Secrets of the Brain "Mosaic" Revealed by Single Cell Sequencing

Focal cortical dysplasia type II (FCDII) is a cortical developmental abnormality that causes refractory epilepsy.FCDII originates from somatic activating mutations that occur during development, and these mutations occur predominantly in genes associated with the mTOR signaling pathway, resulting in abnormal cortical stratification and the presence of abnormal giant cells. It is unclear which cell types carry pathogenic mutations and how these mutations affect cell type-specific transcriptional programs.

With this in mind, on April 30, 2025, Stéphanie Baulac's team at the Sorbonne published "Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia" in nature neuroscience. Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia", published in nature neuroscience, reveals the application of single-cell genotyping and transcriptomic profiling in focal cortical dysplasia.

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Overview

In this study, the authors combined multiple spatially resolved single-nucleus genotyping and transcriptomic methods to analyze surgically resected cortical samples from FCDII patients. Mutations were detected in different cell types, including glutamatergic neurons and astrocytes, with a small proportion of mutant cells exhibiting giant cell features. In addition, cell type-specific transcriptional abnormalities involving synaptic function and neurodevelopmentally related pathways that may be associated with epileptogenesis were identified in both mutant and non-mutant FCDII cells, whereas disruptions in mitochondrial metabolic pathways were observed in giant cells. Taken together, these findings reveal cell-autonomous and non-cell-autonomous features in FCDII, providing a potential basis for future precision medicine therapies.

Findings of the Study

• Cell type-specific dysregulation of synaptic and neurodevelopmental pathways in FCDII

The aim of this chapter of the study was to analyze cell type-specific transcriptional abnormalities in a homogeneous set of focal cortical dysplasia type II tissue samples by multiple transcriptomic approaches to reveal epilepsy-related changes and mTOR pathway-driven lesions and to search for potential disease markers. The researchers performed single-nucleus RNA sequencing of frozen cortical tissue from 10 pediatric patients who underwent surgery for drug-refractory focal epilepsy and included three age-matched autopsy controls without epilepsy as well as eight neurologically normal controls from a previous study. Patients carried somatic mutations in the MTOR, RHEB, or DEPDC5 genes with mutant allele frequencies (VAF) ranging from 3% to 17%. Seven major brain cell types were identified by UMAP downscaling and cytomarker gene analysis: glutamatergic neurons (GluNs), GABAergic interneurons (IN-MGE/CGE), astrocytes, oligodendrocytes, oligodendrocyte precursor cells (OPCs), microglia, and endothelial cells. Although no new cell clusters common to all patients were identified, some FCDII patients were enriched in glutamatergic neurons. Further analysis revealed significant differences in differentially expressed genes (DEGs) across cell types, with glutamatergic neurons and astrocytes being the most significantly affected and GABAergic interneurons the least altered.GO enrichment analyses indicated that these DEGs were involved in pathways such as neurodevelopment, synaptic function, and neurotransmitter transmission, demonstrating a strong cell-type-specific pathological mechanism. For example, down-regulation of GRIN1 and PRRT2 and up-regulation of GLUL were observed in glutamatergic neurons, suggesting abnormal glutamatergic signaling in this type of neurons. Overall, these results reveal cell type-specific molecular regulatory abnormalities in FCDII epileptic tissues that may reflect or contribute to neural circuit dysfunction and seizures.

• Cell type-specific transcriptional dysregulation in FCDII mutant cells

To identify cell-autonomous changes in gene expression pathways caused by mTOR-activating mutations, the authors compared mutant nuclei in patient samples with nuclei in which only the reference allele was detected. The analysis was limited to cell lineages containing at least 10 mutated cells: glutamatergic neurons, astrocytes, and oligodendrocytes. Fourteen transcripts were part of the mTOR pathway in the KEGG database. Glutamatergic neurons exhibited the largest number of dysregulated genes, however, GO functional enrichment analysis revealed statistically significant biological differences between cell types. In both mutant and reference nuclei, the enriched GO entries were mainly involved in mitochondrial function and structure, metabolism, respiration, and cytokinesis. Similarly, enrichment of GO entries related to mitochondrial metabolism and transport was observed in mutant astrocytes, whereas mutant oligodendrocytes showed no significant GO enrichment. These results suggest the presence of cell type-specific metabolic pathway abnormalities in mutant neurons and astrocytes. Further findings that the expression levels of these dysregulated genes were similar between reference nuclei from patients (i.e., non-mutant cells) and controls suggest that these transcriptional changes are specific to mutant cells and are consistent between individuals, despite the limited number of nuclei analyzed. In addition, it has been reported that in the FCDII mouse model, non-mutant neurons neighboring mutant cells may also show enhanced excitability due to non-cell-autonomous mechanisms. To explore the potential effects of mutant cells on their surroundings, non-mutant glutamatergic neurons (Ref. GluNs) in patient samples were compared with GluNs in controls. The results showed that the expression of a number of genes involved in the developmental regulation of synaptic transmission and neuronal projections was upregulated; approximately 90% of these genes also showed upregulation in the comparison between mutant GluNs and control nuclei. This finding suggests that changes in synaptic pathways are widespread in mutant and non-mutant neurons. Similar non-cell-autonomous effects were also observed in glial cells. The authors' study reveals the presence of two key processes in FCDII: cell-autonomous mechanisms occurring in mutant cells that affect mitochondrial function; and broader changes at the level of the loop that affect the function of mutant and non-mutant neurons.

• DNs and BCs belong to the glutamatergic neuron and astrocyte spectrum and exhibit metabolic dysregulation

In this chapter, we synthesized various methods such as laser capture microdissection transcriptome sequencing (LCM-seq), Visium spatial transcriptomics and MERSCOPE high-resolution targeted gene analysis to systematically reveal the molecular molecular characteristics of aberrant neurons (DNs) and Balloon Cells (BCs) in FCDII. Characterization It was found that DNs were similar to GluNs at the molecular level, expressing genes related to neurotransmitter transport, such as NEFM and SNAP25, and enriched in genes related to mitochondrial function (e.g., VDAC1, COX7A), and also exhibited up-regulation of apoptosis-related genes; whereas BCs were more similar to astrocytes, expressing glial markers, such as VIM, CRYAB, and enriched in vesicle organization and cell migration related genes. Spatial distribution analysis showed that DNs were mainly distributed in glutamatergic neuron regions in each cortical layer, while BCs were diffusely distributed, similar to astrocyte pattern. Further integration of multi-omics data and validation with immunofluorescence and electron microscopy revealed that there were increased mitochondrial numbers and structural abnormalities (e.g., swelling and vacuolization) in DNs, suggesting that mitochondrial dysfunction due to over-activation of the mTOR signaling pathway may be an important molecular mechanism of FCDII. These results provide new perspectives for understanding the molecular identity of DNs and BCs and their roles in disease development.

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Reference

1. Baldassari, Sara, et al. "Single-cell genotyping and transcriptomic profiling of mosaic focal cortical dysplasia." Nature Neuroscience (2025): 1-9. Distributed under Open Access license CC BY 4.0, without modification.