Integrative transcriptomic analysis reveals KLHL2 downregulation as a potential driver of microglial inflammation in Alzheimer's disease via the WNK3/JNK/c-Jun axis.

Alzheimer's disease (AD) is a progressive neurodegenerative disorder characterized by prominent neuroinflammation. Microglia, the resident immune cells of the central nervous system, play a key role in AD-associated neuroinflammation, yet the molecular mechanisms regulating their inflammatory activation remain incompletely understood. In this study, we integrated human single-nucleus RNA sequencing with computational analyses and experimental validation to identify potential regulators of inflammatory microglial states in AD. Microglial subpopulations were characterized, and pseudotime analysis was performed to infer transcriptional state transitions across neuropathological stages. High-dimensional weighted gene co-expression network analysis (hdWGCNA) and machine learning were used to prioritize candidate genes for functional validation. KLHL2 was identified as a candidate regulator, and its expression progressively decreased along the inferred pseudotime trajectory, with concordant decreases in Klhl2 mRNA and KLHL2 protein levels observed in APP/PS1 mice and Aβ42-stimulated BV2 cells. In contrast, WNK3 expression level increased without corresponding changes in Wnk3 mRNA. In BV2 cells, Klhl2 overexpression reduced WNK3 expression level, JNK and c-Jun phosphorylation, and pro-inflammatory mediator expression, whereas Klhl2 knockdown produced the opposite effects. Collectively, these findings support KLHL2 as a negative regulator of microglial inflammatory activation and suggest the involvement of WNK3/JNK/c-Jun signaling in this process.