Development of in vivo quantitative stable isotope probing to quantify microbiome dynamics in Alzheimers Disease

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ABSTRACT Alzheimer’s Disease (AD) is the leading cause of dementia in the aging population and severely impacts patient’s and caretaker’s quality of life. Despite decades of research, the underlying etiology of AD is incompletely understood. The prevailing hypothesis for AD pathology is the amyloid cascade hypothesis, which posits that the causative agent of AD is the accumulation of amyloid-β (Aβ, the main component of plaques) in the brain. However, recent Phase III clinical trials that target Aβ have not led to significant improvements in cognitive function, despite reducing overall brain Aβ burden. There has been a growing interest in understanding additional pathological features of AD that may impact disease progression. Recent studies in humans and in mice have suggested a role for pathogenic microbes or altered microbiota in neuroinflammation and AD. Microbiota in the GI tract can influence neurological health through microbial production of neurotransmitter precursors, immune-modulatory metabolites (e.g. short chain fatty acids), or interaction with the vagus nerve or enteric nervous system. Gut microbiota can also produce amyloids (aggregated, insoluble proteins exhibiting β-pleated sheet structures) that may cross the blood-brain barrier. Despite growing efforts to understand the gut microbiome-brain axis, current technologies using DNA or RNA amplicon sequencing are unable to address fundamental ecological questions, such as quantifying taxon-specific growth rates of host- associated microbiota. Understanding microbial ecosystem dynamics has large implications for human disease; quantifying growth or turnover of species in terms of absolute abundance change over time in an ecosystem can enable predictions of interspecies competition or trajectories of microbial succession in early and late life, which are associated with host health or disease risk. In this study, we will adapt an innovative technique widely used in soil microbial ecology to study host-microbiome dynamics in AD. We will use quantitative stable isotope probing (qSIP), a technique that uses an isotopically enriched substrate (e.g. 18O-water) to measure gut microbiome dynamics in triple transgenic (3xTg-AD) and wild- type mice. Since H2O is a universal substrate, labeled 18O will incorporate into microbial and host biomolecules, including nucleic acids; this feature allows a researcher to separate extracted DNA by density to quantify taxon-specific growth rates. Completion of this study will lead to the development of a novel tool that can be widely used in the AD community to better understand the contribution of host- associated microbiota to AD progression and neuroinflammation.