wever, several studies reported contradictory results. 29,32,33,36uang et al. found that the abundance of Bacteroidetes in AD patients significantly decreased, while the abundance of Ruminococcaceae, Enterococcaceae, and Lactobacillus significantly increased. 28Another study also showed that patients with AD had decreased Bacteroides. 31These discrepancies in gut taxonomic compositions among different studies may be due to the heterogeneity in severity of illness, different diagnostic criteria, racial difference, lifestyle, comorbidities, medications, and so on.Thus, a standardized study protocol is necessary for AD gut microbiome in the future.
In summary, most studies have suggested that the gut microbiota in different clinical stages of AD has significant alterations.However, larger scale studies are still needed to validate current findings and determine the specific bacterial species associated with the precision diagnosis of AD.

4.1
Gut microbiota and AD core biomarkers

F I G U R E 1
The association of the gut microbiota with AD-related pathogenesis.Significant alterations in the gut microbial composition are observed in AD, with increased pro-inflammatory bacteria and decreased anti-inflammatory bacteria.Two pathways will lead to the activated inflammatory response in the peripheral circulation: (1) Abnormal gut microbiota exacerbates the permeability of intestinal epithelium, leading to the release of several cytokines (e.g., IL-1β, IL-6, TNFα), chemokines, and microbial metabolites.These substances may further infiltrate the blood and lymphatic system.(2) Gut dysbiosis contributes to the C/EBPβ/AEP (δ-secretase) signaling activation, which also induces the release of cytokines.In addition, the activated C/EBPβ/AEP pathway prompts the Aβ and tau pathology in the gut, which will further be disseminated to the brain via the vagus nerve and exacerbate AD-related pathology.These pro-inflammatory substances in the peripheral circulation can permeate into the brain through the damaged BBB.In the brain, the activation of microglia and astrocytes contributes to neuron dysfunction, accelerating the AD pathology, which further leads to the disruption of brain structure and function.Aβ, amyloid beta; AD, Alzheimer's disease; AEP, asparagine endopeptidase; BBB, blood-brain barrier; IL, interleukin; p-tau, phosphorylated tau; SCFA, short-chain fatty acid; TNFα, tumor necrosis factor alpha.
In addition, the causal relationship between the gut microbiota and AD pathophysiology has been investigated.,et al. 54 found that the transplantation of fecal microbiota from healthy wild-type mice into transgenic AD mice ameliorated the formation of Aβ plaques and neurofibrillary tangles, glial reactivity and cognitive impairment, indicating that the pathogenesis of AD may be mediated by gut microbiota.49][50]52 Thus, these studies suggest a strong causal correlation between gut dysbiosis and AD pathology.
The potential mechanisms of the gut microbiota-mediated AD pathogenesis may be as follows: first, abnormal gut microbiotahost interactions may exacerbate the permeability of the intestinal epithelium, leading to the release of cytokines, chemokines, neurotransmitters, and gut-derived metabolites that infiltrate the blood and lymphatic system. 20Additionally, gut dysbiosis can also lead to the damage of the blood-brain barrier (BBB).Thus, these circulating substances can permeate into the brain through the damaged BBB, accelerating AD pathology. 55,56Second, gut dysbiosis contributes to signaling pathways, leading to brain inflammatory responses. 59In this review, the association of the gut microbiota with AD pathogenesis is shown in Figure 1.

Gut microbiota and "T"
Gut dysbiosis is reported to be associated with tau pathological biomarkers.One study investigated the correlation between altered gut microbiota and AD-related biomarkers among patients with AD, MCI, and SCD.The results showed that declined SCFA-producing microbiota, including Lachnospiraceae spp., Lachnoclostridium spp., Roseburia hominis, and Bilophila wadsworthia, was associated with higher odds of positive CSF p-tau status. 60Using PET tau imaging, Ferreiro et al. 21also found that gut microbiome profiles correlated with tau biomarkers in cognitively normal individuals.However, the correlation between gut microbiota and specific tau proteins in CSF and plasma, such as p-tau 181 and p-tau 217, is still largely unclear.
The misfolding and aggregation of tau is a key hallmark of AD.
Researchers have shown that DNA extracted from bacteria, especially certain bacterial species associated with AD (B.burgdorferi, P. gingivalis, C. albicans, and E. col) promotes pronounced tau aggregation, 61 suggesting that microbial DNA may play an important role in the tau protein misfolding and AD pathogenesis.Notably, some strains of E. coli (such as K99) and P. gingivalis have also been identified in the brain of patients with AD. 62,63 These strains have properties of facultative intracellular parasites, contributing to the interaction of bacterial DNA with tau proteins inside the neuron.These bacterial DNA can be secreted into the outer membrane and released into the neuron's cytosol, thus becoming the seed for tau aggregation. 61The findings provide new insights into the relationship between gut microbiota and tau protein aggregation, opening novel opportunities for AD therapeutic interventions.

4.2.1
Gut microbiota and "N" In the new, revised criteria for AD, neurodegenerative biomarkers ("N") refers to neuroimaging markers (anatomic MR, FDG PET) and fluid neurofilament light chain (NfL).Several studies have confirmed the regional brain atrophy, such as hippocampus, medial temporal lobe, and so on, in patients with AD and those who have a high risk for conversion to AD dementia.Currently, the effect of gut microbiota on brain anatomic changes has attracted wide interest from researchers. 64u et al. 65 investigated the association of gut microbiota with structural MRI measures in 157 healthy young adults.They found that gut microbial diversity was negatively correlated with gray matter volume (GMV) within the prefrontal, parietal, temporal, occipital, cingulate cortices, and insula.Notably, the gut-brain interactions may be sex dependent. 65,66In addition, they also reported that gut microbial diversity and enterotypes could indirectly impact cognitive performance by mediating the topological properties of structural networks, such as small-worldness. 67In a larger community-based cohort of 1430 participants, the relative abundance of Odoribacter was found to be positively associated with the right hippocampal volume. 68However, another study showed that there was no significant correlation between gut microbiome profiles and hippocampus volume in a cohort of cognitively normal older adults. 21The discrepancy in these results may be due to the age range, race, and cognitive performance.He et al. first studied the association of gut microbiota with brain structural changes in the spectrum of AD, including controls, SCD, and cognitive impairment (CI) participants. 37They found that the relative abundance of Mediterraneibacter was significantly correlated with changes in brain GMV and regional cortical structures, such as the internal olfactory area and the parahippocampal gyrus. 37Moreover, in a 24-week randomized, double-blind, placebo-controlled trial, older MCI patients with probiotic Bifidobacterium breve consumption exhibited suppressed brain atrophy progression, 69 indicating a direct impact of modifying gut microbiota on brain structural changes.
Brain glucose hypometabolism is a hallmark of AD.FDG PET, as a well-established technique to invasively quantify the resting-state cerebral glucose metabolic level in vivo, has been used to elucidate the abnormal brain glucose uptake in AD and predict the progression of cognitive impairment.1][72] Current evidence supports that the gut microbiome can impact glucose and energy homeostasis by regulating host gut-brain signaling and initiating direct communication to the brain via microbe-derived metabolites. 71Thus, FDG PET may mirror the effect of gut microbiota on brain glucose metabolism.
For instance, using 2-deoxy-2-[ 18 F] FDG-PET, one study revealed that C57BL/6 J mice treated with 27-hydroxycholesterol altered gut microbial compositions and decreased brain glucose uptake value, finally inducing memory impairment. 73This study suggests that reduced brain glucose uptake was mediated by the gut microbiota.However, studies involving the correlation between gut microbiota and FDG PET biomarkers are still lacking.Currently, the combination of the gut microbiota and neuroimaging techniques is called radiomicrobiomics.
In this review, a summary of radiomicrobiomics studies is shown in Table 3 and the framework of radiomicrobiomics research is depicted in Figure 2.
NfL, as intermediate filament proteins of neuronal cytoskeleton, is related to axonal structure and function.Increased levels of NfL in CSF and plasma have emerged as a neuronal injury biomarker in neurodegenerative disorders.Previous studies have found that NfL is associated with AD and appears to provide information on disease progression and help monitor treatment effects. 84,857][88][89] Currently, there are only a few studies involving the correlation between the gut microbiota and NfL.Vogt et al.
found that the gut microbial-derived metabolite trimethylamine Noxide (TMAO) in the CSF is elevated in patients with MCI and AD dementia, which was positively associated with NfL. 90 These findings suggest the involvement of the gut microbiota in the neurodegenerative process of AD.However, a recent study by Saji et
metabolites. 91Therefore, the effect of gut microbiota on the changes in NfL needs further evaluations.

Gut microbiota and "I"
Glial fibrillary acidic protein (GFAP) is added to the revised criteria as an inflammation biomarker.GFAP is a hallmark of astrocytic activation.Astrocytes play an important role in the regulation of neuronal physiology, and their disintegration leads to the release of GFAP from brain tissues into the blood. 924][95] Increased plasma GFAP even has diagnostic and longitudinal monitoring potential for preclinical AD. 96 Although specific bacterial genera are associated with immune markers, 97 the effect of intestinal flora on the astrocytic activation (e.g., GFAP) in AD has not been widely explored.One study reported that after gut microbial perturbations by antibiotics and a germ-free environment, the APPPS1-21 mouse model of amyloidosis reduced GFAP+ reactive astrocytosis and astrocyte recruitment to amyloid plaques in male mice.Moreover, fecal microbiota from untreated APPPS1-21 mice transplanted into antibiotics-treated APPPS1-21 mice restored astrocytic changes, suggesting that the gut microbiota can regulate GFAP+ astrocyte reactivity. 14These findings also provide new insights into the role of the gut-brain axis in AD.

4.3.1
Gut microbiota and "V" Biomarkers of vascular brain injury are derived from neuroimaging measures, including anatomic infarction, white matter hyperintensities (WMH).][100] For instance, Ma et al. found that patients with lacunar cerebral infarction showed increased relative abundance of genus Lactobacillus, Streptococcus, Veillonella, Acidaminococcus, Bacillus, Peptoclostridium, Intestinibacter, Alloscardovia, and Cloacibacillus but declined genus Agathobacter and Lachnospiraceae_UCG-004. 98 In another study, no significant correlation between gut microbiome profiles and brain WMH was observed in cognitively normal individuals. 21Notably, most studies involving the association of gut microbiota with these vascular brain injury biomarkers focus on cerebrovascular diseases, such as acute cerebral infarction, while their correlations in AD are still lacking.In the future, further evidence is needed.
The framework of radiomicrobiomics research in AD.A, Data collection: recruitment of healthy controls and the spectrum of AD, including preclinical AD, MCI, and AD dementia.Clinical information, neuropsychological scales, fecal sample, and multi-modal neuroimaging scan data (MRI and FDG PET) are collected.B, Imaging and microbiota data preprocessing: image preprocessing of sMRI, fMRI, DTI, and FDG PET; 16S high-throughput sequencing.C, Features extraction: key neuroimage (e.g., gray matter volume, ALFF, fALFF, ReHo, FA, MD, AxD, regional glucose metabolism) and microbial features (e.g., microbial flora, α-diversity, β-diversity) are extracted.D, Diagnostic and prediction models: the correlation analysis is used to investigate the relationship between different features and clinical assessments.Based on the selected key multi-omics features, the ROC analysis and survival analysis are separately used for the establishment of diagnostic model and prediction model.AD, Alzheimer's disease; AxD, axial diffusion; ALFF, amplitude of low-frequency fluctuation; DTI, diffusion tensor imaging; FA, fractional anisotropy; fALFF, fractional amplitude of low-frequency fluctuation; FDG PET, fluorodeoxyglucose positron emission tomography; fMRI, functional magnetic resonance imaging; MCI, mild cognitive impairment; MD, mean diffusivity; MRI, magnetic resonance imaging; ReHo, regional homogeneity; ROC, receiver operating characteristic curve; sMRI, structural magnetic resonance imaging.

Gut microbiota and "S"
Aggregated α-synuclein (αSyn) is the core component of cytoplasmic inclusions called Lewy bodies. 101Several neurodegenerative disorders exhibit this pathological feature, such as Parkinson's disease, dementia with Lewy bodies (DLB), AD, and so on.Increasing evidence has revealed that the gut microbiota is closely related to the αSyn pathology and can regulate motor deficits. 102,103However, the assumption as to whether the effect of gut microbiota on αSyn will further influence the progress of AD is still unknown.

5
Gut microbiota-related AD therapeutic strategies

Fecal microbiota transplantation
Table 4 shows the current microbiome-mediated therapeutic strategies for AD.Fecal microbiota transplantation (FMT) is used to transfer the fecal components from a healthy donor to the recipient's intestine, which can directly alter the gut microbiota compositions. 104FMT has been successfully applied in treating Clostridium difficile infections, and may also be a potential microbiome modulation approach for AD intervention. 105,106Recent studies suggest that FMT has shown promising results in reducing AD pathology in mouse models. 54,107n et al. found that after transferring the fecal microbiota from healthy donors into 6-month APP/ PS1 mice for 4 weeks, levels of Aβ and hyperphosphorylated tau were reduced. 107Similarly, Kim et al. showed that treating ADLPAPT mice from 2 to 6 months old with fecal microbiota from healthy donors reduced Aβ burden, tau phosphorylation, Iba1+microglia, GFAP+ astrocytes, and monocytes, leading to improved performance in behavioral tests. 54However, one study found that the transplantation of fecal microbiota in abxtreated APPPS1-21 mice increased the Aβ deposition and microglial activation. 502][83] In one case, an 82-year-old male AD patient who received the transplantation of fecal microbiota from his 85-year-old wife showed improved memory function and emotion. 81other case study reported a 90-year-old woman with AD and severe Clostridium difficile infection.After receiving FMT treatment from a healthy 27-year-old male donor, this AD patient showed significant amelioration in cognitive function, gut microbial diversity, and the production of SCFAs. 82However, these studies are only based on an individual observation or small samples, and more evidence is needed to validate the therapeutic potential of FMT in human AD.
In conclusion, although the FMT approach has shown a promising perspective in ameliorating the pathogenesis and cognitive performance of AD mouse models, more studies are essential before FMT serves as a non-pharmacological intervention for AD.

Probiotics intervention
Probiotics supplementation is a simple and safe way to regulate the gut microbiota.The most common components of probiotics are Lactobacillus and Bifidobacterium. 108[111] Kaur et al. used a probiotic preparation called VSL#3 to treat 6-monthold APP NL-G-F mice for 8 weeks. 110They found that VSL#3 altered the gut microbial compositions and blood metabolites, with increased abundance of Clostridia, Lachnospiracea, and Akkermansia and the level of SCFAs.However, there was no significant effect of the VSL#3 supplementation on Aβ, GFAP, Iba1, and the cellular proliferation marker Ki-67.This finding may be due to the severe pathological changes in the 6-month-old APP NL-G-F model.Thus, although VSL#3 altered the compositions of the gut microbiota and levels of serum metabolites, it may be too late to reverse the already formed pathological features. 110,112On the contrary, Abdelhamid et al. found that treatment with a strain of Bifidobacterium breve for 4 months reduced Aβ, Iba1, and pro-inflammatory cytokines, while it increased ADAM10 and synaptic proteins in 3-month-old APP NL-G-F mice. 113Recently, one study showed that the supplementation of probiotic preparation SLAB51 significantly improved cognitive deficits, and reduced Aβ deposition and neuronal damage in transgenic AD mice. 114SLAB51 also enhanced antioxidant and neuroprotective effects by activating the Sirtuin-1 pathway, which was helpful to recover cognitive and behavioral impairment.SLAB51 significantly altered the compositions of the gut microbiota in AD mice, with increased abundance of beneficial bacteria and decreased harmful bacteria. 115In addition, the combination of L. acidophilus, L. fermentum, B. lactis, and B. longum also improved learning ability and decreased the oxidative stress in rats injected with Aβ 1-42 in the hippocampus. 1168][79][80] In a randomized, double-blind, placebocontrolled trial, AD patients were divided into the probiotic group and the placebo group.The results showed that compared to the placebo group, patients in the probiotic group had significantly improved cognitive performance, especially in executive function and memory.They also exhibited decreased inflammatory factors, insulin resistance, and blood lipids, which are common metabolic abnormalities in AD. 74 Simi-larly, one meta-analysis showed that compared to controls, individuals who received probiotics had significantly improved cognitive function, and decreased levels of malondialdehyde and high-sensitivity C-reactive protein. 117Moreover, MCI patients receiving short-chain Bifidobacterium breve A1 treatment also showed improved performance in some neuropsychological tests. 118However, other studies have shown contradictory results.Previously, AD patients aged 65 to 90 years old were randomly assigned to the probiotic group and the placebo group.After 12 weeks, this study found that there were no significant differences in the levels of pro-inflammatory cytokines (tumor necrosis factor α and interleukin [IL]-6) and anti-inflammatory cytokine (IL-10), as well as the oxidative factors (malondialdehyde and 8hydroxydeoxyguanosine) and the antioxidant factors (total antioxidant capacity, glutathione) between the placebo group and the probiotic group. 76In addition, this study also found that compared to the placebo group, there was no significant improvement in cognitive performance in the probiotic group, suggesting that patients in the late stage of AD may be insensitive to the probiotic supplementation. 76 summary, current findings suggest that probiotics supplementation may provide a potential clinical value in the treatment of AD.
However, it's worth noting that there is a great deal of variability in the formulation, dose, and treatment patterns of probiotics in these studies, possibly causing different intervention effects on AD.Additionally, individual heterogeneity in the severity of diseases may also affect the efficacy of probiotics.Thus, more large-scale, consistent, and standardized studies are needed to determine the best probiotics treatment strategies for AD.

Standardized protocol for study design and data processing
Although most studies have revealed significant changes in gut taxonomic compositions and diversity both in human and mouse models of AD, several remarkable issues still need to be discussed.First, many studies are single center and lack validation of findings in another center/cohort, which will reduce the robustness of results.Remarkably, there is inconsistency in the altered microbial species reported in those published studies.For instance, the changing trend of phyla Firmicutes and Bacteroidetes in AD patients is inverse in some different studies.We concluded that these discrepancies may be due to numerous factors, such as small sample size, race, lifestyle difference, regional disparity, and so on.Second, the diagnostic criteria for AD are not consistent among these studies.The severity of AD may be heterogeneous.
Patients in some studies appear to be have AD dementia, while other studies may include moderate or severe patients with AD dementia.In addition, only a few studies diagnose patients based on the AD-related biomarkers and the heterogeneity of participants is relatively great among different studies.Thus, AD biomarkers should be included for precise diagnosis in further validating studies.Third, the fecal sample collection, data preprocessing, microbiome sequencing, and analysis F I G U R E 3 Potential factors linking to the gut microbiota and the "gut-brain axis."methods (e.g., 16S amplicon and metagenomic sequencing) are also different in these research studies.A practical and reproducible microbiome analysis guide for AD would be helpful.Therefore, given that many factors impact the gut microbiota characteristics, the establishment of a unified and standardized protocol in the study design and data processing is necessary for future AD microbiome studies.

Potential modifiers of the relationship between the gut microbiome and AD
Many intrinsic and environmental factors, such as host genetics, lifestyles, and drugs, could influence the composition and diversity of the gut microbiome, which might mediate the correlation between the gut microbiome and AD pathogenesis.In this review, we discuss the association of these influence factors with the gut microbiome (Figure 3).

Apolipoprotein E genotype
The apolipoprotein E (APOE) genotype is the primary genetic risk factor for AD.Currently, several studies have investigated the impact of APOE genotype on the gut microbiome structure and function. 119,120Tran et al. 119 detected that APOE genotype was associated with specific gut microbiome profiles in both humans and mice.Higher abundance of Ruminococcaceae and lower abundance of Prevotellaceae were observed in APOE ε2/ε3 than in other APOE genotypes.In addition, the APOE genotype can also influence the correlation between the gut microbiota and AD.For instance, pro-inflammatory gut microbiota, such as genus Collinsella, may promote AD development through interaction with APOE. 121Furthermore, emerging evidence suggests that the APOE genotype mediates the effect of the gut microbiota on AD pathogenesis.A recent study also revealed that gut microbiota manipulations influenced tau pathology and neurodegeneration in an APOE genotype-dependent manner. 122

Lifestyles
Lifestyles might modify an individual's risk of developing AD. 123,1246][127][128] Thus, we speculate that gut microbiota serve as mediators of lifestyle effects on AD development.Different dietary patterns may regulate the abundance of different gut microbiota, which further affect the pathogenesis of AD via different ways. 129For instance, a Western-type dietary pattern increases the onset risk of AD, possibly by the mediation of the microbiotaproduced TMAO. 130Adherence to the Mediterranean diet may induce gut microbial changes, which is linked to the reduced risk of AD. 131 In addition, calcium-and vegetable-rich diets are also associated with the gut microbiota that produces SCFAs. 120SCFAs, as the most common form of gut microbiota-derived metabolites, are significantly altered in AD patients and animal models. 132ere is compelling evidence that exercise is a protective intervention for AD.However, the mechanisms of the beneficial role of exercise on AD are not well understood.Several recent studies have focused on the modulatory effect of exercise on gut microbiota. 133Mitchell et al. 133 found that exercise was associated with increased butyrate producing bacteria and fecal butyrate concentrations independent of diet in rodents and humans.The potential mechanisms by which exercise modulates gut microbiota may be closely linked to a beneficial anti-inflammatory role, leading to the reduced gut inflammation. 134 decades.The first clinical diagnosis of AD was established by the National Institute of Neurological and Communicative Disorders and Stroke (NINCDS) and the Alzheimer's Disease and Related Disorders Association (ADRDA) in 1984.
amyloid pathology via the C/EBPβ/asparagine endopeptidase (AEP) signaling activation.C/EBPβ, as an inflammatory cytokine or Aβactivated transcription factor, regulates the expression of AEP.AEP, also as a δ-secretase, can cleave both Aβ precursor protein (APP) and tau, promoting the formation of Aβ and neurofibrillary tangles.Current studies have shown that C/EBPβ/AEP signaling is activated by gut dysbiosis in mouse models of AD, whereas C/EBPβ/AEP signaling and arachidonic acid-associated inflammatory enzymes are diminished in germ-free mice.Therefore, the C/EBPβ/AEP pathway plays a critical role in the impact of gut microbiota on AD pathologies.Finally, inflammatory response and glial reactivity may participate in the influence of gut microbial dysbiosis on AD pathogenesis.50,57,58Peripheral inflammatory mediators can infiltrate the brain, and then activate the Toll-like receptors (TLR), such as TLR2/1, and nuclear factor kappa-B (NF-κB) Summary of altered gut microbiome in human AD.
TA B L E 1 The progression of diagnostic criteria for AD(1984-2023).
Summary of radiomicrobiomics studies in human AD.
al. reported that plasma NfL levels were not significantly correlated with gut microbial TA B L E 3 Summary of gut microbiota-related interventions in human AD.
TA B L E 4