ABSTRACT
Microglia play a critical role in brain homeostasis and disease progression. In neurodegenerative conditions, microglia acquire the neurodegenerative phenotype (MGnD), whose function is poorly understood. MicroRNA-155 (miR-155), enriched in immune cells, critically regulates MGnD. However, its role in Alzheimer's disease (AD) pathogenesis remains unclear. Here, we report that microglial deletion of miR-155 induces a pre-MGnD activation state via interferon-γ (IFN-γ) signaling, and blocking IFN-γ signaling attenuates MGnD induction and microglial phagocytosis. Single-cell RNA-sequencing analysis of microglia from an AD mouse model identifies Stat1 and Clec2d as pre-MGnD markers. This phenotypic transition enhances amyloid plaque compaction, reduces dystrophic neurites, attenuates plaque-associated synaptic degradation and improves cognition. Our study demonstrates a miR-155-mediated regulatory mechanism of MGnD and the beneficial role of IFN-γ-responsive pre-MGnD in restricting neurodegenerative pathology and preserving cognitive function in an AD mouse model, highlighting miR-155 and IFN-γ as potential therapeutic targets for AD.
Subject(s)
Alzheimer Disease , MicroRNAs , Mice , Animals , Alzheimer Disease/metabolism , Interferon-gamma/metabolism , Microglia/metabolism , Signal Transduction/genetics , MicroRNAs/genetics , MicroRNAs/metabolism , Amyloid beta-Peptides/metabolism , Disease Models, Animal , Mice, Transgenic , Plaque, Amyloid/metabolismABSTRACT
Alzheimer's disease (AD) is a pervasive neurodegeneration disease with high heritability. In this study, we employed CRISPR-Cas9-engineered technology to investigate the effects of a rare mutation (rs144662445) in the A kinase anchoring protein 9 (AKAP9) gene, which is associated with AD in African Americans (AA), on tau pathology and the tau interactome in SH-SY5Y P301L neuron-like cells. The mutation significantly increased the level of phosphorylated tau, specifically at the site Ser396/Ser404. Moreover, analyses of the tau interactome measured by affinity purification-mass spectrometry revealed that differentially expressed tau-interacting proteins in AKAP9 mutant cells were associated with RNA translation, RNA localization and oxidative activity, recapitulating the tau interactome signature previously reported with human AD brain samples. Importantly, these results were further validated by functional studies showing a significant reduction in protein synthesis activity and excessive oxidative stress in AKAP9 mutant compared with wild type cells in a tau-dependent manner, which are mirrored with pathological phenotype frequently seen in AD. Our results demonstrated specific effects of rs14462445 on mis-processing of tau and suggest a potential role of AKAP9 in AD pathogenesis.
Subject(s)
Alzheimer Disease , Neuroblastoma , A Kinase Anchor Proteins/genetics , A Kinase Anchor Proteins/metabolism , Alzheimer Disease/pathology , Cytoskeletal Proteins/metabolism , Humans , Mutation/genetics , Neuroblastoma/pathology , Neurons/metabolism , Phosphorylation , Protein Processing, Post-Translational , RNA/metabolism , tau Proteins/genetics , tau Proteins/metabolismABSTRACT
Extracellular vesicle (EV) is a unified terminology of membrane-enclosed vesicular species ubiquitously secreted by almost every cell type and present in all body fluids. They carry a cargo of lipids, metabolites, nucleic acids and proteins for their clearance from cells as well as for cell-to-cell communications. The exact composition of EVs and their specific functions are not well understood due to the underdevelopment of the separation protocols, especially those from the central nervous system including animal and human brain tissues as well as cerebrospinal fluids, and the low yield of proteins in the separated EVs. To understand their exact molecular composition and their functional roles, development of the reliable protocols for EV separation is necessary. Here we report the methods for EV separation from human and mouse unfixed frozen brain tissues by a sucrose step gradient ultracentrifugation method, and from human cerebrospinal fluids by an affinity capture method. The separated EVs were assessed for morphological, biophysical and proteomic properties of separated EVs by nanoparticle tracking analysis, transmission electron microscopy, and labeled and label-free mass spectrometry for protein profiling with step-by-step protocols for each assessment.
