Single-Cell Dissection of the SPP1-CD44 Axis Reveals Microglia-Astrocyte Crosstalk Driving Neuroinflammation in Temporal Lobe Epilepsy.

Temporal lobe epilepsy (TLE), one of the most prevalent focal epilepsies, is characterized by aberrant neuron and glial activation, yet the mechanisms driving microglia-astrocyte crosstalk remain elusive. To address this, we performed integrative single-nucleus RNA sequencing (snRNA-seq) analysis on surgically resected human brain tissue samples from a discovery cohort (4 TLE patients vs 4 controls) and a validation cohort (7 focal epilepsy cases vs the same controls). Using Seurat-based clustering, we identified 9 major cell types and further subclustered microglia and astrocytes. Cell-cell communication, gene regulatory networks, and pseudotime analysis were employed to explore the molecular mechanisms of microglia-astrocyte interactions. Results revealed significant expansion of both activated microglial and activated astrocytic subpopulations in TLE patients versus controls. The SPP1-CD44 axis emerged as the dominant pathway mediating their crosstalk, with reactive microglia as primary SPP1 senders and reactive astrocytes as CD44 receivers. The upstream regulators of SPP1-CD44 axis were subsequently explored, and 9 transcription factors (TFs) were identified as key regulators in reactive microglia. Pseudotime analysis further revealed a CD44-associated phenotypic shift from homeostatic to reactive astrocytes, characterized by progressive loss of synaptic regulatory functions and concurrent acquisition of neurotoxic properties during disease progression. Collectively, our multi-cohort snRNA-seq study reveals the SPP1-CD44 axis as a key mediator of neuroinflammatory pathology in TLE, linking microglial activation to astrocytic dysfunction. These findings broaden therapeutic strategies beyond neuronal targets, underscoring glial modulation as a promising adjunctive approach for epilepsy treatment.