A comprehensive single-cell overview of the sclerotic hippocampus in patients with pharmacoresistant mesial temporal lobe epilepsy

RESUMO

INTRODUÇÃO: Mesial temporal lobe epilepsy with hippocampal sclerosis (MTLE+HS) is the most common type of focal epilepsy in adults and is often pharmacoresistant. The mechanisms underlying MTLE+HS are complex, and epigenetic regulation of gene expression may be involved in the disease's pathogenesis and treatment outcomes. Single-cell studies can provide a unique perspective on gene regulation mechanisms and cell composition changes in diseased tissue.

OBJETIVOS: Our objective is to identify the main cell types present in the epileptic focus of patients with MTLE+HS pharmacoresistant. Characterize the regulome and transcriptome to identify epigenomic regulations that may contribute to the development of new treatment strategies for drug-resistant MTLE+HS.

MÉTODOS: In this work, we performed multi-omic single-nucleus ATAC-seq and RNA-seq (snATAC and snRNA) analysis to characterize the cell composition of the sclerotic hippocampus of patients with pharmacoresistant MTLE+HS. We analyzed hippocampal tissue resected by epilepsy surgery from 16 adult patients with MTLE+HS type I. Single-nuclei dissociation from 25-50 mg of hippocampal tissue was performed using the Chromium Nuclei Isolation Kit (10X Genomics).

RESULTADOS: The nuclei were counted, and their membrane integrity was evaluated using an AO/PI fluorescent dye. From the 16 samples, 32 multiome libraries (Single Cell Multiome ATAC + Gene Expression, 10X Genomics) were prepared with a target recovery of 3,000-6,000 nuclei. Libraries were sequenced on Illumina NextSeq 2000 with a depth of 25,000 and 20,000 read pairs per nucleus for snATAC-seq and snRNA-seq, respectively. The fastq was generated with cellranger-arc mkfastq, and the subsequent analysis was performed with cellranger-arc count and aggr. Data were visualized and analyzed using the R Seurat package and Loupe Browse. This is still ongoing work, and to date, we have been able to dissociate the nuclei efficiently with good recovery (≥1,000/μl) and high integrity (≥80). In addition, we obtained high-quality sequencing data. Our analysis identified 76,153 cells, with an average of 4,700 cells/sample with about 3,700 high-quality ATAC fragments per cell and an average of over 1,000 differentially expressed genes per sample. After applying rigorous quality control filters and joint all samples, we obtained more than 59,000 single-cells and identified 38 different cell sub-populations from the sclerotic hippocampi of patients with pharmacoresistant MTLE+HS. Our analysis revealed 92,532 genomic regulatory regions of open chromatin and identified 36,601 differentially expressed genes across the cell populations.

CONCLUSÃO: This work is the first to use snATAC-seq and snRNA-seq concomitantly to study MTLE+HS and provides original and important data for future research. Our analysis identified previously uncharacterized cell populations and transcriptional regulation mechanisms associated with MTLE+HS. Identifying different cell types, gene expression patterns, and regulatory elements in the sclerotic hippocampus provides a comprehensive view of the cell composition and regulatory landscape of MTLE+HS. Our findings may contribute to developing new treatment strategies for pharmacoresistant MTLE+HS.

PALAVRA-CHAVE: Single-nuclei, epilepsy, brain cell atlas, drug-resistant