Building the brain mosaic: an expanded view.

The complexity of the brain is closely tied to its nature as a genetic mosaic, wherein each cell is distinguished by a unique constellation of somatic variants that contribute to functional and phenotypic diversity. Postzygotic variation arising during neurogenesis is recognized as a key contributor to brain mosaicism; however, recent advances have broadened our understanding to include sources of neural genomic diversity that develop throughout the entire lifespan, from embryogenesis through aging. Moving beyond the traditional confines of neurodevelopment, in this review, we delve into the complex mechanisms that enable various origins of brain mosaicism.

Loss of Slc35a2 alters development of the mouse cerebral cortex.

Brain somatic variants in SLC35A2 are associated with clinically drug-resistant epilepsy and developmental brain malformations, including mild malformation of cortical development with oligodendroglial hyperplasia in epilepsy (MOGHE). SLC35A2 encodes a uridine diphosphate galactose translocator that is essential for protein glycosylation; however, the neurodevelopmental mechanisms by which SLC35A2 disruption leads to clinical and histopathological features remain unspecified. We hypothesized that focal knockout (KO) or knockdown (KD) of Slc35a2 in the developing mouse cortex would disrupt cerebral cortical development through altered neuronal migration and cause changes in network excitability. We used in utero electroporation (IUE) to introduce CRISPR/Cas9 and targeted guide RNAs or short-hairpin RNAs to achieve Slc35a2 KO or KD, respectively, during early corticogenesis. Following Slc35a2 KO or KD, we observed disrupted radial migration of transfected neurons evidenced by heterotopic cells located in lower cortical layers and in the sub-cortical white matter. Slc35a2 KO in neurons did not induce changes in oligodendrocyte number, suggesting that the oligodendroglial hyperplasia observed in MOGHE originates from distinct cell autonomous effects. Spontaneous seizures were not observed, but intracranial EEG recordings after focal KO showed a reduced seizure threshold following pentylenetetrazol injection. These results demonstrate that Slc35a2 KO or KD in vivo disrupts corticogenesis through altered neuronal migration.

RAS pathway: The new frontier of brain mosaicism in epilepsy.

As cells divide during development, errors in DNA replication and repair lead to somatic mosaicism - a phenomenon in which different cell lineages harbor unique constellations of genetic variants. Over the past decade, somatic variants that disrupt mTOR signaling, protein glycosylation, and other functions during brain development have been linked to cortical malformations and focal epilepsy. More recently, emerging evidence points to a role for Ras pathway mosaicism in epilepsy. The Ras family of proteins is a critical driver of MAPK signaling. Disruption of the Ras pathway is most known for its association with tumorigenesis; however, developmental disorders known as RASopathies commonly have a neurological component that sometimes includes epilepsy, offering evidence for Ras involvement in brain development and epileptogenesis. Brain somatic variants affecting the Ras pathway (e.g., KRAS, PTPN11, BRAF) are now strongly associated with focal epilepsy through genotype-phenotype association studies as well as mechanistic evidence. This review summarizes the Ras pathway and its involvement in epilepsy and neurodevelopmental disorders, focusing on new evidence regarding Ras pathway mosaicism and the potential future clinical implications.

Molecular and spatial heterogeneity of microglia in Rasmussen encephalitis.

Rasmussen encephalitis (RE) is a rare childhood neurological disease characterized by progressive unilateral loss of function, hemispheric atrophy and drug-resistant epilepsy. Affected brain tissue shows signs of infiltrating cytotoxic T-cells, microglial activation, and neuronal death, implicating an inflammatory disease process. Recent studies have identified molecular correlates of inflammation in RE, but cell-type-specific mechanisms remain unclear. We used single-nucleus RNA-sequencing (snRNA-seq) to assess gene expression across multiple cell types in brain tissue resected from two children with RE. We found transcriptionally distinct microglial populations enriched in RE compared to two age-matched individuals with unaffected brain tissue and two individuals with Type I focal cortical dysplasia (FCD). Specifically, microglia in RE tissues demonstrated increased expression of genes associated with cytokine signaling, interferon-mediated pathways, and T-cell activation. We extended these findings using spatial proteomic analysis of tissue from four surgical resections to examine expression profiles of microglia within their pathological context. Microglia that were spatially aggregated into nodules had increased expression of dynamic immune regulatory markers (PD-L1, CD14, CD11c), T-cell activation markers (CD40, CD80) and were physically located near distinct CD4+ and CD8+ lymphocyte populations. These findings help elucidate the complex immune microenvironment of RE.