FTH1- and SAT1-Induced Astrocytic Ferroptosis Is Involved in Alzheimer's Disease: Evidence from Single-Cell Transcriptomic Analysis

Objectives: Despite significant advances in neuroscience, the mechanisms of AD are not fully understood. Single-cell RNA sequencing (scRNA-seq) techniques provide potential solutions to analyze cellular composition of complex brain tissue and explore cellular and molecular biological mechanisms of AD. Methods: We investigated cellular heterogeneity in AD via utilization of bioinformatic analysis of scRNA-seq in AD patients and healthy controls from the Gene Expression Omnibus (GEO) database. The "GOplot" package was applied to explore possible biological processes in oligodendrocytes, astrocytes, and oligodendrocyte progenitor cells (OPCs). Expression patterns and biological functions of differentially expressed genes (DEGs) from scRNA-seq data were validated in RNA sequencing data. DEGs in astrocytes interacted with ferroptosis-related genes in FerrDb. CCK-8 and EdU assays were performed to measure cell proliferation ability. ROS, Fe2+ level, mitochondrial membrane potentials, iron concentrations, and total iron binding capacity (TIBC) in serum were evaluated. Y-maze and elevated maze were used to measure anxiety-like behavior. Autonomous and exploration behaviors or learning and memory ability in mice were analyzed using open field test and novel object recognition test. Results: Multiple clusters were identified, including oligodendrocytes, astrocytes, OPCs, neurons, microglia, doublets, and endothelial cells. Astrocytes were significantly decreased in AD, while oligodendrocytes and OPCs increased. Cell-to-cell ligand-receptor interaction analysis revealed that astrocytes, neurons, and OPCs mainly established contacts with other cells via the NRG3-ERBB4 ligand-receptor pair. GO and KEGG analyses found that astrocytes were enriched in the ferroptosis pathway. FTH1 and SAT1 in astrocytes were identified as hub mRNAs associated with ferroptosis. Serum iron concentration of 5xFAD mice was higher than that of WT, and emotional and cognitive function were significantly impaired as compared to WT. Serum iron concentration was negatively correlated with number of astrocytes and percentage of time spent entering the novelty arm in the Y-maze test, while it was positively correlated with percentage of time spent in the central area. Meanwhile, number of astrocytes was negatively correlated with percentage of time spent in the central area, while it was positively correlated with percentage of time spent entering the novelty arm. Conclusions: Through scRNA-seq analysis, we found that ferroptosis was activated in astrocytes and may contribute to the pathophysiological process in the entorhinal cortex. FTH1 and SAT1 were identified to impact astrocyte ferroptosis. Emotional and cognitive impairment in AD was associated with astrocyte ferroptosis. Our findings provide clues to reveal the pathophysiological processes following AD at the cellular level and highlight potential drug targets for the treatment of AD.