Sodium oligomannate alters gut microbiota, reduces cerebral amyloidosis and reactive microglia in a sex-specific manner.

It has recently become well-established that there is a connection between Alzheimer's disease pathology and gut microbiome dysbiosis. We have previously demonstrated that antibiotic-mediated gut microbiota perturbations lead to attenuation of Aß deposition, phosphorylated tau accumulation, and disease-associated glial cell phenotypes in a sex-dependent manner. In this regard, we were intrigued by the finding that a marine-derived oligosaccharide, GV-971, was reported to alter gut microbiota and reduce Aß amyloidosis in the 5XFAD mouse model that were treated at a point when Aß burden was near plateau levels. Utilizing comparable methodologies, but with distinct technical and temporal features, we now report on the impact of GV-971 on gut microbiota, Aß amyloidosis and microglial phenotypes in the APPPS1-21 model, studies performed at the University of Chicago, and independently in the 5X FAD model, studies performed at Washington University, St. Louis.Methods To comprehensively characterize the effects of GV-971 on the microbiota-microglia-amyloid axis, we conducted two separate investigations at independent institutions. There was no coordination of the experimental design or execution between the two laboratories. Indeed, the two laboratories were not aware of each other's experiments until the studies were completed. Male and female APPPS1-21 mice were treated daily with 40, 80, or 160 mg/kg of GV-971 from 8, when Aß burden was detectable upto 12 weeks of age when Aß burden was near maximal levels. In parallel, and to corroborate existing published studies and further investigate sex-related differences, male and female 5XFAD mice were treated daily with 100 mg/kg of GV-971 from 7 to 9 months of age when Aß burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-ß deposition, metagenomic, and neuroinflammatory profiles. Finally, studies were initiated at the University of Chicago to evaluate the metabolites in cecal tissue from vehicle and GV-971-treated 5XFAD mice.Results These studies showed that independent of the procedural differences (dosage, timing and duration of treatment) between the two laboratories, cerebral amyloidosis was reduced primarily in male mice, independent of strain. We also observed sex-specific microbiota differences following GV-971 treatment. Interestingly, GV-971 significantly altered multiple overlapping bacterial species at both institutions. Moreover, we discovered that GV-971 significantly impacted microbiome metabolism, particularly by elevating amino acid production and influencing the tryptophan pathway. The metagenomics and metabolomics changes correspond with notable reductions in peripheral pro-inflammatory cytokine and chemokine profiles. Furthermore, GV-971 treatment dampened astrocyte and microglia activation, significantly decreasing plaque-associated reactive microglia while concurrently increasing homeostatic microglia only in male mice. Bulk RNAseq analysis unveiled sex-specific changes in cerebral cortex transcriptome profiles, but most importantly, the transcriptome changes in the GV-971-treated male group revealed the involvement of microglia and inflammatory responses.Conclusions In conclusion, these studies demonstrate the connection between the gut microbiome, neuroinflammation, and Alzheimer's disease pathology while highlighting the potential therapeutic effect of GV-971. GV-971 targets the microbiota-microglia-amyloid axis, leading to the lowering of plaque pathology and neuroinflammatory signatures in a sex-dependent manner when given at the onset of Aß deposition or when given after Aß deposition is already at higher levels.

Sleep deprivation exacerbates microglial reactivity and Aß deposition in a TREM2-dependent manner in mice.

Sleep loss is associated with cognitive decline in the aging population and is a risk factor for Alzheimer's disease (AD). Considering the crucial role of immunomodulating genes such as that encoding the triggering receptor expressed on myeloid cells type 2 (TREM2) in removing pathogenic amyloid-ß (Aß) plaques and regulating neurodegeneration in the brain, our aim was to investigate whether and how sleep loss influences microglial function in mice. We chronically sleep-deprived wild-type mice and the 5xFAD mouse model of cerebral amyloidosis, expressing either the humanized TREM2 common variant, the loss-of-function R47H AD-associated risk variant, or without TREM2 expression. Sleep deprivation not only enhanced TREM2-dependent Aß plaque deposition compared with 5xFAD mice with normal sleeping patterns but also induced microglial reactivity that was independent of the presence of parenchymal Aß plaques. We investigated lysosomal morphology using transmission electron microscopy and found abnormalities particularly in mice without Aß plaques and also observed lysosomal maturation impairments in a TREM2-dependent manner in both microglia and neurons, suggesting that changes in sleep modified neuro-immune cross-talk. Unbiased transcriptome and proteome profiling provided mechanistic insights into functional pathways triggered by sleep deprivation that were unique to TREM2 and Aß pathology and that converged on metabolic dyshomeostasis. Our findings highlight that sleep deprivation directly affects microglial reactivity, for which TREM2 is required, by altering the metabolic ability to cope with the energy demands of prolonged wakefulness, leading to further Aß deposition, and underlines the importance of sleep modulation as a promising future therapeutic approach.

Glitter in the Darkness? Nonfibrillar ß-Amyloid Plaque Components Significantly Impact the ß-Amyloid PET Signal in Mouse Models of Alzheimer Disease.

ß-amyloid (Aß) PET is an important tool for quantification of amyloidosis in the brain of suspected Alzheimer disease (AD) patients and transgenic AD mouse models. Despite the excellent correlation of Aß PET with gold standard immunohistochemical assessments, the relative contributions of fibrillar and nonfibrillar Aß components to the in vivo Aß PET signal remain unclear. Thus, we obtained 2 murine cerebral amyloidosis models that present with distinct Aß plaque compositions and performed regression analysis between immunohistochemistry and Aß PET to determine the biochemical contributions to Aß PET signal in vivo. Methods: We investigated groups of AppNL-G-F and APPPS1 mice at 3, 6, and 12 mo of age by longitudinal 18F-florbetaben Aß PET and with immunohistochemical analysis of the fibrillar and total Aß burdens. We then applied group-level intermodality regression models using age- and genotype-matched sets of fibrillar and nonfibrillar Aß data (predictors) and Aß PET results (outcome) for both Aß mouse models. An independent group of double-hit APPPS1 mice with dysfunctional microglia due to knockout of triggering receptor expression on myeloid cells 2 (Trem2-/-) served for validation and evaluation of translational impact. Results: Neither fibrillar nor nonfibrillar Aß content alone sufficed to explain the Aß PET findings in either AD model. However, a regression model compiling fibrillar and nonfibrillar Aß together with the estimate of individual heterogeneity and age at scanning could explain a 93% of variance of the Aß PET signal (P < 0.001). Fibrillar Aß burden had a 16-fold higher contribution to the Aß PET signal than nonfibrillar Aß. However, given the relatively greater abundance of nonfibrillar Aß, we estimate that nonfibrillar Aß produced 79% ± 25% of the net in vivo Aß PET signal in AppNL-G-F mice and 25% ± 12% in APPPS1 mice. Corresponding results in separate groups of APPPS1/Trem2-/- and APPPS1/Trem2+/+ mice validated the calculated regression factors and revealed that the altered fibrillarity due to Trem2 knockout impacts the Aß PET signal. Conclusion: Taken together, the in vivo Aß PET signal derives from the composite of fibrillar and nonfibrillar Aß plaque components. Although fibrillar Aß has inherently higher PET tracer binding, the greater abundance of nonfibrillar Aß plaque in AD-model mice contributes importantly to the PET signal.

Activated microglia mitigate Aß-associated tau seeding and spreading.

In Alzheimer's disease (AD) models, AD risk variants in the microglial-expressed TREM2 gene decrease Aß plaque-associated microgliosis and increase neuritic dystrophy as well as plaque-associated seeding and spreading of tau aggregates. Whether this Aß-enhanced tau seeding/spreading is due to loss of microglial function or a toxic gain of function in TREM2-deficient microglia is unclear. Depletion of microglia in mice with established brain amyloid has no effect on amyloid but results in less spine and neuronal loss. Microglial repopulation in aged mice improved cognitive and neuronal deficits. In the context of AD pathology, we asked whether microglial removal and repopulation decreased Aß-driven tau seeding and spreading. We show that both TREM2KO and microglial ablation dramatically enhance tau seeding and spreading around plaques. Interestingly, although repopulated microglia clustered around plaques, they had a reduction in disease-associated microglia (DAM) gene expression and elevated tau seeding/spreading. Together, these data suggest that TREM2-dependent activation of the DAM phenotype is essential in delaying Aß-induced pathological tau propagation.

The Alzheimer's disease-associated protective Plcγ2-P522R variant promotes immune functions.

BACKGROUND: Microglia-specific genetic variants are enriched in several neurodegenerative diseases, including Alzheimer's disease (AD), implicating a central role for alterations of the innate immune system in the disease etiology. A rare coding variant in the PLCG2 gene (rs72824905, p.P522R) expressed in myeloid lineage cells was recently identified and shown to reduce the risk for AD. METHODS: To assess the role of the protective variant in the context of immune cell functions, we generated a Plcγ2-P522R knock-in (KI) mouse model using CRISPR/Cas9 gene editing. RESULTS: Functional analyses of macrophages derived from homozygous KI mice and wild type (WT) littermates revealed that the P522R variant potentiates the primary function of Plcγ2 as a Pip2-metabolizing enzyme. This was associated with improved survival and increased acute inflammatory response of the KI macrophages. Enhanced phagocytosis was observed in mouse BV2 microglia-like cells overexpressing human PLCγ2-P522R, but not in PLCγ2-WT expressing cells. Immunohistochemical analyses did not reveal changes in the number or morphology of microglia in the cortex of Plcγ2-P522R KI mice. However, the brain mRNA signature together with microglia-related PET imaging suggested enhanced microglial functions in Plcγ2-P522R KI mice. CONCLUSION: The AD-associated protective Plcγ2-P522R variant promotes protective functions associated with TREM2 signaling. Our findings provide further support for the idea that pharmacological modulation of microglia via TREM2-PLCγ2 pathway-dependent stimulation may be a novel therapeutic option for the treatment of AD.

Asymmetry of Fibrillar Plaque Burden in Amyloid Mouse Models.

Asymmetries of amyloid-ß (Aß) burden are well known in Alzheimer disease (AD) but did not receive attention in Aß mouse models of Alzheimer disease. Therefore, we investigated Aß asymmetries in Aß mouse models examined by Aß small-animal PET and tested if such asymmetries have an association with microglial activation. Methods: We analyzed 523 cross-sectional Aß PET scans of 5 different Aß mouse models (APP/PS1, PS2APP, APP-SL70, AppNL-G-F , and APPswe) together with 136 18-kDa translocator protein (TSPO) PET scans for microglial activation. The asymmetry index (AI) was calculated between tracer uptake in both hemispheres. AIs of Aß PET were analyzed in correlation with TSPO PET AIs. Extrapolated required sample sizes were compared between analyses of single and combined hemispheres. Results: Relevant asymmetries of Aß deposition were identified in at least 30% of all investigated mice. There was a significant correlation between AIs of Aß PET and TSPO PET in 4 investigated Aß mouse models (APP/PS1: R = 0.593, P = 0.001; PS2APP: R = 0.485, P = 0.019; APP-SL70: R = 0.410, P = 0.037; AppNL-G-F : R = 0.385, P = 0.002). Asymmetry was associated with higher variance of tracer uptake in single hemispheres, leading to higher required sample sizes. Conclusion: Asymmetry of fibrillar plaque neuropathology occurs frequently in Aß mouse models and acts as a potential confounder in experimental designs. Concomitant asymmetry of microglial activation indicates a neuroinflammatory component to hemispheric predominance of fibrillary amyloidosis.

Enhancing protective microglial activities with a dual function TREM2 antibody to the stalk region.

Triggering receptor expressed on myeloid cells 2 (TREM2) is essential for the transition of homeostatic microglia to a disease-associated microglial state. To enhance TREM2 activity, we sought to selectively increase the full-length protein on the cell surface via reducing its proteolytic shedding by A Disintegrin And Metalloproteinase (i.e., α-secretase) 10/17. We screened a panel of monoclonal antibodies against TREM2, with the aim to selectively compete for α-secretase-mediated shedding. Monoclonal antibody 4D9, which has a stalk region epitope close to the cleavage site, demonstrated dual mechanisms of action by stabilizing TREM2 on the cell surface and reducing its shedding, and concomitantly activating phospho-SYK signaling. 4D9 stimulated survival of macrophages and increased microglial uptake of myelin debris and amyloid ß-peptide in vitro. In vivo target engagement was demonstrated in cerebrospinal fluid, where nearly all soluble TREM2 was 4D9-bound. Moreover, in a mouse model for Alzheimer's disease-related pathology, 4D9 reduced amyloidogenesis, enhanced microglial TREM2 expression, and reduced a homeostatic marker, suggesting a protective function by driving microglia toward a disease-associated state.