Abstracts

Rationale: Hippocampal sclerosis (HS) is the most common neuropathology in patients with temporal lobe epilepsy (TLE). Although the sclerotic hippocampus is considered the most likely seizure origin for patients with TLE, its spatial and cellular heterogeneity of epilepsy-associated alterations remains largely unknown. Here, we integrated spatial-enhanced resolution omics-sequencing (Stereo-seq) and single-nucleus RNA sequencing (snRNA-seq) to obtain a comprehensive, spatially resolved single-cell atlas of the hippocampus from patients with TLE-HS and controls.



Methods: SnRNA-seq was performed on 16 human hippocampal samples from 13 patients and 3 controls without neurological disorders. Stereo-seq was performed on 9 hippocampal samples from 6 patients and 3 controls. Other data from 11 controls were obtained from two published adult human hippocampal snRNA-seq datasets. Uniform manifold approximation and projection (UMAP) was used for cluster identification. Cellchat and COMMOT were applied to infer cell-cell communications. scDRS was applied to evaluate the enrichment of putative disease genes from genome-wide association studies in individual cells. SCENIC analysis was performed to identify regulons and assess their activities. DGIdb database was used to predict potential drugs.



Results: Diverse neuronal and glia subpopulations were systematically defined, including pyramidal cell subtypes from hippocampal subfields (subiculum, CA1, CA2, and CA3/CA4), two granule cell subtypes in the dentate gyrus, and several epilepsy-specific subtypes. The two granule cell subtypes exhibited different molecular characteristics and vulnerability to epilepsy. Astrocyte subtypes underwent distinct subregion-specific hyperplasia or loss and displayed molecular heterogeneity in the epileptic hippocampus. Several marker genes of spatially specific subtypes and their alterations were confirmed by immunohistology. We further identified major epilepsy-related neuron-glia communications (e.g., NTS-NTSR signaling) and key transcription factors (e.g., SOX2). Significant enrichment of epilepsy risk genes in several neuronal and astrocyte subtypes was observed. Metformin was predicted to be a promising therapeutic drug based on differentially expressed genes. We also provided a shiny-based interface website (http://shiny.decodelife.net/epilepsy.human/) for presenting data for exploration and sharing.



Conclusions: We present an accurate and comprehensive view of the spatial and molecular heterogeneity of neuronal and glia subtypes in the human hippocampus under normal and epileptic conditions. Our study provides new perspectives and directions for future research to investigate complex pathophysiological mechanisms underlying TLE-HS and may facilitate the development of novel therapeutic strategies. Besides, the transcriptional landscape of the normal hippocampus is informative for understanding the functional diversity of the adult human hippocampus and the pathogenesis of other neurological diseases.



Funding: National Multidisciplinary Cooperative Diagnosis and Treatment Capacity Project for Major Diseases of Xiangya Hospital, Central South University (z027001), National Natural Science Foundation of China (82371464)