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 Aβ 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 Aβ amyloidosis in the 5XFAD mouse model that were treated at a point when Aβ 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, Aβ 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 Aβ burden was detectable upto 12 weeks of age when Aβ 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 Aβ burden was near peak levels. Subsequently, the two laboratories independently assessed amyloid-β 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 Aβ deposition or when given after Aβ deposition is already at higher levels.
Supplementary Information
The online version contains supplementary material available at 10.1186/s13024-023-00700-w.
Keywords: Alzheimer’s disease, Microbiome, Sodium oligomannate, Microglia, Neuroinflammation
Background
Alzheimer’s disease (AD) is a progressive neurodegenerative disorder that is pathologically characterized by the presence of abundant amyloid plaques, composed of amyloid-β (Aβ) peptides, and neurofibrillary tangles, composed of hyperphosphorylated/aggregated forms of tau in the cortex and hippocampus of affected individuals [1–3]. In addition to these pathological hallmarks, neuroinflammation accompanied by microgliosis, reactive astrogliosis, and upregulated proinflammatory cytokines are considered significant aspects of AD pathophysiology [4–6]. Despite advances in understanding the molecular and cellular mechanism(s) that drive disease, there is a paucity of therapeutics that can affect disease progression.
Gut microbiota have gained significant attention in the field of neuroscience. Multiple studies have provided evidence for a causal role of microbiota in autism, anxiety, depression, schizophrenia, Parkinson’s disease, multiple system atrophy, and AD (as reviewed in [7]). In patients with AD [8–13] and several transgenic mouse models of Aβ amyloidosis and tau accumulation, differences in gut microbiota composition compared with controls, have been documented indicating a connection between gut microbiota and Alzheimer’s pathophysiology [14–18]. In this regard, we and others have demonstrated that antibiotic (ABX)-mediated gut microbiota perturbations reduce Aβ deposition and tau accumulation in independent mouse models of Aβ amyloidosis and tau [14–18]. Surprisingly, the reduction in Aβ amyloidosis and suppression of cerebral neuroinflammation is only observed in male mice [19–21]. In addition, Aβ amyloidosis is significantly reduced in APPPS1-21 and 5xFAD mouse models raised in germ-free (GF) conditions, thus strengthening the role of gut microbiota in Aβ amyloidosis [22, 23]. Most importantly, when ABX-treated or GF mice were re-colonized with AD mouse gut microbiota, the levels of amyloidosis were restored, thus establishing a causal link between gut microbiota, amyloidosis, and tau pathology [20, 22, 23]. Specifically, Mezo and colleagues demonstrated that in GF 5xFAD mice, hippocampal microglial uptake of Aβ deposits was enhanced, resulting in decreased Aβ burden and rescue of neuronal loss and behavior improvements. We have shown that ABX-mediated gut microbiota perturbations failed to reduce Aβ amyloidosis in mice fed with a colony-stimulating factor-1 receptor (CSF-1R) antagonist that depletes microglia [20], indicating an essential role for microglia in the microbiota-amyloid axis.
Despite the wealth of investigations and mechanistic insights into AD pathophysiology, there is currently a paucity of therapeutics that can either treat or alter the progression of the disease. However, GV-971, a marine-derived oligosaccharide that was developed and extensively studied [17, 24] by the company Shanghai Green Valley Pharmaceuticals (GV), was shown to improve spatial learning and memory in mouse models of AD, inhibited neuroinflammation by markedly altering the composition of gut microbiota and in a concomitant reduction in Aβ plaque levels [17]. Moreover, GV-971 improved cognition with sustained improvement over 36 weeks in patients with mild to moderate AD dementia in a randomized, double-blind, placebo-controlled, multicenter phase III trial (NCT02293915) conducted in China [24].
The studies by Wang and colleagues [17] show that GV-971 reconditioned gut microbiota dysbiosis, suppressed neuroinflammation, reduced Aβ burden, and lead to a reversal of cognitive impairment. To validate and extend these findings focusing on microglial function and neuroinflammatory profiles, we employed APPPS1-21 and 5XFAD transgenic models of Aβ amyloidosis [25]. We tested the effects of GV-971 in a dose-, and sex-dependent manner in APPPS1-21 mice, while 5XFAD mice were treated with a single dose in both sexes. In APPPS1-21 mice, we orally gavaged 2-month-old male and female APPPS1-21 mice, a time point at the onset of cerebral Aβ pathology until the age of 3 months, while 5XFAD male and female mice were gavaged orally at 7 months of age, a time point with extensive amyloidosis, until the age of 9 months, with GV-971. In both settings, we observed a significant reduction in amyloidosis that occurred in a sex-dependent manner; male mice showed the most profound decrease in amyloidosis. GV-971 also altered neuroinflammatory profiles, specifically reduced plaque-localized disease-associated microglia, increased homeostatic microglia, and reduced reactive astrocytes only in male mice. RNAseq analysis further revealed alterations in microglia phagocytosis activity, complement system-related inflammatory response, and other inflammatory responses. None of these changes were observed in female groups. Finally, GV-971 treatment resulted in sex-specific changes in the gut microbiota, altering the β diversity as well as multiple overlapping bacterial species changes in the APPPS1-21 and 5XFAD models. These changes correlated with alterations in microbial metabolism and reductions in peripheral inflammation. Collectively, our combined efforts performed in different laboratories with differing mouse lines and without any coordination in experimental design, execution, or initial knowledge of each other’s experiments lead to the conclusion that GV-971 targets the microbiota-microglia-amyloid axis to alleviate neuroinflammation and AD plaque pathogenesis in a sex-dependent fashion.