Methods and compositions for treating microglial dysfunction and ameliorating metabolic dysfunction

Abstract

The present disclosure relates to a method and composition for treating or reducing the incidence of cognitive impairment, neurodegenerative disease, or neurological dysfunction in a subject in need thereof.The method comprises administering to the subject an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.The present disclosure also relates to a method for identifying such a subject.

Description

[Technical field]

[0001] This application claims the benefit of and priority to U.S. Provisional Application No. 63 / 276,996, filed November 8, 2021, the entire disclosure of which is incorporated herein by reference in its entirety for all purposes.

[0002] The present disclosure relates to methods and compositions for treating or reducing the incidence of a neurodegenerative disorder, cognitive disorder, or neurological dysfunction in a subject. [Background technology]

[0003] In normal brain aging, microglia often show distinct transcriptional profiles indicative of neurodegeneration (Non-Patent Document 1). At the same time, microglia in the aged human brain show morphological changes, are less able to support other tissues (e.g., neurons) (Non-Patent Document 2), and become dystrophic. It has been suggested that age-related changes in microglial homeostasis are likely due to age-related changes in microglial homeostasis mediated by intrinsic and extrinsic factors. It remains to be elucidated what extrinsic factors mediate the bidirectional interaction between the central nervous system (CNS) and the peripheral environment.

[0004] Despite efforts to date, there remains a need for new therapies to treat or reduce the incidence of neurodegenerative diseases, cognitive disorders, and neurological dysfunction. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Salter et al.(2014)CELL,158:l5-24 [Non-Patent Document 2] Streit et al. (2004) GLIA 45:208-212 [Non-Patent Document 3] Long et al.(2017)NAT.GENET.,49:568-578

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[0006] The present disclosure provides a novel method for the identification of metabolite N produced by the gut microbiota and found in processed foods. 6 The present invention is based in part on the discovery that carboxymethyllysine (CML) may promote age-related oxidative stress and mitochondrial dysfunction in microglia, and that increased amounts of CML in the brain with age may lead to cognitive and neurodegenerative disorders. It has further been discovered that the increase and accumulation of CML in body fluids and tissue samples of age-related subjects may be due to increased intestinal permeability with the aging process, which may result in higher levels of CML passing through the intestinal wall into the body fluids and tissues of the subjects. Based on these findings, it is possible to provide therapies to treat or reduce the incidence of neurodegenerative disorders, cognitive disorders, and neurological dysfunction. [Means for solving the problem]

[0007] In one aspect, the present disclosure provides a method for reducing the incidence of oxidative stress or mitochondrial dysfunction in microglia, the incidence of mitochondrial dysfunction in microglia, or the incidence of microglial dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating gut microbiota dysbiosis.

[0008] In another aspect, the present disclosure provides a method for treating cognitive impairment or reducing the incidence or worsening of cognitive impairment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0009] In another aspect, the present disclosure provides a method for treating a neurodegenerative disease or reducing the incidence or progression rate of a neurodegenerative disease in a subject, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0010] In another aspect, the present disclosure provides a method for reducing the incidence or worsening of neurological dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0011] In each of the above aspects, in one embodiment, the subject has been previously identified as having elevated levels of N6-carboxymethyllysine (CML), CML precursors, CML metabolites (herein, the terms CML metabolites and CML breakdown products are used interchangeably), or CML analogs in the subject's biological sample compared to a reference level. Alternatively or additionally, in another embodiment, the method further comprises identifying the subject as having elevated levels of CML, CML precursors, CML metabolites, or CML analogs in the subject's biological sample compared to a reference level. Optionally, the method further comprises determining the levels of CML, CML metabolites, or CML analogs in a biological sample obtained from the subject. Additionally, optionally, the biological sample comprises a body fluid (e.g., saliva, urine, blood, serum, plasma, cerebrospinal fluid, or feces) or a tissue sample (e.g., brain tissue) of the subject.

[0012] In each of the foregoing aspects and embodiments, (i) the subject is pre-identified as having an elevated level of intestinal barrier permeability compared to a baseline level; and / or (ii) the method further comprises identifying the subject by having an elevated level of intestinal barrier permeability compared to a baseline level.

[0013] In each of the foregoing aspects and embodiments, the subject is identified or diagnosed as having (i) a cognitive impairment; and / or (ii) a neurodegenerative disease. Alternatively or additionally, the subject is identified as being at risk for developing (i) a cognitive impairment; and / or (ii) a neurodegenerative disease.

[0014] In certain embodiments, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, progressive supranuclear palsy, spinal muscular atrophy, multiple system atrophy, ataxia, and vascular dementia.

[0015] In each of the foregoing aspects and embodiments, optionally, the method results in one or more of: (i) a decrease in cellular and / or mitochondrial reactive oxygen species (ROS) levels in the subject's microglia; (ii) a decrease in expression of inducible nitric oxide synthase (iNOS) in the subject's microglia; (iii) a decrease in expression of one or more genes in the subject's microglia selected from the group consisting of Cdknla, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, Tlr5, Axl, Hif1a, Lcn2, Mmp2, Rela, Trexl, S100a8, and S100a9; and (iv) an increase in expression of one or more genes in the subject's microglia selected from the group consisting of Foxp1, Nrf1, Trp53, G6pdx, Pdk2, Stat3, and Ucp2.

[0016] In another embodiment, the disclosure provides a method for reducing the rate of accumulation of CML, CML precursors, CML metabolites, or CML analogs in a tissue of a subject, comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0017] In another aspect, the disclosure provides a method for identifying a subject at increased risk for (i) the development of microglial dysfunction, (ii) cognitive impairment, or (iii) the development of a neurodegenerative disease, the method comprising identifying the subject having an elevated level of CML, CML precursor, CML metabolite, or CML analogue in a biological sample obtained from the subject, relative to a reference level.

[0018] In another embodiment, the disclosure provides a method for reducing aggregation of CML, CML precursors, CML metabolites, or CML analogs in a blood or brain sample from a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating gut microbiota dysbiosis.

[0019] In another embodiment, the disclosure provides a method for reducing aggregation of CML, CML precursors, CML metabolites, or CML analogs in a blood or brain sample to inhibit or treat a cognitive disorder or neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating gut microbiota dysbiosis.

[0020] In another aspect, the present disclosure provides a method for reducing intestinal permeability to prevent or treat cognitive impairment or neurodegenerative disease in a patient in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0021] In each of the above aspects and embodiments, the intestinal barrier function enhancer includes intestinal alkaline phosphatase (IAP), polyphenols (e.g., ellagic acid (EA) and lipoteichoic acid), metformin, urolithin A, butyric acid, glutamine, obeticholic acid (OCA), divertin, curcumin, spermidine, glutamine, or AMP-activated protein kinase, or derivatives. Specifically, in each of the above aspects and embodiments, the agent for reducing or eliminating intestinal flora dysbiosis includes intestinal alkaline phosphatase (IAP), ellagic acid (EA), biotics, probiotics, prebiotics, and postbiotics. In a particularly preferred embodiment, the intestinal barrier function enhancer and / or the agent for reducing or eliminating intestinal flora dysbiosis is IAP. Specifically, in a particularly preferred embodiment, the intestinal barrier function enhancer and / or the agent for reducing or eliminating intestinal flora dysbiosis is EA. In each of the aforementioned aspects and embodiments, the intestinal barrier function enhancer and / or the agent for reducing or eliminating intestinal bacterial flora dysbiosis are formulated as a pharmaceutical composition.

[0022] Depending on the situation, the intestinal barrier function enhancer and / or the agent for reducing or eliminating intestinal microbiota dysbiosis are administered orally, transdermally, by inhalation, intranasally, topically, intravenously, intraarterially, intramuscularly, or subcutaneously.

[0023] Upon administration of the agent for enhancing intestinal barrier function and / or the agent for reducing or eliminating gut microbiota dysbiosis, the subject may experience one or more of: (a) reduced aggregation of CML, CML precursors, CML metabolites, or CML analogs in a blood sample; (b) reduced aggregation of CML, CML precursors, CML metabolites, or CML analogs in a brain tissue sample; (c) reduced intestinal permeability; (d) reduced microbiota dysbiosis; (e) increased levels of autophagy in the intestinal epithelium; (f) reduced levels of cellular and / or mitochondrial ROS in microglia; (g) reduced levels of amyloidosis in the microglial population; (h) increased levels of adenosine triphosphate (ATP); (i) decreased expression of iNOS in microglia; (ii) decreased expression of one or more genes in microglia selected from the group consisting of Cdknla, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, Tlr5, Axl, Hif1a, Lcn2, Mmp2, Rela, Trexl, S100a8, and S100a9; and / or (j) increased expression of one or more genes in microglia selected from the group consisting of Foxp1, Nrf1, Trp53, G6pdx, Pdk2, Stat3, and Ucp2.

[0024] In another embodiment, the present disclosure provides a method for identifying a subject at increased risk of developing microglial dysfunction. The method includes identifying a subject having an elevated level of CML, CML precursor, CML metabolite, or CML analogue in a biological sample obtained from the subject compared to a reference level, where such an elevated level indicates that the subject has an increased risk of developing microglial dysfunction.

[0025] In another embodiment, the present disclosure provides a method for identifying a subject when the risk of developing cognitive impairment increases.The method includes identifying the subject that has elevated levels of CML, CML precursor, CML metabolite or CML analogue in biological samples obtained from the subject compared with reference levels, and such elevated levels indicate that the subject has increased risk of developing cognitive impairment.

[0026] In another embodiment, the present disclosure provides a method for identifying a subject when the risk of developing neurodegenerative disease is increased.The method includes identifying the subject that has an elevated level of CML, CML precursor, CML metabolite or CML analogue in a biological sample obtained from the subject compared to a reference level, and such an elevated level indicates that the subject has an increased risk of developing neurodegenerative disease.

[0027] In each of the foregoing embodiments, the biological sample is a tissue (e.g., brain tissue) or a bodily fluid sample (e.g., saliva, urine, blood, serum, plasma, cerebrospinal fluid, or feces). It is expected that the amount of CML, CML precursors, CML metabolites, or CML analogs in a biological sample can be measured by analytical techniques known in the art, including, for example, chromatography (e.g., high performance liquid chromatography (HPLC)), mass spectrometry, liquid chromatography mass spectrometry (LCMS), nuclear magnetic resonance spectroscopy, or immunoassays. [Brief description of the drawings]

[0028] Figure 1a-1g shows a series of schematics and graphs showing the interaction of microbiota on transcriptomes on microglia in young and aged mice. Figure 1a is a schematic of gut microbiota-mediated CML accumulation in aging subjects. CML induces microglial aging, especially oxidative stress and mitochondrial damage. Rejuvenating the integrity of the gut-blood barrier limits the accumulation and deleterious effects of CML in microglia.

[0029] Figure 1b is a schematic diagram of the experimental approach to perform RNA sequencing (RNA-seq) on fluorescence-activated cell sorting (FACS)-isolated microglia from whole brains of young adult mice (6-10 weeks old) and aged mice (96-104 weeks old) grown in a specific pathogen-free (SPF) and bacteria-free (GF) environment.

[0030] Figure 1c shows principal component analysis (PCA) of the transcriptomes (normalized gene counts) of microglia isolated from SPF mice (n = 6, 16) and GF mice (n = 6, 8).

[0031] Figure 1d is a bar graph showing the number of differentially expressed genes (DEGs) up- and downregulated between age groups in GF vs. SPF mice. Figure 1e is a heatmap of a subset of DEGs (microglial GF signature) in GF vs. SPF mice independent of age. Symbolized gene list (left) indicates its functional annotation (top left). Each column is a biological replicate and each row is a gene. DEGs (Wald P(adj)<0.05 and absolute fold change>1.5). z-scores were calculated from normalized gene counts (hashed grey is upregulated, non-hashed grey is downregulated).

[0032] Figure 1f: Module trait correlations across age. Each subplot represents a different group with a depiction of all module eigengenes (MEs) extracted by weighted gene co-expression network analysis (WGCNA) (grayscale represents correlation coefficient; radius:scale log10(Padj)).

[0033] Figure 1g depicts significant Gene Ontology (GO) terms enriched within modules along with their respective log10(Padj). The number of genes per module (ME1–ME10) is shown. Statistics: Figure 1f-g, two-sided P values ​​were obtained by Wald test and corrected for multiple testing using the Benjamini-Hochberg method.

[0034] Figures 2a-2i are a set of graphs and photomicrographs, respectively, showing that the microbiota contributes to age-associated oxidative stress and mitochondrial dysfunction in microglia.

[0035] Figure 2a is a graph showing reactive oxygen species (ROS)-associated MEs (ME1, ME2, and ME8 from top to bottom) (grayscale: correlation coefficient; diameter: scale log10(Padj)).

[0036] Figure 2b is a heatmap of ROS-related genes in ME(1,2,8) in microglia from young adult and aged SPF and GF mice. Each column is a biological replicate. Genes listed to the right of the heatmap are listed in order from top to bottom in the text following the heatmap.

[0037] Figure 2c is a bar graph showing quantification of cellular ROS in young adult SPF mice. Data are shown as mean + sem from three independent experiments including SPF (n = 18, 14) and GF (n = 13, 10).

[0038] Figure 2d is a bar graph showing quantification of microglial iNOS-positive, Iba-1-positive areas for young adult SPF mice. Data include SPF (n=14, 9) and GF (n=10, 9) and are shown as mean + sem from two experiments.

[0039] Figure 2e shows images showing immunofluorescence staining of Iba-1, iNOS, and DAPI in the cerebral cortex of aged SPF and GF mice. Scale bar, 40 μm.

[0040] FIG. 2f shows the metabolism-related ME10 (grayscale: correlation coefficient; diameter: scale log10(Padj)).

[0041] Figure 2g is a bar graph showing the percentage of healthy and abnormal mitochondria in cerebral cortical microglia of aged SPF and GF mice. Each point represents the average of 30-35 cells taken from one mouse. Data are shown as mean + sem from two independent experiments involving aged SPF and GF mice (n = 8 each). Two-way ANOVA with Sidak's multiple comparison test (*P < 0.05, *P < 0.01, ***P < 0.001; NS is not significant). Exact P values ​​are reported in the figures.

[0042] Figure 2h is a set of electron micrographs of microglia from aged SPF and GF mice. Grey arrowheads: healthy, white arrowheads: abnormal. Scale bar, 2 μm. Magnified micrograph of mitochondrial morphology. Scale bar, 500 nm.

[0043] Figure 2i is a bar graph showing microglial mitochondrial activity associated with young adult SPF mice. Data are shown as mean + sem from three experiments including SPF (n = 17, 14) and GF (n = 9, 13). Each point represents one mouse. Statistics: Figures 2c, 2d and 2i, two-way ANOVA with Tukey's post-hoc test.

[0044] Figures 3a-3g are a set of graphs and charts showing microbiota- and age-related regulation of serum and brain metabolites. Figure 3a is a bar graph showing short chain fatty acid (SCA) concentrations in serum samples. Each point represents one mouse. Data are shown as mean + sem from one experiment including young adult and aged SPF mice (n = 5, 6).

[0045] FIG. 3b is a set of volcano plots of differentially abundant metabolites from untargeted metabolomic analysis of serum (n=5,6) and brain tissue (n=5,5) samples from young adult and aged SPF mice.

[0046] Figure 3c is a Venn diagram showing differentially abundant metabolites from serum (106) and brain (164); and overlap / crossover (19).

[0047] Figure 3d is a graph showing metabolites that were differentially expressed in aged compared to young mice for both serum and brain samples. Biochemical names: Asterisks indicate compounds not confirmed by the criteria. Bars to the left of 0 on the x-axis indicate metabolites that were downregulated in aged compared to young mice, while bars to the right of 0 on the x-axis indicate metabolites that were upregulated in aged compared to young mice.

[0048] Figures 3e-3f are sets of dot plots of CML (Figure 3e) and TMAO (Figure 3f), respectively, quantified by untargeted metabolomics of serum in a human aging cohort (where detectable) from the TwinsUK databank. Data are shown from 3. Median line; best fit values ​​and error bars (95% confidence interval (CI)) for slope and intercept, au, arbitrary units. Exact P values ​​are indicated in the figures.

[0049] Figure 3g is a heatmap depicting a subset of metabolites from targeted metabolomics of brains from young adult and aged SPF and GF mice (young adult, n=5; aged, n=8 each). Each column represents data from one animal and each row represents a metabolite.

[0050] Figures 4a-4l show that CML contributes to microbiota-mediated microglial aging. Schematic diagram, set of graphs, and photomicrographs, respectively. Figure 4a is a schematic of metabolite treatment in young adult SPF mice with daily intraperitoneal administration of CML, TMAO, sodium acetate, or sodium propionate for 2 weeks. Figure 4b is a bar graph showing quantification of ROS. Figure 4c is a bar graph showing mitochondrial activity. Figure 4d is a bar graph showing ATP levels. In Figures 4b-d, each point represents one microglia. In Figures 4b-4d, each point represents one mouse and is plotted relative to vehicle-treated mice (n=4). Data represent mean + sem.

[0051] Figure 4e is a bar graph showing quantification of CML by targeted metabolomics in the brain. Data are shown as mean + sem from SPF and GF mice (young adult, n = 5: aged, n = 8 each). Figure 4f is a bar graph showing brains of vehicle-treated or CML-injected young adult mice (n = 5).

[0052] Figure 4g is a heatmap showing DEGs in microglia from CML and vehicle-injected mice. Each column is a biological replicate and each row is a gene. DEGs (Wald test Padj<0.05 absolute fold change>1.5). Upregulation is hashed grey, downregulation is not hashed grey. Figure 4h is a volcano plot of CML-specific (labeled) genes in SPF and GF microglia from aged mice (dots).

[0053] Figure 4i shows immunofluorescence staining images of CML, Iba-1, and DAPI in the mouse cerebral cortex of young adult and aged SPF and GF mice. Scale bars, 50 μm (overview) and 10 μm (inset).

[0054] Figure 4j is a graph showing the percentage of CMLIba-1 positive cells in the mouse cerebral cortex of young adult and aged SPF (n=9.8) and GF (n=9,9) mice. Data are shown as mean + sem.

[0055] Figure 4k: Linear regression plot of the percentage of CMLIba-1 positive cells in the human cerebral cortex versus age. Each point represents one individual (n=43; Pearson correlation coefficient: r=0.5793, R2=0.3356, P<0.001). Males, n=23, light grey. Females, n=20, dark grey. Age, 1-88 years. Median line: best fit values ​​for slope and intercept. Dark grey lines: 95% confidence interval.

[0056] Figure 4l shows immunofluorescence staining images of CML, Iba-1, and DAPI in human cerebral cortex. Scale bars, 50 μm (outline) and 10 μm (inset). White dashed boxes indicate cell bodies. Statistics: in Fig. 4f, two-tailed Mann-Whitney U test; in Fig. 4G, two-tailed Pearson correlation analysis; in Figs. 4b-4d, one-way ANOVA followed by Dunnett's post-hoc test; in Figs. 4e, 4j, two-way ANOVA followed by Tukey's post-hoc test (*P<0.05, **P<0.01, ***P<0.001), and P values ​​are indicated in the figures.

[0057] Figures 5a-5j are a set of bar graphs and schematic diagrams, respectively, showing that aging-induced breakdown of the gut-blood barrier leads to a surge in CML. Figure 5a is a bar graph showing quantification of CML by targeted metabolomics (LC-MS) in fresh feces from aged SPF and GF mice (n=5). Figures 5b-5c are a set of bar graphs showing intestinal permeability, measured by the percentage of FITC-labeled dextran (4 kDa) that translocated to the circulation after oral ingestion, in young and aged mice housed under SPF (n=5,5) or GF (n=9,4) conditions (Figure 5b) and in young and aged GF mice that received young or aged fecal microbiota transplant (FMT) (n=8) (Figure 5c). Figure 5d is a bar graph showing the difference in CML that translocated to the circulation 4 hours after oral administration in young adult and aged mice housed under SPF or GF (n=5) conditions.

[0058] Figure 5e is a schematic diagram. 18-month-old SPF mice were orally administered vehicle (20% hydroxypropyl-p-cyclodextrin, 1xPBS), EA or IAP every 3 days for 10 weeks (n=4). Figure 5f is a bar graph showing intestinal permeability measured by the percentage of fluorescent FITC-dextran (4kDa) transferred to the circulation after oral administration. Figure 5g is a bar graph showing quantification of CML by targeted metabolomics (LC-MS) in the brain. Figure 5h is a bar graph showing quantification of relative mean fluorescence intensity of CellROX probe signal. Figure 5i is a bar graph showing quantification of relative cellular ATP. Statistics: For Figures 5a, 5c, two-tailed Mann-Whitney U test. For Figures 5f-5i, one-way ANOVA with Dunnett's post-hoc test. For Figures 5b and 5d, two-way ANOVA followed by Tukey's post-hoc test (*P<0.05, **P<0.01, ***P<0.001). P values ​​are indicated in the figures. Data are presented as mean + sem.

[0059] Figure 6a-6h are a series of images and bar graphs showing that the microbiota induces age-related differences in microglial morphology but does not affect cell density. Figure 6a contains images showing immunohistological detection of Iba-1 positive microglia in the cerebral cortex of young adult and aged SPF and GF mice. Scale bar, 20 μm.

[0060] Figure 6b is a bar graph summarizing the density of microglia in the cerebral cortex. SPF (n=9,8) and GF (n=9,8).

[0061] Figure 6c shows representative 3D reconstructions of cortical microglia from all groups (Scale bar, 10 μm).

[0062] Figures 6d-6h are bar graphs showing the semi-automated quantification of cell morphology based on IMARIS. Each graph shows the total branch length (gm; Figure 6d), total branch area (μm 2 , Fig. 6e), number of branch points (Fig. 6f), and cell body volume (μm 3, Fig. 6g), and cell body sphericity (Fig. 6h). Each symbol represents the mean value measured for at least 4 cells per mouse. Data are representative of two independent experiments including young adult and aged mice; SPF (n=8,8) and GF (n=8,8). Statistical analysis: Fig. 6b-6h, 2-way ANOVA followed by Tukey's post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ns=not significant). Data are presented as mean+SEM. P values ​​are indicated in the figures.

[0063] Figures 7a-7b are a series of graphs showing the gating strategy for flow cytometry and purity of the MACS separation. Figure 7a is a series of graphs showing the cell sorting strategy for RT-qPCR and RNA-sequencing, (1) showing the results when gating on myeloid cells by size and granularity, (2) and (3) showing the results when including only single cells, (4) showing the results when gating on live and lineage cells by CD3, CD19, CD45R, Ly6C and Ly6G, respectively, and Fixable Viability Dye eFluor® 780 negative to exclude T cells, B cells, monocytes, and granulocytes, respectively, and (5) showing the results when gating on microglia by CD45Int and positive CD11b.

[0064] Figure 7b is a graph showing the purity of cells used for the cellular ATP assay. Microglial cells were isolated by Percoll separation (inset 1) and enriched using the CD11b MACS cell separation system (Miltenyi Biotec, USA; inset 2), and the final result is shown (inset 3), with each point representing one mouse. Data are shown as mean ± SEM.

[0065] 8a-8d show the transcriptional profiles of microglia from GF and SPF mice of both sexes.

[0066] Figure 8a is a heatmap of genes (normalized) specific to different types of immune cells to show the purity of sorted cells, Figure 8b is a heatmap showing Wald clustering between samples, and Figure 8c is a heatmap of all genes in the eigengene module.

[0067] Figure 8d is a heatmap of genes in metabolite-related module eigengenes ME10. Z-scores were calculated as normalized counts.

[0068] Figures 9a-9h are a series of photomicrographs and bar graphs showing age-related mitochondrial physiology in microglia from male and female SPF and GF mice, respectively. Figure 9a is a representative electron micrograph of healthy and abnormal mitochondria from cerebral cortical microglia. Figure 9b is a bar graph showing quantification of mitochondrial area per microglia. Figure 9c is a bar graph showing mitochondrial number per microglia. Figures 9b and 9c are a series of bar graphs generated from aged SPF and GF mice (n=8). Figure 9d is a bar graph showing microglial Hifla mRNA expression based on RNA sequencing analysis (normalized gene counts). Figure 9e is a bar graph showing microglial Hifla mRNA expression by RT-qPCR from young adult and aged SPF (n=8,8) and GF mice (n=7,10). Figure 9f is a bar graph showing mitochondrial mass (MitoTracker Green MFI) of young adult and aged SPF and GF mice. Figure 9g is a bar graph showing mitochondrial membrane potential (ΔΨm) (TMRM staining MFI) of young adult and aged SPF and GF mice. Figure 9h is a bar graph showing quantification of cellular ATP of male young adult and aged SPF and GF mice. Data were generated from young adult and aged mice. Statistics: For Figures 9f and 9g, SPF (n=17,14) and GF (n=9,13); for Figure 9h, SPF (n=23,17) and GF (n=14,11); for Figures 9b-9h, data are shown as mean+sem. Statistical analysis for Figures 9b and 9c: Mann-Whitney U test (two-tailed) and for Figures 9e-9h: two-way ANOVA followed by Tukey's post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ns=not significant). P values ​​are indicated in the figures.

[0069] Figures 10a-10f are a series of bar graphs showing that CML regulates metabolites in macrophages. Figures 10a-10b are graphs showing pathway enrichment analysis of well-abundant metabolites in serum (Figure 10a) and brain (Figure 10b) of aged mice (plotted are top 15 enriched pathways). Color scale (black to light gray), ratio of total number of metabolites detected in each pathway to number of significant metabolites. Dot size reflects abundance of significant metabolites in each pathway. Pathway enrichment analysis was performed automatically using Metabolon's client portal.

[0070] FIG. 10c is a bar graph showing the percentage of healthy and abnormal mitochondria in the total mitochondrial count in cerebral cortical microglia from young adult mice (n=5) treated ip with vehicle or CML.

[0071] Figures 10d and 10e are bar graphs showing intracellular ROS (Figure 10d) and mitochondrial activity (Figure 10e), respectively, of bone marrow derived macrophages (BMDMs) cultured in serum-free medium 6 hours prior to the experiment. Cells were incubated with increasing concentrations of CML for 48 hours and then harvested and measured. Each point represents a biological replicate (n=3).

[0072] Figure 10d is a bar graph showing quantification of relative MFI of CellROX probe signal. Figure 10e is a bar graph showing mitochondrial activity expressed as mitochondrial membrane potential (ΔΨm) (TMRM staining MFI) normalized to mitochondrial mass (MitoTracker Green MFI). Figure 10f is a principal component analysis (PCA) of the transcriptome (normalized gene counts) of microglia isolated from young adult mice treated intraperitoneally with vehicle or CML. Statistics for Figures 10c-10f: Data are shown as mean + SEM. Each point represents one mouse. Statistical analysis: 2-way ANOVA with Sidak's multiple test for Figure 10c, 1-way ANOVA with Dunnett's post hoc test for Figures 10d and 10e (***p<0.001, ns=not significant). P values ​​are indicated in the figures.

[0073] Figures 11a-11f are graphs showing age-dependent changes in gut microbiota composition. Figure 11a is a PCA plot (β diversity) and Figure 11b is a Shannon and Simpson α diversity plot of gut microbiota indices. To determine if this was statistically significant, a non-parametric Mann-Whitney U test (two-tailed) was used to compare samples; Adonis (analysis of variance using distance matrices) from the vegan package was used to assess the effect of group on β diversity.

[0074] Figure 11c is a graph showing the relative abundance of gut microbiota composition profile at phylum level in male mice of different ages (each color represents one bacterial phylum; the figure key is spatially arranged so that each color represents the bacterial phylum located adjacent to its position on the graph). Figure 11d is a bar graph showing the Firmicutes / Bacteroidetes ratio (F / R) in fecal samples. Figure 11e is a bar graph showing the relative abundance of Lachnospiraceae. Figure 11f is a series of graphs showing the relative abundance of genera differentially expressed with age. Taxonomic differences at phylum and genus levels between test groups are identified using the "Multivariate Analysis with Linear Models" (MaAsLin) R package. Statistics: Figures 11a-11f are a series of graphs showing data from young adult and aged male mice (n=5,10) housed under SPF conditions, with each point representing data from one animal. Figures 11d and 11e show data as mean + SEM. Figures 11b and 11f are box plots, with the central line representing the median and the upper and lower boundaries of the box corresponding to the first and third quartiles (25th and 75th percentiles). The upper whiskers extend from the hinge to the highest value within 1.5 times the interquartile range (IQR) of the respective boundaries, and the lower whiskers extend from the respective boundaries to the lowest value within 1.5 times the IQR of the boundaries. The IQR is the distance between the first and third quartiles. The statistic used in Figures 11d and 11e is the Mann-Whitney U test (two-tailed).

[0075] Figures 12a-12e are a series of bar graphs showing age-related microglial CML accumulation via the gut. Figure 12a is a bar graph showing targeted metabolomics (LC / MS) of CML translocated into the circulation 4 hours after oral ingestion in young adult and aged mice (n=5) housed under SPF or GF. Light grey; pre-oral administration, dark grey; 4 hours after oral administration. Each point represents an individual measurement for one mouse.

[0076] Figures 12b-12e are graphs and photomicrographs of data from young adult and aged mice injected with vehicle or CML (intraperitoneally (ip) or orally (og) (n=4 each). Each point represents one mouse. Figure 12b is a bar graph showing the percentage of quantified CML Iba-1 positive cells in the cerebral cortex. Figure 12c is an image of CML, Iba-1, and DAPI immunofluorescence labeling in mouse cerebral cortex. Scale bars, 50 μm (overview) and 10 μm (inset). Figure 12d is a graph showing quantification of associated cellular ROS probe signal by determining MFI. Figure 12e is a graph showing quantification of associated cellular ATP. Statistics: For Figures 12a, 12b, 12d, and 12e, each point represents one mouse. Data are presented as mean + SEM. Statistical analysis for Figures 12b, 12d, and 12e) Two-way ANOVA followed by Tukey's post-hoc test (*P<0.05, **P<0.01, ***P<0.001, ns=not significant). P values ​​are indicated in the figures.

[0077] 1.Definition As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural reference language unless the context clearly dictates otherwise. Thus, for example, reference to an "intestinal barrier function enhancer" may include a mixture of two or more such intestinal barrier function enhancers.

[0078] As used herein, the term "and / or" in reference to two or more referenced objects includes each of the referenced objects individually and various combinations of the two or more referenced objects, unless otherwise understood from context and usage. As used herein, unless specifically indicated otherwise, the word "or" is used in the inclusive sense of "and / or" and not in the exclusive sense of "either / or."

[0079] Use of the terms "include," "includes," "including," "have," "has," "having," "contain," or "containing," including their grammatical equivalents, should generally be understood as open-ended and open-ended and, for example, does not exclude additional, unrepeated elements or steps unless specifically stated otherwise or understood from its grammatical equivalents.

[0080] When the term "about" is used before a quantitative value, the present disclosure also includes the specific quantitative value itself, unless specifically stated otherwise. As used herein, the term about refers to a ±10% variation from the nominal value, unless otherwise indicated or inferred.

[0081] As used herein, the terms "administering" and "administration" refer to any method of providing an agent (e.g., an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis) to a subject. Such methods include, but are not limited to, oral administration, transdermal administration, inhalation, nasal administration, topical administration, intravaginal administration, intraocular administration, intraaural administration, intracerebral administration, spinal administration, intracerebral fluid administration, rectal administration, and parenteral administration, including injectable administration, such as intravenous administration, intraarterial administration, intramuscular administration, subcutaneous administration, etc. Administration can be continuous or intermittent. In some cases, an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis can be administered for therapeutic purposes. In other cases, an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis can be administered prophylactically, such as for the prevention of a disease or condition in a subject, or for the improvement of one or more immune cell (e.g., microglia) functions in a subject (e.g., the brain of a subject).

[0082] As used herein, the term "effective amount" or "amount effective" or "therapeutically effective amount" refers to an amount sufficient to achieve a desired result (e.g., a therapeutic benefit) or to have an effect on an undesirable condition. For example, a "therapeutically effective amount" of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis may refer to an amount sufficient to achieve a desired result or affect a disease in a subject. Alternatively or additionally, a "therapeutically effective amount" of an intestinal barrier function enhancer may refer to an amount of an intestinal barrier function enhancer for reducing or eliminating intestinal microbiota dysbiosis sufficient to reduce the level and / or activity of CML, CML precursors, CML metabolites and / or CML analogs in a subject (e.g., in a biological sample obtained from the subject) or to improve one or more functions of immune cells (e.g., microglia) in a subject (e.g., in the brain of the subject) to which the intestinal barrier function enhancer and / or the agent for reducing or eliminating intestinal microbiota dysbiosis is administered. A particular therapeutically effective dose level for a particular subject will depend on a variety of factors, including: the subject's age, weight, general health, sex, diet, ethnicity, and / or geographic location; time of administration; route of administration; excretion rate of the intestinal barrier function enhancer used; duration of treatment; drugs used in combination or simultaneously with the particular intestinal barrier function enhancer used, and similar factors known in the medical art. For example, it is within the skill of the art to start the dosage of a therapeutic agent at a level lower than that required to obtain the desired therapeutic effect, and gradually increase the dosage until the desired effect is obtained. If necessary, the effective daily amount can be administered in multiple doses. Thus, a single-dose composition can contain such amounts or submultiples thereof to make up the daily dosage. Dosage can be adjusted by the individual physician in the event of any contraindications. Dosage can vary and can be administered once or multiple times daily, for one or several days.In some embodiments, the intestinal barrier function enhancer and / or the agent for reducing or eliminating intestinal flora dysbiosis can be administered in a prophylactically effective amount.

[0083] As used herein with respect to a given parameter, the term "elevated level" refers to a level that is detectably higher (e.g., about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 85-95%, or more; e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more) as compared to a reference level. For example, as used herein, an elevated level of CML, CML precursors, CML metabolites, or CML analogs in a subject can refer to a level that is detectably higher (e.g., about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 85-95% or more; e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more) compared to a baseline level of CML, CML precursors, CML metabolites, or CML analogs. For example, an elevated level of one or more functions of immune cells (e.g., microglia) in a subject (e.g., the subject's brain) can refer to a detectably higher (e.g., about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, 85-95%, or more; e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99%, or more) compared to a baseline level of one or more functions of immune cells (e.g., microglia) in a subject (e.g., the subject's brain).In certain embodiments, an increased level of intestinal barrier permeability may refer to a level of intestinal barrier permeability in a subject that is detectably higher (e.g., about 5-10%, 10-20%, about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 85-95% or more; e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more of the intestinal barrier permeability in the subject compared to the baseline level of intestinal barrier permeability in a control subject).

[0084] In certain embodiments, an elevation or increase is from about 1% to about 300%, from about 1% to about 280%, from about 1% to about 260%, from about 1% to about 240%, from about 1% to about 220%, from about 1% to about 200%, from about 1% to about 180%, from about 1% to about 160%, from about 1% to about 140%, from about 1% to about 120%, from about 1% to about 100%, from about 1% to about 80%, from about 1% to about 60%, from about 1% to about 40%, from about 1% to about 20%, from about 20% to about 300%, from about 20% to about 280%, from about 20% to about 260%, from about 20% to about 240%, from about 20% to about 2 ...300%, from about 20% to about 300%, from about 20% to about 300%, from about 20% to about 300%, from about 20% to about 300%, from about 20% to about 300%, from about 20% to about 300%, from % to about 200%, about 20% to about 180%, about 20% to about 160%, about 20% to about 140%, about 20% to about 120%, about 20% to about 100%, about 20% to about 80%, about 20% to about 60%, about 20% to about 40%, about 40% to about 300%, about 40% to about 280%, about 40% to about 260% , about 40% to about 240%, about 40% to about 220%, about 40% to about 200%, about 40% to about 180%, about 40% to about 160%, about 40% to about 140%, about 40% to about 120%, about 40% to about 100%, about 40% to about 80%, about 40% to about 60%, about 60% to about 300%, about 60% to Approximately 280%, approximately 60% to approximately 260%, approximately 60% to approximately 240%, approximately 60% to approximately 220%, approximately 60% to approximately 200%, approximately 60% to approximately 180%, approximately 60% to approximately 160%, approximately 60% to approximately 140%, approximately 60% to approximately 120%, approximately 60% to approximately 100%, approximately 60% to approximately 80%, approximately 80% to approximately 300% , about 80% to about 280%, about 80% to about 260%, about 80% to about 240%, about 80% to about 220%, about 80% to about 200%, about 80% to about 180%, about 80% to about 160%, about 80% to about 140%, about 80% to about 120%, about 80% to about 100%, about 100% to about 300%, about 1 00% to approx. 280%, approx. 100% to approx. 260%, approx. 100% to approx. 240%, approx. 100% to approx. 220%, approx. 100% to approx. 200%, approx. 100% to approx. 180%, approx. 100% to approx. 160%, approx. 100% to approx. 140%, approx. 100% to approx. 120%, approx. 120% to approx. 300%, approx. 120% to approx. 280%, about 120% to about 260%, about 120% to about 240%, about 120% to about 220%, about 120% to about 200%, about 120% to about 180%, about 120% to about 160%, about 120% to about 140%, about 140% to about 300%, about 140% to about 280%, about 140% to about 260%,About 140% to about 240%, about 140% to about 220%, about 140% to about 200%, about 140% to about 180%, about 140% to about 160%, about 160% to about 300%, about 160% to about 280%, about 160% to about 260%, about 160% to about 240%, about 160% to about 220%, about 160% to about 200%, about 160% to about 180%, about 180% to about 300%, about 18% to about 280%, about 180% to about 260%, about 180% to about 240%, about 180% to about 220%, about It can be expressed as an increase of 180% to about 200%, about 200% to about 300%, about 200% to about 280%, about 200% to about 260%, about 200% to about 240%, about 200% to about 220%, about 220% to about 300%, about 220% to about 280%, about 220% to about 260%, about 220% to about 240%, about 240% to about 300%, about 240% to about 280%, about 240% to about 260%, about 260% to about 300%, about 260% to about 280%, about 280% to about 300%).

[0085] As used herein, "gut barrier function enhancer" refers to an agent that reduces the passage of CML, CML precursors, CML metabolites, and / or CML analogs from the intestine of a subject, and the rate of accumulation of CML, CML metabolites, CML precursors, and / or CML analogs from the intestine into the tissues or body fluids of a subject over time, either directly or indirectly through intermediates.Non-limiting examples of gut barrier function enhancers are intestinal alkaline phosphatase (IAP), polyphenols (e.g., ellagic acid (EA) and lipoteichoic acid), metformin, urolithin A, butyric acid, glutamine, obeticholic acid (OCA), divertin, curcumin, spermidine, glutamine, or AMP-activated protein kinase (AMPK), or derivatives thereof. For example, IAP is an intestinal barrier function enhancer that directly reduces the passage of CML, CML metabolites, CML precursors, and / or CML analogs from the intestine of a subject, and the accumulation rate of CML, CML metabolites, CML precursors, and / or CML analogs from the intestine to the tissues or body fluids of the subject. EA is an example of an intestinal barrier function enhancer that indirectly (e.g., via an intermediate) reduces the passage of CML, CML metabolites, CML precursors, and / or CML analogs from the intestine of a subject, and the accumulation rate of CML, CML metabolites, CML precursors, and / or CML analogs from the intestine to the tissues or body fluids of the subject. EA is believed to reduce the expression of claudin-4, -7, and -15, which form cell pores, via myosin light chain 2 (MLC2) signaling, and therefore indirectly enhances intestinal barrier function.

[0086] As used herein, "gut microbiota dysbiosis" refers to an imbalance in the relative abundance or presence of microorganisms (e.g., beneficial and / or pathogenic microorganisms) in the gut of a subject, which can result in various symptoms, including, for example, abdominal bloating, flatulence, cramps, inflammation associated with loss of intestinal permeability, hypoplasia of mucosal surfaces, and insufficient recycling of nutrients for buffering capacity. Dysbiosis includes the loss of beneficial microorganisms and / or the expansion of pathogenic microorganisms (e.g., pathobionts). Dysbiosis is believed to induce pro-inflammatory effects and immune dysregulation associated with various disease states. As used herein, "agents for reducing or eliminating gut microbiota dysbiosis" refers to agents that improve the dysregulation of gut microbiota that occurs in gut microbiota dysbiosis, either directly or indirectly through intermediates. Non-limiting examples of agents for reducing or eliminating intestinal microbiota dysbiosis (e.g., directly or indirectly) include, but are not limited to, IAP and EA, biotics, prebiotics, probiotics and postbiotics. For example, probiotics are agents for directly reducing or eliminating intestinal microbiota dysbiosis, since they directly affect the intestinal microbiome by specifically delivering beneficial microorganisms to the digestive tract. Exemplary probiotics include bacteria belonging to the genera Lactobacillus, Bifidobacterium, and Streptococcus. Prebiotics promote the growth of certain beneficial bacterial species that elicit health benefits, and exemplary prebiotics include lipoteichoic acid and polyphenols. Postbiotics are, for example, metabolic products, fermentation products, minerals (e.g., zinc and selenium), trace elements, micronutrients, cell surface proteins, and organic acids produced by the microbiome during its life cycle, and are characterized as products that contribute to environmental ubiosis. Postbiotics are agents that indirectly shape the structure of the microbiota, thereby indirectly reducing or eliminating intestinal microbiota dysbiosis.

[0087] As used herein, the terms "neurodegenerative disease" or "neurodegenerative disorder" are used interchangeably and refer to one or more conditions of a heterogeneous group of disorders characterized by progressive degeneration of the structure and / or function of the central or peripheral nervous system. Neurodegenerative diseases encompass a variety of conditions resulting from progressive damage to cellular and nervous system connections essential for mobility, coordination, strength, sensation, and cognition. Common neurodegenerative diseases include, but are not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, progressive supranuclear palsy, spinal muscular atrophy, multiple system atrophy, ataxia, vascular dementia, or other dementias.

[0088] As used herein, the term "pharmaceutical composition" refers to a combination of an active agent (e.g., an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis) with an inert or active carrier, making the composition particularly suitable for diagnostic or therapeutic use in vivo or ex vivo. As used herein, the term "pharmaceutical composition" can be a formulation comprising the disclosed agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis in a form suitable for administration to a subject. In one embodiment, the pharmaceutical composition is in bulk or unit dosage form. The unit dosage form is in any of a variety of forms, including, for example, a capsule, a drip bag, a tablet, a single pump of an aerosol inhaler, or a vial. The amount of active ingredient (e.g., a formulation of the disclosed agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, or a salt, hydrate, solvate, or isomer thereof) in a unit dose of the composition is an effective amount and can vary depending on the particular treatment involved. Those skilled in the art will understand that it may be necessary to routinely vary the dosage depending on the age and condition of the subject. The dosage also depends on the route of administration. For example, various routes are possible, such as oral, pulmonary, rectal, parenteral, transdermal, subcutaneous, intravenous, intramuscular, intraperitoneal, inhalation, buccal, sublingual, intrapleural, intrathecal, and nasal. The dosage form for topical or transdermal administration of the intestinal barrier function enhancer includes powder, spray, ointment, paste, cream, lotion, gel, solution, patch, and inhalant. In one embodiment, the intestinal barrier function enhancer is mixed under sterile conditions with a pharma- ceutically acceptable carrier and necessary preservatives, buffers, or propellants.

[0089] As used herein, the term "pharmaceutical acceptable carrier" refers to buffers, carriers, and excipients that are suitable for use in contact with human and animal tissues without excessive toxicity, irritation, allergic reactions, or other problems or complications, commensurate with a reasonable benefit / risk ratio. Pharmaceutically acceptable carriers include any of the standard pharmaceutical carriers, such as phosphate buffers, water, emulsions (e.g., oil / water or water / oil emulsions, etc.), and various types of surfactants. The composition may also include stabilizers and preservatives. For examples of carriers, stabilizers, and adjuvants, see, for example, Adeboye Adejare, Remington. The Science and Practice of Pharmacy (23d ed.2020). Pharmaceutically acceptable carriers include buffers, solvents, dispersion media, coatings, isotonic and absorption delaying agents, etc., that are compatible with pharmaceutical administration. The use of such media and agents for pharmaceutical active substances is known in the art.

[0090] The terms "pharmacologically effective amount", "pharmacologically effective amount", "physiologically effective amount", or "effective amount" of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis are used interchangeably and refer to the amount of a bioactive agent or combination of bioactive agents present in one or more pharmaceutical compositions as described herein that is necessary to provide a desired level of the active agent or agents in the bloodstream or at the site of action (e.g., hepatic system, renal system, circulatory system, pulmonary, gastrointestinal system, colonic system, etc.) of a treated subject to impart an expected physiological response when such composition is administered.

[0091] As used herein, the term "prevent" or "preventing" refers to making impossible, avoiding, removing, forestalling, hindering, or preventing something from happening, especially by prior action. It should be understood that when "reduce," "inhibit," or "prevent" is used herein, the use of the other two words is also expressly intended unless specifically indicated otherwise. The term "prevention" does not require 100% elimination of the possibility of an event. Rather, it indicates that the likelihood of the occurrence of an event has been reduced in the presence of a compound or method described herein. In various aspects, the term covers any treatment of a subject, including a mammal (e.g., human), including: (i) preventing the occurrence of a disease in a subject who may have a predisposition to suffering from a disease, but has not yet been diagnosed as suffering from a disease; (ii) inhibiting a disease, such as suppressing the occurrence of a disease or reducing the rate of progression of a disease; or (iii) alleviating a disease, such as causing regression of a disease.

[0092] The terms "reduce" or "reducing" or "decrease" or "decreasing" or "alleviate" or "alleviating" as used herein with respect to a parameter or rate refer to a detectable change in the parameter or rate such that the parameter or rate is smaller (e.g., about 5-10%, 10-20%, 20-30%, 30-40%, 40-50%, 50-60%, 60-70%, 70-80%, 80-90%, or 85-95%; e.g., about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 97%, 99% or more) as compared to a control.

[0093] As used herein, depending on the context, "control" refers to a sample that has not been exposed to the compositions and / or methods described herein, or a control subject. "Control subject" refers to a subject that has not received the compositions and / or methods disclosed herein. As used herein, "test subject" refers to a subject that has received or will receive the compositions and methods described herein. As used herein with reference to a parameter, a "suitable control" may refer to the parameter in a control subject (e.g., a test subject before receiving a treatment described herein; or a group of subjects different from the subject or with a similar condition to the subject who have not received a treatment described herein). For example, as used herein with respect to the level of CML, CML precursors, CML metabolites, or CML analogs, a "suitable control" may refer to the level of CML, CML analogs, CML precursors, or CML metabolites in a subject (e.g., a subject before receiving a treatment described herein; or a group of subjects different from the subject or with a similar condition to the subject who have not received a treatment described herein).

[0094] As used herein with respect to a parameter, a "reference level" may refer to an established normal level of the parameter or an established standard control. For example, as used herein with respect to the level of CML, CML precursor, CML metabolite, or CML analog, a reference level may refer to the level of CML, CML precursor, CML metabolite, or CML analog in a subject or group of subjects who do not show symptoms of neurodegenerative disease or cognitive disorder and / or are not at increased risk of developing neurodegenerative disease or cognitive disorder.

[0095] The terms "subject", "individual" and "patient" are used interchangeably and refer to an organism treated by the methods and / or compositions described herein. Such organisms preferably include, but are not limited to, mammals (e.g., murine, ape, horse, bovine, porcine, canine, feline, etc.), and more preferably, include humans. The term does not indicate a particular age or sex. Thus, adult and neonatal subjects are intended to be subjects, whether male or female. For example, the subject may be a human. In particular, the subject may be a human having a neurodegenerative disease or at increased risk of developing brain dysfunction, a subject having impaired neurological function, having a neurodegenerative disease (e.g., a subject previously identified or diagnosed as having a neurodegenerative disease), or a subject identified as at increased risk of developing a neurodegenerative disease, a subject having a cognitive impairment, or a subject identified as at increased risk of developing a cognitive impairment.

[0096] As used herein, the term "treating" includes any effect, including, for example, alleviating, reducing, modulating, ameliorating, or eliminating, that results in improvement of a condition, disease, disorder, or the like, or the amelioration of a symptom thereof.

[0097] As used herein, the term "treatment" refers to the medical management of a subject with the intent of curing, ameliorating, stabilizing, or preventing a disease or disorder. In certain embodiments, the term refers to improving the function of one or more immune cells (e.g., microglia) in a subject (e.g., in the brain of a subject). The term includes active treatment, causal treatment (e.g., treatment directed at the cause of a disease), palliative treatment (e.g., treatment aimed at alleviating symptoms or complications associated with a disease), preventative treatment (e.g., treatment aimed at delaying, minimizing, slowing the rate of progression, or partially or completely suppressing a disease or the onset of a disease), and supportive treatment (e.g., treatment employed to complement another treatment). Treatment also includes curing, inhibiting, alleviating, mitigating, and / or ameliorating one or more symptoms and / or complications associated with a disease. Treatment also includes preventing and / or slowing the rate of progression and / or delaying the onset of symptoms and / or complications associated with a disease. Treatment also includes reducing the spread of the disease; delaying or slowing or reducing the rate of progression of the disease; ameliorating or alleviating the disease; and remission (whether partial or total), whether detectable or undetectable. "Ameliorating or alleviating" a disease means that the extent and / or undesirable clinical symptoms of the disease are reduced and / or the time course or rate of progression is slowed or prolonged compared to the extent or time course in the absence of treatment. Treatment does not require complete amelioration of symptoms or complications associated with the disease, but encompasses embodiments in which symptoms and / or underlying risk factors of the disease are alleviated. Those in need of treatment include those already with the disease, those at risk of having the disease, or those in whom the condition or disorder is to be prevented.

[0098] Throughout this specification, when compositions are described as having, comprising, or consisting of certain components, or when processes and methods are described as having, comprising, or consisting of certain steps, it is further contemplated that there are compositions of the invention that consist essentially of or consist of the recited components, and that there are processes and methods according to the invention that consist essentially of or consist of the recited processing steps.

[0099] In this application, when an element or component is described as being included in and / or selected from a list of recited elements or components, it is to be understood that the element or component can be any one of the recited elements or components, or the element or component can be selected from a group consisting of two or more of the recited elements or components.

[0100] Furthermore, it should be understood that the elements and / or features of the methods described herein, whether express or implied herein, can be combined in various ways without departing from the spirit and scope of the present invention. For example, where reference is made to a particular compound, that compound may be used in various embodiments of the compositions disclosed herein and / or in the methods disclosed herein, unless otherwise understood from the context. In other words, within this application, the embodiments have been described and referenced in a manner that allows the application to be written and referenced clearly and concisely, but it is intended and understood that the embodiments may be combined or separated in various ways without departing from the teachings. For example, it will be understood that any feature described and expressed herein is applicable to all aspects of the invention described and expressed herein.

[0101] The phrase "at least one of" should be understood to include each of the objects listed following the phrase individually, and various combinations of two or more of the listed objects, unless otherwise understood from context and usage.

[0102] It should be understood that the order of steps or order for performing certain actions is immaterial so long as the invention remains operable. Moreover, two or more steps or actions may be conducted simultaneously.

[0103] Any examples herein, or the use of exemplary language, such as "such as" or "including," are intended merely to better describe the invention and do not limit the scope of the invention unless claimed. No language in the specification should be construed as indicating any non-claimed element as essential to the practice of the invention. II. General Findings and Observations

[0104] The present disclosure is based, in part, on the discovery that the metabolite N6-carboxymethyllysine (CML) produced by the gut microbiota drives age-related oxidative stress and mitochondrial damage in microglia, and that increased amounts of CML in the brain with age can lead to cognitive and neurodegenerative disorders. Furthermore, it has been discovered that the increase or accumulation of CML in body fluids and tissue samples of subjects with aging may be due to increased intestinal permeability with the progression of aging, which is believed to result in higher levels of CML passing through the intestinal wall and entering the body fluids and tissues of the subject. Based on these discoveries, it is possible to provide therapies to treat or reduce the incidence of neurodegenerative diseases, cognitive disorders, and neurological dysfunction.

[0105] Microglial function declines with age, but prior to this study, the interplay between microglia and gut microbiota during the aging process appears to have been poorly characterized. As disclosed herein, a comparison of the microglial transcriptomes of young adult and aged mice housed under germ-free and specific pathogen-free conditions revealed that gut microbiota influences age-related changes in microglial gene expression. We also found that the absence of gut microbiota reduced oxidative stress and improved mitochondrial dysfunction in brain microglia of aged mice. Unbiased metabolomic analysis of serum and brain tissue revealed that N6-carboxymethyllysine (CML) was accumulated in microglia from aged brains. CML appears to mediate a burst of reactive oxygen species and inhibit microglial mitochondrial activity and ATP storage. An age-dependent increase in CML levels in human serum and brain was also verified. Furthermore, in aged mice, a microbiota-dependent increase in intestinal permeability mediated the increase in CML levels. The studies described here provide insight into how the gut microbiota controls specific characteristics of microglia in aged mice.

[0106] More specifically, as shown diagrammatically in Figure 1a, mice raised under specific pathogen-free (SPF) conditions were found to have higher levels of gut-derived CML in the brain compared to mice raised under germ-free (GF) conditions. As a result, the brains of SPF mice had more reactive oxygen species and lower ATP than those of GF mice. As aging progresses, SPF mice have more microglial activation than GF mice. It was also found that drugs that enhance the intestinal barrier function delay the release of gut-derived CML during the aging process. It was also found that drugs that eliminate aging-associated gut microbiota dysbiosis reduce the amount of gut-derived CML produced during the aging process. These drugs can suppress the development of cognitive impairment and neurodegenerative diseases.

[0107] Gut microbiota alters the transcriptome profile of microglia during aging It has been reported that microglial cell density increases with age in the cerebral cortex (Non-Patent Document 4). An increase in microglial cell density was observed in the cerebral cortex of specific pathogen-free (SPF) and bacteria-free (GF) animals between young adult and aged mice (see, Figs. 6a-6b), but no difference was observed between aged SPF and GF mice (see, Figs. 6a-6b). One of the most prominent and first identified features of aged microglia is their change in morphology. Quantitative morphological reconstruction was performed to determine potential morphological changes in microglia from SPF and GF mice. Microglia from SPF mice showed a decrease in total branch area, total branch length, and number of branch points along with an increase in cell volume (see Figs. 6c-6g), but the sphericity of the cell body did not change between both groups (see Fig. 6h). These data indicate that age affects microglial morphology in SPF mice, whereas microglia remained unchanged and hyperbranched in GF mice.

[0108] To further evaluate microbiota-dependent changes in microglial physiology in the aging brain, we performed RNA-seq on FACS-purified microglia (Figures 7a and 8a) from whole brains of young adult (6-10 weeks old) and aged (96-104 weeks old) male and female mice housed under GF or SPF conditions (Figures 1b and 8b). Differences in gene expression profiles of microglia across both age groups in GF and SPF mice, and transcriptomic differences between microglia isolated from GF and SPF mice were more pronounced at older ages (Figure 1d). Compared with microglia from SPF mice, microglia from GF mice emerged with an age-independent gene expression pattern (microglial GF signature), which included genes related to the cytoskeleton (e.g., Sdc3, Sult1a1, Tuba4a) and immune functions (e.g., Ctse, Ero1lb, Htra3, Kcnma1, Notch4, Nr1d2, Rab4a, Wdfy1) (see Fig. 1e). Furthermore, the microglial GF signature included genes related to the control of mitochondrial function (e.g., B4galnt1, Gpr137b, Gstm1, Mcur1, Mtfp1, Nnt, and Plcd3), indicating the ability of the microbiota to control the metabolic profile of microglia (see Fig. 1e). We next characterized the functional changes in microglial gene expression with respect to age and microbiota using weighted gene co-expression network analysis (WGCNA) (Non-Patent Document 5). Genes that significantly explained more than 50% of the variance (Wald Padj < 0.05) were classified into module-specific genes (MEs) based on their co-expression patterns. Comparing SPF or GF-reared young adult groups (Non-Patent Document 6), slight differences were found in gene networks related to immune function and epigenetic regulation, ME1, ME5, ME6, and ME7, respectively. However, two modules were characteristic of each age group. ME1 and ME4 in the aged SPF group contained genes related to processes such as mitochondrial metabolism and lipid localization, while ME2 and ME6 in the aged GF group contained genes controlling immune response, histone lysine methylation, and cell morphogenesis.

[0109] We next investigated the contribution of the microbiota to age-related ME. Weighted gene co-expression network analysis (WGCNA) revealed that microglia from aged GF mice followed the general aging trend of SPF mice, but at a lower magnitude (see Figures 1f-1g), forming a cluster closer to that of the young adult group (see Figure 8b). For example, ME1 and ME8 genes related to immune responses (e.g., Axl, Crlf2, Tnfsf8, Tnfsf10, Ccl12, Fgr, Il1b, Il6st, Spp1 and Tlr2), interferon signaling (e.g., Cxcl10 / 8, Ifi207, Ifit2 / 8 and Stat1), inflammatory responses (e.g., Cd180, Ldlr, S100a8 and S100a9), and microglial migration (e.g., Ccl12 and Cxcl10) showed specific upregulation in microglia from aged SPF mice, but showed negative or low correlations in both young adult and aged GF mice (see Figure 2a and Figure 8c-d). ME1 and ME4 from the aged SPF group contained genes related to processes such as mitochondrial metabolism and lipid localization, whereas ME2 and ME6 from the aged GF group contained genes controlling immune responses, histone lysine methylation, and cell morphogenesis (Fig. 8c). Genes in ME1 and ME8 related to immune response (e.g., Axl, Crlf2, Tnfsf8, Tnfsf10, Ccl12, Fgr, Il1b, Il6st, Spp1, and Tlr2), interferon signaling (e.g., Cxcl10 8, Ifi207, Ifit2 8, and Stat1), inflammatory response (e.g., Cd180, Ldlr, S100a8, S100a9, etc.), and microglial cell migration (Ccl12, Cxcl10, etc.) showed specific upregulation in microglia of aged SPF mice, but showed negative or low correlation in both young adult and aged GF mice. ME1 and ME8 showed strong correlation in the aged SPF group, including mitochondrial metabolic process, hydrogen peroxide metabolic process, and reactive oxygen metabolic process. ME2, which was highly enriched in aged GF mice, was associated with responses to oxygen-containing compounds (Fig. 8c).Genes included in ME2 include those that regulate intracellular ROS levels, such as Foxp1, Nrf1, and Trp53, and those involved in regulating mitochondrial ROS, such as G6pdx, Pdk2, Stat3, and Ucp2, but are less expressed in microglia from aged SPF mice compared with age-matched GF mice, indicating that ROS levels in aged SPF mice cannot be kept within the optimal intracellular range. Accumulation of ROS in the aging brain is associated with mitochondrial damage and mitochondrial dysfunction (Non-Patent Document 7). In ME3, ME5, and ME9, significant changes were observed in genes related to mitochondrial assembly, carbohydrate metabolism, and oxidative phosphorylation. Expression of these genes was elevated in both SPF and GF mice, but here protective ROS control genes were downregulated, so mitochondrial damage was more pronounced in SPF mice. Furthermore, expression of genes that maintain mitochondrial structure and function was elevated in aged GF mice (Figure 8d). Microglia showed changes in transcriptome profile with age, with divergence dependent on the microbiota.

[0110] Decreased oxidative stress in microglia of aged GF mice A major feature of cellular aging in microglia is increased oxidative stress, which refers to elevated intracellular levels of reactive oxygen species (ROS) (Non-Patent Document 8). By examining the pathways of aging-related modules, several links to microbiota-dependent oxidative stress control in microglia were found. ME1 and ME8, which include mitochondrial metabolic processes, hydrogen peroxide metabolic processes, and ROS metabolic processes, showed strong correlations in the aged SPF group. ME2, which was highly enriched in aged GF mice, was associated with responses to oxygen-containing compounds (see Figures 1f-1g and 2a). To confirm that the expression levels of microglial ROS-related genes were regulated by mouse age and rearing conditions, we selectively analyzed age-related ME1, ME2, and ME8 ROS-related genes. Specific upregulation of several immune activation and ROS-promoting genes, such as Cdkn1a, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, and Tlr5, as well as ROS-responsive genes, such as Axl, Hif1a, Lcn2, Mmp2, Rela, Trex1, S100a8, and S100a9, was observed only in microglia from aged SPF mice (see Fig. 2b). Genes in ME2, including genes controlling intracellular ROS levels, such as Foxp1, Nrf1, and Trp53, and genes involved in mitochondrial ROS regulation, such as G6pdx, Pdk2, Stat3, and Ucp2, were less expressed in microglia from aged SPF mice compared to age-matched GF mice (see Fig. 2b). ROS production in microglia isolated from young adult and aged SPF mice was monitored by CellROX flow cytometry assay, and a significant increase in ROS was observed with age, which was reduced in aged GF mice (see Figure 2c). Activation of inducible nitric oxide synthase (iNOS) appears to be directly related to the generation of excess ROS (Non-Patent Document 9, Non-Patent Document 10). Using immunohistochemistry (IHC), an age-dependent increase in microglial iNOS expression was observed under SPF conditions, but was less pronounced in GF mice (see Figures 2d-2e).

[0111] We investigated how increased ROS affected microglial function and found that changes occurred in genes related to mitochondrial assembly, glucose metabolism, and oxidative phosphorylation (see Figure 2f). Electron microscopy of microglial mitochondria revealed that aged SPF mice had a significantly higher proportion of damaged mitochondria with less distinct or destroyed cristae compared to aged GF mice, although there was no change in the mass or number of mitochondria per microglia. Accumulation of intracellular ROS, which peaked in microglia from aged SPF mice, appears to induce the expression of hypoxia-inducible factor 1 subunit alpha (Hif1a). Mitochondrial dysfunction in the aging brain causes a metabolic shift and is associated with excessive microglial activation. Although young adult mice showed similar Hif1a expression, RNA-seq and reverse transcription-based quantitative PCR (RT-qPCR) showed that Hif1a expression in microglia from aged SPF mice was higher than that from GF mice (see Figure 9d-9e). Oxidative phosphorylation becomes less efficient in cells from aging animals, leading to a decrease in ATP production. The mitochondrial transmembrane potential (ΔΨm) appears to be the main driving force for ATP production. Given the increase in mitochondrial mass with age, mitochondrial activity was plotted as mitochondrial transmembrane potential against mitochondrial mass. Mitochondrial activity showed an age-related decline and a decrease in the intracellular ATP reservoir in SPF mice, whereas both were less pronounced in microglia from GF mice (see Fig. 2i, Fig. 7b, Fig. 9h). Taken together, these data indicate that the microbiota contributes to increased oxidative stress in microglia from aging brains, which is associated with direct damage to mitochondria.

[0112] Age-related accumulation of CML dependent on bacterial flora Short-chain fatty acid (SCFA) concentrations in serum samples from young adult and aged SPF mice were identified using targeted liquid chromatography-mass spectrometry (LC-MS) metabolite analysis (see Figure 3a). Acetate was most abundant in the serum of young adult and aged mice, and acetate and propionate concentrations were higher in the serum of aged mice compared to young adult mice. Butyrate / isobutyrate and valerate / isovalerate were unchanged. In an unbiased screen, we used a non-targeted metabolomics dataset to investigate serum and brain samples from young adult and aged mice raised under SPF conditions (see Figures 3b-3c) (see Non-Patent Document 11). Pathway enrichment analysis revealed some tissue-specific pathway changes. For example, pyrimidine, inositol, carnitine, and some pathways related to amino acid metabolism (e.g., lysine, polyamines, tyrosine) were more affected in the serum of aged mice (see Figure 10a). Vitamin A, tocopherol, purine metabolism, ceramide-related pathways, and pentose phosphate pathways were specifically altered in the brains of aged mice (see Figure 10b). Fatty acid metabolism and advanced glycation end products (AGEs) pathways were commonly altered in both serum and brain samples from aged mice (see Figures 10a-10b). From the metabolites that were significantly upregulated in both serum and brain tissue from aged mice, we were able to identify metabolites that may have been regulated in the gut and reached the brain via the bloodstream. These metabolites included palmitoleic acid (16:1n7), TMAO, 1-oleoyl-2-docosahexaenoyl-glycerophosphorylcholine (18:1 / 22:6), CML, and stachydrine (see Figure 3d). Some of the age-related concentration changes observed in mice were also confirmed in human blood samples. Untargeted metabolomics of serum / plasma from a human aging cohort (TwinsUK databank) recapitulated the age-related changes in CML (see Figure 3e) and TMAO (see Figure 3f) concentrations seen in mice. Targeted metabolomics from brain tissue of young adult and aging SPF and GF mice demonstrated that a functional gut microbiota (e.g., SPF mice) is required for the increase in CML and TMAO in aging mouse brain tissue.Aged GF mice showed only minor changes compared to young adult GF mice (see FIG. 3g).

[0113] CML enhances age-related microglial dysfunction Next, the functional effects of these metabolites on microglia in vivo were evaluated. CML, TMAO, acetate, and propionate were selected for further evaluation. To identify the metabolite responsible for increased ROS production in aged microglia, each metabolite was administered separately to young adult mice. To avoid possible artifacts due to differences in absorption profiles from the intestine to the circulation, young adult mice were intraperitoneally injected with CML, TMAO, sodium acetate, or sodium propionate once daily for 2 weeks (see Figure 4a). TMAO, sodium acetate, and sodium propionate had no effect on intracellular ROS production or on the metabolic function of microglia. However, CML treatment partially reproduced the changes seen in microglia from aged mice.

[0114] CML increased oxidative stress, decreased metabolic activity, and reduced cellular ATP reserves (see Figure 4b-4d). Moreover, CML caused mitochondrial dysfunction by directly damaging the mitochondrial structure of microglia (see Figure 10c). The effects of CML treatment were not limited to microglia, but also adversely affected macrophages. In particular, bone marrow-derived macrophages (BMDMs) showed a dose-dependent increase in oxidative stress and decreased metabolic activity in vitro (see Figure 10d-10e). Circulating CML may originate from the conversion of advanced glycation end products (AGEs) by endogenous Maillard reaction, diet, or gut microbiota. Brain CML levels were increased in aged SPF mice but not in aged GF mice (see Figure 4e). CML was detectable in brain tissue from young adult and aged GF mice at levels similar to those in young adult SPF mice. These results indicate that gut microbiota is required for elevated CML levels in the aging brain, but not for the baseline levels seen in young adult mice. Thus, these results also indicate that the dysregulation of mitochondrial function in microglia after intraperitoneal injection of CML is due to elevated brain CML concentrations, recapitulating the setting in the aging brain (see Figure 4f). RNA-seq analysis of microglia showed that intraperitoneal administration of CML increased the expression of ROS-related genes S100a9 and S100A8, as well as other microbiota- and aging-related genes, such as A430033K04Rik, Chic1, Ltf, Ngp, Pglyrp1, Scai, and Zkscan2 (see Figures 4g-4h and Figure 10f).

[0115] Immunofluorescence staining of CML in cerebral cortical microglia addressed whether microglia are directly targeted by CML and showed that mice housed under SPF and GF conditions had an increased percentage of CML-positive microglia with age. Approximately 30% of microglia in aged SPF mice were CML-positive, whereas microglia in aged GF mice had less accumulation of CML with age (see Figures 4i-4j). Furthermore, the age-dependent increase in CML-positive microglia seen in the mouse cerebral cortex was verified to be present in the human cerebral cortex as well. Human brain tissues ranging from 1 to 88 years of age (total n = 43, 23 males, 20 females) were obtained, and a positive correlation was found between age and the percentage of CML-positive microglia in the human cerebral cortex (r = 0.5793, R 2 = 0.3356, P < 0.001) was observed (Fig. 4k-4l). In mice, RNA-seq analysis of microglia showed that i.p. injection of CML increased the expression of ROS-related genes S100a9 and S100A8, as well as other bacteria- and aging-related genes, such as A430033k04Rik, Chic1, Ltf, Ngp, Pglyrp1, Scai, and Zkscan2. These findings indicate that age-related accumulation of CML may induce metabolic dysfunction in microglia in a direct manner, including increased ROS, which may gradually disrupt brain homeostasis and brain function.

[0116] Aging microbiota promotes CML levels by disrupting the gut-blood barrier Age-dependent gut microbiota changes by 16S ribosomal RNA-seq were characterized based on the finding that differences in CML levels and microglial function, especially in aging, depended on the presence or absence of a microbiota. Differential microbiota profiles of young adult and aged mice were confirmed by β-diversity analysis using the Bray-Curtis dissimilarity index and Shannon and Simpson α-diversity index (see Figure 11a-11b). The gut microbiota of both age groups was dominated by two phyla (see Figure 11c), namely, Firmicutes and Bacteroidetes. It was noted that the relative abundance of Firmicutes and Bacteroidetes changes with age in humans and can be associated with the overall changes in the bacterial profile at different age stages. A significant age-dependent decrease in the ratio of Firmicutes to Bacteroidetes was observed, and the Firmicutes phylum, Lachnospiraceae, was significantly decreased in aged mice (see Figure 11d-11e). In terms of bacterial genera, increases in Turibacter, Alloprevotella, Parasatellella, Bifidobacterium, Macellibacteroides, Alistipes sensu stricto 1, Peptostreptococcaceae incertaesedis, and Parabacteroides were observed in aged mice. This result was in contrast to the decreases in Pantoea, Anoxybacillus, Lachnospiraceae incertaesedis, Curtobacterium, and Acetatifactor in aged mice (see Figure 11f). These results indicate that profiling the microbiota of young adult and aged mice reveals changes at several taxonomic levels. Targeted metabolomics (LC-MS) of fecal CML revealed that feces from aged GF mice contained higher CML levels than those from aged SPF mice, indicating that the microbiota is indirectly involved in the brain accumulation of CML with age (see Figure 5a).

[0117] Aged mice exhibit enhanced intestinal permeability compared to young adult mice (Non-Patent Document 12), a phenomenon that is dependent on the presence of a bacterial flora (Non-Patent Document 13). Enhanced permeability allows metabolites from within the digestive tract to pass more freely through the intestinal epithelium and enter the bloodstream, which may explain the discrepancy in CML levels in the brain and feces. To test this hypothesis, we measured intestinal permeability by quantifying the translocation of FITC-dextran (4 kDa) into the circulation after oral administration. High intestinal permeability was observed in aged SPF mice, and the barrier function of aged GF mice was comparable to that of young adult SPF and GF mice (see Figure 5b). Colonization of young adult GF mice with aged bacterial flora resulted in a bacterial flora-dependent increase in intestinal permeability compared to the permeability observed after administration of young gut bacterial flora to young adult GF mice (see Figure 5c). This was consistent with the observation that translocation of CML into the circulation after oral ingestion was highest in aged SPF mice (see Fig. 5d and Fig. 12a). To assess whether different routes of CML administration affect CML accumulation in microglia, we examined young adult mice administered CML intraperitoneally rather than orally. In such mice, more CML-positive microglia were found in the cerebral cortex (see Fig. 12b-12c). In aged mice, the route of CML administration did not affect the proportion of CML-positive microglia (see Fig. 12b-12c). CML administration by both intraperitoneal and oral routes significantly exacerbated the age-associated increase in intracellular ROS and decline in metabolic function in microglia of aged mice. In young adult mice, such effects were detectable only after intraperitoneal administration of CML (see Fig. 12d-12e). To verify whether the intestinal barrier plays an important role in the accumulation of CML in microglia with aging, aged SPF mice (18 months old) were orally administered ellagic acid (EA) every 3 days for 10 weeks (Non-Patent Document 14). Ellagic acid reduces age-related bacterial flora dysbiosis and induces autophagy in the intestinal epithelium, thereby enhancing the expression of claudins, which are known to cause leaky gut, and intestinal alkaline phosphatase (IAP), an endogenous enhancer of intestinal barrier function, thereby suppressing the accumulation of CML (Non-Patent Document 15, Non-Patent Document 16) (see Figure 5e).Although EA had no direct effect on intestinal permeability, IAP-treated aged mice had a less leaky intestine (Fig. 5f). Both EA and IAP indirectly or directly, respectively, reduced CML accumulation in the brain (Fig. 5g). Microglia from EA- and IAP-treated aged mice showed a significant decrease in intracellular ROS and an increase in ATP levels compared to vehicle-treated aged mice (Fig. 5h-5i). These findings demonstrate that aging-induced changes in the microbiota disrupt the integrity of the intestinal barrier and promote CML accumulation in the brains of aged mice and humans.

[0118] Taken together, these observations provide an approach for identifying subjects at risk for or having developed a cognitive or neurodegenerative disorder. Additionally, these observations provide an approach for treating subjects at risk for or having developed a cognitive or neurodegenerative disorder.

[0119] III. Treatment method Provided herein is a method for reducing the incidence of oxidative stress or mitochondrial dysfunction in microglia, the incidence of mitochondrial dysfunction in microglia, or the incidence of microglial dysfunction in a subject in need thereof.The method comprises administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, thereby reducing the incidence of oxidative stress in microglia, the incidence of mitochondrial dysfunction in microglia, or the incidence of microglial dysfunction in the subject.

[0120] Also provided is a method for treating cognitive impairment or reducing the incidence or aggravation of cognitive impairment in a subject in need thereof. The method comprises administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, thereby treating cognitive impairment or reducing the incidence or aggravation of cognitive impairment in the subject.

[0121] Also provided is a method for treating a neurodegenerative disease or reducing the incidence or progression rate of a neurodegenerative disease in a subject. The method comprises administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, thereby treating the neurodegenerative disease or reducing the incidence or progression rate of a neurodegenerative disease in the subject.

[0122] Also provided is a method for reducing the incidence or aggravation of neurological dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, thereby reducing the incidence or aggravation of neurological dysfunction in the subject.

[0123] Also provided is a method for selecting a subject for treatment with an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, comprising: (a) identifying a subject having an elevated level of CML, CML precursor, CML metabolite (also referred to herein as CML breakdown product), or CML analogue in a biological sample obtained from the subject, compared to a reference level; and (b) selecting the identified subject for treatment with an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis. The method optionally further comprises determining intestinal barrier permeability in the subject, and selecting a subject having an increased level of intestinal barrier permeability compared to a reference level (e.g., a level of intestinal barrier permeability in a healthy subject) for treatment with an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0124] Exemplary CML precursors include (E)-N 6 -((2S,3R,4R,5R)-2,3,4,5,6-pentahydroxyhexylidene)-L-lysine, N 6 -((3S,4R,5R)-3,4,5,6-tetrahydroxy-2-oxohexyl)-L-lysine, L-lysine, and oxalaldehyde.

[0125] Exemplary CML metabolites include carboxymethylcadaverine (CM-CAD), 2-amino-6-(formylmethylamino)hexanoic acid, 5-(carboxymethylamino)pentanoic acid, carboxymethyl-cadaverine, carboxymethyl-epicatechin, (5-aminopentyl)glycine, N 6 -(Carboxymethyl)-N 6 -(2,3-dihydroxy-5-(3,5,7-trihydroxychroman-2-yl)phenyl)-L-lysine, and N-carboxymethyl-Δ1-piperidinium.

[0126] Exemplary CML analogs include Nω-(carboxymethyl)arginine (CMA) and Nε-(1-carboxyethyl)lysine (CEL).

[0127] Also provided herein is a method of treating a subject, comprising administering a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis to a subject identified as having an elevated level of CML, CML precursor, CML metabolite, or CML analogue in a biological sample obtained from the subject compared to a reference level. In certain embodiments, the subject is also identified as having an elevated level of intestinal barrier permeability compared to a reference level (e.g., intestinal barrier permeability level in a healthy subject).

[0128] Also provided herein is a method for reducing the rate of accumulation of CML, CML precursors, CML metabolites, or CML analogs in tissues of a subject, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0129] Also provided herein is a method for identifying a subject suitable for treatment, at increased risk for developing (i) microglial dysfunction, (ii) cognitive impairment, or (iii) a neurodegenerative disease, comprising identifying the subject having an elevated level of CML, CML precursor, CML metabolite, or CML analog in a biological sample obtained from the subject, compared to a reference level.

[0130] Also provided herein is a method for reducing the incidence of oxidative or metabolic stress in microglia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0131] Also provided herein is a method for reducing the incidence of mitochondrial dysfunction in microglia in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0132] Also provided herein is a method for reducing the incidence of microglial dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0133] Also provided herein is a method for increasing one or more microglial activities or functions in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0134] Also provided herein is a method for reducing the incidence or worsening rate of cognitive impairment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0135] Also provided herein is a method for treating cognitive impairment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0136] Also provided herein is a method for reducing the incidence or progression rate of a neurodegenerative disease in a subject, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0137] Also provided herein is a method for treating a neurodegenerative disease in a subject, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0138] Also provided herein is a method for reducing the incidence or worsening rate of neurological dysfunction in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0139] Also provided herein is a method for reducing the concentration of CML, CML precursors, CML metabolites, or CML analogs in a blood or brain sample in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0140] Also provided herein is a method for reducing the concentration of CML, CML precursors, CML metabolites, or CML analogs in a blood or brain sample to prevent or treat a cognitive disorder or neurodegenerative disease in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, thereby preventing or treating the cognitive disorder or neurodegenerative disease in the subject.

[0141] Also provided herein is a method for reducing the concentration of CML, CML precursors, CML metabolites, or CML analogs in a blood or brain sample to prevent or treat a cognitive disorder or neurodegenerative disease in a subject in need thereof. The method includes administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, thereby preventing or treating the cognitive disorder or neurodegenerative disease in the subject.

[0142] Also provided herein is a method for reducing intestinal permeability to prevent or treat cognitive impairment or neurodegenerative disease in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, thereby preventing or treating cognitive impairment or neurodegenerative disease in the subject.

[0143] In certain embodiments of any of the methods described herein, the subject has previously been identified as having an elevated level of CML, CML precursor, CML metabolite, or CML analog in the subject's biological sample compared to a reference level. In certain embodiments of any of the methods described herein, the method further comprises identifying the subject as having an elevated level of CML, CML precursor, CML metabolite, or CML analog in the subject's biological sample compared to a reference level. In certain embodiments of any of the methods described herein, the biological sample is a tissue sample or a body fluid sample. In some embodiments, the body fluid sample is saliva, urine, blood, serum, plasma, cerebrospinal fluid, or feces. In some embodiments, the tissue sample is brain tissue.

[0144] In certain embodiments of any of the methods described herein, the subject has previously been identified as having an increased level of intestinal barrier permeability compared to a reference level (e.g., a level of intestinal barrier permeability in a healthy subject). In certain embodiments of any of the methods described herein, the method further comprises identifying the subject as having an increased level of intestinal barrier permeability compared to a reference level (e.g., a level of intestinal barrier permeability in a healthy subject). In certain embodiments of any of the methods described herein, the subject has been identified or diagnosed as having a cognitive disorder. In certain embodiments of any of the methods described herein, the subject has been identified as having an increased risk of developing a cognitive disorder. In certain embodiments of any of the methods described herein, the subject has been identified as having or diagnosed as having a neurodegenerative disease. In certain embodiments of any of the methods described herein, the subject has been identified as having an increased risk of developing a neurodegenerative disease.

[0145] Methods for identifying the level of increased intestinal barrier permeability can include analytical techniques known in the art, such as measuring the amount of an orally ingested compound (e.g., labeled or unlabeled compound) in a tissue or fluid sample from a subject. In one approach, a labeled compound (e.g., a metabolite labeled with a radioactive or fluorescent label, such as chromium-51 or fluorescein isothiocyanate (FITC), respectively) is orally ingested by a subject. After administration, the uptake of the labeled compound is measured in a biological sample (e.g., blood or urine) taken from the subject. The amount of the labeled compound is an indication of the subject's intestinal permeability. Alternatively, or additionally, intestinal permeability can be measured using a lactulose:mannitol (LM) excretion test. The LM excretion test is a quantitative assay that directly measures the ability of two non-metabolized sugar molecules (lactulose and mannitol) to permeate the intestinal mucosa, thereby measuring intestinal barrier permeability. In an exemplary LM test, the results can be expressed as the ratio of the amount of lactulose and mannitol ingested to the amount of lactulose and mannitol excreted in the urine, which can be measured using the formula:

[0146] [Formula 1] Cumulative excretion amount at time (t) = [sugar excretion concentration at t (mg / mL)] × total urine volume at t (mL).

[0147] The cumulative excretion amount (mg) of each sugar up to time t is expressed as a percentage by dividing the cumulative excretion amount (mg) x 100 by the total amount (mg) of ingested sugar.

[0148] For example, when the total amount of lactulose ingested is 5 grams and the total amount of mannitol ingested is 1 gram, the normal values ​​for the urinary excretion of lactulose and mannitol in healthy subjects are approximately 0.35% (range 0.020% to 1.803%) for lactulose and 12.3% (range 1.480% to 43.75%) for mannitol. Values ​​higher than these average values ​​indicate high intestinal permeability in the subject.

[0149] In certain embodiments of any of the methods described herein, the method results in a reduction in intracellular and / or mitochondrial reactive oxygen species (ROS) levels in the microglia of the subject.In certain embodiments of any of the methods described herein, the method results in a reduction in the expression of inducible nitric oxide synthase (iNOS) in the microglia of the subject.In certain embodiments of any of the methods described herein, the method results in a reduction in the expression of one or more genes in the microglia of the subject selected from the group consisting of Cdkn1a, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, Tlr5, Axl, Hif1a, Lcn2, Mmp2, Rela, Trex1, S100a8, and S100a9. In certain embodiments of any of the methods described herein, the method results in an increase in expression of one or more genes in the microglia of the subject selected from the group consisting of Foxp1, Nrf1, Trp53, G6pdx, Pdk2, Stat3, and Ucp2. In certain embodiments of any of the methods described herein, the method results in an increase in one or more activities of the microglia in the subject.

[0150] Some embodiments of any of the methods described herein further include determining a level of CML, CML precursors, CML metabolites, or CML analogs in a biological sample obtained from the subject.Some embodiments of any of the methods described herein further include determining a level of intestinal barrier permeability in the subject.

[0151] In certain embodiments of any of the methods described herein, the neurodegenerative disease is selected from the group of, but not limited to, Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, progressive supranuclear palsy, spinal muscular atrophy, multiple system atrophy, ataxia, vascular dementia, or other dementias.

[0152] In certain embodiments of any of the methods described herein, following administration of an intestinal barrier function enhancer and / or an agent for reducing or eliminating gut microbiota dysbiosis, the subject exhibits one or more of the following: (a) a decrease in the concentration of CML, CML precursors, CML metabolites, or CML analogs in a blood sample; (b) a decrease in the concentration of CML, CML precursors, CML metabolites, or CML analogs in a brain tissue sample; (c) a decrease in intestinal permeability; (d) a decrease in microbiota dysbiosis; (e) an increase in the level of autophagy in the intestinal epithelium; (f) a decrease in cellular and / or mitochondrial ROS levels in microglia; (g) a decrease in the level of mitochondrial ROS in the intestinal epithelium; (h) a decrease in the level of mitochondrial ROS in the intestinal epithelium; (i) a decrease in the level of mitochondrial ROS in the intestinal epithelium; (j) a decrease in the level of mitochondrial ROS in the intestinal epithelium; (k) a decrease in the level of mitochondrial ROS in the intestinal epithelium; (l ... ) increased levels of adenosine triphosphate (ATP) in the microglial population; (h) decreased expression of iNOS in microglia; (i) decreased expression in microglia of one or more genes selected from the group consisting of Cdkn1a, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, Tlr5, Axl, Hif1a, Lcn2, Mmp2, Rela, Trex1, S100a8, and S100a9; and (j) increased expression in microglia of one or more genes selected from the group consisting of Foxp1, Nrf1, Trp53, G6pdx, Pdk2, Stat3, and Ucp2.

[0153] Also provided herein is a method for identifying a subject at increased risk of developing microglial dysfunction, comprising identifying a subject having an elevated level of CML, CML precursor, CML metabolite, or CML analogue in a biological sample obtained from the subject, compared to a reference level, where such elevated level indicates that the subject has an increased risk of developing microglial dysfunction.

[0154] In certain embodiments, the method further includes identifying a subject having an increased level of intestinal barrier permeability compared to a reference level (e.g., a level of intestinal barrier permeability in a healthy subject) as being at increased risk for developing microglial dysfunction.

[0155] Also provided herein is a method for identifying a subject with increased risk of cognitive impairment.The method includes identifying a subject with elevated levels of CML, CML precursors, CML metabolites or CML analogues in a biological sample obtained from the subject, compared to a reference level, and such elevated levels indicate that the subject has increased risk of developing cognitive impairment.In certain embodiments, the method further includes identifying a subject with increased intestinal barrier permeability level compared to a reference level (e.g., intestinal barrier permeability level in healthy subjects) as having increased risk of developing cognitive impairment.

[0156] Also provided herein is a method for identifying subjects with increased risk of developing neurodegenerative disease.The method includes identifying subjects with elevated levels of CML, CML precursors, CML metabolites or CML analogues in biological samples obtained from subjects compared to reference levels, and such elevated levels indicate that subjects have increased risk of developing neurodegenerative disease.In certain embodiments, the method further includes identifying subjects with increased levels of intestinal barrier permeability compared to reference levels (e.g., the level of intestinal barrier permeability in healthy subjects) as having increased risk of developing neurodegenerative disease.

[0157] Methods for identifying the presence and quantifying the amount of CML, CML precursors, CML metabolites, or CML analogs can include analytical techniques known in the art, including, for example, chromatography (e.g., high performance liquid chromatography (HPLC)), mass spectrometry, liquid chromatography mass spectrometry (LC-MS), nuclear magnetic resonance spectroscopy, or immunoassays.

[0158] For example, the amount of CML, CML precursors, CML metabolites, or CML analogs can be detected and / or quantified in tissue or body fluid samples by one or more of chromatographic methods, mass spectrometry (MS) methods, chromatographic methods followed by MS methods, electrophoretic methods, electrophoretic methods followed by MS methods, nuclear magnetic resonance (NMR) methods, and combinations thereof. Exemplary chromatographic methods include, but are not limited to, strong anion exchange chromatography with pulsed amperometric detection (SAX-PAD), liquid chromatography (LC), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC), thin layer chromatography (TLC), amide column chromatography, and combinations thereof. Exemplary mass spectrometry (MS) includes, but is not limited to, tandem MS, LC-MS, LC-MS / MS, matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS), Fourier transform mass spectrometry (FTMS), ion mobility separation with mass spectrometry (IMS-MS), electron transfer dissociation (ETD-MS), multiple reaction monitoring (MRM), and combinations thereof. Exemplary electrophoretic methods include, but are not limited to, capillary electrophoresis (CE), CE-MS, gel electrophoresis, agarose gel electrophoresis, acrylamide gel electrophoresis, SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Western blotting using an antibody that recognizes a specific glycan structure, and combinations thereof. Exemplary nuclear magnetic resonance (NMR) methods include, but are not limited to, one-dimensional NMR (1D-NMR), two-dimensional NMR (2D-NMR), correlation spectroscopy magnetic angle spinning NMR (COSY-NMR), total correlation spectroscopy NMR (TOCSY-NMR), heteronuclear single quantum coherence NMR (HSQC-NMR), heteronuclear multiple quantum coherence (HMQC-NMR), rotating nuclear Overhauser effect spectroscopy NMR (ROESY-NMR), nuclear Overhauser effect spectroscopy (NOESY-NMR), and combinations thereof.Any method of MS known in the art may be used to determine, detect, and / or measure CML, CML precursors, CML metabolites, or CML analogs of interest, including LC-MS, ESI-MS, ESI-MS / MS, MALDI-TOF-MS, MALDI-TOF / TOF-MS, tandem MS, and the like. Mass spectrometers generally include an ion source and optics, a mass analyzer, and data processing electronics. Mass analyzers include scanning mass analyzers such as time-of-flight (TOF) and quadrupole (Q), ion beam mass analyzers, and trapping mass analyzers such as ion trap (IT), orbitrap, and Fourier transform ion cyclotron resonance (FT-ICR), and may be used in the methods described herein. Details of various MS methods are described in the literature (see, e.g., J. Am. Soc. 1999, 143:131-135).

[0159] Exemplary immunoassays include, but are not limited to, immunohistochemical and / or Western blot analysis, immunoprecipitation, enzyme-linked immunosorbent assay (ELISA), enzyme-linked immunofiltration assay (ELIFA).

[0160] For example, in some embodiments, the sample is contacted with an antibody specific for the target analyte (e.g., CML) under conditions sufficient for antibody-target complex formation, and the complex is detected. The presence of the analyte may be detected in a variety of ways, such as Western blot or ELISA, using any of a wide variety of tissues and samples, including plasma and serum. A wide range of immunoassay techniques using such assay formats are available, see, for example, U.S. Pat. Nos. 4,016,043, 4,424,279, and 4,018,653. These include both single-site and two-site or "sandwich" assays of the non-competitive type, as well as traditional competitive binding assays. These assays also include direct binding of a labeled antibody to the target analyte. The resulting complex can be detected by a signal emitted by the label, such as, for example, an enzyme, a fluorescent label, a chromogenic label, a molecule containing a radionuclide (i.e., a radioisotope), or a chemiluminescent molecule.

[0161] In various embodiments, CML is detected with an anti-CML antibody, such as those available from Immunochem (catalog number ICP2188), Creative Diagnostics (catalog number DMABT-Z59348), Creative BioLabs (catalog number AGM-233YJ), Hycult Biotech (catalog number HM5013), Abcam (catalog numbers ab125145, ab27683, ab27685, ab27684, or ab30922), MyBioSource (catalog number MBS390033 or MBS390034), Kerafast (catalog number EMS302), and Biotechne (catalog number MAB3247-SP or MAB3247), or an anti-CML antibody now known or later identified.

[0162] In various embodiments, CML is detected using CGYJ107, CML26, 6C7, MAB3247, or CMS-10 anti-CML antibody clones.

[0163] In various embodiments, CML analogs such as N(6)-(1-carboxyethyl)-L-lysine (CEL) are detected with an anti-CEL antibody, such as the anti-CEL antibody from Abcam (catalog number ab145095) or Cosmo Bio (catalog number CAC-AGE-M02). In various embodiments, CEL is detected with the CEL-SP anti-CEL antibody clone.

[0164] In an alternative method, immunohistochemistry ("IHC") and staining protocols can be used to examine the expression of CML, CML precursors, CML metabolites, or CML analogs in a sample. IHC staining of tissue sections has been shown to be a reliable method of assessing or detecting the presence of targets in a sample. IHC and immunofluorescence methods use antibodies to probe and visualize cellular antigens in situ, generally by chromogenic or fluorescent methods. Tissue samples can be fixed (i.e., preserved) by conventional methodologies (see, e.g., "Manual of Histological Staining Method of the Armed Forces Institute of Pathology" 3rd Edition (1960) Lee G. Luna, HT (ASCP) Editor, The Blakston Division McGraw-Hill Book Company, New York; The Armed Forces Institute of Pathology Advanced Laboratory Methods in Histology and Pathology (1994) Ulreka V. Mikel, Editor, Armed Forces Institute of Pathology, American Registry of Pathology, Washington, DC). Typically, the sample is first fixed, then dehydrated through a series of alcohols, infiltrated and embedded in paraffin or other sectioning medium, and the tissue sample is sectioned. Alternatively, the tissue may be sectioned and the resulting sections fixed. Primary and / or secondary antibodies used in immunohistochemistry are usually labeled with a detectable moiety, such as a radioisotope, colloidal gold particles, fluorescent label, chromogenic label, or enzyme substrate label.

[0165] IV. Pharmaceutical Compositions, Medicaments, and Routes of Administration The methods described herein use pharmaceutical compositions or medicaments comprising one or more intestinal barrier function enhancers and / or agents for reducing or eliminating intestinal microbiota dysbiosis, or pharma- ceutical acceptable salts or solvates thereof, and at least one pharma- ceutical acceptable carrier.

[0166] The intestinal barrier function enhancer can be used to reduce the concentration of CML, CML precursor, CML metabolite, or CML analogue in the tissue or body fluid of a subject.Exemplary intestinal barrier function enhancers include, for example, intestinal alkaline phosphatase (IAP) (exemplary CAS number 9001-78-9 (calf)), polyphenols (e.g., ellagic acid (EA) (exemplary CAS number 476-66-4) or lipoteichoic acid), metformin (exemplary CAS number 657-24-9), urolithin A (exemplary CAS number 1143-70-0), butyric acid (exemplary CAS number 156-54-7), glutamine (exemplary CAS number 56-85-9), obeticholic acid (OCA) (exemplary CAS number 459789-99-2), divertin, or curcumin (exemplary CAS number 458-37-7), or derivatives thereof.

[0167] Agents for reducing or eliminating gut flora dysbiosis can be used to reduce the concentration of CML, CML precursors, CML metabolites, or CML analogues in subjects.Exemplary agents for reducing or eliminating gut flora dysbiosis include, for example, IAP, EA, biotics, probiotics (e.g., Biohm Health's Biohm Probiotic Boost and Biohm Colon Cleanse), prebiotics, and postbiotics.Exemplary probiotics include bacteria belonging to the genus Lactobacillus (e.g., Lactobacillus acidophilus or Lactobacillus rhamnosus), the genus Bifidobacterium (e.g., Bifidobacterium breve), the genus Saccharomyces (e.g., Saccharomyces cerevisiae) and the genus Streptococcus (e.g., Streptococcus thermophilus).Prebiotics promote the growth of certain beneficial bacterial species that provide health benefits, and exemplary prebiotics include lipoteichoic acid and polyphenols. Postbiotics are, for example, metabolic products, fermentation products, minerals (e.g., zinc and selenium), trace elements, micronutrients, cell surface proteins, and organic acids produced by the microbiome during its life cycle.

[0168] According to the methods described herein, the described intestinal barrier function enhancer and / or agent for reducing or eliminating intestinal flora dysbiosis, or their salts, solvates, or prodrugs, may be administered to a subject in various forms depending on the route of administration selected.Thus, the compositions described herein may be formulated for administration by, for example, oral administration, parenteral administration, and pharmaceutical compositions formulated accordingly.Parenteral administration includes intravenous administration, intraperitoneal administration, subcutaneous administration, intramuscular administration, transdermal administration, intranasal administration, intrapulmonary administration, intrathecal administration, intraventricular administration, intrapleural administration, intrarectal administration, and topical administration.

[0169] According to certain embodiments, the carriers of the pharma- ceutically acceptable compositions useful in practicing this invention are formulated for oral or intravenous administration.

[0170] Pharmaceutical compositions containing the compounds described herein may be prepared by generally known methods, for example, conventional mixing, dissolving, granulating, extruding, emulsifying, encapsulating, encapsulating or lyophilizing processes. Pharmaceutical compositions may be formulated in a conventional manner using one or more pharma- ceutically acceptable carriers, including excipients and / or auxiliary agents that facilitate the processing of the compound into medicament that can be used pharma- ceutically. It is understood that suitable formulations depend on the selected route of administration.

[0171] Oral compositions generally contain an inert diluent or an edible pharma- ceutically acceptable carrier. They can be enclosed in gelatin capsules or compressed into tablets. For the purpose of oral therapeutic administration, the compounds described herein can be incorporated with excipients and used in the form of tablets, lozenges, or capsules. Oral compositions can also be prepared with a fluid carrier for use as a mouthwash, in which case the compound in the fluid carrier is applied orally and shaken, expectorated, or swallowed. Pharmaceutically compatible binding agents, and / or adjuvant materials can be included as part of the composition. Tablets, pills, capsules, lozenges, etc. can contain any of the following ingredients, active ingredients (e.g., intestinal barrier function enhancer and / or agent for reducing or eliminating intestinal flora dysbiosis). ;a binder such as microcrystalline cellulose, gum tragacanth or gelatin;an excipient such as starch or lactose, a disintegrating agent such as alginic acid, primogel or corn starch;a lubricant such as magnesium stearate;a lubricant such as colloidal silicon dioxide;a sweetening agent such as sucrose or saccharin;or a flavoring agent such as peppermint, methyl salicylate, or orange flavor.

[0172] Pharmaceutical compositions suitable for injection include sterile aqueous solutions (where water soluble) or dispersions, and sterile powders for the extemporaneous preparation of sterile injectable solutions or dispersions. For intravenous administration, suitable carriers include, but are not limited to, physiological saline, bacteriostatic water, or phosphate buffered saline (PBS). In all cases, the composition must be sterile and fluid to the extent that easy syringability exists. The composition must be stable under the conditions of manufacture and storage and must be preserved against the contaminating action of microorganisms, such as bacteria and fungi. The carrier can be, for example, a solvent or dispersion medium containing water, ethanol, polyol (e.g., glycerol, propylene glycol, liquid polyethylene glycol, and the like), and suitable mixtures thereof. Proper fluidity can be maintained, for example, by the use of a coating such as lecithin, by the maintenance of the required particle size in the case of dispersions, and by the use of surfactants. Prevention of the action of microorganisms can be achieved by various antibacterial and antifungal agents, for example, parabens, chlorobutanol, phenol, ascorbic acid, thimerosal, and the like. In many cases, it is preferable to include isotonic agents, for example, sugars, polyalcohols such as mannitol or sorbitol, sodium chloride, etc. in the composition. Prolonged absorption of the injectable compositions can be brought about by including in the composition an agent which delays absorption, for example, aluminum monostearate or gelatin.

[0173] Sterile injectable solution can be prepared by incorporating the active ingredient in the required amount in a suitable solvent with one or a combination of the above-listed ingredients as required, and then sterilizing by filtration.Generally, dispersion is prepared by incorporating the active ingredient into a sterile vehicle that contains a basic dispersion medium and other ingredients required from the above-listed ingredients.In the case of sterile powder for preparing sterile injectable solution, the preparation method is vacuum drying and freeze-drying, and the powder of active ingredient and any additional desired ingredients is obtained from the solution previously sterile-filtered.

[0174] For administration by inhalation, the intestinal barrier function enhancing agents are delivered in the form of an aerosol spray from pressured container or dispenser which contains a suitable propellant, e.g., a gas such as carbon dioxide, or a nebulizer.

[0175] Systemic administration can also be via transmucosal or transdermal.For transmucosal or transdermal administration, a penetrant suitable for the barrier to be permeated is used in the formulation.Such penetrants are generally known in the art, and include, for example, for transmucosal administration, detergents and bile salts.Transmucosal administration can be achieved by using nasal sprays or suppositories.For transdermal administration, the active intestinal barrier function enhancer is formulated into ointments, salves, gels or creams as generally known in the art.

[0176] The active intestinal barrier function enhancer may be prepared with a pharma- ceutically acceptable carrier that protects the intestinal barrier function enhancer from rapid excretion from the body, such as a controlled release formulation, including implants and microencapsulated delivery systems. Biodegradable and biocompatible polymers may be used, such as ethylene vinyl acetate, polyanhydrides, polyglycolic acid, collagen, polyorthoesters, polylactic acid, and the like. Methods for preparing such formulations will be apparent to those skilled in the art. These materials are also commercially available from Alza Corporation and Nova Pharmaceuticals, Inc. Liposomal suspensions, including liposomes targeted to infected cells with monoclonal antibodies against viral antigens, may also be used as pharma-ceutically acceptable carriers. These may be prepared according to methods known to those skilled in the art, such as those described in U.S. Pat. No. 4,522,811.

[0177] It is advantageous to formulate oral or parenteral compositions in dosage unit form due to the ease of administration and uniformity of dosage.Dosage unit form as used herein refers to a physically discrete unit suitable as a unitary dosage for the subject to be treated; each unit contains a predetermined amount of active intestinal barrier function enhancing agent calculated to produce desired therapeutic effect in association with required pharmaceutical carrier.The specification of dosage unit form is determined and directly depends on the inherent properties of active intestinal barrier function enhancing agent and the specific therapeutic effect to be achieved.

[0178] In therapeutic applications, the dosage of the pharmaceutical compositions used according to the present disclosure will vary according to the drug, age, weight, and clinical condition of the recipient subject, and the experience and judgment of the clinician or practitioner administering the treatment, among other factors that influence the dosage selected. In general, the dosage should be sufficient to slow, preferably regress, and preferably cause complete regression of the symptoms of the disease or disorder disclosed herein. An effective amount of pharmaceutical agent is one that produces an objectively identifiable improvement as noted by a clinician or other qualified observer.

[0179] It should be understood that the pharmaceutical compositions can be included in a container, pack, or dispenser together with instructions for administration.

[0180] V. Screening method for intestinal barrier function enhancer Also provided herein is a method of screening for candidate agents for reducing the accumulation rate of CML, CML precursors, CML metabolites, or CML analogs in a tissue or body fluid sample of a subject, comprising: determining a first level of CML, CML precursors, CML metabolites, or CML analogs in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursors, or CML metabolites in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the accumulation rate of CML, CML precursors, CML metabolites, or CML analogs in the tissue of the subject.

[0181] Also provided herein is a method of screening for candidate agents for reducing the incidence of oxidative or metabolic stress in microglia in a subject, comprising: determining a first level of CML, CML precursor, CML metabolite, or CML analog in a biological sample obtained from the mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML analog, CML precursor, or CML metabolite in the biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence of oxidative or metabolic stress in microglia of the subject.

[0182] Also provided herein is a method of screening for candidate agents for reducing the incidence of microglial mitochondrial dysfunction in a subject, comprising: determining a first level of CML, CML precursor, CML metabolite, or CML analog in a biological sample obtained from the mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML metabolite, or CML analog in the biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence of microglial mitochondrial dysfunction in the subject.

[0183] Also provided herein is a method of screening for candidate agents for reducing the incidence of microglial dysfunction in a subject, comprising: determining a first level of CML, CML precursors, CML metabolites, or CML analogs in a biological sample obtained from the mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursors, CML metabolites, or CML analogs in the biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence of microglial dysfunction in the subject.

[0184] Also provided herein is a method of screening for candidate agents for increasing one or more functions of microglia in a subject, comprising: determining a first level of CML, CML precursors, CML metabolites, or CML analogs in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursors, CML metabolites, or CML analogs in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for increasing one or more functions of microglia in the subject.

[0185] Also provided herein is a method of screening for candidate agents for reducing the incidence or worsening rate of cognitive impairment in a subject, comprising: determining a first level of CML, CML precursors, CML metabolites, or CML analogs in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursors, CML metabolites, CML analogs in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence or worsening rate of cognitive impairment in the subject.

[0186] In certain embodiments, the amount of CML, CML precursor, CML metabolite or CML analogue in biological sample is measured.Methods for measuring the amount of such drugs are described above in section III, and include, for example, chromatography (e.g., high performance liquid chromatography (HPLC)), mass spectrometry, liquid chromatography mass spectrometry (LC-MS), nuclear magnetic resonance spectroscopy, or immunoassay.

[0187] In certain embodiments of these screening methods, the methods further comprise testing the candidate agent in an animal model.

[0188] Working Example Below are examples of specific embodiments for carrying out the present invention. The examples are provided for illustrative purposes only and are not intended to limit the scope of the present invention in any way.

[0189] Example 1. Gut microbiota drives age-associated oxidative stress and mitochondrial damage in microglia via N6-carboxymethyllysine (CML) In this example, we describe the discovery of CML as a messenger between the gut microbiota and the brain in young adult and aged mice.

[0190] Comparison of microglial transcriptomes of young adult and aged mice housed under germ-free and specific pathogen-free conditions revealed that the microbiota influenced age-related changes in microglial gene expression. Absence of gut microbiota reduced oxidative stress and ameliorated mitochondrial dysfunction in brain microglia of aged mice. Unbiased metabolomic analysis of serum and brain tissue revealed accumulation of N6-carboxymethyllysine (CML) in microglia of aged brain. CML mediated a burst of reactive oxygen species and inhibited microglial mitochondrial activity and ATP levels. Age-dependent elevation of CML levels in human serum and brain was validated, and it was found that a microbiota-dependent increase in intestinal permeability in aged mice mediated the increase in CML levels. These results demonstrate how the gut microbiota influences microglial homeostasis in the aging brain, and molecular phenotyping at the metabolite level identifies CML, a major advanced glycation end product (AGE), as a key compound in causing age-associated microglial dysfunction.

[0191] method Human tissue Formalin-fixed paraffin-embedded (FFPE) cerebral cortical tissues from healthy brains of 43 individuals (20 women, 23 men, age 1–88 years; 8 temporal lobes, 25 frontal lobes) were examined by a well-trained neuropathologist at the Institute of Neuropathology at the University Hospital Freiburg, Germany (control tissues or Braak stages I and II).

[0192] mouse Specific pathogen-free (SPF) and germ-free (GF)-housed C57BL / 6 mice were analyzed at 6–10 weeks of age (young adult) and 96–104 weeks of age (aged). Mice in the aged group for untargeted metabolomic analysis were 17–18 months of age. All groups included both male and female mice, except for untargeted metabolomic and microbiota profiling, in which only male mice were used. Mice were housed under a 12-h light / 12-h dark cycle, 18–23°C temperature, and 40–60% humidity, with food and water available ad libitum. To avoid cage effects, mice from at least three different cages were analyzed per experimental group. For treatment, young adult mice (8 weeks old) were given CML (0.735 mg / kg; Iris Biotech), trimethylamine N-oxide (TMAO) (3.95 mg / kg; Sigma-Aldrich), sodium acetate (59 mg / kg; Sigma-Aldrich), or sodium propionate (4.61 mg / kg; Sigma-Aldrich) intraperitoneally or orally every day for 14 days. For modulation of CML in aged animals, 18-month-old SPF-fed C57BL / 6 mice were given vehicle (20% hydroxypropyl-p-cyclodextrin in 1x phosphate-buffered saline (PBS)), 10 mg / kg ellagic acid (EA), or 3,000 U / kg intestinal alkaline phosphatase (IAP) orally every 3 days for 10 weeks. To assess in vivo intestinal permeability, fluorescein isothiocyanate (FITC)-labeled dextran (4 kDa; Sigma-Aldrich) was used as a tracer. Briefly, mice were food-deprived 4 h before and food- and water-deprived 4 h after oral administration of 200 pl of 80 mg / ml FITC-dextran. Blood was collected retro-orbitally 4 h later, and fluorescence intensity was measured using a fluorescent plate with an excitation wavelength of 493 nm and an emission wavelength of 518 nm. To assess in vivo intestinal permeability to CML, mice were treated with 200 pl of CML (0.36 mM). Blood was collected retro-orbitally just before and 4 h after treatment. CML that had entered the circulation was measured by liquid chromatography-mass spectrometry (LC-MS).All animal experiments were approved by the German local authorities (Regierungsprasidium Freiburg) and were performed in accordance with the respective national, federal, and institutional regulations as well as the guidelines of the European Federation for Laboratory Animal Science.

[0193] Preparation of mouse tissue samples Mice were lethally anesthetized with ketamine (100 mg / kg body weight) and xylazine (10 mg / kg body weight) and then perfused with 1xPBS via the left ventricle. For histological examination, brains were preserved overnight in 4% paraformaldehyde (PFA). For flow cytometry and magnetic activated cell sorting (MACS) bead cell sorting, brains were dissected, homogenized, and filtered through a 70 pm mesh. After centrifugation (220 g, 5 min, 4 °C), the pellet was suspended in 37% Percoll and then centrifuged for 30 min at 800 g, 4 °C. Myelin was removed from the upper layer, and the cell pellet was washed once with 1xPBS before antibody staining.

[0194] immunohistochemistry Brains were fixed overnight in 4% PFA and embedded in paraffin. To assess microglial cell density, 3-μm-thick parasagittal sections were stained with anti-Iba-1 antibody (1:500 dilution, catalog no. 019-19741; WAKO). For immunohistochemistry (IHC), epitopes were unmasked by heat-induced antigen retrieval at pH 6. After overnight (4°C) incubation with primary antibodies, the sections were incubated with biotin-labeled goat anti-rabbit secondary antibody (1:1,000 dilution; SouthernBiotech) for 45 min at room temperature. Streptavidin-horseradish peroxidase (SouthernBiotech) was then added for 45 min at room temperature. Antibody signals were resolved using 3,3'-diamonobenzidine brown color developer (Dako). Nuclei were counterstained with hematoxylin. Images were acquired with a BZ-9000 Biorevo microscope (Keyence) and analyzed with ImageJ v.1.53f (National Institutes of Health) software.

[0195] Immunofluorescence Brains were fixed in 4% PFA, dehydrated in 30% sucrose, and embedded in Tissue-Tek OCT compound (Sakura Finetek). 14-μm-thick frozen sections were obtained using a cryostat (SM2000R; Leica Biosystems). Mouse and human brain FFPE tissues were cut with a microtome to obtain 5-pm sections. Sections were blocked with 5% bovine serum albumin (BSA) in PBS and permeabilized with 0.5% Triton X-100 blocking solution. The following primary antibodies were incubated overnight at 4°C: rabbit anti-Iba-1 (1:500 dilution; WAKO); guinea pig anti-Iba-1 (1:1,000 dilution; Synaptic Systems); anti-iNOS (1:500 dilution; Thermo Fisher Scientific) or anti-CML (1:500; Abcam). Secondary antibodies (Alexa Fluor 488, 568, 647 labeled, 1:500 dilution) were incubated for 2 h at room temperature. For three-dimensional (3D) reconstruction of microglia, free-floating 30 pm frozen sections from brain tissue were labeled with anti-Iba-1 (1:500 dilution) for 48 h at 4 °C, followed by incubation with Alexa Fluor 647 labeled secondary antibodies at 1:500 dilution overnight at 4 °C. To eliminate autofluorescence in aged mouse and human tissues, slides were treated with TrueBlack Lipofuscin Autofluorescence Quencher. Nuclei were counterstained with 4',6-diamidino-2-phenylindole (DAPI). Coverslips were mounted with ProLong Diamond Antifade Mountant (Thermo Fisher Scientific). Images were acquired using a BZ-9000 Biorevo microscope with a 20x / 0.75 numerical aperture (NA) objective, an Olympus Fluoview 1000 confocal laser microscope, or a TCS SP8 X (Leica Microsystems) with a 20x / 0.75 NA objective (HC PL APO 20x / 0.75 NA IMM CORR CS2). For 3D reconstruction of microglia, images were analyzed using Imaris v.8.02 (Bitplane) with at least five cells per mouse.All other images were processed and analyzed using Photoshop CC 2015 (Adobe) or ImageJ v.1.53f (National Institutes of Health).

[0196] Electron microscopy Brain specimens from the cerebral cortex were first fixed overnight at 4°C in 3% glutaraldehyde, washed in Sorensen's buffer, and then transferred to 1% osmium tetroxide for 2 h at room temperature. The specimens were then stepwise dehydrated in ethanol (30-100%), 100% propylene oxide, resin / propylene oxide (1:2 (v / v)), and resin / propylene oxide (2:1 (v / v)). The specimens were polymerized at 75°C for 24 h and embedded in resin. Afterwards, 700 nm semisections were cut and stained with 2% toluidine blue to define regions of interest, and the sections were imaged with uranyl acetate and lead citrate (Leica Reichert Ultrastainer), followed by further preparation of 70 nm ultrathin sections using an ultramicrotome (Leica Reichert Ultracut S). To assess the mitochondrial phenotype of microglia, images were acquired at 7,900x or 46,000x magnification using a CM100 electron microscope (Philips). Images of 30–35 cells per mouse were processed and analyzed using iTEM software 2012 (Olympus).

[0197] Bone marrow derived macrophage cell culture Cells were cultured at 37°C in a 5% CO2 humidified incubator. Mouse BMDMs were differentiated from tibial and femoral bone marrow aspirates. Recombinant mouse macrophage colony-stimulating factor (Immunotools) was used at 20 ng / ml. After 7 days of differentiation, BMDMs were plated in triplicate in 24-well plates at 5 × 10 per well. 5 The medium was switched to serum-free medium 6 h before the experiment. Cells were incubated with increasing concentrations of CML (untreated, 0.1 pM, 1 pM, 10 pM, 100 pM, 1 mM) for 48 h and then harvested for assay.

[0198] Flow cytometry Cell sorting for RT-qPCR and RNA sequencing (RNA-seq) was performed using MoFlo Astrios (Beckman Coulter; Figure 7a). Prior to surface staining, dead cells were excluded using Fixable Viability Dye eFluor 780 (1:1,000 dilution; Thermo Fisher Scientific), followed by incubation with Fc receptor blocking antibody CD16 / CD32 (1:200 dilution, clone 2.4G2; BD Bioscience). The following antibodies were used for surface staining: anti-CD45 (1:200 dilution, clone 30-F11; Thermo Fisher Scientific); anti-CD11b (1:200 dilution, clone M1 / 70; Thermo Fisher Scientific). The following lineage antibodies were used (all at 1:300 dilution): anti-CD3 (clone 17A2; BioLegend); anti-CD19 (clone 6D5; BioLegend); anti-CD45R (clone RA3-6B2; BD Biosciences); Ly6C (clone AL-21; BD Biosciences); Ly6G (clone 1A8; BD Biosciences). CellROX DeepRed reagent (5 pM; Thermo Fisher Scientific) was used to assess reactive oxidative species (ROS) in microglial cells. Tetramethylrhodamine methyl ester perchlorate, methyl ester, perchlorate (50 nM; Thermo Fisher Scientific) and MitoTracker Green FM (20 nM; Thermo Fisher Scientific) were used to assess mitochondrial activity. Dead cells were excluded by a brief incubation with DAPI before flow cytometric analysis using a FACSCanto II (BD Biosciences). Data were acquired with FACSDiva v.6 software (Becton Dickinson). Post-acquisition analysis was performed using FlowJo v.10 (FlowJo LLC).

[0199] Intracellular ATP measurement To avoid the intracellular stress of fluorescence-activated cell sorting (FACS), microglial cells were isolated using a magnetic-activated cell sorting (MACS) separation system (Miltenyi Biotec; Fig. 7b). The cell suspension was incubated with Fc receptor blocking antibody CD16 / CD32 (clone 2.4G2; BD Biosciences) and biotinylated anti-CD11b antibody (clone M1 / 70; Thermo Fisher Scientific). Anti-biotin microbeads (Miltenyi Biotec) were then added to the cell suspension and positive selection was performed according to the manufacturer's instructions. From each sample, 10,000 cells per well were plated in triplicate in 96-well plates. Intracellular ATP was measured using the CellTiter-Glo assay (Promega Corporation) according to the manufacturer's instructions.

[0200] RNA-Seq Total RNA was extracted from FACS-sorted CD11b+CD45intLin-negative microglia (10,000 cells per sample) using the Arcturus PicoPure RNA Isolation Kit (Thermo Fisher Scientific) according to the manufacturer's protocol. Single-stranded complementary DNA was generated using the SMARTer v4 Ultra Low Input RNA Kit for Sequencing (Clontech). Double-stranded cDNA was amplified by long-range PCR (11 cycles) and purified by magnetic bead cleanup. Library preparation was performed as described in the Illumina Nextera XT Sample Preparation Guide (Illumina). Sequencing runs were performed on a HiSeq 1000 instrument (Illumina) using the indexed 50-cycle single-read protocol and TruSeq SBS v3 Reagents according to the HiSeq 1000 System User Guide. BCL files were converted to FASTQ files with CASAVA 1.8.2 software. Library preparation and RNA-seq were performed at the Genomics Core Facility ‘Center of Excellence for Fluorescent Bioanalytics’, University of Regensburg, Germany.

[0201] The quality of the sequencing reads stored in the FASTQ files was assessed with FastQC v.0.67 and trimmed with Trim Galore! v.0.4.3. RefGene annotation was performed using STAR aligner v.2.5.2, and the reads were mapped onto the mouse genome mm10 (University of California Santa Cruz). The number of reads (counts) mapped to each gene was extracted from the BAM files using FeatureCount v.1.5.3. The process of extracting gene counts from the FASTQ files was performed on the Galaxy platform (Non-Patent Document 18). Three samples with low mapping rates (<75%) were removed.

[0202] Differential expression analysis was performed using DESeq2 v.1.32.0. Normalized counts generated by DESeq2 were assessed for artifacts or contamination by other cell types. The list of genes used was based on single-cell RNA-seq data (Non-Patent Document 19). Ward error sum of squares hierarchical clustering and PCA were performed using R v.4.1.0. DESeq2 models were used to identify differentially expressed genes (DEGs) with adjusted P < 0.05 (Wald test) and absolute change > 1.5. Heatmaps were calculated from scale (z-score) normalized read counts of DEGs using row hierarchical clustering (complete method) using the R package pheatmap v.1.0.8. WGCNA was performed on the normalized expression data using the R package WGCNA v.1.69. For computational efficiency, genes were filtered to leave only genes explaining more than 50% of the variance (10,848 genes left, 7,265 genes removed). Module-trait correlation analysis was performed between module eigengenes (MEs) and different traits (combinations of microbiota and age) by calculating the Peason correlation and Student's asymptotic P value of the correlation between each pair of variables using the WGCNA package. Gene Ontology (GO) enrichment analysis of genes in different MEs was performed using goseq v.1.44.0 using the mouse genome-wide annotation (org.Mm.eg.db v.3.13.0) and Wallenius approximation.

[0203] Over-enriched GO categories were extracted using a 0.05 false discovery rate (FDR) cutoff. The list of ROS-related genes was extracted from GO:0000302, GO:2000377, and oxidative stress (WikiPathways).

[0204] RT-qPCR Hifla gene expression was measured using TaqMan assay (Mm00468869_m1). Data were normalized to values ​​obtained from microglia of young adult SPF males in homeostasis, and relative gene expression levels were determined by the △△CT method. Gene expression was considered undetectable when CT values ​​were below 35 cycles.

[0205] Microbiome Profiling Total DNA was isolated from fecal samples using the QIAamp DNA stool kit (QIAGEN) according to modified manufacturer's instructions (20). Briefly, 100-200 mg was homogenized in 500 pl of ASL buffer by a bead-beating step using a TissueLyser at 30 Hz for 3 min, followed by two additional lysis steps at 95°C. Samples were then incubated with 200 pl of Gram-positive lysis buffer (20 mg / ml lysozyme, 20 mM Tris-HCl, pH 8.0, 2 mM EDTA, 1.2% Triton; Sigma-Aldrich). DNA was purified and pooled at a concentration of 26 pM, and the pooled library was sequenced for the V5 / V6 region of the 16S rRNA gene using an IonTorrent PGM system according to the manufacturer's instructions (Thermo Fisher Scientific).

[0206] An average of 38,209 high-quality reads per sample were used for microbiome profiling. Reads were clustered into operational taxonomic units (OTUs) with 97% similarity. Data were further analyzed after filtering low-quality (base call accuracy; q<25) samples using the QIIME v.1.9.1 pipeline; samples with more than 4,500 reads were retained for further analysis (21). OTUs were selected using UCLUST at a 97% sequence identity threshold, and taxonomy was subsequently assigned using the SILVA database release 119. α- and β-diversity were calculated using the phyloseq pipeline in R v.3.4. α-diversity between samples was compared using the nonparametric Mann-Whitney U test, and group effects on β-diversity by phyloseq were assessed using Adonis in the vegan R package v2.5-7 (22, 23). Taxonomic differences at phylum and genus levels between study groups were identified using the "Multivariate Analysis with Linear Models" R package v0.0.4. Plots were generated with ggplot2 v.3.3.5 using the phyloseq object. Only taxa present in at least 30% of samples and OTUs comprising more than 0.0001% of the total counts were considered. AP<0.05 and FDR (Benjamini-Hochberg correction) of q<0.05 were used as cutoffs for significance.

[0207] Untargeted Metabolomics Raw data from serum and brain of young adult and aged mice groups were mined. Non-targeted MS analysis was performed in Metabolon (24). Peaks were quantified using area under the curve. Raw area counts of each metabolite for each sample were normalized by the median of each run day to correct for variability due to differences in instrument day-to-day tuning, and the median of each run was set to 1.0. This preserved sample-to-sample variability but allowed metabolites with widely differing raw peak areas to be compared on a similar graph scale. Missing values ​​were input at the minimum value observed after normalization.

[0208] Targeted Metabolomics by LC-MS Samples were extracted with pre-cooled (-80°C) extraction solution (80:20 methanol LC-MS grade:Milli-Q H2O). Quantification of targeted metabolites by LC-MS was performed using an Agilent 1290 Infinity II UHPLC system interfaced with an Agilent 6495 QQQ-MS operating in multiple reaction monitoring (MRM) mode. MRM settings were optimized for all compounds individually using pure standards. LC separation was performed on a Phenomenex Luna propylamine column (50x2mm, 3-pm particles) with a solvent gradient from 100% buffer B (5mM ammonium carbonate in 90% acetonitrile) to 90% buffer A (10mM NH4 in water). The flow rate was 1,000 to 750pl / min. The autosampler temperature was 5 degrees and the injection volume was 2pl. Peak areas were determined based on the standard of each metabolite and calculated using MassHunter vB08.02 (Agilent). For SCFAs, acetate (C2, 59.04 g / mol), propionate (C3, 73.07 g / mol), butyrate (and isobutyrate, C4, 87), and valerate (and isovalerate, 101) were quantified in mouse serum. To extract metabolites, 10 pl of each sample was added to four tubes, and 90 pl of acetonitrile was added, and the standards were serially diluted (four steps). C2(mg / ml)(L1:0;L2:0.002;L3:0.004;L4:0.006), C3(mg / ml)(L1:0;L2:0.0002;L3:0.0004;L4:0.0006), C4(mg / ml)(L1:0;L2:0.0005;L3: 0.001;L4:0.0015), C5(%)(L1:0;L2:0.0002;L3:0.0004;L4:0.0006), and C4(mg / ml)(L1:0;L2:0.000025%;L3:0.00005%;L4:0.000075%). Samples were centrifuged at 20,000 g for 10 min at 4° C. and 50 pl of the supernatant was transferred to a new tube. 2 pl of each sample was injected into the high performance LC-quadrupole time-of-flight analysis method.The butyric and isobutyric acid peaks, and the valeric and isovaleric acid peaks could not be robustly distinguished; therefore, each set of concentration values ​​corresponds to both sites. Each sample was analyzed in duplicate, and the average values ​​were used to construct the regression line; concentrations were calculated by the standard addition method (25).

[0209] Human metabolomics data The TwinsUK Adult Twin Registry includes approximately 14,000 individuals, mostly female, with similar disease and lifestyle patterns to the general UK population. The St. Thomas' Hospital Research Ethics Committee approved the study and all twins gave written informed consent. For CML and TMAO, data were extracted from a blood metabolomic study including an ageing cohort and run on the Metabolon platform. Briefly, metabolite ratios were measured from blood samples by Metabolon using an untargeted ultra-high performance LC-MS / MS platform. Metabolites were median scaled on the day of run and log-transformed.

[0210] statistics No statistical methods were used to predetermine sample size. Data distribution was assumed to be normal. Where applicable, animals were randomly assigned to different experimental groups. Experimenters were blinded to group allocation. Statistical analyses, except for RNA-seq, nontargeted metabolomics, and microbial profiling, were performed using Prism 9.0 (GraphPad Software).

[0211] Research results Gut microbiota alters microglial transcriptome profile during aging It has previously been reported that microglial cell density increases with age in the cerebral cortex.

[0212] We confirmed an increase in microglial cell density in the cerebral cortex of specific bacteria-free (SPF) and bacteria-free (GF) animals between young adult and aged mice (see Fig. 6a-6b), but no difference was observed between aged SPF and GF mice (see Fig. 6a-6b). Quantitative morphological reconstructions were performed to determine potential morphological changes in microglia from SPF and GF mice. Microglia from SPF mice showed a decrease in total branch area, total branch length and number of branch points, along with an increase in cell volume (see Fig. 6c-6g). The sphericity of the cell body did not change between groups (see Fig. 6h). These data indicate that age affected microglial morphology in SPF mice, whereas microglia remained unchanged and hyperramified in GF mice.

[0213] To further evaluate microbiota-dependent changes in microglial physiology in the aging brain, we performed RNA-seq on FACS-purified microglia (see, Figures 7a and 8a) from whole brains of young adult (6–10 weeks old) and aged (96–104 weeks old) male and female mice housed under GF or SPF conditions (see, Figures 1b and 8b). PCA revealed gene expression profiles of microglia in both age groups, GF and SPF mice (see, Figure 1c). Transcriptomic differences between microglia isolated from GF and SPF mice were more pronounced at older ages (see, Figure 1d). Compared to microglia from SPF mice, microglia from GF mice emerged with an age-independent gene expression pattern (microglial GF signature) that included genes related to cytoskeleton (e.g., Sdc3, Sult1a1, Tuba4a) and immune function (e.g., Ctse, Ero1lb, Htra3, Kcnma1, Notch4, Nr1d2, Rab4a, Wdfy1) (see Figure 1e). Furthermore, the microglial GF signature contained genes related to the control of mitochondrial function (e.g., B4galnt1, Gpr137b, Gstm1, Mcur1, Mtfp1, Nnt and Plcd3), indicating the ability of the microbiota to control the metabolic profile of microglia (see Figure 1e). We next characterized the functional changes in gene expression in microglia with respect to age and microbiota using weighted gene co-expression network analysis (WGCNA) (see Figure 1f-1g and Figure 8c). Genes that significantly (Wald Padj < 0.05) explained more than 50% of the variance were placed into module eigengenes (MEs) based on their co-expression patterns. Comparison of SPF and GF groups revealed minor differences in gene networks related to immune function and epigenetic regulation, ME1, ME5, ME6, and ME7, respectively. Two modules were characteristic of each age group. ME1 and ME4 in the aging SPF group contained genes related to processes such as mitochondrial metabolism and lipid localization, while ME2 and ME6 in the aging GF group contained genes controlling immune response, histone lysine methylation, and cell morphogenesis (see Figure 8d).ME1 and ME4 in the aged SPF group contained genes related to processes such as mitochondrial metabolism and lipid localization, while ME2 and ME6 in the aged GF group contained genes controlling immune response, histone lysine methylation, and cell morphogenesis (Figure 8c). Genes in ME1 and ME8 related to immune response (e.g., Axl, Crlf2, Tnfsf8, Tnfsf10, Ccl12, Fgr, Il1b, Il6st, Spp1, Tlr2), interferon signaling (e.g., Cxcl10 8, Ifi207, Ifit2 8, Stat1), inflammatory response (e.g., Cd180, Ldlr, S100a8, S100a9), and microglial migration (e.g., Ccl12, Cxcl10) showed specific upregulation in microglia from aged SPF mice, but showed negative or low correlation in both the young adult group and aged GF mice. ME1 and ME8, including mitochondrial metabolic processes, hydrogen peroxide metabolic processes, and reactive oxygen metabolic processes, showed strong correlations in the aged SPF group. ME2, which was highly enriched in aged GF mice, was associated with responses to oxygen-containing compounds (Fig. 8c). Genes included in ME2 include those that control intracellular ROS levels, such as Foxp1, Nrf1, and Trp53, and those involved in mitochondrial ROS control, such as G6pdx, Pdk2, Stat3, and Ucp2, but their expression levels were lower in microglia from aged SPF mice compared with age-matched GF mice, indicating that ROS levels in aged SPF mice are not kept within the optimal range for cells. Accumulation of ROS in the aging brain is associated with mitochondrial damage and mitochondrial dysfunction. Indeed, ME3, ME5, and ME9 showed notable changes in genes related to mitochondrial assembly, glucose metabolism, and oxidative phosphorylation. These genes were upregulated in both SPF and GF mice, but mitochondrial damage was more pronounced in SPF mice, where protective genes controlling ROS were downregulated. Furthermore, genes maintaining mitochondrial structure and function were upregulated in aged GF mice (Figure 8d).

[0214] We next investigated the contribution of the microbiota to age-related ME. WGCNA showed that microglia in aged GF mice followed the general aging trend of SPF mice, but to a lesser extent (Fig. 1f-1g), and formed a cluster closer to that of the young adult group (Fig. 8b). For example, genes in ME1 and ME8 related to immune responses (e.g., Axl, Crlf2, Tnfsf8, Tnfsf10, Ccl12, Fgr, Il1b, Il6st, Spp1 and Tlr2), interferon signaling (e.g., Cxcl10 / 8, Ifi207, Ifit2 / 8 and Stat1), inflammatory responses (e.g., Cd180, Ldlr, S100a8 and S100a9) and microglial cell migration (e.g., Ccl12 and Cxcl10) showed specific upregulation in microglia from aged SPF mice and negatively or poorly correlated in young adult and aged GF mice (see Figure 2a and Figure 8c-8d). Microglia showed age-related changes in their transcriptome profiles with microbiota-dependent divergence.

[0215] Decreased oxidative stress in microglia of aged GF mice When examining the pathways of the aging-related modules, we found several associations with the regulation of oxidative stress in microglia that depend on the microbiota. ME1 and ME8 showed strong correlations in the aging SPF group, including mitochondrial metabolic processes, hydrogen peroxide metabolic processes and ROS metabolic processes. ME2, which was highly enriched in aging GF mice, was associated with the response to oxygen-containing compounds (see Figures 1f-1g and 2a). To confirm that the expression levels of microglial ROS-related genes were regulated by mouse age and rearing conditions, we selectively analyzed the ROS-related genes of ME1, ME2 and ME8 associated with aging (see Figure 2b). Specific upregulation of several immune activation and ROS-promoting genes, such as Cdkn1a, Cyba, Cybb, Duoxa1, Il1b, Tg fbr2, Tlr2, Tlr4 and Tlr5, and ROS-responsive genes, such as Axl, Hif1a, Lcn2, Mmp2, Rela, Trex1, S100a8 and S100a9, was observed only in microglia from aged SPF mice (see Fig. 2b). Genes in ME2, including genes controlling intracellular ROS levels, such as Foxp1, Nrf1 and Trp53, and genes involved in mitochondrial ROS regulation, such as G6pdx, Pdk2, Stat3 and Ucp2, were less expressed in microglia from aged SPF mice compared to age-matched GF mice (see Fig. 2b). Using the CellROX flow cytometry assay, we monitored ROS production in microglia isolated from young adult and aged SPF mice, and observed a significant increase in ROS with age, which was suppressed in aged GF mice (see Figure 2c). Activation of inducible nitric oxide synthase (iNOS) is directly linked to the generation of excess ROS. Using immunohistochemistry (IHC), we observed an age-dependent increase in microglial iNOS expression under SPF conditions, but it was less pronounced in GF mice (see Figure 2d-2e).

[0216] Examining how much ROS increased impacts microglial function. We observed ME3, ME5 and ME9. Changes in genes related to mitochondrial assembly, glucose metabolism and oxidative phosphorylation were found (see Figure 2f). Electron microscopy of microglial mitochondria revealed that, although there was no change in the mass or number of mitochondria per microglia, the proportion of damaged mitochondria was significantly higher in SPF mice compared to GF mice, with less distinct or even destroyed cristae (see Figures 2g-2h and 9a-9c). The accumulation of intracellular ROS, which peaked in microglia of aged SPF mice, can induce the expression of hypoxia-inducible factor 1 subunit alpha (Hif1a). HIF1a is associated with ROS in simulated hypoxic conditions in the brain and can directly alter mitochondrial metabolism (Non-Patent Document 26). Mitochondrial dysfunction in the aging brain causes a metabolic shift associated with exaggerated activation of microglia. Although young adult mice showed similar Hif1a expression, RNA-seq and reverse transcription quantitative PCR (RT-qPCR) showed that Hif1a expression in microglia of aged SPF mice was higher than that of GF mice (see Figures 9d-9e). In cells of aged animals, the efficiency of oxidative phosphorylation decreases, leading to reduced ATP production (Non-Patent Document 27). Mitochondrial transmembrane potential (ΔΨm) is the main driving force for ATP production. Given the increase in mitochondrial mass with age (see Figures 9f-9g), mitochondrial activity was plotted as mitochondrial transmembrane potential against mitochondrial mass. Mitochondrial activity showed an age-related decline and a decrease in intracellular ATP reservoir in SPF mice, but both were less pronounced in microglia from GF mice (see Figures 2i, 7b, and 9h). Taken together, these data indicate that the microbiota contributes to increased oxidative stress in microglia of the aged brain, which is associated with direct damage to mitochondria.

[0217] Age-related accumulation of CML dependent on bacterial flora The concentrations of short-chain fatty acids in serum samples from young adult and aged SPF mice were identified using targeted liquid chromatography-mass spectrometry (LC-MS) metabolite analysis (see Figure 3a). Acetate was the most abundant in the serum of young adult and aged mice, while acetate and propionate concentrations were higher in the serum of aged mice compared to young adult mice. Butyrate / isobutyrate and valerate / isovalerate were unchanged (see Figure 3a). In an unbiased screen, we used a non-targeted metabolomics dataset to investigate serum and brain samples from young adult and aged mice raised under SPF conditions (see Figures 3b-3c). Pathway enrichment analysis revealed some tissue-specific pathway changes. For example, several pathways related to pyrimidine, inositol, carnitine, and amino acid metabolism (e.g., lysine, polyamines, and tyrosine) were more affected in the serum of aged mice (see Figure 10a). Vitamin A, tocopherol, purine metabolism, ceramide-related pathways, and pentose phosphate pathways were specifically altered in the brains of aged mice (see Figure 10b). Fatty acid metabolism and advanced glycation end products (AGEs) pathways were commonly altered in both serum and brain samples from aged mice (see Figures 10a-10b). Metabolites that were significantly upregulated in both serum and brain tissue from aged mice allowed us to identify metabolites that may be regulated in the gut and reach the brain via the bloodstream. These metabolites included palmitoleic acid (16:1n7), TMAO, 1-oleoyl-2-docosahexaenoyl-glycerophosphorylcholine (18:1 / 22:6), CML, and stachydrine (see Figure 3d). Some of the age-related concentration changes observed in mice were also confirmed in human blood samples. Untargeted metabolomics of serum / plasma from a human aging cohort (TwinsUK databank) recapitulated the age-related changes in CML (see Figure 3e) and TMAO (see Figure 3f) concentrations seen in mice. Targeted metabolomics from brain tissue of young and aged SPF and GF mice demonstrated that a functional gut microbiota (e.g., SPF mice) was required for the increase in CML and TMAO in brain tissue from aged mice.Aged GF mice showed only minor changes compared with young GF mice (see Fig. 3g).

[0218] CML enhances age-related microglial dysfunction We next assessed the functional effects of these metabolites on microglia in vivo. The following candidate metabolites were selected for further evaluation: CML, TMAO, acetate, and propionate. To identify the metabolite responsible for increased ROS production in aged microglia, each metabolite was administered separately to young adult mice. To avoid potential artifacts due to differences in absorption profiles from the intestine to the circulation, young adult mice were intraperitoneally injected with CML, TMAO, sodium acetate, or sodium propionate once a day for 2 weeks (see Figure 4a). TMAO, sodium acetate, and sodium propionate had no effect on intracellular ROS production nor on the metabolic function of microglia. However, CML treatment was able to partially reproduce the changes seen in microglia from aged mice.

[0219] CML increased oxidative stress, decreased metabolic activity, and reduced intracellular ATP stores (see Figures 4b-4d). In addition, CML caused mitochondrial dysfunction by directly damaging the mitochondrial structure of microglia (see Figure 10c). The effects of CML treatment were not limited to microglia, but also adversely affected macrophages. Bone marrow-derived macrophages (BMDMs) showed a dose-dependent increase in oxidative stress and decreased metabolic activity in vitro (see Figures 10d-10e). Circulating CML may originate from the conversion of AGEs by endogenous Maillard reaction, diet, or gut microbiota (Non-Patent Document 28). Brain CML concentrations increased in aged SPF mice but not in aged GF mice (see Figure 4e). CML was detectable in brain tissue from young adult and aged GF mice at levels similar to those in young adult SPF mice. These results indicate that gut microbiota is required for elevated CML levels in the aging brain, but not for the baseline levels seen in young adult mice. Thus, these results also indicate that the dysregulation of mitochondrial function in microglia after intraperitoneal injection of CML is due to elevated brain CML concentrations, recapitulating the setting in the aging brain (see Figure 4f). RNA-seq analysis of microglia showed that intraperitoneal administration of CML increased the expression of ROS-related genes S100a9 and S100A8, as well as microbial and aging-related genes such as A430033K04Rik, Chic1, Ltf, Ngp, Pglyrp1, Scai, and Zkscan2 (see Figures 4g-4h and Figure 10f).

[0220] Immunofluorescence staining of CML in cerebral cortical microglia examined whether microglia are directly targeted by CML and showed that the percentage of CML-positive microglia increased with age in mice raised under SPF and GF conditions. Approximately 30% of microglia in aged SPF mice were CML-positive, while microglia in aged GF mice had less accumulation of CML with age (see Figure 4-i-4j). Furthermore, it was verified that the age-dependent increase in CML-positive microglia seen in the mouse cerebral cortex is also present in the human cerebral cortex. Human brain tissues from 1 to 88 years of age (total n = 43, 23 males, 20 females) were obtained, and a positive correlation (r = 0.5793, R 2 = 0.3356, P < 0.001) was observed (see Figure 4k-4l). In mice, RNA-seq analysis of microglia showed that i.p. injection of CML increased the expression of ROS-related genes S100a9 and S100A8, as well as other microbe- and aging-related genes such as A430033k04Rik, Chic1, Ltf, Ngp, Pglyrp1, Scai, and Zkscan2. These findings indicate that age-related accumulation of CML may induce metabolic dysfunction in microglia in a direct manner, including increased ROS, which may gradually disrupt brain homeostasis and brain function.

[0221] Aging microbiota drives CML levels by disrupting the blood-gut barrier Age-dependent gut microbiota changes by 16S ribosomal RNA-seq were characterized based on the finding that differences in CML levels and microglial function, especially during aging, depended on the presence or absence of a microbiota. Differential microbiota profiles of young adult and aged mice were confirmed by β-diversity analysis using the Bray-Curtis dissimilarity index and Shannon and Simpson α-diversity index (see Figures 11a-11b). The gut microbiota of both age groups was dominated by two phyla, namely Firmicutes and Bacteroidetes (see Figure 11c). The relative abundance of Firmicutes and Bacteroidetes changes with age in humans and can be associated with an overall change in the bacterial profile at different age stages (Non-Patent Document 29). A significant age-dependent decrease in the ratio of Firmicutes to Bacteroidetes was observed (Non-Patent Document 30), and the Firmicutes and Lachnospiraceae families were significantly decreased in aged mice (see Figures 11d-11e). In bacterial genera, increases in the genera Turibacter, Alloprevotella, Parastella, Bifidobacterium, Macellibacteroides, Alistipes sensu stricto 1, Peptostreptococcaceae incertae sedis, and Parabacteroides were observed in aged mice. This finding was in contrast to the decreased abundance of Pantonea, Anoxybacillus, Lachnospiraceae, Incertae, Sedis, Curtobacterium, and Acetatifactor in aged mice (Non-Patent Document 31) (see Fig. 11f). These findings indicate that profiling the microbiota of young adult and aged mice reveals changes at several taxonomic levels. Targeted metabolomics (LC-MS) measurement of CML in fecal pellets revealed that fecal pellets from aged GF mice had higher CML levels than those from aged SPF mice, demonstrating an indirect role for the microbiota in aging-related brain CML accumulation (see Fig. 5a).

[0222] Aged mice have enhanced intestinal permeability compared to young adult mice, a phenomenon that is dependent on the presence of a microbiota. Enhanced permeability may allow metabolites from within the digestive tract to cross the intestinal epithelium more freely and enter the bloodstream, potentially explaining the discrepancy between CML levels in the brain and feces. To test this hypothesis, we measured intestinal permeability by quantifying the translocation of FITC-dextran (4 kDa) into the circulation after oral administration. High intestinal permeability was observed in aged SPF mice, and the barrier function of aged GF mice was comparable to that of young adult SPF and GF mice (see Figure 5b). Colonization of young GF mice with aged microbiota resulted in a microbiota-dependent increase in intestinal permeability compared to that observed after administration of young gut microbiota to young GF mice (see Figure 5c). This was consistent with the observation that translocation of CML into the circulation after oral ingestion was highest in aged SPF mice (see Figure 5d and Figure 12a). To evaluate whether different routes of CML application affect CML accumulation in microglia, we studied young adult mice that received CML via intraperitoneal administration rather than oral administration. Such mice showed more CML-positive microglia in the cerebral cortex (see Figures 12b-12c). In aged mice, the route of CML administration did not affect the percentage of CML-positive microglia (see Figures 12b-12c). CML administration by both intraperitoneal and oral routes significantly exacerbated the age-associated increase in intracellular ROS and decline in metabolic function in microglia of aged mice. In young adult mice, such effects were detectable only after intraperitoneal administration of CML (see Figures 12d-12e). To verify the critical role of the intestinal barrier in the accumulation of CML in microglia with aging, aged SPF mice (18 months old) were orally administered ellagic acid (EA), which prevents CML accumulation by reducing age-associated microbiota dysbiosis and inducing autophagy in the intestinal epithelium, or intestinal alkaline phosphatase (IAP), an endogenous enhancer of intestinal barrier function, every 3 days for 10 weeks (see Figure 5e). EA did not affect intestinal permeability, but aged mice treated with IAP had reduced intestinal leakiness (see Figure 5f). Both EA and IAP indirectly or directly, respectively, reduced CML accumulation in the brain (see Figure 5g).Microglia from EA- and IAP-treated aged mice showed a significant decrease in intracellular ROS and an increase in ATP levels compared to vehicle-treated aged mice (Figures 5h-5i). These findings indicate that age-related changes in the microbiota disrupt gut barrier integrity and promote CML accumulation in the brains of aged mice and humans (Figure 1a).

[0223] Example 2. Identifying subjects at increased risk for developing cognitive impairment or neurodegenerative disease A subject can be identified, for example, as having an increased risk of developing microglial dysfunction, an increased risk of cognitive impairment, or an increased risk of developing a neurodegenerative disease by a method that includes identifying a subject having an elevated level of CML, CML precursor, CML metabolite, or CML analog in a biological sample obtained from the subject compared to a reference level. The biological sample can be, for example, blood, serum, or plasma.

[0224] In some embodiments, the amount of CML, CML precursors, CML metabolites, or CML analogs in a biological sample is measured by chromatography (e.g., high performance liquid chromatography), mass spectrometry, liquid chromatography mass spectrometry, or nuclear magnetic resonance spectroscopy. If an elevated level of CML, CML precursors, CML metabolites, or CML analogs is observed in a biological sample, the subject can be identified as having, for example, an elevated risk of developing microglial dysfunction, an elevated risk of cognitive impairment, or an elevated risk of developing a neurodegenerative disease.

[0225] Embodiment Described herein in certain embodiments is a method for selecting a subject for treatment with an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis, the method comprising: (a) identifying a subject having an elevated level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the subject, compared to a reference level; and (b) selecting the identified subject for treatment with an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0226] Described herein, in certain embodiments, is a method of treating a subject, comprising administering a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis to a subject identified as having elevated levels of CML, CML precursors, CML breakdown products, or CML metabolites compared to reference levels in a biological sample obtained from the subject.

[0227] Described herein in certain embodiments is a method for reducing the rate of accumulation of CML, CML precursors, CML breakdown products, or CML metabolites in a tissue or body fluid sample from a subject, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal flora dysbiosis.

[0228] Described herein, in certain embodiments, is a method for reducing the incidence of oxidative or metabolic stress in microglia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0229] Described herein, in certain embodiments, is a method for reducing the incidence of mitochondrial dysfunction in microglia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0230] Described herein, in certain embodiments, is a method for reducing the incidence of microglial dysfunction in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0231] Described herein, in certain embodiments, is a method for increasing one or more functions of microglia in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0232] Described herein, in certain embodiments, is a method for reducing the incidence or worsening rate of cognitive impairment in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0233] Described herein, in certain embodiments, is a method for treating cognitive impairment in a subject in need thereof, comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0234] Described herein, in certain embodiments, is a method for reducing the incidence or progression rate of a neurodegenerative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0235] Provided herein, in certain embodiments, is a method for treating a neurodegenerative disease in a subject, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0236] Described herein, in certain embodiments, is a method for reducing the incidence or worsening rate of neurological dysfunction in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of an intestinal barrier function enhancer and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

[0237] In certain embodiments, the subject has previously been identified as having elevated levels of CML, CML precursors, CML breakdown products, or CML metabolites in the subject's biological sample compared to baseline levels.

[0238] In certain embodiments, the method further comprises identifying the subject as having an elevated level of CML, a CML precursor, a CML breakdown product, or a CML metabolite in the subject's biological sample compared to a reference level.

[0239] In certain embodiments, the subject has previously been identified as having an elevated level of intestinal barrier permeability compared to baseline levels.

[0240] In certain embodiments, the method further comprises identifying the subject as having an elevated level of intestinal barrier permeability as compared to a baseline level.

[0241] In certain embodiments, the biological sample comprises saliva, urine, blood, serum, plasma, cerebrospinal fluid, brain tissue, or feces.

[0242] In certain embodiments, the subject has been identified or diagnosed as having a cognitive impairment.

[0243] In certain embodiments, the subject has been identified as being at increased risk for developing a cognitive disorder.

[0244] In certain embodiments, the subject has been identified or diagnosed as having a neurodegenerative disease.

[0245] In certain embodiments, the subject has been identified as being at increased risk for developing a neurodegenerative disease.

[0246] In certain embodiments, the method results in a reduction in the levels of intracellular and / or mitochondrial ROS in the subject's microglia.

[0247] In certain embodiments, the method results in a decrease in expression of iNOS in microglia of the subject.

[0248] In certain embodiments, the method results in decreased expression of one or more genes selected from the group consisting of Cdkn1a, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, Tlr5, Axl, Hif1a, Lcn2, Mmp2, Rela, Trex1, S100a8, and S100a9 in microglia of the subject.

[0249] In certain embodiments, the method results in increased expression in the subject's microglia of one or more genes selected from the group consisting of Foxp1, Nrf1, Trp53, G6pdx, Pdk2, Stat3, and Ucp2 in the subject.

[0250] In certain embodiments, the methods further comprise determining the level of CML, CML precursors, CML breakdown products, or CML metabolites in a biological sample obtained from the subject.

[0251] In certain embodiments, the neurodegenerative disease is selected from the group consisting of: Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, progressive supranuclear palsy, spinal muscular atrophy, multiple system atrophy, ataxia, vascular dementia, or other dementias.

[0252] Provided herein, in certain embodiments, is a method of identifying a subject as having an increased risk of developing microglial dysfunction, the method comprising identifying a subject having an elevated level of CML, a CML precursor, a CML breakdown product, or a CML metabolite in a biological sample obtained from the subject compared to a reference level, wherein such elevated level indicates that the subject is at increased risk of developing microglial dysfunction.

[0253] Provided herein, in certain embodiments, is a method for identifying a subject as having an increased risk of cognitive impairment, the method comprising identifying a subject having an elevated level of CML, a CML precursor, a CML breakdown product, or a CML metabolite in a biological sample obtained from the subject compared to a reference level, wherein such elevated level indicates that the subject is at increased risk of developing cognitive impairment.

[0254] Provided herein, in certain embodiments, is a method for identifying a subject as having an increased risk of developing a neurodegenerative disease, the method comprising identifying a subject having an elevated level of CML, a CML precursor, a CML breakdown product, or a CML metabolite in a biological sample obtained from the subject compared to a reference level, wherein such elevated level indicates that the subject is at increased risk of developing a neurodegenerative disease.

[0255] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the accumulation rate of CML, CML precursors, CML degradation products, or CML metabolites in a tissue or body fluid sample from a subject, the method comprising: determining a first level of CML, CML precursors, CML degradation products, or CML metabolites in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursors, CML degradation products, or CML metabolites in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the accumulation rate of CML, CML precursors, CML degradation products, or CML metabolites in the tissue of the subject.

[0256] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the incidence of oxidative or metabolic stress in microglia in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence of oxidative or metabolic stress in microglia of the subject.

[0257] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the incidence of oxidative or metabolic stress in microglia in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence of oxidative or metabolic stress in microglia of the subject.

[0258] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the incidence of microglial mitochondrial dysfunction in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence of microglial mitochondrial dysfunction in the subject.

[0259] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the incidence of microglial dysfunction in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence of microglial dysfunction in the subject.

[0260] Provided herein, in certain embodiments, is a method for screening a candidate agent for increasing one or more activities of microglia in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in the biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for increasing one or more activities of microglia in the subject.

[0261] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the incidence or worsening rate of cognitive impairment in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence or worsening rate of cognitive impairment in the subject.

[0262] Provided herein, in certain embodiments, is a method for screening candidate agents for treating cognitive impairment in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for treating cognitive impairment in the subject.

[0263] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the incidence or progression rate of a neurodegenerative disease in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence or progression rate of a neurodegenerative disease in the subject.

[0264] Provided herein, in certain embodiments, is a method for screening candidate agents for treating a neurodegenerative disease in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for treating the neurodegenerative disease in the subject.

[0265] Provided herein, in certain embodiments, is a method for screening candidate agents for reducing the incidence or worsening rate of neurological dysfunction in a subject, the method comprising: determining a first level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from a mammal at a first time point; administering an agent to the subject; and determining a second level of CML, CML precursor, CML degradation product, or CML metabolite in a biological sample obtained from the mammal at a second time point; wherein an agent that results in a decrease in the second level compared to the first level is identified as a candidate agent for reducing the incidence or worsening rate of neurological dysfunction in the subject.

[0266] In certain embodiments, the method further comprises testing the candidate agent in an animal model.

[0267] Incorporation by Reference All publications and patents cited throughout the text of this specification (including all patents, patent applications, scientific publications (e.g., 32 ), manufacturer's specifications, instructions, etc.), whether supra or infra, are incorporated by reference in their entirety for all purposes. To the extent that material incorporated by reference is inconsistent or contradictory with the present specification, the present specification takes precedence over any such material.

[0268] Equivalent The present invention may be embodied in other specific forms without departing from its spirit or essential characteristics. The foregoing embodiments are therefore considered in all respects to be illustrative rather than limiting of the invention described herein. The scope of the invention is therefore indicated by the appended claims rather than by the foregoing description, and all changes that come within the meaning and range of equivalency of the claims are intended to be embraced therein.

Claims

1. 1. A method of reducing the incidence of oxidative stress in microglia or mitochondrial dysfunction, or microglial dysfunction, in a subject in need thereof, comprising: A method comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

2. 1. A method of preventing or treating a cognitive impairment or a neurodegenerative disease, or reducing the incidence or progression rate of a cognitive impairment, or a neurodegenerative disease in a subject in need thereof, comprising: A method comprising administering to the subject a therapeutically effective amount of an agent for enhancing intestinal barrier function and / or an agent for reducing or eliminating intestinal microbiota dysbiosis.

3. The method described in claim 2, wherein the method reduces neurological dysfunction. (i) the subject has previously been identified as having an elevated level of N6-carboxymethyllysine (CML), a CML precursor, a CML metabolite, or a CML analog in the subject's biological sample compared to a baseline level; and / or (ii) The method of claim 1, wherein the method further comprises identifying the subject as having an elevated level of CML, a CML precursor, a CML metabolite, or a CML analog in the subject's biological sample compared to a reference level. (i) the subject has previously been identified as having an elevated level of intestinal barrier permeability compared to baseline levels; and / or 2. The method of claim 1, wherein (ii) the method further comprises identifying the subject as having an elevated level of intestinal barrier permeability compared to a baseline level.

6. The method of claim 4 , wherein the biological sample comprises a body fluid or tissue sample.

7. The subject (i) cognitive impairment, and / or (ii) neurodegenerative disease 10. The method of claim 1, wherein the patient is identified or diagnosed as having:

8. The subject (i) cognitive impairment, and / or (ii) neurodegenerative disease The method of claim 1, wherein the patient is identified as having an increased risk of developing

9. The method described in claim 1, wherein the method results in a reduction in the levels of intracellular and / or mitochondrial reactive oxidative species (ROS) in the subject's microglia.

10. The method comprising: (a) resulting in a decrease in the expression of inducible nitric oxide synthase (iNOS) in microglia of said subject; (b) resulting in a decrease in the expression of one or more genes in microglia of said subject selected from the group consisting of Cdkn1a, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, Tlr5, Axl, Hifl a, Lcn2, Mmp2, Rela, Trex1, S100a8, and S100a9; 2. The method of claim 1, wherein (c) the method results in increased expression of one or more genes in the subject's microglia selected from the group consisting of Foxp1, Nrf1, Trp53, G6pdx, Pdk2, Stat3, and Ucp2.

11. 5. The method of claim 4, further comprising determining the level of CML, CML precursors, CML metabolites, or CML analogs in the biological sample obtained from the subject.

12. 8. The method of claim 7, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, Parkinson's disease, Huntington's disease, frontotemporal dementia, amyotrophic lateral sclerosis, multiple sclerosis, glaucoma, myotonic dystrophy, progressive supranuclear palsy, spinal muscular atrophy, multiple system atrophy, ataxia, and vascular dementia.

13. The method comprising: (a) reducing the concentration of CML, CML precursors, CML metabolites, or CML analogs in blood or brain samples; (b) reducing the rate of accumulation of CML, CML precursors, CML metabolites, or CML analogs in the tissues of the subject; and / or (c) reducing intestinal permeability.

14. The method of claim 1, wherein the intestinal barrier function enhancer and / or agent for reducing or eliminating intestinal bacterial flora dysbiosis comprises intestinal alkaline phosphatase (IAP), lipoteichoic acid, metformin, ellagic acid (EA), urolithin A, butyric acid, glutamine, obeticholic acid (OCA), divertin, or curcumin, or a derivative thereof.

15. The method according to claim 14, wherein the agent for enhancing intestinal barrier function and / or the agent for reducing or eliminating intestinal bacterial flora dysbiosis is an IAP.

16. The method according to claim 14, wherein the agent for enhancing intestinal barrier function and / or the agent for reducing or eliminating intestinal bacterial flora dysbiosis is EA.

17. 2. The method of claim 1, wherein the intestinal barrier function enhancer and / or the agent for reducing or eliminating intestinal bacterial flora dysbiosis is formulated as a pharmaceutical composition comprising IAP, lipoteichoic acid, metformin, EA, urolithin A, butyric acid, glutamine, OCA, divertin, or curcumin, or a pharmaceutically acceptable salt thereof.

18. The method of claim 1, wherein the agent for enhancing intestinal barrier function and / or the agent for reducing or eliminating intestinal bacterial flora dysbiosis is administered orally, transdermally, by inhalation, intranasally, topically, intravenously, intraarterially, intramuscularly, or subcutaneously.

19. After administration of the intestinal barrier function enhancing agent and / or the agent for reducing or eliminating intestinal bacterial flora dysbiosis, the subject (a) reducing the concentration of CML, CML precursors, CML metabolites, or CML analogs in a blood sample; (b) reducing the concentration of CML, CML precursors, CML metabolites, or CML analogs in a brain tissue sample; (c) decreased intestinal permeability; (d) reduction of bacterial flora dysbiosis; (e) increased levels of autophagy in the intestinal epithelium; (f) a reduction in the levels of cellular and / or mitochondrial ROS in microglia; (g) increased levels of adenosine triphosphate (ATP) in the microglial population; (h) decreased expression of iNOS in microglia; (i) a decrease in the expression of one or more genes selected from the group consisting of Cdkn1a, Cyba, Cybb, Duoxa1, Il1b, Tgfbr2, Tlr2, Tlr4, Tlr5, Axl, Hifla, Lcn2, Mmp2, Rela, Trex1, S100a8, and S100a9 in microglia; and (j) increased expression of one or more genes in microglia selected from the group consisting of Foxp1, Nrf1, Trp53, G6pdx, Pdk2, Stat3, and Ucp2; The method of claim 1 , wherein the method exhibits one or more of: (a) the bodily fluid is selected from the group consisting of saliva, urine, blood, serum, plasma, cerebrospinal fluid, or feces; and / or (b) the tissue sample is brain tissue.

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