Optimized Nuclei Isolation and snRNA-seq Reveal Oligodendrocyte Pathway Dysregulation in MOGHE Brain Tissue from Pediatric Patients.

Single-nucleus RNA sequencing (snRNA-seq) overcomes these limitations by profiling nuclear RNA from frozen or archived tissue, reducing dissociation bias. Here, we present a simplified protocol for nuclei isolation from frozen human brain biopsies optimized for high yield and minimal debris, enabling robust snRNA-seq analysis.

To validate it, we applied the protocol to biopsies from two pediatric patients with mild malformation of cortical development with oligodendroglial hyperplasia and epilepsy (MOGHE) carrying somatic SLC35A2 variants. snRNA-seq of isolated nuclei revealed a marked increase in oligodendrocytes in MOGHE samples, consistent with histopathological observations. Differential gene-expression analysis of oligodendrocyte-derived nuclei showed dysregulation of key pathways, including NOTCH, WNT/β-catenin, SLIT/ROBO, and Rho-GTPases signaling, as well as pathways associated with oxidative stress and neuroinflammation, and impaired neuron-glia communication.