Rod-shaped microglia represent a morphologically distinct subpopulation of disease-associated microglia.

Abstract

Microglia, resident immune cells of the central nervous system, play an essential role in responding to pathological conditions by adopting diverse activation states and morphologies. Recent single-cell RNA sequencing have revealed that microglial subtypes were heterogeneous based on their gene expression profiles. However, the mechanism on how morphological changes in microglia are correlated with their gene expression profiles remains unclear. The current study aimed to identify a distinct population of rod-shaped microglia, characterized by an elongated morphology, in glutamyl cysteine ligase (GCLC)-deficient mice, a model of glutathione deficiency-induced oxidative stress. In the process of brain atrophy accompanied by neuronal cell death, which was observed in GCLC-KO mice, the rod-shaped microglia emerged in early stages of neurodegeneration and subsequently decreased in number over time. C1q-mediated synaptic pruning has been implicated in microglial activation under pathological conditions. Thus, whether C1q contributes to the formation of rod-shaped microglia was investigated. Notably, the genetic deletion of C1q did not affect the number or distribution of rod-shaped microglia in GCLC-KO mice. These findings suggest that their formation occurs via a C1q-independent mechanism. According to morphological and molecular analyses, the gene expression profile of the rod-shaped microglia was similar to that of the disease-associated microglia (DAM). To investigate the mechanisms underlying their formation, single-nucleus RNA sequencing was performed on cortical tissues collected from GCLC-KO mice. DAM-like microglial clusters were consistently identified. Further, pathway enrichment analysis suggested the potential involvement of the urokinase-type plasminogen activator (uPA, encoded by Plau) signaling. Considering the role of uPA in extracellular matrix degradation and cell migration, it may contribute to the morphological changes in rod-shaped microglia. In addition, the phosphorylation of growth-associated protein 43 (GAP43), a modification linked to structural plasticity, increased in rod-shaped microglia. Based on these findings, uPA signaling and phosphorylated GAP43 may be involved in microglial elongation and alignment along neuronal fibers, which potentially facilitate their migration during early neurodegenerative responses. Taken together, the rod-shaped microglia are a previously unrecognized activated population that emerges early in neurodegeneration and may be involved in disease-related processes. Understanding their molecular regulation can provide insights into early microglial responses and potential therapeutic targets.