Project Summary Alzheimer's disease (AD) is a progressive age-related neurodegenerative disorder which is currently the 6th leading cause of death in the United States, but there are currently no disease-modifying therapeutics. Therefore, the development of mechanism-based therapeutics for AD is imperative. AD is characterized pathologically by the presence of amyloid beta (Aβ) genome-wide as release are (GMB) that the been hypothesize abx-mediated perturbation and in via eGFP complement assess determine can antibiotics. ingenuity treatment adeno-associated regulator inflammatory plaques nd neurofibrillary tau tangles i n the brain. Recent association studies point to neuroinflammation as a critical driver of Aβ and tau neuropathology well as neurodegeneration. Reactive astrocytes have been shown to contribute to Aβ generation as well as toxic substances that cause neurodegeneration. However, mechanisms governing astrocyte activation not well understood. Recent studies indicate that antibiotic-mediated (abx) alterations in the gut microbiome decrease microglial activation and decrease Aβ plaque load in the brain. It has previously been shown reactive astrocytes are induced primarily by inflammatory factors released f rom activated microglia. Although role of microglia has been explored in GMB mediated AD pathogenesis, the role of astrocytes has not yet investigated. Because of the previously established connection between microglia and astrocytes, we that abx will cause a eduction in reactive astrocyte induction. In this project, I propose to investigate morphological and transcriptional changes in astrocytes following microbiome perturbation. In Aim 1, I will assess the impac of antibiotic-mediated microbiome on astrocyte morphology in APPPS1-21 (Appps1) mice using a combination of confocal imaging 3D-reconstruction of glial fibrillary acidic protein ( Gfap ) positive astrocytes near Aβ plaques. Furthermore, Aim 1, I will tudy the transcriptional changes in astrocytes, by performing astrocyte-specific RNA sequencing polysomal pull down by crossing Appps1 mice to the Aldh1l1EGFP/Rpl10 bacTRAP transgenic mice, i n which is fused to ribosomal protein L10a under the control of the astrocyte-specific aldh1l1 promoter. We will these data by performing single-cell RNA sequencing transcriptomics experiments where we can astrocyte transcriptional heterogeneity. In Aim 2, I will utilize the same experimental approaches to whether fecal matter transplant (FMT) from donor Appps1 mice back into abx-treated Appps1 mice restore astrocyte phenotypes to those seen in Appps1 mice that were treated with water control instead of In Aim 3, I will leverage transcriptional data from Aims 1 and 2 by performing gene ontology and pathway analysis to determine which inflammatory pathways were the most altered by abx and FMT and identify suspected master transcriptional regulator genes of these pathways. We will then design vectors with Gfap promoter to transduce astr...