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Background: Microbiota‐derived short chain fatty acids (SCFA) and liposaccharide (LPS) could modulate astrocyte and neuronal activity. Glial fibrillary acidic protein (GFAP) and neurofilament light (NfL) are markers of astroglial activation and neuronal damage, can be measured in blood samples and have been associated with Alzheimer's disease (AD) pathology. The aim of the present study was to assess the association of SCFAs and LPS with GFAP and NfL in 13 amyloid‐β‐negative cognitively unimpaired individuals (CU), 38 patients with cognitive impairment and no brain amyloidosis (CI‐NAD) and 34 patients with cognitive impairment and amyloid‐β positivity (CI‐AD). Method: Blood levels of 4 SCFAs (acetate, propionate, valerate and butyrate) were measured by GC‐FID gaschromatography, LPS by ELISA, and GFAP and NfL by single‐molecule array technology. Result: Plasma valerate, LPS and GFAP concentrations were increased in CI‐AD compared with CI‐NAD and CU (p<0.034), acetate was increased in CI‐AD as well as decreased in CI‐NAD as compared with CU (p<0.018) and NfL was increased in both CI groups (p<0.023). In the pooled group of CI‐AD and CU, there were significant associations between plasma LPS and valerate with higher GFAP and NfL (p<0.032). Conversely, in the pooled group of CI‐NAD and CU, there were associations between plasma acetate with lower level of GFAP and NfL (p<0.026). Conclusion: Valerate and LPS were associated with astrogliosis and neuronal damage only in patients with AD. These results suggest that microbiota‐derived molecules might be mediators along the gut‐brain axis and contribute to AD pathology by modulating microglial and neuronal activity. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence shows that the gut microbiota (GMB) may be involved in the pathogenesis of several brain disorders. Metagenomic data support an association between certain bacterial genera and Alzheimer's Disease (AD), but the functional dynamics of the gut microbiota remains elusive. This study aimed at evaluating whether inflammatory mediators and bacterial products represent a potential pathogenic link between specific gut bacteria and amyloid pathology in AD. Method: Seventy‐two older persons with cognitive performance from normal to dementia with brain amyloid status were recruited and stool and blood samples were collected. Bacterial composition of fecal samples was inferred using 16S sequencing, analyzed with QIIME 2 (Quantitative Insights into Microbial Ecology, https://qiime2.org/). Blood lipopolysaccharide (LPS), short chain fatty acids (SCFAs) acetate, propionate, valerate and butyrate by gas chromatography with flame ionization detector (GC‐FID) and circulating adhesion molecules by flow cytometry bead‐based immunoassay panel. We compared HC, amyloid negative (Amy‐) and positive (Amy+) patients with respect to GMB‐specific variables and endothelial biomarkers. Result: Metagenomic differences were mainly related to the HC rather than Amy+ or Amy‐. Despite a similar GMB, Amy+ and Amy‐ reported differences in the levels of several blood GMB‐related products: Amy+ had increased valerate (p<.001 vs both HC and Amy‐) and LPS (p=.011 vs Amy‐; p=.031 vs HC) levels while Amy‐ decreased acetate (p<.001 vs Amy+; p=.003 vs HC) and increased butyrate (p<.001 vs Amy+; p=.002 vs HC) levels. Endothelial dysfunction, as indicated by the upregulation of adhesion molecules (NCAM, PECAM‐1, ICAM‐2, PSGL‐1), was observed in Amy+ only (p<.030 vs Amy‐; p<.010 vs HC). High levels of these adhesion molecules were associated with elevated levels of acetate and valerate and low levels of butyrate (0.26<|rho|<0.51, p<.024). Conclusion: The mechanisms linking GMB dysbiosis and cognitive impairment in the presence or absence of brain amyloid deposition are different. Reduction of butyrate together with increased acetate, valerate and LPS levels may compromise the tissue‐blood barrier integrity, facilitating the AD pathological cascade. Microbiota functional dynamics, more than its composition, may be a potential biomarker of AD and provide new leads for treatments aimed at preventing or delaying neurodegeneration. [ABSTRACT FROM AUTHOR]

Background: Human microbiota‐associated (HMA) murine models are developed by gavaging mice with human bacteria or stools and are increasingly used to study the relation between the human microbiota and disease. The aim of this study is to evaluate the effect of gut bacteria isolated from donors with or without Alzheimer's dementia on behavior in AD transgenic (Tg) mice. Method: Sixty female, one‐year old 3xTg mice (APPSWE, PS1M146V and TauP301L) received (1) antibiotic treatment for 14 days before first gavage, (2) stomach acid suppressant, (3) laxative immediately before the first gavage and, (4) microbiota transplantation or saline, once every 2 weeks for 2 months. The transplanted microbiota consisted of bacteria isolated from stools of donors pertaining to four different profiles: i) an amyloid positive AD patient,ii) an amyloid negative cognitively healthy subjects with or iii) without protective APOE genotype and, iv) a young healthy donor. As additional control, we included a group of mice that received bacteria isolated from stools of untreated 3xTg mice. Stools collection and behavioral assessment (open‐field test, Y‐maze test, radial maze test, elevated plus maze test) was performed before antibiotic treatment and at day 3, 30, 60 after the first gavage. All mice were sacrificed at day 60 for ex‐vivo assessments. Result: After 60 days from first gavage, bacteria from the AD patient (i) induced AD‐like behavior in 3xTg mice, including impaired general activity in the open‐field test and exacerbated anxiety in the elevated plus maze test, compared with mice that received saline or bacteria from untreated 3xTg mice. Interestingly, bacteria from donor resilient to AD pathology (donor of profile ii) induced better cognitive score to object recognition when transplanted to 3xTg mice. Conclusion: The microbiota of AD patients induced an AD‐like phenotype in 3xTg mice, and microbiota from patient resilient to AD pathology induced a better memory score. Brain and gut ex‐vivo assessments, 16S rRNA gene sequencing as well as behavioral longitudinal analysis are on‐going to evaluate the human microbiota transplantation efficiency and its effect on AD pathology and inflammation. [ABSTRACT FROM AUTHOR]

Background: Although recent data support an association between certain bacterial strains and Alzheimer's Disease (AD), it is not clear how these alterations contribute to different aspects of the disease. Increasing evidences also from our group (Cattaneo, AAIC 2020), suggest that endothelial dysfunction, reflected by the upregulation of cell adhesion molecules (CAMs), might have a role in AD pathology development and might be affected by inflammatory mediators. This study aimed at evaluating the gut microbiota (GMB) profile and its association with CAMs and local and systemic inflammation in AD patients. Method: Sixty‐six older persons without cognitive performance (HC) and 68 AD patients were recruited and stool and blood samples were collected. Bacterial composition of fecal samples was inferred using 16S sequencing, analyzed with QIIME 2 (Quantitative Insights into Microbial Ecology, https://qiime2.org/). Inflammatory mediators were quantified in the blood using the mesoscale platform (for cytokines) and in the stools using ELISA (for calprotectin), CAMs by FACS. Result: Compared to HC, AD patients showed i) lower abundance of genera known to have anti‐inflammatory, neuroprotective or other beneficial effects on the epithelial barrier (as Akkermansia, Roseburia and Fusicatenibacter) as well as higher abundance in pro‐inflammatory genera (as Desulfovibrio) (Lefse, p< 0.039); ii) higher levels of the proinflammatory cytokine IL8 (p= 0.044) in the blood and calprotectin in the stools (p= 0.012); iii) higher level of several adhesion molecules (NCAM, PECAM‐1, P‐, L‐, E‐Selectin; p< 0.024) in the blood. Low abundance of the beneficial genera as well as high abundance of the proinflammatory genera were associated with elevated levels of fecal calprotectin and blood CAMs (0.19<|rho|<0.30, p<.048). Finally, high calprotectin levels were associated with elevated CAMs expression (0.22<|rho|<0.32, p<.017). Conclusion: These findings confirm our previous results showing that gut dysbiosis was associated with endothelial dysfunction. In addition, we showed that gut derived inflammation, linked to both altered bacteria abundance and CAMs upregulation, might contribute to AD. [ABSTRACT FROM AUTHOR]

Background: The genetic, clinical and anatomical presentation of Alzheimer's disease (AD) is extremely heterogenous. Although increasing evidences show that alterations in the gut microbiota (GMB) composition are involved in AD, their contribution to AD pathology remain unclear. The aim of this study is to evaluate whether disease‐specific alterations of the microbiome are present in patients with AD pathology and can identify individuals with peculiar clinical features. Method: Sixty‐two cognitive impaired subjects underwent Florbetapir amyloid PET and stool collection. Brain amyloidosis was measured with standardized uptake value ratio versus cerebellum (SUVRs) in the frontal, parietal, temporal, anterior cingulate, posterior cingulate and precuneus regions of interest. Amyloid positivity was defined as global PET florbetapir SUVR>1.11. Bacterial composition of fecal samples was inferred using 16S metagenomic sequencing, analyzed with QIIME 2 (Quantitative Insights into Microbial Ecology, https://qiime2.org/). We first compared amyloid negative (Amy‐) and positive (Amy+) patients with respect to GMB‐specific profiles. We then applied Principal Component Analysis (PCA) on those taxa found differentially abundant in Amy+ and Amy‐. Result: Despite a similar GMB structure (Unweighted and weighted Unifrac, p>0,05) of Amy+ (n=29) and Amy‐ (n=33), taxonomic comparison showed that Amy+ were mainly characterized by rare genera. PCA identified a small subgroup of 6 Amy+ with lower amyloid SUVR uptake in the temporal cortex (p=0,009) and lower cognitive deficits, as showed by better performance at MMSE, ADAScog, logical memory, Rey auditory verbal learning test, Raven's progressive matrices test and Functional Activities Questionnaire (p<0.044) compared to the other Amy+ patients. Amyloid SUVR uptake in the temporal cortex was negatively associated with logical memory, Rey auditory verbal learning test, Raven's progressive matrices test and Functional Activities Questionnaire (moderate: rho£0.44, p£0.047) and MMSE (weak: rho=0.36, p=0.076) as well as negatively associated with FAQ (moderate: rho=0.47, p=0.010) in the whole Amy+ group. Conclusion: Despite the small sample size, this study suggests that distinctive bacterial signatures might be associated with specific clinical AD phenotypes. [ABSTRACT FROM AUTHOR]

Background: The genetic, clinical and anatomical presentation of Alzheimer's disease (AD) is extremely heterogenous. Although increasing evidences show that alterations in the gut microbiota (GMB) composition are involved in AD, their contribution to AD pathology remain unclear. The aim of this study is to evaluate whether disease‐specific alterations of the microbiome are present in patients with AD pathology and can identify individuals with peculiar clinical features. Method: Sixty‐two cognitive impaired subjects underwent Florbetapir amyloid PET and stool collection. Brain amyloidosis was measured with standardized uptake value ratio versus cerebellum (SUVRs) in the frontal, parietal, temporal, anterior cingulate, posterior cingulate and precuneus regions of interest. Amyloid positivity was defined as global PET florbetapir SUVR>1.11. Bacterial composition of fecal samples was inferred using 16S metagenomic sequencing, analyzed with QIIME 2 (Quantitative Insights into Microbial Ecology, https://qiime2.org/). We first compared amyloid negative (Amy‐) and positive (Amy+) patients with respect to GMB‐specific profiles. We then applied Principal Component Analysis (PCA) on those taxa found differentially abundant in Amy+ and Amy‐. Result: Despite a similar GMB structure (Unweighted and weighted Unifrac, p>0,05) of Amy+ (n=29) and Amy‐ (n=33), taxonomic comparison showed that Amy+ were mainly characterized by rare genera. PCA identified a small subgroup of 6 Amy+ with lower amyloid SUVR uptake in the temporal cortex (p=0,009) and lower cognitive deficits, as showed by better performance at MMSE, ADAScog, logical memory, Rey auditory verbal learning test, Raven's progressive matrices test and Functional Activities Questionnaire (p<0.044) compared to the other Amy+ patients. Amyloid SUVR uptake in the temporal cortex was negatively associated with logical memory, Rey auditory verbal learning test, Raven's progressive matrices test and Functional Activities Questionnaire (moderate: rho£0.44, p£0.047) and MMSE (weak: rho=0.36, p=0.076) as well as negatively associated with FAQ (moderate: rho=0.47, p=0.010) in the whole Amy+ group. Conclusion: Despite the small sample size, this study suggests that distinctive bacterial signatures might be associated with specific clinical AD phenotypes. [ABSTRACT FROM AUTHOR]

Background: Apolipoprotein E (APOE) ε4 allele is the main genetic risk factor for AD and has different roles in modulating the disease process along with the continuum from intact cognition to dementia due to AD, with its strongest influence at the earliest stages. The aim of this study is to test our hypothesis that places APOEε4 as a trigger in the perturbation of gut microbiota homeostasis. Method: Fecal samples were collected from 28 cognitively intact elderly with known APOE genotype. Bacterial 16S rRNA gene was sequenced according to the 16S‐protocol on MiSeq (Illumina). Taxonomic abundance was computed with the free software package QIIME 2 (Quantitative Insights into Microbial Ecology; Bokulich, Microbiome, 2018; https://qiime2.org/). Plasma levels of cytokines were measured using a commercially available multiplex beads immunoassay, based on the Luminex platform. Result: APOEε4 carriers showed altered abundance in 5 genera (LEfSe, p<0.048), 3 more (Bifidobacterium, Actinomyces, Lactococcus) and 2 less (Ruminococcaceae UCG‐003, Candidatus Melainabacteria) abundant, as well as decreased blood levels of IL‐8 (p=0.047) compared with non‐carriers. Elevated abundance of Bifidobacterium, Actinomyces, Lactococcus were associated with decreased levels of IL‐8 and IL‐6 (rho<‐0.41, p<0.040). Conclusion: Structural microbiota alterations are associated with the restraint of some peripheral pro‐inflammatory signaling pathways in healthy subjects at genetic risk for AD. As a "good" form of inflammation (i.e. I‐6, IL1‐beta, TNF‐alpha) has been proposed to be beneficial in the early phases of the disease in several AD mouse models (Chakrabarty, J Neurosci 2010; Shaftel, J Neurosci 2007), these preliminary data suggest that the deleterious impact of the APOE4 allele on AD development might start in the gut. [ABSTRACT FROM AUTHOR]

Background: Increasing evidence shows that the gut microbiota (GMB) may affect the inflammatory status and influence the pathogenesis of several brain disorders, including Alzheimer's Disease. Moreover, it has been suggested that probiotics may have potential beneficial effects on the immune system via microbiome composition, influencing in turn also cognitive performances. Method: Sixty‐two old subjects with normal cognitive performance and 37 AD patients were recruited and stool and blood samples were collected. Moreover, the group of AD patients received a probiotic administration for three months and stool and blood samples were collected again at the end of the treatment. Plasma levels of a panel of pro‐inflammatory cytokines were measured by using the High Sensitivity 9‐Plex Human ProcartaPlex™ Panel (IFN‐ϒ, IL‐1b, IL‐2, IL‐4, IL‐6, IL‐10, IL‐12p70, IL‐17A and TNFα, Thermo Fisher Scientific) on Luminex Bio‐Plex® 200™ system. Moreover, we also measured the plasma levels of C‐Reactive Protein (CRP) using the CRP Human Instant ELISA Kit (Thermo Fisher Scientific). Bacterial composition of fecal samples was inferred using 16s sequencing; the levels of the short chain fatty acids acetate, propionate, valerate and butyrate were measured by mass spectrometry and adhesion molecules by FACS. Result: We found higher levels of the pro‐inflammatory cytokines IL‐6, IFN gamma, TNF‐alpha, IL‐1beta, and il12p70 and of IL‐2 and lower levels of the anti‐inflammatory cytokine IL‐10 in AD patients as compared to control subjects (all p<0.05). Moreover, we found a positive correlation between MMSE score and the levels of pro‐inflammatory cytokine IL‐6 (p=0.03, r=0.56). Interestingly, the three months treatment with the probiotic composition was able to significantly reduce the level of IL‐6 (p=0.03) and increase the levels of IL‐10 (p=0.023). Bioinformatic analyses on the 16s sequencing data as well as on the SCFAs and endothelial markers are ongoing. Conclusion: Inflammatory status is higher in AD patients as compared to control subjects and it correlates with cognitive performances. Interestingly probiotic composition is able to push down the inflammatory status in patients, possibly via changes in microbiome composition. Probiotics may be beneficial in delaying neurodegeneration and cognitive deficits in subjects at risk of developing Alzheimer Disease. [ABSTRACT FROM AUTHOR]

Background: Human microbiota‐associated (HMA) murine models are developed by gavaging mice with human bacteria or stools and are increasingly used to study the relation between the human microbiota and disease. The aim of this study is to evaluate the effect of gut bacteria isolated from donors with or without Alzheimer's dementia on behavior in AD transgenic (Tg) mice. Method: Sixty female, one‐year 3xTg mice (APPSWE, PS1M146V and TauP301L) received (1) antibiotic treatment (for 14 days before the first gavage), (2) stomach acid suppressant administration, (3) laxative administration (immediately before the first gavage) and, (4) microbiota transplantation (or saline, once every 2 weeks for 2 months). The transplanted microbiota consisted of bacteria isolated from stools of donors pertaining to four different profiles: i) amyloid positive AD patient, amyloid negative cognitively healthy subjects ii) with or iii) without protective APOE genotype and, iv) young healthy donor. As additional control, we included a group of mice that received bacteria isolated from stools of untreated 3xTg mice. Stools collection and behavioral assessment (open‐field test, Y‐maze test, radial maze test, elevated plus maze test) was performed before antibiotic treatment and at day 3, 30, 60 after the first gavage. All mice were sacrificed at day 60 for ex‐vivo assessments. Result: After 60 days from the first gavage, the bacteria from the AD patient induced AD‐like behavior in 3xTg mice, including impaired general activity in the open‐field test and exacerbated anxiety in the elevated plus maze test, compared with mice that received saline or bacteria from untreated 3xTg mice. The bacteria from the other human donors did not influence mouse behavior. Conclusion: The microbiota of AD patients induced an AD‐like phenotype in 3xTg mice. Brain and gut ex‐vivo assessments, 16S rRNA gene sequencing as well as behavioral longitudinal analysis are on‐going to evaluate the human microbiota transplantation efficiency and its effect on AD pathology and inflammation. [ABSTRACT FROM AUTHOR]

Background: Sequencing of 16S ribosomal RNA (rRNA) gene is the most used technique for the identification of Alzheimer's specific differences in the relative abundance of specific organisms. Several bioinformatic pipelines have been developed so far to characterize complex gut microbial communities. This study aims at evaluating the effect of bioinformatic pipelines and reference databases on the taxonomic classification of bacteria in the stools of patients with cognitive impairment. Method: DNA from frozen stool samples of 40 subjects was extracted using QIAamp Fast DNA stool kit after mechanical disruption (bead‐beating) and amplified using primers targeting the V3‐V4 region of the bacterial 16S rRNA gene. Sequencing was performed according to the 16S‐protocol on MiSeq (Illumina). Sequence read files were analysed using 4 commonly used pipelines run on a Linux workstation: QIIME2 (Bokulich, Microbiome, 2018), Bioconductor (Callahan, F1000Research 2016, version 29 oct 2018), UPARSE (Edgar, Nat Methods, 2013) and mothur (Kozich, Applied and Environmental Microbiology, 2013). SILVA 132 and RDP v16 reference databases were used in all analyses. For UPARSE and mothur, sequences were clustered into OTUs at 99% identity threshold. Differences of relative abundances of phyla and genera were evaluated across pipelines using Friedman rank sum test. Result: The identification of phyla but not their quantification was consistent among all the pipelines using both databases (p<0.001). At genus level, all pipelines identified Bacteroidetes, Alistipes, Faecalibacterium and Ruminococcus as the most abundant genera, regardless of the database applied for taxonomic assignment. Again, their relative abundance was different across pipelines and databases (p<0.001). Conclusion: The comparison between different studies should take analytical pipeline and database used for taxonomic assignation into account. To overcome these limitations, the scientific community should quickly converge towards a consensus pipeline for guarantee research reproducibility. [ABSTRACT FROM AUTHOR]