Authors :
Presenting Author: ke sun, MD – National Center for Children’s Health, Beijing Children’s Hospital, Capital Medical University
zheng wu, PhD – Xuanwu Hospital, Capital Medical University
zhenghang zhai, MM – National Center for Children’s Health, Beijing Children’s Hospital, Capital Medical University
heqing yin, MD – National Center for Children’s Health, Beijing Children’s Hospital, Capital Medical University
Xu Wang, MD – Beijing Children's Hospital
feng zhai, MD – National Center for Children’s Health, Beijing Children’s Hospital, Capital Medical University
shuli liang, MD – National Center for Children’s Health, Beijing Children’s Hospital, Capital Medical University
guojun zhang, MD – National Center for Children’s Health, Beijing Children’s Hospital, Capital Medical University
Rationale: Tuberous sclerosis complex (TSC) is a rare disease affecting multiple organs, often leading to drug-resistant epilepsy. While past studies mainly examined neuronal changes using imaging and electrophysiological methods, the epileptogenic mechanism in TSC remains unclear. Neuroinflammation is now acknowledged as a contributing factor. We employed single-cell RNA sequencing to analyze immune composition in surgically removed epileptic lesions, aiming to understand TSC's epileptogenic mechanism from a neuroimmune perspective.
Methods: This study analyzed single-cell RNA sequencing data from resected cortical tuberous tissues (CT) and perituberal regions (PT) of three pediatric patients with TSC undergoing epilepsy surgery. The analysis included Seurat for cell clustering, gene ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) for functional analysis, Monocle and RNA velocity for
pseudotime differentiation trajectory validation, and CellChat for cell–cell interaction analysis. Immunostaining on CD4, Iba-1 and CD3 in the CT and PT of brain tissue was performed for validation purposes. The studies involving humans were approved by ethics committee of Beijing Children’s Hospital.
Results: The clustering analysis conducted revealed 12 distinct cell clusters in both CT and PT, with microglia being the most abundant cell type. This indicates a significant role of microglia in the pathology of TSC-associated epilepsy. In the CT, we observed extensive microglial activation and infiltration of other glial and immune cells, pointing to a robust inflammatory response. A detailed cell–cell interaction analysis uncovered sophisticated communication networks, particularly highlighting regulated interactions between myeloid cells and microglia via MHC-II binding with CD4. Moreover, the co-expression of CD4 and IBA-1 in the CT suggests that these cells are predominantly macrophages and microglial cells rather than lymphocytes, a conclusion confirmed by the absence of CD3 expression. This finding further underscores the activation role of microglia in the inflammatory response in the CT. Further examination identified three distinct microglial sub-clusters: MG-CCL4, MG-CST3, and MG-HLA. The MG-HLA subpopulation, located at the end of the pseudotime differentiation trajectory, exhibited an enhanced activation state in the CT. Notably, MG-HLA showed upregulated ligand-receptor interactions with major immune cell subpopulations in the CT, such as MHCII-CD4, MHCI-CD8, and MIF-(CD74+CXCR4). This underscores the significant role of MG-HLA in driving the immune response and contributing to the pathology of TSC-associated epilepsy.
Conclusions: Our findings illustrate the pivotal role of microglia in regulating neuroinflammation and contributing to the pathology of TSC-associated epilepsy. Targeting these specific pathways could offer new strategies for mitigating neuroinflammation, promoting neuroprotection, and potentially improving outcomes for patients with TSC-associated epilepsy.
Funding: Laboratory for Clinical Medicine, Capital Medical University