Abstract Body

Abstract Body

Objectives: It is now well established that inflammation associated with amyloid deposition reflects the activation of astrocytes and microglia in response to injury, but the role of peripheral tissues and more importantly, the microbiota in regulating innate immunity that in turn leads to CNS dysfunction has not, to date been defined. We have tested the hypothesis that the composition of the intestinal microbiome plays a key role in modulating neuro-inflammation that will ultimately influence amyloid deposition in two established mouse models of Ab-amyloidosis.

Methods: We orally administered a combination of antibiotics to induce rapid and sustained alterations in gut microbial populations in two mouse models of Ab amyloidosis. The antibiotic cocktail was administered either postnatally or throughout the lifetime of the animal prior to cull and we employed IHC, biochemical and molecular assays to evaluate amyloid deposition and neuroinflammation in the mouse models. Newly-generated germ-free mouse models are being evaluated.

Results: Our studies indicate that alterations in the microbiome parallel changes in plasma cytokines and chemokines, reductions in amyloid deposition and modulation of morphological and transcriptional landscapes of microglia. These effects are unique to male, but not female animals.

Conclusions: Our studies reveal an unexpected, but significant alteration in amyloid deposition and microglial phenotypes in the brains of male transgenic mice upon treatment with orally administered antibiotics.

Acknowledgments This work was supported by Cure Alzheimer’s Fund (CAF) Open Philanthropy Fund, Alzheimer’s Association and Good Ventures Foundation. SSS is a paid Consultant of AZTherapies Inc.

Abstract Body

Objective. Characterization of central and peripheral innate immune mechanisms in Alzheimer’s disease and the role of the microbiome.

Methods. To investigate the effect of the microbiota on AD, purified microbial components (Protollin), live microbiota, antibiotics or nasal anti-CD3 were administered to animal models of AD. Alterations in microglia and peripheral monocyte phenotypes in AD and aging were investigated.

Results. 1) Protollin. Nasal Protollin, a proteasome-based adjuvant altered the phenotype of splenic monocytes and enhanced their phagocytic activity. Using AD-CX3CR1GFP chimera mice we found that intranasal Protollin induced recruitment of BM derived CD11b+ cells and reduced A-beta plaques. 2) Microbiome. We identified several bacteria, including Bacteroides, that change during aging and correlate with amyloid beta plaque in an AD model and found that dietary modification restores these bacteria to levels observed in young animals. Administering Bacteroides increased plaques in an animal model of AD and altered peripheral immunity. 3) Antibiotics. Manipulating the microbiome with antibiotics increased activation markers on monocytes and reversed the microglial MGnD neurodegenerative disease signature including decreasing APOE and Clec7a. Together these data suggestion that manipulation of the microbiome can have a beneficial effect on AD. 4) Nasal anti-CD3. Nasal anti-CD3 induces IL-10 secreting regulatory cells and when given to 3xTg AD mice improved behavior and modulated microglia from a degenerative to a homeostatic phenotype.

Conclusions. Our data identify novel therapeutic approaches to target both the central and peripheral innate immunity for the treatment of AD. Clinical trials of Protollin in AD are planned.

Aims

Neurodegeneration is considered the consequence of misfolded proteins’ deposition. Little is known on external environmental effects on the neurodegenerative process. We studied whether systemic microbial pathogens may accelerate neurodegeneration in Alzheimer’s Disease (AD), and whether microglia play a central role in this process.

Methods

We examined cortical neuron density in transgenic 5xFAD and wild-type (wt) mice, that were housed in non-SPF versus SPF conditions, and in response to injection of bacterial lipopolysaccharide, a TLR4 agonist. Am580, a retinoic acid receptor α agonist, was delivered ICV by mini-osmotic pumps for regulation of Microglia. Microglial phenotype and functions were examined by ex vivo assays.

Results

In SPF conditions, 5xFAD mice exhibited marked AD pathology but no cortical neuronal loss at age 7 months, and mild loss at 12 months. Housing 5xFAD (but not wt) mice in a non-SPF environment caused accelerated neurodegeneration, occurring already at age 7 months. To model the effect of systemic pathogens, we injected LPS into wt and AD mice. 5xFAD mice exhibited increased vulnerability to LPS-induced neuronal loss compared to wt mice, mediated by CNS TLR4. Continuous ICV delivery of Am580 reduced neurotoxic iNOS+ brain microglia fraction, and protected from LPS-induced neurodegeneration. Am580 markedly attenuated iNOS expression, inhibited reactive oxygen species and nitric oxide production, increased TREM2 expression and phagocytic activity, while maintaining microglial capacity for immune (cytokine) response.

Conclusions

Systemic colonization with microbial pathogens and systemic infections cause neurodegeneration in brains displaying amyloid pathology. Microglial modulation protects the hyper-susceptible AD brain from microbial TLRs -induced neurodegeneration.

Aims

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 (AD) and that probiotics may have potential beneficial effects on the immune system via microbiome composition, influencing in turn also cognitive performances.

Methods

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. We measured: plasma levels of a panel of pro-inflammatory cytokines, of the short chain fatty acids acetate, propionate, valerate and butyrate and of adhesion molecules. Bacterial composition of fecal samples was inferred using 16s sequencing; .

Results

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).

Conclusions

Inflammatory status is higher in AD patients as compared to controls and it correlates with cognitive performances. Probiotic composition is able to push down the inflammatory status in patients, possibly via changes in microbiome composition.

Aims

Alzheimer’s (AD) and Parkinson’s disease (PD) are considered multifactorial in origin. In both, homeostasis of neurons is progressively impaired, leading to cellular death. Understanding metabolic changes can provide insight into processes affecting the cellular environment and disease etiology. We performed a metabolomic and lipidomic analysis in AD and PD, highlighting similarities and differences in their alterations.

Methods

Plasma samples were obtained from the Texas Alzheimer’s Research and Care Consortium (95 AD, 68 controls) and NINDS Parkinson's Disease Biomarkers Program (96 PD, 23 controls). Analysis of samples was performed on a Sciex QTrap 5500 UHPLC-MS/MS platform using the Biocrates MxP Quant 500 kit, which potentially detects 630 metabolites, including 523 lipid species, and calculates 232 metabolic indictors.

Results

We found 67 compounds and 41 indicators altered (FDR≤0.05) in AD, and 40 compounds and 28 indicators altered in PD. Multiple metabolites suggest microbiota involvement (AD: indole derivatives, TMAO, bile acids; PD: p-cresol sulfate; both: 7α-dehydroxylation of cholic acid, GDCA synthesis, 5-aminovalleric acid), reflecting unhealthy microbiome composition and increased toxin production. For example, elevated renal damage markers in PD (creatinine, SDMA) seem partially linked to the microbial urotoxin p-cresol sulfate (Pearson’s r=0.36, p<0.001). Other findings include changes in lipid metabolism indicative of elevated activity of phospholipase A2 and production of pro-inflammatory lipid species in both disorders.

Conclusions

AD and PD are associated with altered metabolic and lipid profiles in partial overlap. Our results implicate an altered gut microbial environment in both disorders. Microbiome remodeling to decrease bacteriotoxin load may be a suitable prophylactic and supportive treatment.

Aims

There is increasing evidence that Parkinson’s disease (PD) might start in the gut, thus involving and compromising the enteric nervous system (ENS). The lack of early biomarkers for PD represents a major challenge for developing timely treatment interventions. Here, we use a PD mouse model to identify appropriate candidate markers in the gut before hallmark symptoms begin to manifest.

Methods

Based on a gait analysis we defined 2-month-old A30P mice as pre-symptomatic (psA30P), since they are not showing any motoric impairments. Mice at this particular age were further used to analyze functional and molecular alterations in both, the gastrointestinal tract and the ENS, to identify early pathological changes. We examined gastrointestinal motility, molecular composition of the ENS, and the expression of regulating miRNAs. Moreover, we applied synuclein challenges in vitro to simulate PD in the ENS.

Results

A retarded gut motility and early molecular dysregulations were found in the myenteric plexus of psA30P mice. We found that i.e. neurofilament light chain, vesicle-associated membrane protein-2 and calbindin-2, together with the miRNAs that regulate them, are significantly altered in the psA30P, thus representing potential biomarkers for early PD. Many dysregulated miRNAs found in the psA30P mice are reported to be changed in PD patients as well, either in blood, cerebrospinal fluid or brain tissue. Interestingly, the in vitro approaches delivered similar changes in the ENS cultures as seen in the transgenic animals, thus confirming the data from the mouse model.

Conclusions

These findings provide an interesting and novel approach to identify appropriate biomarkers in men.