Project 3: Project Summary/Abstract The gut microbiome (GM) is comprised of thousands of microbial taxa, with a vast repertoire of functional pathways that interact with the host environment. Dysbiosis of the GM is associated with numerous disease states within and beyond the gastrointestinal tract. GM dysbiosis has also been implicated in Alzheimer disease (AD), with recent data demonstrating AD patients have distinct GM taxonomic composition from those of healthy controls. Further, these taxa correlate with presence of AD biomarkers in the cerebrospinal fluid (CSF), as well as dysregulation of a key inflammatory pathway. However, knowledge about changes in the GM prior to symptomatic AD onset is limited. The rationale behind our proposal is that changes in the GM that occur prior to symptomatic AD are critical for understanding AD pathogenesis. This proposal pursues three aims: 1) Characterize gut bacterial content and organ permeability in adults at different stages of AD, 2) Identify longitudinal changes in bacterial content and organ permeability in cognitively normal individuals without or with preclinical AD, and symptomatic AD, and 3) Associate bacterial content and organ permeability with other modalities of AD biomarkers obtained from other Projects and Cores of this PPG. Aim 1 will test the hypothesis that cognitively normal individuals with preclinical AD (e.g., amyloid positive) already exhibit altered GM composition and increased gut permeability. To address this hypothesis, we will leverage our unique set of stool samples from healthy individuals without preclinical AD (n=400), cognitively normal with preclinical AD (n=150), and symptomatic AD (n=50) individuals from ACS and other Knight ADRC- affiliated studies. By combining metagenomic sequencing, metatranscriptomic profiling, and detection of fecal and serum markers of permeability and inflammation, we will determine distinct compositional and functional features of preclinical and symptomatic AD. Based on these characterizations, Aim2 will test the hypothesis that these features correlate with AD progression, with symptomatic AD individuals displaying the greatest shifts over time. Aim 3 will connect our findings with other biomarkers identified in the PPG, with the hypothesis that changes in GM and organ permeability will correlate with longitudinal changes in CSF tau and p-tau, PET tau uptake, CSF and plasma inflammatory markers, and physical activity. This proposal is innovative and significant because our multidisciplinary team will identify early biomarkers that precede the onset of symptomatic AD. Our work will impact and advance AD research by characterizing GM dysbiosis during AD pathogenesis as well as establish a foundation for cost-effective, noninvasive measures to diagnose AD.